IMMUNODIAGNOSIS OF HUMAN FILARIASIS BASED ON ANTIGEN

DETECTION

DISSERTATION SUOMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

Muittv of ^dtloiB^opl^p IN

BIOTECHNOLOGY

B7

HUMA MUSTAFA M.$c. (Biofch.)

Dated

Approved:

Saad Tayyab [Supervisor)

INTERDISCIPLINARY BIOTECHNOLOGY UNIT ALIGARH M U S U M UNIVERSITY

ALIGARH (INDIA)

1997

DS2978

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^^X irrsrsr, ^t'Z g iw T» 173

^mn^i — 226 001 ( HfT^ )

CENTRAL DRUG RESEARCH INSTITUTE

Cbattar Manzil, Post Box No. 173

Lu€knovv-22600I (INDIA)

JT o /No. r^^r^/Datc

C E I I T I F I C A . T E

r/ i is i s to certify that the work embodied in this thesis entitled

"ImmuBodiagnosis of human filariasis based on antigen detection" has been

carried out by Ms Huma Mustafa, M.Sc.(Biotechnology) under my supervision.

She has fulfilled the requirements of the Aligarh Muslim University

regarding the prescribed period of invesigational work for the award of M.

Phil, degree in Biotechnology.

The work included in this thesis is original unless stated otherwise,

and has not been submitted for any other degree.

bKj)uuyirKj^JL^

S c i e n t i s t E-1 Divis ion of Biochemistry

fiR : ^:jii l^^ : 053J-286 '^^ ' 91-0522-243405 "^ • 232411-18 'Tt.iT.«rl.rT . l » l » r i » : CENDRUG Ttlix ; Fi« ; r k m ; P. A. B. X.

INTERDISCIPLINARY BIOTECHNOLOGY UNIT A L I G A R H M U S L I M UNIVERSITY. ALIGARH 202 002 (INDIA)

CERTIFICATE

This is to certify that the work embodied in this dissertation entitled

- iMMUNODIAGNOSIS OF HUMAN FUMRIASJS BASED ON ANTIGEN

DETECnON*'^is an original work carried out by Ms. Huma Mustafa^, under the

guidance of Dr. (Mrs.) Nuzhat A. Kaushal, Assistant Director, Division of Biochemistry,

Central Drug Research Institute, Lucknow, far the award of the Degree of Master of

Philosophy in Biotechnology.

Ms. Huma Mustt^a was registered for her M. Phil Degree in Biotechnology in

0K InierKAsdj^inary Biotechnology Unit, Aligarh Muslim University, Aligarh wuier my and

Dr. (Mrs.) Nuzhat A. Kaushal's joint supervision. The candidate has fiilfilled all the

requirements of the Ahgqrh Muslim University, Aligarh fiir the award of the degree of

Master K^ Philosof^ in Biotechnology.

%

Dr. SAAD TAYYAB Lecturer, Interdisciplinary Biotechnology Unit Aligarh Muslim University, Aligarh.

Telephone : (0571)401718 Telex : 564—230 AMU IN Telegram : BIOTECHNOLOGY. UNIVERSITY, ALIGARH

TO MUMMY

WITH LOVE

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dkamm am OJM to mo- \xm- (mmnmA, 3^. ill. III. Xhwn, lumu, (AmumfKi,

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to mo/ mAMUA iW ImA uymmnamO' a/ruL amdmwm wMimiA mxmm/ rrw to axxmnmm

i/uAf aAwywaiimh.

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'lie amd dnduAinial mmiAm, lum- Solm, (A OAMMU' aAmmmamcL.

(HUMA MUSTAFA)

(i)

CONTENTS

Page No.

Acknowledgement (1) Abbreviations (ii. - iii)

Preface (iv - V )

CHAPTER 1 Introduction and Review of Literature 1 -•24

CHAPTER II Materials and Methods 25-•34

CHAPTER III Results and Discussion 35-•69

CHAPTER IV Summary and Conclusions 70--74

Bibliography 75--92

ABBREVIATIONS

BBS PME BSA CIE CUE CNBr DMEM EDTA ELISA E-S HCl HEPES

H2O2 H2SO4 HRP h lEP I^Cr I M niM min ml

Ml MoAb N NBS ng NHS O.D. OPD PBS PBS-Tween

PC rpni

Borate buffered saline Beta-mercaptoethannl Bovine serum albumin Cross Immunoelectrophoresis Crossed line Immunoelectrophoresis Cyanogen Bromide Dulbecco 's modified eagle medium Ethylene diamine tetra acetic acid Enzyme-linked immunosorbent assay Excretory-secretory Hydrochloric acid Hydroxyl ethyl piperazine-N '-2-ethane sulphonic acid Hydrogen peroxide Sulphuric acid Horse radish peroxidase Hour Immunoelectrophoresis Immunoglobulin G Litre Molarity Mi Hi Molar Minute Millilitre Microlitre Monoclonal antibody Normality Normal buffalo serum Nanograms Normal human serum Optical Density Orthophenylene diamine Phosphate buffered saline Phosphate buffered saline containing Tween-20 Phosphorylcholine revolution per minute

i i ) ( i i )

ScES Setaria cervi excretory secretory antigens SDS Sodium dodecyl sulphate SDS-PAGE : SDS-polyacylamide gel electrophoresis tCIE : Tandem crossed Immunoelectrophoresis TEhAED : NNN^N^ Tetra methyl ethylene diamine V : Voltage

( i l l )

PREFACE

Filariasis is a major parasitic disease found in the tropical and

subtropical regions of the world. The major filarial infection in India is

due to Wuchereria bancrofti. Every third person in India faces the risk

of exposure to filariasis. The disease causes severe physical discomfort,

morbidity, loss of man-hours and even social and psychological

disturbances.

A National Filaria Control Programme (NFCP) was launched in

India in 1966. It had two primary objectives: one was to carry out

surveys to determine the extent of prevalence offilariases and the other

was to undertake large-scale pilot studies to evaluate the known

methods of its control. Unfortunately, the principle vector of

bancroftian filariases (Culex quinquifasciatus) is predominantly

resistant to chlorinated hydrocarbons. Apart from this the diagnosis of

filariasis also poses a problem. An unequivocal diagnosis of filarial

infection is still based on the detection of the circulating microfilariae

in the blood. In early infections, when the circulating microfilariae are

few, one could miss detecting the parasite by the above method. This

led to the use of immunological methods for the diagnosis of filariasis.

The antibody based diagnosis shows extensive cross-reactivity.

Therefore, the immunodiagnostic method based on antigen detection

(Iv)

could provide an effective and active diagnosis of filanasis, has been

used in the recent past

The work reported in this dissertation is oriented towards the

immunological studies on the excretory-secretory (E-S) products of the

Setaria cervi (a bovine filarial parasite) which is closely related to the

human filarial parasites. The study deals with the preparation and

characterization of S^ cervi ES products, raising of polyvalent

hyperimmune rabbit sera, quantitation of parasite specific antibodies

and the evaluation of immune sera for the detection of filarial

circulating antigen in patient sera. These studies also provides useful

information about the protein and antigenic make-up of S^ cervi E-S

products. Thus the information obtained from the present studies may

be regarded as baseline and may further be explored to identify and

isolate the relevant antigen equivalent to the filarial circulating

antigen.

(v)

CHAPTER I

INTRODUCTION AND

REVIEW OF LITERATURE

Filariasis is a chronic disabilitating disease affecting more than 600 milh'on

people in the world (WHO, 1987). The disease is prevalent mainly in the tropical

and subtropical countries, and causes a serious health problem. Although,

filariasis is not fatal but it carries a social stigma and causes considerable

morbidity. In India, the incidence of the disease has increased to alarming

proportions due to the natural increase in population of the endemic areas and

migration of the population as a result of improvement in communication, increase

in urbanization and industrialization.

Filariasis in man is caused by the three species Wiichereha hancwfti, Burgia

nialayi, and B. thmoh. About 90% of the infection is caused by W. hancrofti.

Besides these, the filarial species infecting animals and birds are Bnigia pahangi

(cats), Dirofilaria immitis (dogs), Setaria cervi, S. digitata (catties), S. equina

(horse), Acanthocheilonema vitae (rodents), Litomosoides carinii (cotton rat) and

Chandlerella hawkingii (Jungle crow).

The life cycle of the filarial parasite is complex and an intenriediary host

(i.e. mosquito) is necessary to complete their development (Fig. 1). The most

widely distributed vectors are species of Culex, Anopholese, Aedes and Mansoni.

The adult filarial worms are fine fihform found in the lymphatic vessels and glands.

The females (6.5-10 cm x 0.2-2.8 mm) are longer than the males (4 cm x 0.1 mm).

The female has a tapering anterior end with a slight rounded swelling. The

reproductive activity of the adults leads to the release of microfilariae (mf)

which are characteristically present in circulation.

Vi<i,. 1: Sclieinntic life cycle ofniarinl parnsitc

The microfilariae exhibits nocturnal periodicity i.e. they are present in the

peripheral blood in greater numbers during the night as compared to the day. The

maximum concentration is during 22.00 to 02.00 hr. When the mosquito bites an

infected individual, the salivary secretion causes the concentration of

microfilariae near the site of bite (Strong et ai, 1934). In the vector, the

microfilariae undergoes metamorphosis and reaches the mature infective larval

stage. Wlien this infected mosquito bites another person, the larvae breaks free

from the labium and penetrates through the skin thus causing infection. The

infective larvae passes through the peripheral blood vessels to the lymphatics

where they become mature in an estimated period of three months to a year.

In most cases of filarial infection the parasite does not excersise any

manifestation. There are 70% microfilariamic cases which does not show any

clinical and pathological sign of filarial infection. The pathological changes in

W. hancrofti and B. malayi are related to the developmental and adult stages of the

wonns, whereas, in tropical pulmonary eosinophilia they are caused by the

microfilariae. The pathological changes in the lymphatics and lymph glands are the

result of an immunological reaction by the host. The manifestation of the disease

are hydrocoele, elephantiasis, tropical eosinophilia and chyluria.

The effective control of filariasis depends on early and specific diagnosis of

filariasis. The certain diagnosis of filarial infection can be made only by

demonstrating the presence of microfilariae in the night blood smears of the

patient. However, this method is very inconvenient to both the patient as well as

the investigator and it often fails to detect the disease in the very early stage when

the microfilariae are sparse and the late stages of the disease when they are absent

or sequestered inside the tissues.

This has put forward more emphasis for the development of immuno-

diagnostic tests. The immunodiagnosis of filariasis is one of the major challenges

(Kagan, 1981). The earlier studies on immunodiagnosis of filariasis were based on

antibody detection. But, the antibody based diagnosis showed two major

drawbacks, firstly, it shows cross-reactivity with other helminth parasites and

secondly, it cannot differentiate the past exposure fi"om the current active infection

(Ambroise-Thomas, 1974).

The detection of antigen in biological fluids such as serum or urine has been

suggested to be a better approach as it gives definitive diagnosis for detection of

filarial infection. In most of the previous studies, the antigen based

immunological test, employing polyclonal antibodies raised against crude somatic

extracts, could not provide specific diagnosis due to the extensive antigenic

cross-reactivity among nematode parasites.

Specific antibodies against defined antigens are required for developing

antigen detection assays. Excretory-secretory (E-S) products are known to be less

complex in nature and more specific in defining infection as compared to the

somatic antigens/extracts. Therefore, the study was undertaken for the

characterization of the E-S antigens and the evaluation of the antibodies against

these E-S antigens which could provide better reagents for developing

immunodiagnostic test.

REVIEW OF LITERATURE

The definitive diagnosis of filarial infections in humans solely depends on an

assessment of the patient's clinical signs and symptoms in addition to microscopic

conformation for the presence of microfilariae (mf) circulating in peripheral blood

{W. hancroffi, B. malayi) or dwelling in the skin {O. volvulus) (Sasa, 1978). The

microfilariae of W. bancrofti and B. malayi shows nocturnal periodicity. However,

this method is inconvenient and fails to detect the disease when mf are sparse or

sequestered in the tissues or in inaccessible sites. This method is also

insensitive because of the well recognized phenomenon of filariasis without

microfilariae (Beaver, 1970). The parasitological detection of microfilariae is

found to be relatively insensitive, giving emphasis to the development of

immunodiagnostic techniques for filariasis.

I. IMMUNODIAGNOSIS

Immunodiagnosis of filariasis is one of the important area in filarial

immuno-logy (Kagan, 1963; 1981; Ottesen, 1984; Taylor and Denham, 1986). A

number of immunodiagnostic tests have been developed for human and animal

filarial infections (Ambroise-Thomas, 1974). Immunological methods such as

immimodiffi-ision, complement fixation, counter current immunoelectro-phoresis,

heamagglutination test, immunoflouresence, enzyme-linked immuno-sorbent

assay, immunoradiometric assay, skin test etc., have been explored for the

diagnosis of filariasis. A pracfical and sensitive assay would improve under­

standing of the epidemology of filariasis and would be usefiil for monitoring the

success of vector control efforts and drug trials (WHO, 1984).

A. Antibody Detection

A number of immunological tests based on gel diffusion (Petithory et ai,

1972; KhaXoon e( al., 1987), immunoelectrophoresis (Capron e/o/., 1968; 1970),

counter current immunoelectrophoresis (Desowitz and Una, 1976; Weil et ai,

1984; 1986; Dasgupta et al., 1980), indirect haemagglutination test (Takahashi and

Sato, 1976; Kaliraj et al., 1981a;b; Das et al., 1987), indirect immunoflourescent

antibody test (Diesfeld e a/., 1973; Forsyth er a/., 1985; Kaliraj, e( a/., I98lc;

Das et al., 1987; Dissanayake et al., 1984) and the hypersensitivity skin reaction

(Swada et al., 1969; 1975; Swada and Sato, 1969; Gupta and Ansari, 1989) have

been used for detecting filarial antibodies employing antigens from the

homologous and heterologous filarial parasites.

The skin test has earlier been used for the diagnosis of filarial infecfion by

detecting antibodies in human patient sera. The test employed by researchers has

been the intracutaneous injection of antigen in the arms of the individuals. The

response is of immediate type and is usually read within 15 to 30 min. Twenty four

hours or delayed reaction has also been noted. According to Wharton (1947) many

of the reports of delayed reaction which was due to the use of antigen in very high

concentrations, were in reality immediate skin reactions which persisted and

produced exacerbation of symptoms in the infected individuals. False positive

reactions have been attributed to cross-reaction with intestinal helminths

(Bozicevich and Hutter, 1944; King, 1944; Hunter, 1958). Filarial antigens have

been used for skin test reactivity in people infected with hookworms and

strongyloides (Woodruff et al., 1958). A number of workers reported that the

skin test is negative in some cases of elephantiasis, chyluria and hydrocoele

(Frank ef c//., 1947). Chandra e/cr/., 1974) developed a filaria skin test kit using

B. malayi infective larval antigen for antibody detection. The test has been

evaluated for its sensitivity and specificity in the immunodiagnosis of filariasis

(antibody based) covering over 2500 cases from filaria endemic and non-endemic

regions of India (Katiyar et al., 1985 and Sircar et ai, 1990). The major limitation

of this kit was that the antigen has to be procurred from the mosquito and it showed

cross-reactivity with other helminth parasites. Acton and Rao (1933) used

hydrocoele fluid as antigen in skin test for the diagnosis of human filariasis.

Complement fixation, a widely employed test (Kagan, 1963) has been shown

to be more sensitive than skin test for the diagnosis of filarial infection. The test

showed fairly high sensitivity with sera from individuals with Loa loa but was

less sensitive with sera from individuals with W. bancrofti and other human filarial

infections (Scofield, 1957). Cross-reactions were also reported against

ancylostomiasis, schistosomiasis and stronglyoidiasis and in infection with other

intestinal parasites (Stemplen, 1944).

The occurrance of precipitating antibody in the senim of humans and

animals infected with filarial worms have been reported by several investigators

(Biguet et ai, 1962; Desowitz and Una, 1976; Kliatoon et ai, 1987). The precipitin

test has limited use in the diagnosis of the filarial infection. The antigen employed

were the saline extracts of the whole worms and the results varied considerably.

The precipitin test showed a sensitivity equivalent to that of complement fixation

and skin test by Culbertson et al (1944). The precipitin test was found to be less

sensitive than the skin test for the diagnosis of Mansonella ozzardi (Biagi, 1956).

Ellsworth and Johnson (1973) observed precipitate formation when sera of

Diwfilaria immitis infected dogs were reacted with a crude antigen extract from

adult heart worms by diffusion capillary tubes. Whereas, Khatoon, et ai (1987)

used a simple and reliable test for the diagnosis of Setaria cervi by gel dififiision

method. The sensitivity and reactivity of the precipitin test are so much lower

than other conventional serologic tests that its use for routine diagnosis can be

hardly recommended.

The technique of immunoelectrophoresis (lEP) has been used by Wheeling

and Hutchinson (1971) for tentative diagnosis of human filariasis employing

antigens prepared from D. immitis microfilariae and adults. Kaeuffer et al. (1974),

however, did not detect any positive reactions when the sera of 14 Tahitians

infected with W. bancrofti were tested in lEP with adult D. immitis antigen. lEP

has also been successfiiUy employed for the diagnosis of onchocerciasis with

antigen extracted from aduU worms of OA7c/7ocerca (Biguet e/a/., 1962; 1964;

Capron e? ai, 1968; 1970). By employing this technique of lEP, Gentilini et al.

(1973) obtained positive reactions with the sera of 8 out of 9 patients of

onchocerciasis but no precipitin bands were observed when the sera of 3

patients with bancroftian filariasis were tested against the Onchocerca volvulus

antigen. The average number of arcs produced are considered by D'Haussy et al.

(1972) to be related to the clinical severity of the onchocercal infection.

The specificity of these precipitin reactions is not known to any degree of

certainty, whereas, Wheeling and Hutchinson (1971) obtained a positive reaction to

D. immitis antigen in a case of human filariasis, while the results were entirely

negative to the same antigen, i.e., D. immitis, in the series of Tahitian patients with

W. bancrofti microfilaraemia as investigated by Kauffer et al, (1974). The

possibility of broad spectrum of antigenic specificity was indicated by the

investigation of Niel et al, (1972) who found that the antigens prepared from

Setaria labiato-papillosa and Dipetalonema vitae produced precipitin arcs, by

double diffusion and lEP, when reacted against the serum of patients with loiasis,

onchocerciasis, bancroftian filariasis and dracuncuhasis. These authors have also

reported positive precipitin reactions with an antigen of Ascaris suum and the sera

from the filarial patients. However, since intestinal helminthiasis is usually highly

prevalent in areas of endemic filariasis, it is difficult to determine whether the

positive heterologous precipitin reaction represents a common antigen relationship

or a reaction with A. suum antibody.

Counter current immunoelectrophoresis (CIEP) was first used by Desowitz

and Una (1976) for the detection of filarial antibodies using D. immitis antigen

and seems to be fairly sensitive. CIEP has the advantage of simplicity of

performance, rapidity and the capability of processing relatively large numbers of

serum samples at the same time.

The indirect haemagglutination test (IHAT) for the diagnosis of filarial

antibody have also been employed. Jung and Harris (1960) used a haemagglu­

tination procedure for the detection of human filarial infection by using somatic

antigen from D. immitis adult wonns. Indirect haemagglutination test (IHAT)

employing heterologus antigen (D. immitis) showed apparent sensitivity in the

diagnosis of filariasis due to Achanthocheilonema perstans (Kagan et al., 1963)

and W. bancrofti (Fujita et al., 1970), but also gave false positive reactions with

sera from normal individuals as well as other helminth infections.

The indirect flourescent antibody test (IFAT) employing an adult filarial

worm antigen of ^. vitae (Ambroise-Thomas and Kein Trong, 1974) and S. digitata

(Tan et al., 1988) or sonicated W. bancrofti microfilarial and larval antigens (Hedge

and Ridley, 1977; Kaliraj et al., 1979b; 1981c; Das et al., 1987; 1988c) has

been employed for the immunodiagnosis of filariasis. However, large scale

iiTimuno-flourescence testing is tedious and needs special equipments.

The enzyme-linked immunosorbent assay (ELISA) was found to be

simple, sensitive and suitable for mass screening of most parasitic infections (VoUer

et ai, 1974; 1975a; 1975b; 1976a,b; Kaliraj et ai, 1981b,c). Since its

development (Engvall and Pearlman, 1972) ELISA has been successfully used

for detecting antibodies and antigens of a variety of organisms (Sever and

Madden, 1977).

In recent years, ELISA had been found very useful in the diagnosis of

many parasitic diseases including filariasis (VoUer et ai, 1976a; Bartlett et ai,

1975; Bartlett and Bidwell, 1976; Barakat et ai, 1983; Gueglio et ai, 1995). Both

homologous and heterologous antigens were used for measuring antibody response

in filarial infection (Ruitenberg et ai, 1975; Spencer et ai, 1981). Bartlett et al

(1975; 1976) have used a micro ELISA for the detection of antibody in Onchocerca

volvulus and found that contaminants of host origin are present in homologous

antigen reacted non-specifically with the conjugate. However, no such problem

was encountered while using the same antigen after purification (Marcoullis et ai,

1978). ELISA employing W. bancrofti mf antigen and mf excretory-secretory (mf

E-S) antigen was used for the detection of filariasis by a number of workers (Kaliraj

etai, 1981b; 1981c; Kharat e/cr/., 1982; Malhotra e/ ai, 1982; Malhotra and

Harinath, 1984; Harinath et ai, 1984; 1986; Dissanayake and Ismail, 1980a) and

has been found to have better sensitivity than night blood examination. Furthe;r,

human urinary filarial antigen and hydrocoele filarial antigen from W. bancrofti

infected individuals were also used for the diagnosis, and the purified antigens

showed high sensitivity and specificity when tested in sandwich ELISA (sELISA)

10

(Malhotra et ai, 1985a,b; Singh et al., 1993; Ramaprasad and Harinath, 1995)

and stick enzyme immunoassay (Ramaprasad and Harinath, 1989) for the detection

of filarial antibody in the sera of filarial patients. Besides, the use of homologous

antigens, heterologous antigens were also used by many workers for the

diagnosis of filariasis, i.e., B. malayi (Spencer et al., 1981; Cheirmaraj et al., 1990;

Kumar and Sarthaman, 1990, Chanteau et al., 1991, Yuan et al., 1992; Li et al,

1993), D. immitis (Weil et al., 1985; 1987) and . . digitata (John et al., 1995; Dhas

and Raj, 1995). The lack of acceptable specificity and sensitivity of antibody

detection assays in the serodiagnosis of filarial infections has led to the development

of anfigen based diagnosis. (Dissanayake and Ismail, 1987).

B. Antigen Detection

Although, antibody based immunodiagnosis has been usefiil in defining

humoral immune responses during various stages of filarial infections but could not

correlate with the concurrent levels of microfilariae in the blood (Ottesen et al.,

1982). The major shortcomings of the antibody detection are firstly, due to the

extensive antigenic cross-reactivity among different nematode parasites (Oliver-

Gongalez and Morales, 1945; Kagan, 1963), and secondly, the inability to

distinguish the past exposure firom the current infection (Ottesen et al., 1982, Weil

et al., 1987). Detecdon of antigen has been shown to be more useftil than antibody

detecfion for the diagnosis of active parasitic infecfions (Hamilton, 1985).

It has been reported by several workers that the parasite antigen detection is

more sensitive and specific for diagnosing active filarial infection than clinical

examinafion or detection of mf in peripheral blood (Harinath et al., 1984; Forsyth et

al., 1985; Weil et al., 1985; 1987; Lai et al., 1987; Anon, 1989). The evidence for

11

the presence of circulating antigen was first demonstrated in bancroftian filariasis

in 1946 by passive cutaneous anaphylaxis (Frank, 1946). The filarial antigen

obtained fi"om infected patients have been employed in immunological test for the

detection of circulafing filarial anfigen in parasific infections (Kaliraj et al., 1979a;

Forsyth et a/., 1985; Weil et al., 1986; Weil, 1988, 1990). After that considerable

emphasis has been given to the detection of circulating antigen in the blood and

other body fluids of filarial patients (WHO, 1992; Weil, 1990). In most of the

earlier attempts, methods employing the antisera raised against somatic extracts of

the heterologous filarial parasites, i.e., L carinii (Dasgupta and Bala, 1978,

Kaliraj et al., 1981a), D. inimitis (Tanabe, 1959; Weil et al., 1984; 1986) and

Setaria cervi (Gupta and Ghosh, 1984; Tandon et al., 1983; Kaushal, 1995) have

been used for the detection of filarial antigen in sera as well as in urine of infected

humans and animals.

The ClEP was used for the detection of circulating antigen using rabbit anfi-

filarial sera (Kaliraj e/o/., 1979a; Dasgupta e a/., 1980; 1984; Das et al., 1988c;

Kumar et a/., 1991; Dumenigo e/a/., 1993). Antiserum raised against L. carinii

antigen in rabbit was used for the detection of antigen in human filarial sera and it

was observed that 5.7% of cases diagnosed as bancroftian filariasis by parasito-

logical examination can be increased to 62.8% by CIEP (Dasgupta et al., 1980).

The CIEP detecfion of D./mm/Y/5 anfigenemia represents a significant improve­

ment over previously available diagnostic techniques because it is more sensitive

than microfilarial test and can be related to the active infection (Kaliraj et al.,

1981a; Weil et al., 1984; 1986) and can be used as pracfical diagnosfic test for

active W. bancrofti infection (Weil et al., 1986). The simplicity of CIEP using anfi

/.. carinii iiyperimmune senim, rapidity in obtaining results, comparability with

12

IHAT, and increased sensitivity over immunodiffusion for the detection of

circulating antigen have been estabhshed in various studies (Krupp, 1974: Dasgiipta

and Bala, 1978; 1980; Shariff and Parija, 1991). Therefore, CIEP may be useful

in the demonstration of circulating antigen in filarial patient sera.

The circulating antigen has also been demonstrated in W. hancrofti infected

individuals (Reddy et al., 1984a,b; Zheng et al., 1987b; Lutsch ei al., 1987; 1988;

Ramaprasad and Harinath, 1995) and also in patient infected with onchocer­

ciasis (Ouaissi et al., 1981; Des Montis e( al., 1983; Petralanda ct al., 1988;

Schlie-Guzman and Rivas Alcala, 1989; Chandrashekhar ct al., 1990) using a

variety of polyclonal and monoclonal antibody. Detection of filarial antigen

with specific rabbit sera, raised against somatic extract of IV. hancrofti

microfilariae, was possible in few cases of microfilaremic positive patient sera

(Kaliraj ct al., 1979a). Presence of circulating antigens in the filarial infected

individuals has further been shown by a number of workers (Au ct al., 1981;

Dissanayake ct al., 1982; 1984; Lambert ct al., 1978; Verroust ct al., 1979;

Karavodin& Ash, 1980; Paranjape e/o/., 1986; Weil and Liftis, 1987; Dasgupta

ctal., 1978; 1984).

Due to the non-availability of adult W. hancrofti antigen the use of antigens

fiom heterologous filarial parasites to raise polyclonal antibodies in mice and

rabbits and monoclonal antibodies for the detection of circulating antigens in

filarial infection has been employed by several workers (Dasgupta ct al., 1980,

1984; More and Copeman, 1990; 1991; Kumar et al., 1991; Jaoko, 1995; Forsyth

ct al., 1985; Weil et al., 1984, 1985, 1986; Maizels et al., 1983; Phillip et al.,

1984a; Cabrera and Parkhouse, 1986; Egwang and Kazura, 1987; Prasad and

Harinath, 1988; Rajasekhariah ct al., 1991; John ct al., 1995). Besides these,

13

materials from the microfilariae and infective larvae of W. hancrofti and of related

filarial parasites were also used by a number of researchers to produced antisera

for the detection of filarial circulating antigens in pafient sera (Almeida et al.,

1990;Reddy et o/., 1984b,c; Lutsch e/a/., 1987; Cheirmaraj e/o/., 1990; 1992;

More and Coperman, 1991; Dumenigo et al., 1993; Zheng et al 1987; Lai 1991, Li

ct al 1993;Chenthamarakshane/a/., 1995; 1996).

In the above studies the problems of specificity and sensitivity were observed

due to the use of polyclonal anUbodies raised against somatic antigens, for the

detection of circulating filarial antigen. Therefore, monoclonal antibodies have

been employed in ELISA by several investigators for the detection of circulating

antigen in the filarial infected individuals (Weil et al., 1985; 1987; Lai et al., 1987;

Weil and Liftis, 1987; Zheng et al., 1987; Santhanam et al., 1989; Li et al., 1993;

Ramaprasad and Harinath, 1995). Antigen detection have also been developed for

the diagnosis of onchocerciasis (Cabrera and Parkliouse, 1987; More and Copeman,

1990; 1991) and was found to be highly sensitive and specific. Monoclonal

antibody based sandwich ELISA (sELISA) has also been used for the detecfion of

circulating filarial antigen using rabbit anti-microfilarial immuno-globulin as first

antibody and monoclonal antibodies, against W. bancrofti mf E-S antigen and B.

malayi L3 antigen, as second antibody, the positivity rates were 94.5 and 89.0%

respectively in microfilareamic patients. (Zheng et al., 1987a;b; 1990). An

immunoradiometric assay (IRMA) for the detection of filarial antigen was

developed and it was based on a monoclonal antibody to 0. gihsoni egg anfigen

(Gib-13) (Forsyth et al., 1985; Dissanayake et al., 1984). The assay detected

parasite antigen in 93% and 75% of the sera from subjects with W. hancrofti

infection, collected in two different series. It takes two days to perform the Gib-

14

13 IRMA, however, and as it is an radioimmunoassay, it is not likely to be

widely applied in endemic areas. Initially, the monoclonal antibodies, employed

to detect the parasite antigens in W. hancrofli infected patients sera, were directed

against phosphorylcholine (PC) determinant (Forsyth et al., 1985; Lai et ai, 1987).

PC determinants were found to be widespread in nature and shows strong cross-

reactivity with 5. malayi (Maizels er a/., 1985).

Later on monoclonal antibodies were developed that bind to a repeated, non-

PC determinant on a 200 Kd circulating W. hancrofti antigen (Weil et al., 1987;

Weil and Liftis, 1987; Ramzy et al., 1991). These assays have been successfiilly

used for monitoring efficacy of chemotherapeutic trials (Weil, 1988). Another

moAb (E-S 34) which is against W. hancrofti mf E-S antigens (55-63 Kd) has

shown promising results in field studies conducted in India and China (Zheng et

al., 1987; Reddy et al., 1989). This monoclonal antibody also did not detect antigen

in clinical cases and specificity data is insufficient. The monoclonal antibody

produced against O. gibsoni adult antigen (Og4C3) have been found to be relatively

better than all the other monoclonal antibodies produced earlier and has advantage

of detecting filarial circulating antigen also in amicrofilareamic clinical cases

(More and Copeman, 1990). However, most of these MoAbs are of IgM isotypes

and not very suitable for use in the field test. Kaushal et al. (1994a) produced

moAb against a common epitope between the bovine (Setaria cervi) and human

filarial parasites. This monoclonal (Scl) showed high reactivity with filarial

antigen while very little or no reacfivity was observed with the non-filarial antigen

(Kaushal et a!., 1994a; Kaushal, 1995). This Scl moAb is an IgGl isotype and

directed against a heat stable, repefitive pepfide epitope. This assay has high

specificity, whereas, lacks sufficient sensitivity.

15

II. CHARACTERIZATION OF ANTIGENS

The antigens of greater practical importance in filariasis are those related to

immunodiagnosis, immunopathology and protective immunity. Since these are the

molecules that are defined functionally (by eliciting an immune response), their

analysis should also be closely integrated with ftmctional studies. Thus, the

characterization of parasitic-derived antigen appears to be especially important for

understanding the fimctional immunity to the parasite (Thorson, 1951, Poulain et ai,

1976) and also for establishing specific diagnostic techniques. Filarial antigens

such as somatic extracts, surface antigens along with the excretory-secretory

(E-S) antigens were analysed by different workers, using a number of

biochemical, immunochemical and radiolabelling techniques (Forsyth e o/1981a;

Maizels et al, 1982; Kaushal et al 1982; Malhotra et al 1987).

Crude somatic extracts/antigens of the adult filarial parasites were

characterized by several workers (Neilson 1975; Disanayake and Ismail, 1980b;

1983; Ho el al 1986; Morgan et al., 1986; Das et al., 1987; Kaushal et al.., 1987;

Maizels et a!., 1987; Lai and Ottesen, 1988; Lammie et a!., 1990; Lobos et al.,

1992; Bradley et cr/., 1993a,b). In view of the non-availability of human filarial

parasites in sufficient quantities, antigens from related animal filarial parasites

including S. digitata (Dissanayake and Ismail, 1980b), O. gibsoni (Forsyth et

al., 1981a, Catmull et al., 1994), B. pahangi (Maizels et al., 1982), D. immitis

(Weil et al., 1984; 1985; 1987) and S. cervi (Malhotra et al., 1986; Srivastava et

al., 1996) have been employed in different studies for the characterization of

somatic antigens.

16

Several tecliniques were employed for the characterization of somatic

antigens such as, gel diffusion, immunoprecipitation, SDS-polyacrylamide gel

electrophoresis, (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA),

western blot/immunoblotting. Chatterjee et al., (1978) analysed the somatic

antigens of Chanderella hawkingii by gel diffiision and showed the presence of

7 antigenic components. The antiserum were raised in rabbits against the

somatic antigens showed high titre of precipitating and complement fixing

antibodies against the homologous antigens. The somatic extracts of the B. malayi

adult worms gave 30-35 protein bands by SDS-PAGE analysis, in the molecular

weight range of 10-200 Kd (Kaushal e/cr/., 1982). The adult somatic antigen of 5.

cervi showed the presence of 35-40 protein bands in the range of 10-200 Kd, while

the microfilarial antigenic extracts revealed 25-29 protein band in SDS-PAGE in

molecular weight range of 10-70 Kd (Malhotra et al., 1986).

Immunoelectrophoretic techniques were employed to analyse the

antigenic pattern of somatic extracts of bovine filarial parasite (Malhotra et al.,

(1986) and onchocerca (Lobos and Weiss, 1985). The immunoelectrophoretic (lEP)

analysis of X cervi somatic antigen revealed the presence of 9-10 antigenic

components, while crossed immunoelectrophoresis (CIE) showed 22-24 antigenic

peak (Malhotra et al., 1986), whereas, same number of antigenic components

were observed in O. voluvlus (Lobos and Weiss (1985). Farrar et al., (1991)

showed the presence of 42 protein bands in the molecular weight range of 12-160

Kd in western blot analysis of the B. pahangi adult somatic antigens. The somatic

extracts were also analysed in ELISA

Considerable efforts have been made to analyse the surface antigens of

filarial parasites as they appear to play a role in protective immunity as well as

17

immunodiagiiosis (Maizels et al., 1982; Sutanto el al., 1985; Selkirk e( ai, 1986;

Philipp and Davis, 1986; Theodore and Kaliraj, 1990; Devaney et a/., 1990;

Kwan-Lim and Maizels, 1990; Petralanda and Piessens, 1991). Analysis of the

surface proteins by radiolabelling has also suggested that nematode surfaces might

be antigenically simple as well as distinct (Philipp et al., 1984; Storey and

Philipp, 1990). The surface radioiodination and immunoprecipitation studies

of O. volvulus revealed a 22 Kd antigen which showed reactivity with sera from

W. hancrofti infected patients (Philipp et ai, 1984). A 30 Kd surface antigen of B.

pahangi was studied by radioiodination method (Devaney, 1987; 1988).

Studies with lectins to analyse the carbohydrate and glycoprotein moieties

have indicated no appreciable carbohydrate exposed on the surfaces of either adult

or third stage larvae (L. ) forms of B. malayi (Kaushal et al., 1984,1989). These

studies also showed that there is stage specificity of the carbohydrate-containing

antigens on the parasite surface and a dynamic changes of the antigens on these

surface molecules may be either masking by host proteins or actual loss of surface

antigens, as microfilariae matures in vivo (Kaushal et ai, 1984; Ottesen, 1984). A

major surface antigen of 35 Kd was observed by 2-dimensional gel electrophoresis

on the surface of D. immitis L3 (Philipp and Davis, 1986).

The filarial parasites reside in lymphatics where they eat, excrete and

procreate, therefore, the parasite products are detectable in the blood of infected

individuals. Such materials have been collectively termed as "excretory-secretory

(E-S) products". Consequently, the E-S products come in close contact with the host

biological system, tending to provoke an immune response. The nature and

properties of the E-S products is one of the most intriguing and highly speculated

area of research on parasitic nematodes (Kaushal e/o/., 1984; Piessens et ai.

18

1982; Maizels et al., 1986; 1987b; Sugunan and Raj, 1990). In filariasis, these

materials have been reported to play a significant role in the survival of the

parasites, immunopathological reactions in the host as well as in immunodiagnosis

(Sasa, 1976; Maizels et al., 1986; 1987a; Piessens et al., 1982; Mak et al., 1989;

Parklieercr/., 1990).

The use of cnide somatic antigens of the different filarial parasites resulted in

low sensitivity and specificity of the diagnostic tests because of the complex nature

of these antigenic preparations and their cross-reactivity with the other helminth

parasite antigens (Forsyth er a/., 1981 a,b, Maizels e/ al., 1983a,b; 1985b; Philipp

L'( al., 1984b; Kaushal et al., 1984; 1987; Rajasekhariah et al., 1986). The

excretory-secretory (E-S) antigens of the filarial parasites were found to be less

complex in nature and more specific in defining infection as compared to the

somatic antigens (Desavigney and Tizard, 1977; Kaushal et (7/., 1982; Malhotra

et al., 1987). The importance of filarial excretory-secretory antigens has also

been shown in relation to diagnosis and immunization in Onchocerciasis

(Schiller et al., 1980) and bancroftian filariasis (Kharat et al., 1982; Malhotra et al.,

1982; Reddy et al., 1984c; Kaushal et al., 1984; Harinath et al., 1984; Kaushal and

Ottesen, 1987; Malhotra et al., 1987; Dumenigo et al., 1993; Espino and Finlay,

1994). However, relatively little efforts were made to identify and characterize the

excretory-secretory products of hiunan filarial parasites (Kaushal et al., 1984;

Maizels et al., 1986; 1987; Kwan Lim et al., 1989). E-S antigens from animal

filarial parasites were analysed by employing immunochemical techniques

(Parkliouse et al., 1985; Maizels et al., 1985a; Harnett et al., 1989; Thilagavathy

et al., 1990; Kaneko et al., 1990). The main reason is the practical difficulties in

obtaining sufficient amount of E-S products (Kaushal et al., 1982).

19

The characterization of the antigenic components of the E-S products of

filarial parasites may help to identify suitable target antigen and is essential for the

development of sensitive and specific immunodiagnostic tests (Morgan et al.,

1986; Ottesen, 1984; Selkirk et al., 1986; Harinath, 1984; 1986). A variety of

tecliniques have been used by several workers for the characterization of adult E-S

antigens of the filarial parasites (Kaushal et al., 1982; Malhotra et al., 1987;

Reddy et al., 1984c; Parkliouse etal., 1985; Maizels et al., 1985a, 1987b;

Thilagavathy etal., 1990; Wisrewshi e/o/., 1990; Mizuno e/(7/., 1991; Zamowska

and Jastrzebka, 1994). The in vitro released E-S products from Burgia (Kaushal et

al., 1982; Maizds et al., ]9S5a;Wei\, \9SSb) and Wuchereria (Kaushal et al.,

1982) have been assayed for diagnostic specificities as have those for both adults

(Daveau and Ambroise-Thomas, 1982) and microfilariae (Schiller et al., 1980; Ngu

etal., 1981) ofO. volvulus.

The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) have been

effectively applied for the characterization of the antigenic components of the E-S

products. The E-S products from adult B. malayi have been identified and

characterized by using ' I radiolabelling method (Kaushal et al., 1982; Kaushal

and Ottesen, 1987). The autoradiography revealed the presence of 11 radioactive

protein bands in the ' I E-S products. The molecular weight (m.w.) range of

these bands ranged from 10,000 to 70,000 d. The presence of few protein bands

were observed in L. carinii E-S products as compared to numerous protein bands

in the somatic preparations (Rajasekhariah et o/., 1986a). Similarly, analysis of

B. malayi E-S products by radioiodination employing lodobead method found few

major protein bands of the m.w. of 30, 55 and 150 Kd (Egwang and Kazura,

(1987; Kwan-Lim et al (1989) have identified mainly low m.w. range of 10-22

20

Kd along with few high molecular weight proteins of upto 120 Kd in B. malayi E-S

products. Out of these, 2 antigens were found to be glycoprotein in nature,

identified as 29 and 51 Kd m.w. protein bands. The 29 Kd protein band identified

as one of the major surface antigen in B. pahangi (Lai and Ottesen, 1988; Devaney,

1987; 1988; Maizel et ai, 1985; Flecher and Wu, 1992). Studies on the E-S

products by various workers using different filarial parasites found that the E-S

products are low m.w. proteins ranging between 10 to 200 Kd (Kaushal et ai,

1982; Maizels et ai, 1986; Weil, 1988; Kwan-Lim et ai, 1989; Parkhe et ai,

1990; Lobos et ai, 1992). The E-S products of adult D. immitis male and female

worms revealed the presence of 16 and 21 protein bands respectively, while 7 and

10 protein bands were found in male and female D. immitis E-S products when

immimoblotting was employed (Kaneko et ai, 1990).

The E-S products were also characterized immunochemically by

employing techniques such as crossed immunoelectrophoresis (CIE) by various

workers (Kaushal et ai, 1982; Malhotra et ai, 1987; Bright and Raj, 1991).

Kaushal et al (1982) found 11 antigenic proteins in B. malayi E-S' products,

whereas, the S. digitata E-S products revealed the presence of 4-5 precipitin peaks

(Sugunan and Raj, 1990).

The E-S antigens are formed in the utems during embryonic develop­

ment (Decmize and Raj, 1988) and are released during moulting as secreted or

excreted products (Maizels and Selkirk, 1988a,b; Kaushal et ai, 1982; Sugunan and

Raj, 1986; Dhas et ai, 1993). Sugunan and Raj (1986) have also shown a direct

relationship between E-S materials and the number of mf released.

21

Presence of host serum proteins in the filarial E-S products

The filarial parasites reside in the lymphatics of the host. To protect

themselves from the immunologically hostile environment of the host and to evoke

the immune response of the host, the filarial parasites are known to adsorb some of

the host serum proteins on their surfaces. Several workers have reported the

presence of albumin on the surface of the parasites such as the infective larvae of

Trichenella spiralis (Parkhouse et al-., 1981). O. gibsoni (Mitchell et al., 1982), mf

of IV. bancrofti (Maizels et al., 1984a,b; Kar et a/., 1993; Mama and Kar, 1994),

infective larvae of W. bancrofti (Maizds et al., \9S6) and L cor//?//(Phillip et

al., 1984a). The evidence for the presence of host semm proteins has also been

shown in the E-S preparation of B. malayi (Kaushal et al., 1982), B. pahangi

(Parkhouse et al., 1985) and a bovine filarial parasite S. cervi (Malhotra et al.,

1987).

22

SCOPE AND PLAN OF WORK

Improved methods for diagnosing active filarial infections are needed to

monitor control efforts and to evaluate new drugs. The presently used method for

the diagnosis of filariasis, is based on demonstration of mf in the night blood

smears of the patient. This test is inconvenient for both the patients as well as the

investigator and fails to detect the amicrofilareamic stage of the disease or when

mf are present in very small numbers or sequestered in the tissues. This has put

impetus for the immunodiagnostic test. Earlier studies on immunodiagnosis of

filariasis were focussed mainly on the detection of antibodies, but the major

shortcoming is their inability to distinguish past exposure from current infection.

Therefore, in the recent past considerable emphasis has been given to the detection

of circulating antigen in blood or other body fluids of the infected individuals. The

demonstration of the parasite antigen in circulation of the infected individual appears

to be an usefiil indicator of active filarial infecfion. For establishing specific

diagnostic techniques, identificafion and characterizafion of parasite-derived

antigen is important. Due to the complex nature and extensive cross-reactivity

of the crude somatic antigens with the other nematode parasites, antigens

present in the excretory-secretory (E-S) products (obtained by the in vitro

maintenance of filarial parasites) of these parasites were analysed and evaluated

for the immunodiagnosis of filariasis.

Due to the non-availability of human filarial parasite, antigens ft'om Setaria

cervi (a bovine filarial parasite) have been used in the present study. The E-S

products were obtained by maintaining S. cervi adult wonn by short-tenn in vitro

incubation in culture. An effort was made to isolate the X ccrvi E-S products.

23

raise polyclonal antibodies against E-S antigens and to test the potential of anti-E-S

antibodies for the detection of circulating antigen in human filarial patient sera. The

present study deals with the following aspects:

I. Preparation of excretory-secretory (E-S) products from S. cervi adult

wonns.

II. Raising of polyvalent hyperimmune antibodies against S. cervi E-S products

in rabbits.

III. Immunochemical characterization of E-S products.

IV. Evaluation of rabbit anti-E-S antibodies for the detection of filarial

circulating antigen in filarial patient sera.

24

CHAPTER II

MATERIALS AND

METHODS

PARASITES

Setaria cervi, a bovine filarial parasite, was collected from the peritoneal

folds of fi-eshly slaughtered Indian water buffaloes from the local abbatoir in

normal saline. The motile worms (both males and females) were washed

thoroughly with normal saline to remove all the adhering materials and were used

immediately for antigen preparation or stored at -20°C until used.

EXPERIMENT ANIMALS

Albino rabbits of either sex (weighing 1.5 to 2.0 Kg) from the inbred

colony of Central Drug Research Institute's animal house, were used. All the

animals were kept in separate cages and fed on balanced diet and housed in an air

conditioned room.

CHEMICALS USED

Acrylamide, agarose, ammonium persulphate, anti-rabbit Ig horse radish

peroxidase conjugate, 2 beta mercaptoethanol (PME), bisacrylamide, bovine

senim albumin (BSA), bromophenol blue, coomassie brilliant blue (R~250),

ethylene diamine tetra acetic acid (EDTA), molecular weight markers,

orthophenylene diamine (OPD), sodium dodecyl sulphate (SDS), N N NT I'

tetramethyl ethylene diamine (TEMED) were procurred from Sigma Chemical

Company, St. Louis, MO, USA. Freund's complete and incomplete adjuvants were

obtained from Difco Defroit, MI, USA. The CNBr activated sepharose CL-4B

was procurred from Pharmacia Biotech AB, Uppsala, Sweden. The powdered

Duibecco's modified eagle medium (DMEM) was obtained from Gibco

25

Laboratories, NY, USA. All other chemicals used were of analytical grade. Triple

distilled water was used in all the experiments.

PREPARATION OF ANTIGEN

The excretory-secretory (E-S) antigens were prepared by short term in vitro

maintenance of bovine filarial parasites as described by Malhotra e/ al. (1987).

The medium used for the culture was prepared by dissolving one packet of

Dulbecco's modified eagle medium (DMEM), 2.38g of N-2 hydroxylethyl

piperazine-N'-2- ethane sulphonic acid (HEPES), 3.8g sodium bicarbonate and

0.3g glutamine to make 1 Itr with triple distilled water. This solution is filtered

through 0.22|im millipore filter for sterilization and dispensed into sterile screw

capped flask.

The adult motile worms of S. cervi (both males and females) were main­

tained asceptically at 37°C for 32 hr in a protein free defined medium i.e.

DMEM containing 1000 U/ml, penicillin and 100 ng/ml streptomycin. The medium

was changed at regular intervals. The spent medium was centrifiiged at 1000 X g

(3000 rpm) for 10 min to remove the microfilariae released in the mediimi. The

supernatant was kept at -70"C until used. The E-S products were concentrated by

lyophilization. The lyophilized E-S materials were reconstituted in minimal volume

of normal saline and were dialysed against the nonnal saline. This was again

centrifiiged at 1000 X g (3000 rpm) for 10 min and the E-S products finally

prepared were stored at -70°C until used.

26

PROTEIN ESTIMATION

The protein contents of the E-S products were determined by the Biorad

microassay procedure of Bradford (1979) while the procedure of Lowry's et al

(1951) was used to measure the protein contents of somatic antigen and antibodies.

Bradford's method: prepared several dilutions of 0.8 ml of protein standard

(BSA 1 mg/ml) from 1 to 25 |ig/ml and 0.8 ml of sample buffer for blank, then

0.2 ml of Biorad dye reagent (Coomassie brilliant blue in phosphoric acid). It

was mixed gently and after 5 min optical density (CD.) was measured at 595

nm in spectrophotometer.

Lowry's method: several dilutions of 0.4 ml of protein standard (BSA, 500 jig/ml)

from 10 to 50 |ig/ml or above were made and 0.4 ml of sample buffer for blank was

taken. Then, 2.0 ml copper reagent was added (0.5 ml of 2% Na-K tartarate, 0.5 ml

of 1% copper sulphate. 5H2O and 50 ml of 2% sodium carbonate dissolved in 0.1 N

sodium hydroxide), after 10 mins 0.2 ml of Folin's reagent (IN) was added and the

mixture was mixed and optical density was measured at 625 nm after 30 min.

IMMUNIZATION OF RABBITS

Polyvalent hyperimmune serum against the S. cervi E-S antigens were raised

in albino rabbits of either sex. The rabbits were immunized intramuscularly with

an emulsion of 0.5 mg of 5. cervi E-S product in Freund's complete adjuvant. One

month after the first injection, three subsequent injections were given at seven

days interval with E-S products emulsified in Freund's incomplete adjuvant. The

rabbits were bled one week after each injection starting from third injection. The

serum was obtained by centrifugation of blood at 900 x g (2500 rpm) for 10 min.

27

The immunization and bleeding of rabbits were continued and done alternatively

at 15 days interval in order to get the hyperimmune sera. The semm obtained was

stored at -20"C until used.

Similarly hyperimmune sera were raised in rabbits against bovine serum

albumin (2 mg BSA/rabbit).

PREPARATION OF GAMMA GLOBULIN FRACTION

The hyperimmune rabbit anti-.S'. cervi E-S sera and anti-BSA sera were

pooled separately and used to prepare the gamma globulin fractions (y-g) by

ammonium sulphate precipitation method. The required amount of the ammonium

sulphate was added slowly with continous gentle stirring to obtain 45% saturation.

The pH was adjusted between 7-8 with IN NaOH and left overnight at 4°C for

precipitation. The precipitate was recovered by centrifiigation at 10,000 x g (10,000

rpm) for 30 min, resuspended in minimal volume of normal saline and was

dialysed extensively against normal saline. The y-globulin fraction collected was

stored at -20"C in aliquotes until used.

ENZYME-LINKED IMMUNOSORBENT ASSAY

Enzyme-linked immunosorbent assay (ELISA) was performed according to

Voller et al. (1974), with some modifications, in a 96 well polystyrene plates.

Briefly, the microtitre ELISA plate was coated with different concentrations of .S".

cervi E-S antigens (7.5 ng-1000 ng/well) diluted in phosphate buffered saline (PBS,

0.05M, containing 0.04M disodium hydrogen phosphate, 9.5mM sodium

dihydrogen phosphate, and 0.15M sodium chloride, adjusted to pH 7.4) and

incubating the plate for 18-20 hr at 37"C. The plate was washed with PBS to

28

remove the unbound antigen. The uncoated sites of the plate were blocked with

5% non-fat dry milk in PBS for 2hr at 37"C. The plate was washed with PBS

containing 0.05% Tween-20 (PBS-Tween), tlirice. Then the plate was incubated

with antibody by adding 100 \x\ of fixed dilutions to each well and keeping for

2hr at 3TC. Again the plate was washed with PBS-Tween and incubated with

peroxidase conjugated secondary antibody at 1|1000 dilution in 1% milk for 90 min

at 37"C. Finally, the plate was washed with PBS-Tween and was developed with

substrate i.e. orthophenylene diamine (OPD Img/ml) in 0.05M citrate buffer

(0.02M citric acid and 0.05M disodium hydrogen phosphate), pH 5.2, containing

hydrogen peroxide (H2O2, l|il/ml). The reaction was stopped with 5N H2SO4 after

10 min and the colour intensity was read at 490nm in an ELISA reader.

SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS

The SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of .S". cerv/E-S

antigen was done according to Laemmli (1970) using Phannacia slab gel

eletrophoresis apparatus. A separating gel of 10% acrylamide was used. The

composition of the mixture was 10 ml of stock acrylamide (30%) and

bisacrylamide (0.8%), 7.5ml of 1.5M Tris-HCl (pH 8.8), 300 f.il of 10% sodium

dodecyl sulphate (SDS), 100 \x\ of 10% ammonium persulphate (APS), and 10 ^1 of

N N N'N' tetra methyl ethylene diamine (TEMED) in a total volume of 30 ml made

up with triple destilled water. The stacking gel (4.5%) contained 1.49 ml of

acrylamide stock solution (30% acrylamide and 1% bisacrylamide), 2.63 ml of 0.5

M Tris-HCl (pH 6.8), 0.1 ml of 10% SDS, 75 ^1 of 10% APS and 10 ^l of

TEMED in a total volume of 10 ml made up with distilled water. The samples

were prepared by mixing equal volumes of the E-S antigen with SDS-PAGE

29

sample buffer (containing 12.5 ml of IM Tris-HCl buffer, pH 6.8; 20 ml of

glycerol; 10 gms of SDS; 2 ml of 0.1% bromophenol blue; 10 ml of 2^-

mercaptoethanol (BME) in a total volume of 100 ml) in 1:1 ratio and keeping for 5

min in boiling water bath.

Electrophoresis was carried out at a constant current of 25 mA per gel for

about 4-5 hr till the tracking dye has reached 1 cm above the bottom of the gel.

After electrophoresis, the gel was stained with 0.25% coomassie brilliant blue

R~250 (dissolved in 30% methanol and 10% acetic acid) and destained with

30% methanol and 7% acetic acid. The gel was also stained by silver staining

method (Merril et al 1981). Briefly, the gel was fixed with 50% methanol and 12%

acetic acid, excess SDS was removed by washing three times with 200 ml of 10%

ethanol and 5% acetic acid. The gel was soaked for 10 min in 200 ml of 0.0034

M potassium dichromate and 0.0032N nitric acid. The gel was then washed 4 times

with 200 ml of distilled water and placed in 0.2% silver nitrate solution for 30 min.

The gel was again washed twice with distilled water, followed by rapid staining

with two 300 ml of the image developing solution, containing 0.28 M sodium

carbonate and 0.5 ml formalin per Itr. Gel was gently agitated in a third portion

of the developing solution until the image has reached the desired intensity. The

reaction was stopped by putting the gel in 100 ml of 1% acetic acid.

IMMUNOELECTROPHORESIS

Immunoelectrophoresis (lEP) was carried out according to the method of Graber

and Williams (1953). The S. cervi E-S antigens were separated in 1.5 mm thick

layer of 1% agarose in 0.02M barbital buffer, pH 8.6, (20 mM diethyl barbituric

acid, 80 mM Tris, 20 mM sodium azide, 0.8 mM calcium lactate) for 1 hr at a

30

constant voltage of 150 volts. After the separation of antigens, electrophoresis was

stopped and antigen was allowed to react with immune rabbit semm for 18-20 hrs

in a moist chamber at room temperature (RT). The plate was washed three times

with isotonic saline and finally with distilled water, and alternate pressing of the

plates were done under several sheets of filter paper. The plate was air dried,

stained with 0.25% w/v of Coomasie Brilliant Blue (R~250) made in 45% ethanol

and 10% acetic acid and destained with the same.

CROSSED IMMUNOELECTROPHORESIS

Crossed immunoelectrophoresis (CIE) was performed according to Axelsen

cl a/. (1973). The antigens were separated in the first dimension electrophoretically

as described for lEP. A 1x6 cm agarose strip containing the separated antigens

was transfered to another plate (6x9 cm). Anodic (5x6x0.12 cm) and cathodic

(2x6x0.12 cm) gel containing 5-10% immune rabbit semm was poured onto the

plate. An mtennediate gel, without antibody, was poured between the anodic gel

and the first dimension gel. The plate was electrophoresed at right angle to the first

dimension for 18-20 hr at 2-3 V/cm. The CIE plate was washed, dried and stained

in the same way, as for lEP.

CROSSED LINE IMMUNOELECTROPHORESIS

Crossed line immunoelectrophoresis (CLIE) is the modification of CIE and was

carried out in the similar manner except that in CLIE the test antigen (50-100 ^g)

was introduced into the intermediate gel. After electrophoresis the plate was

washed and stained as described eariier for lEP. On staining, the common antigen

gave a line at the base of the antigenic peaks.

31

TANDEM CROSSED IMMUNOELECTROPHORESIS

Tandem crossed immunoelectrophoresis (tCIE) is almost similar to that of

CIE and is carried out in the same maimer except that in tCIE the test antigen

was mixed with the antigen (1:1 ratio) and electrophoresed togather in the first

dimension. The second dimension electrophoresis is similar to that of CIE. After

electrophoresis the plate was washed, dried and stained as described earlier in

lEP. On staining the common antigen showed the increase in the height of the

precipitin peaks as compared with the control CIE.

ABSORPTION OF S. CERVIE-S PRODUCTS ON ANTI-BSA y-GLOBULIN

SEPHAROSE

The anti-BSA sera raised in rabbits were used to prepare y-globulin fraction

by ammonium sulphate fi^actionation method. The anti-BSA y-globulin fraction

was coupled to cyanogen bromide activated sepharose CL-4B (Pharmacia) as

follows: 100 mg anti-BSA y-globulin fraction was mixed with 10 ml of the

preswellon sepharose CL-4B in 45 ml coupling buffer (O.IM NaHC03 and 0.5M

NaCl, pH-8.3) by shaking end over end overnight at 4°C. The unbound protein

was removed by washing the beads with Tris-HCl buffer (pH 8.0) and the

residual active groups were blocked with IM diethanolamine (pH 8.0),

overnight at 4°C. The sorbent was washed alternatively with 0.1 M acetate buffer

(IM NaCl, 0.012 M sodium acetate.3H20 and 0.096M glacial acetic acid), pH

4.0, and 0.1 M borate buffer (0.1 M boric acid, 0.025 M sodium tetraborate and

0,075M NaCl), pH 7.9, with 10 min minimum exposure to each buffer. Finally the

32

8.0. The eliited antibodies were dialysed and concentrated to 1.0 ml (eluted). Both

the absorbed and eluted antibodies were stored at -20"C until used

COUNTER CURRENT IMMUNOELECTROPHORESIS

The counter current immunoelectrophoresis (CIEP) was performed accord­

ing to Siber and Skapriwsky (1978) by pouring 1% agarose, w/v (1,5 mm thick) on

a glass plate. The wells were cut in parallel rows 5 mm apart. Five microlitre of

patient sera was placed in cathodic well and 10 fil of rabbit anti-E-S serum in the

anodic well. Electrophoresis was performed at a constant voltage of 100 volts for

30 min. The plate was placed in a moist chamber at 37"C for 1 lir and then in

normal saline for overnight at RT. The plate was washed, dried and stained

similarly as described in lEP. The presence of a precipitin line after staining,

indicates a positive reaction.

34

CHAPTER in

RESULTS AND

DISCUSSION

RESULTS

The demonstration of circulating filarial antigen in the blood of the patient is

an useful tool for the conformation of an active infection. The somatic antigens used

for the detection of filariasis showed extensive stage and species cross eactivity

among cmde somatic extracts/antigens. The excretory-secretory antigens are

comparatively less complex in nature and more specific in defining infection.

Therefore, the present study was undertaken on the preparations, identification and

characterization of the excretory-secretory (E-S) products, from the adult bovine

filarial parasites, Setaria cervi. The antibodies against 5. cervi E-S products were

also evaluated for their diagnostic potential.

CHARACTERISATION OF EXCRETORY-SECRETORY PRODUCTS OF

S. CERVI

I. Protein analysis of the S. cervi E-S products

The protein pattern of the S. cervi E-S antigen was analysed on SDS-

polyacrylamide gel electrophoresis (SDS-PAGE) along with the .S'. cervi adult

somatic antigens. Out of the different concentrations of the gel tried, 10% of the

polyacrylamide gave the best resolution. The SDS-PAGE pattern of the E-S antigen

is shown in Fig. 2. The S. cervi E-S product showed the presence of 14-15

protein bands, whereas, the adult somatic antigen showed much greater number of

protein bands, i.e., 35-40 protein bands. Standard molecular weight markers were

used in order to determine the molecular weights. The protein bands of the E-S

product lie in the molecular weight range of 10-200 Kd.

35

A H

6 6 -

4i~

36-

2 9 -

2D-

B0B^^

f

Fig.2: SDS-polyacPyiamide gel electrophoretic analysis of Sctana ccrvi excreton'-secreton.' products

A: Somatic antigen

B: Excretory-secretory products

36

II. Immunization of rabbits against S". cervi E-S products and quantitation

of parasite specific antibodies

The hyperimmune rabbit sera were raised in rabbits against the S. cervi E-S

products. Three rabbits (R-1, R-2 and R-3) were immunized intramuscularly for a

period of 6-7 months. The immune sera collected from these rabbits after each

injection was tested for its parasite specific antibodies in enzyme-linked

immunosorbent assay (ELISA).

ELISA was standardized in terms of optimum antigen concentration,

antibody conjugate and dilutions of antisemm. Different concentrations of .S'. cervi

E-S products (1, 0.5, 0.25, 0.125, 0.062 and 0.03 |ig per well) were tried using

hyperimmune sera at a fixed dilution. Out of the different antigen concentrations,

0.25 ]xg of S. cervi E-S product per well was found to be the optimum antigen

concentration and the results are shown in Fig. 3. The optimum antibody conjugate

was standardized by using different dilutions (1:250, 1:500, 1:1000 and 1:2000).

The 1:1000 dilution of antibody conjugate gave the optimum results (Fig. 4). The

other assay conditions such as the optimum orthophenylene diamine (OPD)

and hydrogen peroxide (H2O2) were previously standardized in our laboratory.

The 0.25 |ig/well antigen concentration and 1:1000 dilutions of antibody

conjugate were used fiirther in all the experiments for detennining the antibody

titre of the three immune rabbit sera, collected after each immunization (from Ilf''

injection to the last injection). The antibody titre was found to be increasing upto

the VII* injection in all the three rabbits and became constant after that. The data is

shown in Fig. 5 and summarized in Table 1,

37

O.D. at 490 nm

3.000

2.000

1.000

IBS 1 H - m S 2 -5t^lRS 3

,Jfc=--• - - ^ >

0.000 0.015 0.031 0.082 0.125 0.25 0.50

Antigen concentration (ug/iwell)

1.0

Fig. 3: Optimum antigen concentration of 5. cervi excretory -secretory products using immune rabbit sera from three rabbits

IRS 1-Immune rabbit sera 1; IRS2-Iinmune rabbit sera 2;

IRS3-Immune rabbit sera 3

4.00-O.D. at 490 nm

' • - ^ - - : ' • - ^ - - : - ^ • - - ' — -

3.00 " ~ j i

2.00-

1.00

1 - ^ 1:1000 --1- 1:S000 1 1 0.00-

1 260 500 1000

Reciprocal antibody conjugate dllullona 2000

Fig. 4: Titration of antibody conjugate in ELISA using S. ccrvi excretory-secretory products against rabbit anti-E-S senim

38

Table 1: Antibody titres of rabbit anti-5. cerv/ E-S serum obtained after different injections in enzyme-linked immunosorbent assay using excretory-secretory products.

Number of injections IRSl IRS2 IRS3

3rd 1:8000 1:16000 1:16000

4th 1:16000 1:32000 1:32000

5th 1:32000 1:64000 1:64000

6th 1:64000 1:64000 1:128000

7th 1:64000 1:128000 1:128000

8th 1:128000 1:128000 1:256000

9th 1:128000 1:256000 1:256000

10th 1:128000 1:256000 1:256000

Pooled serum 1:128000 1:256000 1:256000

The sera showing the same titre values were pooled and the pooled rabbit

anti-E-S semm from rabbit 1 (R-1) gave a titre of 1:128,000 whereas, serum from

rabbit 2 (R-2) and rabbit 3 (R-3) gave a titre of 1:250,000 as shown in Fig. 6

and summarized in Table 1.

39

300

250

200

160

100

SO

Reciprocal antibody titre

JIRSI [Z3lR82 E Z J I R S S

4 u B

k

[• V

ri ^ K'

11 l!

H

H r ti

x5-

^ 0

1 S

pp h H ^

ti H

H N

'- s

11 3 5 7 9 11 3 Sera after different injections

9 11

Fig. 5: Titration of antibodies in rabbit anti- S". ceA-v/E-S serum obtained after different injections

O.D. at 490 nm

•^ IRS I -^ - IRS i - X - IRS 3

a = = ^ SOO 1000 16000 129000

Reciprocal antibody dilutions

Fig. 6: Titration rabbit anti-S'. cervi E-S sera for the parasite specific antibodies against S. cervi excretory-secretory products in enzyme-linked immunosorbent assay

40

III. Antigenic analysis of S. ceni E-S products

The antigenic analysis of the .V. ccrvi excretory-secretor)' products were

done by immunochemical techniques such as immunoelectrophoresis, crossed

immunoelectrophoresis using hyperimmune rabbit anti-E-S sera.

A) Immunoelectrophoresis: The S. ccrvi E-S products were analysed in

immimoelectrophoresis (lEP). The E-S products were electrophoretically separated

on an agarose gel and then allowed to react with the rabbit anti-E-S serum

(collected during the course of inmiunization). The numbers of precipitin bands

were found to increase with subsequent injections and became constant after the

fifth injection. The sera collected after the third injection gave 3-4 precipitin bands

in all the tliree rabbits, whereas, the number of bands increased with the number of

Table 2: Summary of immunoelectrophoretic analysis of S. cervi excretory-secretory products using rabbit anti-E-S serum.

Number of injections

Number of precipitin bands Number of injections

IRSl IRS2 IRS3

3rd 3 4 3

4th 4 5 4

5th 6 7 6

6th 6 7 8

Pooled serum 6 7 8

41

A

Fig. 7: Immunoelectrophoretic analysis ofSctaria ccrvi excretoiA'-secreton,-products using rabbit anti-E-'S sera

A: Immune rabbit sera 1: B: Immune rabbit sera2;

C: Immune rabbit sera 3

1: 3rd mjection; 2: 4th injection, 3: 5th injection;

4: 6th iniection: 5: Pooled scrum

42

injections and after the fifth injection 6-8 precipitin bands were observed. The

pooled rabbit semm from rabbit 1 (Rl) gave 6 precipitin bands, whereas, rabbit 2

(R-2) and rabbit 3 (R-3) pooled semm gave 7 and 8 precipitin bands respectively.

Results are shown in Fig. 7 and summarized in Table 2.

B) Crossed immunoelectrophoresis: The S. cervi E-S products were further

analysed by two dimensional gel electrophoresis or crossed immunoelectrophoresis

(CIE), in order to get a better resolution of the antigenic components.

Different concentrations of the rabbit anti-E-S semm (2,5%, 5% and 10%) were

tried in CIE. The number of precipitin peaks remained same in all the three

concentrations but greater resolution of the peaks were found in 10% semm

concentration in the sera from rabbit 1 while the sera from rabbits 2 and 3 gave

optimum results with 5%) semm concentration. The hyperimmune rabbit anti-E-S

semm pools were also used to analyse the S. cervi E-S products in CIE and gave

10 precipitin peaks with immune rabbit semm pool 1 (R-1). However, 11 and 12

precipitin peaks were seen with immune rabbit semm pool 2 (R-2) and serum 3 (R-

3), respectively (Fig. 8 and Table 3).

The anti-^. cervi E-S semm pools from the three rabbits were pooled

togather and was used to prepare y-globulin fraction by ammonium sulphate

fractionation method. In order to get a better resolution, different concentrations of

y-globulin fractions were tried (2.5%, 5% and 7.5%)) in CIE to analyse the 5.

cervi E-S products. The number of peaks in all the three concentrations were same

except that it showed difference in the heights of the peaks. To get the same

resolution of the peaks as shown by the semm pool, 2.5% concentration of y-

43

u

CQ

E 2

I

U i

bX) C CO 3 r^ o E 3

t3 2 O !/5 u O. -*—»

b ^ ^

o rs -4—' i - i

o (U 3 c/1 C

ar g o »—<

• ^ ^

Li o . / X f N (U E

2 00

(>5 ' ^ < 4 - l ^ o C3

E « OJ C

• • — • 3 C3 E O- E o >—<

• • — »

(U 6a k> o . rv

-C 1—i

Q. P O

00 2 o D •*—» O u. ^ c ID x> 3 <U rt E E

u.

c • »— d) 3 -a -C OJ - 4 — * H

O E o

H—^

u <i: <

oo

bJD U.

44

globulin fraction was found to be optnnum and it showed the presence of 13-14

precipition peaks as shown in Fig. 9 and summarized m Table 3.

Table 3: Summary of crossed immunoelectrophoretic analysis of S. cervi excretory-secretory products using immune rabbit sera and y-globulin fractions.

Anti-E-S serum Number of precipitin bands Anti-E-S serum

Rabbit 1 Rabbit 2 Rabbit 3

Immune rabbit serum

y-globulin fraction

10 11 12

11 12 14

EVALUATION OF THE ANTI-E-S ANTIBODIES FOR THE DETECTION

OF CIRCULATING ANTIGEN IN HUMAN FILARIAL SERUM

The pooled rabbit anti-E-S antibodies (raised in rabbits against .V. ccrvi E-S

products) were used for the detection of circulating antigen in human filariasis

using counter current Immunoelectrophoresis (CIEP) technique. The anti-E-S

serum was tested in CIEP against the filarial patient pooled serum (pool of the

filarial patient serum found positive for the presence of circulating antigens). This

immune rabbit sera could detect the circulating antigen in patient pooled serum,

however, it showed non-specificity against the serum fiom normal healthy

individuals, which was used as a negative control (Fig. 10). This non-specificity

may be due to the presence of host senim proteins in the .V. cervi E-S products.

Therefore, further efforts were made to identify these host serum proteins and to

45

/

^ii.

u

CQ

^

4-

< / N

3

_o I

CUD

-4—*

o 3

-a o

G.

O -4—•

1) o I

l-l o X

? .2

C>0

o

-a

^ a

o I

• S O

[/I O n cd

u

c o

; c o c o o

3

u cT o

)-< c (U o c o o

J21

I

CQ

o

- 4 — * c 1) o c o o

3

I

0\ W)

46

Pt.Pool * .J

Fig. 10: Counter current immunoelectrophoresis for the detection of filarial circulating antigen using pooled anti-.V. ccrvi excretory-secretory products

NHS : Nonnal human serum

Pt. Pool: Patient pooled sera

IRS : Pooled immune rabbit sera

47

remove them from the S. cervi E-S products as well as the antibodies against them

from the rabbit anti-E-S serum.

IDENTIFICATION OF HOST SERUM PROTEINS PRESENT IN THE S.

CE/?^7 E-S PRODUCTS

In order to detect the presence of the host senim proteins, the S. cervi E-S

products were analysed in CIE using rabbit antibodies against nonnal buffalo serum

(NBS). The results showed the presence of 3-4 precipitin peaks (Fig. 11), indicating

the presence of host semm components in the E-S products. Further, the

hyperimmune rabbit semm was raised in rabbits against BSA, The anti-BSA

senim was used to analyse the E-S products and this showed 3 precipitin peaks

(Fig. 12A). The bovine semm albumin (BSA) was also used in CIE against the

rabbit anti-E-S semm and this revealed the presence of 2 precipitin peaks (Fig.

I2B).

The above results were ftirther confirmed using different immuno-

electrophoretic techniques such as crossed line immunoelectrophoresis and

tandem crossed immunoelectrophoresis. Crossed line immunoelectrophoresis

(CLIE) is similar to CIE except that 50 |.ig of BSA was introduced in the inter­

mediate gel and this gave precipitin line at the base of two precipitin peaks of the

E-S products when tested against rabbit anti-.S'. cervi E-S semm (Fig. 13A). Another

CLIE was done using E-S products against anti-E-S semm and 5% rabbit anti-BSA

semm was introduced in the intermediate gel and this showed the reduction of two

precipitin peaks (Fig. 13B).

Tandem crossed immunoelectrophoresis (tCIE) is again similar to CIE

except that in the first dimension. BSA was mixed along with the X cervi E-S

48

Fig. 11: Crossed immimoelectrophoretic pattern of .S. cervi excretory-secretory products using rabbit anti-nonnal buffalo s'erum

49

B

/ • ^ /

Fig. 12: Crossed immunoelectrophoretic pattern ofS. cervi excretory-secretory products using rabbit anti-BSA senim (A); and' BSA using anti-E-S semm (B)

50

B

i

ffy

Fig. 13: Crossed line immunoelectrophoretic pattern of excretory-secretory products using anti-E-S senim witli intemieaiate gel containing 5% rabbit anti-BSA senim (A) and 50 ^g BSA (B)

51

products and electrophoresed togather, and run against rabbit anti-IvS serum in the

second dimension. Out of the 12 antigenic components of the E-S products two

precipitin peaks showed considerable increase in the height of the peaks as seen in

Fig. 14. These two antigenic components are the same as seen by the above results

i.e. CLIE, tCIE and CIE of BSA thus indicating the presence of BSA as one of the

major host protein contaminant of the .S". cervi E-S products.

REMOVAL OF HOST SERUM PROTEINS FROM THE S. CERVI

PRODUCTS

An effort was made to remove the host semm components present in the. .S.

ccrvi E-S products, by using anti-BSA sepharose sorbent. Thus, preabsorbed E-S

products were analysed in SDS-PAGE and this showed the removal of a major

protein band of 66 Kd present in the S. ccrvi E-S products, as shown m Fig. 15.

Further, the preabsorbed E-S products were analysed in CIE using anti-E-

S serum. Two major antigenic peaks (detected as host serum albumin earlier in

Fig. 13) were found missing in the preabsorbed E-S products and thus revealed

the presence of 10 precipitin peaks, whereas, in the unabsorbed E-S products

12 precipitin peaks were found with 5% rabbit anti-E-S semm (Fig. 16 and Table 4).

To quantitate the parasite specific antibodies in the anti E-S serum was

tested in ELISA using preabsorbed preabsorbed X ccrvi E-S products. The

preabsorbed E-S products gave a parallel cur\'e with the unabsorbed E-S products,

but the reciprocal antibody titre of the absorbed E-S product was found to be

120,000 while the unabsorbed E-S products gave a reciprocal antibody titre of

250,000 as seen in Fig. 17 and summarized in Table 4.

52

B

Fig. 14: Tandem crossed immunoelectrophoretic pattern oi\S. ccrvi excretory-secretory products and BSA (A) crossed imnuinoelectrophoretic pattern of E-S products (B), using rabbit anti-E-S serum

53

A B lap-

ee-

3 6 -

29-

20-

Fig. 15: SDS-polyacrylamide gel electrophoretic analysis of the .S". cervi excretory-secretory products absorbed on anti-BSA sepharose beads

A: Unabsorbed excretory-secretory products

B: Absorbed excretory-secretory products

54

B

Fig. 16: Crossed immunoeleclrophoretic pattern of .S'. ccrvi excretory-secretory products before (A) and after (B) absorbtion on anti-BSA sepharose beads, using rabbit anti -li-S serum

55

O.D, at A90 rm

3.00

2.00

1.00

0.00-

Unibaorbst] Ab»ofbed J

800 1000 4000 16000 8<000 268000

Rec ip roca l a n t i b o d y d i l u t i o n s

Fig. 17: Antibody titration of rabbit anti-E-S serum usinu unabsorbed and absorbed S. cervi excretorv-secretorv products on anti-BSA sepharose beads, m ELISA

Table 4: Summary of the crossed immunoelectrophoretic analysis and enzyme-linked immunosorbent assay of the absorbed S. cervi excretory-secretory products using rabbit anti-E-S serum and S. cenu excretory-secretory products using absorbed rabbit anti-E-S serum.

Assay Absorbed E-S products

Unabsorbed E-S products

CIE (Number of precipitin bands)

ELISA (Reciprocal 1 antibody titres)

10

120,000

12

250 000

56

REMOVAL OF ANTIBODIES AGAINST THE HOST SERUM COMPO­

NENTS FROM THE RABBIT ANTI-S. CERVI E-S SERUM

The hyperimmune rabbit anti-E-S semm has the possibihty of having

antibodies against host semm components, as they were raised against the whole S.

cervi E-S products. Therefore, an effort was made to remove these antibodies

against host serum components by absorbing the rabbit anti-E-S semm with

albumin sepharose beads.

These absorbed anti-E-S sera were used for the analysis of S. cervi E-S

products in CIE. Both the absorbed and unabsorbed anti-E-S serum was used at

5% concentration. The CIE analysis of the S. cervi E-S products usmg absorbed

anti-E-S serum revealed the same resuU as of preabsorbed E-S product except ten

precipitin peaks with preabsorbed sera and 12 precipitin with the unabsorbed anti-E-

S semm were observed (Fig. 18).

The parasite specific antibodies were quantitated in both the absorbed and

unabsorbed anti-E-S semm in ELISA using .S". cervi E-S products. Both the sera also

gave parallel curves and the antibody titre of the absorbed anti E-S semm was

found to be 1:120,000 while the unabsorbed anti-E-S semm gave antibody titre as

1.250,000, as shown in Fig. 19.

EVALUATION OF THE ANTI-E-S ANTIBODIES (ABSORBED ON BSA-

SEPHAROSE SORBENT) FOR THE DETECTION OF CIRCULATING

ANTIGEN IN HUMAN FILARIAL SERUM

The anti-E-S antibodies were used for the detection of filarial circulating

antigen in patient sera after diluting it with normal human serum (NHS). The

57

A B

;sed immunoelectrophoretic analysis of .V. ccrvi tAv iCtorv-secretorv proaucts using absorbed (A) and iinabsoiBed (B) -- -'-- --'- " ^ ---

18: Crossea immunoeieciropnorenc anai> excretorv-secretorv proaucts using ab

labsorBed (B) rabbit anti-E-S serum

58

O.D. at 490 nm

I

J

600 1000 4000 leOOO e<000 ZSOOOO lOOOOOO

Reciprocal antibody dilutions

Fig. 19: Titration curves of unabsorbed and absorbed anti-E-S serum against S. cervi excretory-secretory products using ELISA.

59

immune rabbit semm was incubated with different ratios of NHS (1:2.5; 1:5; 1:10;

1:20) at 37"C for 30 min. The absorbed rabbit anti-E-S semm, when tested in CIEP

against the filarial patient semm pool, could detect the circulating filarial antigen

and did not show cross-reactivity with the nonnal human serum upto 1:10 dilutions.

The results obtained with the three rabbit semm pools are shown in Fig. 20.

Further, attempts were made to absorb the rabbit anti-E-S semm with

albumin sepharose beads. The preabsorbed anti-E-S semm was used to detect the

circulating filarial antigen in the patient pooled semm by using CIEP. The

preabsorbed anti-E-S semm gave no cross-reactivity with the nomial human semm,

whereas, it could detect the circulating antigen in the filarial patient pooled sera

(Fig. 21). On testing small number of individual human patient sera, the

preabsorbed immune rabbit anti-^. cervi E-S semm could detect the circulating

filarial antigen in all the 15 microfilaria! positive patient sera but not in the 8 non-

endemic control human sera (Table 5).

60

Fig. 20: Counter current immunoelectrophoresis for the detection of filarial circulating antigen using anti-.V. cervi E-S products diluted with nonrianuiman senim

a: Nonnal human serum (NHS) b: Patient pooled senim

61

V 1

+

a • o b O

1 2

• R l

b Q

a O

fc O

I

2

1 >R2

' X »R3

b y\ J - - -

l-ig. 21; Detection of filarial circulating antigen in CIEP using absorbed and unabsorbed anti-E-S'serum

a: Patient pooled serum b: Nomial human serum (NHS) 1: Absorbed serum 2: Unabsorbed serum Rl: Immune rabbitserum 1 R2: Immune rabbit serum 2 R3: Immune rabbit serum 3

62

Table 5: Detection of circulating antigen in filarial patient sera by CIEP using rabbit anti-E-S antibodies.

Sera No. Tested Positive by CIEP

Filariasis (mf+ve) 15 15

Other infections

Ascaris 3 0

Malaria 10 0

Amoeba 5 0

Non endemic control 10 0

63

DISCUSSION

Filariasis is a serious parasitic disease of the tropics and the subtropics. The

causative agents in humans are Wuchereria bancrofti, Bnigia malayi and B.

trimori. The demonstration of the circulating antigen in the body fluids (serum and

urine) of microfilareamic and clinical filarial patients has been suggested to be a

better approach for the immunodiagnosis of filariasis (Ottesen, 1980; 1984;

Harinath, 1986) as it is much more effective for assessing the status of an early and

active infection. The E-S products, released by the living metabolically active

parasites in the host, have been known to be less complex in nature and more

specific in defining filarial infection as compared to the somatic antigens or the

whole worm extract (Kaushal et ai, 1982; 1984, 1989; Prasad et al., 1983; Kharat

et al., 1983; Malhotra et ai, 1987).

The demonstration of the E-S antigens in the circulation of the infected

host could provide a better indicator of active filarial infection (Kaliraj et al.,

1979a). Inspite of the less complex nature of the E-S antigens not much efforts

have been made to critically characterize and identify the diagnostically

important antigen present in E-S products, this may probably be due to the

limitations in obtaining sufficient amount of E-S products from human filarial

parasites (Kaushal et al., 1982; 1984; Kharat et al., 1983).

Setaria cervi, a bovine filarial parasite, was used as an alternate source for

human filarial parasite antigens because the earlier studies fi^om our laboratory on

the characterization of S. cervi antigens, has identified some common antigenic

epitopes between the bovine and human filarial parasites (Kaushal et al., 1987) and

the monoclonal antibodies against the common epitope of high molecular weight

64

antigen showed reactivity with the filarial circulating antigen (Kaushal et ai,

1994). In the present study the adult S. cervi worms were maintained in vitro in

serum free culture medium for 32 hrs at 37"C in order to obtain the E-S products.

The protein released during the preparation of E-S products by the adult filarial

parasites are in substantial amount (12-15 mg of E-S products per 100 S. cervi adult

parasites). The protein released by the other filarial parasites are in insufficient

quantity. Kaushal et a/., (1982) obtained as low as 18 |igs of protein after

incubating 100 adult B. malayi active parasites in a tissue culture medium for a

duration of 48 hours, whereas, Rotmans et al., (1981) have reported the release of

100 [Lg protein/100 Schistosoma mansoni-worms in lAhvs. The release of large

amounts of E-S products by the S. cervi adult parasite, fascilitated

characterization using conventional immunochemical methods.

The S. cervi E-S products were analysed on SDS-polyacrylamide gel

electrophoresis and showed the presence of 14-15 protein bands in the molecular

weight range of 10-200 Kd, whereas, the somatic antigen contained 35-40 protein

bands thus, indicating the less complex nature of the S. cervi E-S products as

compared to the somatic antigen/whole worm extract of 5. cervi. The B. malayi

products were also found to be less complex in nature having eleven protein bands

in a molecular weight range of 10 Kd to 70 Kd (Kaushal et al., 1982) and also few

protein bands having low molecular weight were reported in the E-S product of L

carina (Rajasekhariah et al., 1986).

In order to test the antigenicity of S. cervi E-S products, rabbits were

immunized and. the immune sera obtained from these rabbits were tested in

ELISA using E-S products as antigens. The S. cervi E-S products gave the

65

optimum concentration of 0.25 |i.g/well, and the immune rabbit sera gave high

antibody titles in the range of 1:100,000 to 1:250,000. The increase m titre value

with increasing number of injections were seen in all the three rabbits, which

became constant after the Vllth injection. This study indicates that the .S". cervi E-S

antigens are highly immunogenic and they elicits the formation of antibodies.

The B. pahangi E-S products showed the optimum antigen concentration as

low as 10 ng/ml when tested in ELISA (Maizels e/o/., 1985). Detection of

specific antibodies to E-S products are of particular interest for understanding

the immunologic relevance to the infected host. Conventional immunoassay has

been used for detecting IgG antibodies to secretory antigens in other parasitic

disease (DeSavigney and Tizard, 1975, 1977; DeSavigney et a/., 1979; Yang and

Kermedy, 1979). The Staphylococcus aureus radio-immunoprecipitation assay

(Staph-A RIA) was used to quantitate IgG antibodies to B. malayi E-S antigens and

this gave 5 ng antigen/test (Kaushal et al., 1982; Weil, 1988). The filarial sera also

showed high reactivity with anti B. malayi E-S serum, thus, indicating that natural

filarial infection also evokes high antibody titres (Kaushal et al., 1982).

The antigenic components of .S. cervi E-S products were analysed by employing

the techniques of immunoelectrophoresis (lEP) and crossed immunoelectrophoresis

(CIE) using the rabbit anti-iS. cervi E-S antibodies. The immunoelectrophoresis

showed the presence of 8-10 parasite specific antigen in S. cervi E-S products. The

lEP techniques has been used earlier by a number of workers for the antigenic

analysis of the filarial antigen (Kaushal et al., 1982; Malhotra et al., 1987;

Sv/ada etal., 1969).

66

The antigenic components of the S. cervi E-S products were

flirthermore, analysed by using a more sophisticated and sensitive technique,

i.e., crossed immunoelectrophoresis, CIE, which gave better resolution of the

antigenic components of the S. cervi E-S products. The immune rabbit sera

obtained from the three rabbits showed comparable antigenic pattern of S. cervi E-

S antigen in CIE except for the slight differences in the intensity and the heights

f certain peaks. The number of peaks remained same in all the different

concentrations of the serum tried. But the optimum resolution of the precipitin

peaks were found in 10% serum concentration in rabbit 1 (R-1), which showed

comparatively lower antibody titre, whereas, in rabbit 2 and 3 only 5% of serum

concentration was required. A careful examination of CIE pattern revealed the

presence of 10-14 antigenic peaks in S. cervi E-S product, whereas, when the y-

globulin fractions of the pooled rabbit anti-E-S serum was tested in CIE, 13-14

precipitin peaks were found with 2-5% concentrations of y-globulin fractions.

Earlier the technique of CIE has been employed by various workers to analyse the

antigenic components of filarial and other parasites (Kaushal et ai, 1982; Malhotra

et al.,\9Sl; Mohan e?cr/., 1988; Haider e/o/., 1990, Sugunan et al., 1990). The

CIE analysis revealed the presence of 10-11 antigenic peaks in B. malayi E-S

antigens (Kaushal et al., 1982) while the S. digitata E-S products revealed the

presence of 3-4 antigenic peaks (Sugunan and Raj, 1990).

The parasitic worms are known to adsorb host proteins on their surfaces to

protect themselves from the immunologically hostile environment of the host,

(Smither et al., 1969). In the present study, the S. cervi E-S products were

analysed for the presence of host proteins and with the help of rabbit anti-normal

buffalo serum and it indicated the presence of 3-4 host proteins contamination. One

67

of the major component was found to be albumin which was confirmed by

absorbing the anti-5. cervi E-S serum on BSA-sepharose sorbent. Host serum

albumin has also been identified as a major component on the surface of adult and

microfilariae (mf) as well as in the excretory-secretory products fi-om adults of

Brugia sp. (Kaushal et al., 1982; Ewang et al., 1987; Maizels et al., 1985a) and

other filarial parasites (Phillip et al., 1984a,b; Maizels et al., 1984a,b; 1986;

Malhotra et al., 1987; Kar et al., 1993; Mania and Kar 1994).

Various immunodiagnostic techniques based on the detection of

circulating antigen has been employed by a number of workers (Au et al., 1981;

Desmoutis et al., 1983; Kaliraj e/cr/., 1981a; Ouassi er o/., 1981; Phillip et al.,

1982; Dissanayake et al., 1984; Forsyth et al., 1985; Hamilton et al., 1984; Reddy

et al., 1984b,c) and the counter current immunoelectrophoresis (CIEP) was one of

them. The counter current immunoelectrophoresis (CIEP) has earlier been used

for the detection of filarial circulating antigen by a number of workers (Kaliraj et

al., 1979; 1981a; Dasgupta et al., 1984; Weil et al., 1984a; 1986a) and this

approach promises to be of considerable diagnostic help. This method appears to be

sensitive and conveniant to perform as compared to night blood examination for the

presence of microfilariae, but the problem of specificity was encountered as

antibodies used in these studies were against the cnide somatic extracts of the

parasite (Weil et al., 1984a, 1986a; Hamilton et al., 1984).

Detecfion of circulating antigen, however, can be complicated by the binding of

antigen into immune complexes (Hamilton et al., 1984). Such complexes are

common in sera fi-om patients with filariasis (Au e/o/., 1981; Steward et al.,

1982; Chandershekhar et al., 1990; Das et al., 1988a; Dissanayake et al., 1982;

Lutsch et al., 1988; Thambiah et al., 1992). Variability in the amount of serum

68

antigen in free and bound state may make difficult the detection of circulating

antigen. Therefore, in the present study the filarial patient sera was pretreated with

EDTA and heat to release the antigen from the immune complexes, thus, in turn

increasing the sensitivity of the assay.

In the present study, the polyclonal antibodies raised in rabbits against S. cervi

E-S antigens were used for the detection of circulating antigen in filarial patients

using small number of serum samples. The hyperimmune semm from all the three

rabbits were found to have antibodies against filarial circulating antigen. Inifially,

these immune rabbit sera showed non-specific reactions with normal human serum

in CIEP. However, this non-specificity was removed by preabsorbing the immune

rabbit sera with albumin-sepharose. The preabsorbed anti-E-S serum could detect

the circulating antigen in all the microfilariae positive patient sera but not in the

non-endemic confrol sera from healthy individuals used in the study.

In conclusion, the excretory-secretory products from S. cervi adults can be

obtained by short term in vitro maintenance of filarial adult worms in protein free

defined medium i.e. DMEM. The rabbit anti-E-S serum gave high titres of parasite

specific antibodies, and showed the presence of 10-14 antigenic proteins in the E-S

products, out of these 2-3 anfigens are derived from the host has also been

evaluated. The potential of anti-E-S antibodies for the detection of circulating

antigen in filarial patient sera was tested by counter current

immunoelectrophoresis. Initially, the immune rabbit serum gave non-specific

reaction with the normal human serum. This non-specificity was removed by

preabsorption of the anti-E-S serum with albumin-sepharose. The preabsorbed anti-

E-S serum has been successfully used to detect the circulating antigen in filarial

patients using a small number of serum samples.

69

CHAPTER rV

SUMMARY AND

CONCLUSIONS

Filariasis is a major health problem in the tropical and subtropical

countries. It is caused by parasites belonging to class filariodea, which have

complex life cycle that alternates between a vertebrate and invertebrate host.

These parasites cause lymphatic damage that leads to elephantiasis and hydrocoele.

In India, over 90% of the disease is caused by Wuchereria bancrofti. Even though

the disease is not fatal, it is responsible for considerable morbidity and disability.

The reproductive activity of the adult leads to the release of microfilariae (mf)

which circulates in blood.

The presently used method for the diagnosis of filariasis, is based on

demonstration of mf in the night blood smears of the patients. This test is

inconvenient for both the patient as well as the investigator and fails to detect the

amicrofilaraemic stage of the disease or when mf are present in very small

numbers. This has put forward an emphasis for the immunodiagnostic test as there is

no early and definitive diagnostic test for filariasis.

Earlier studies on immunodiagnosis of filariasis was focussed mainly on the

detection of antibodies. The major shortcomings of the antibody detection are

firstly, it shows cross-reactivity with other helminth parasites and secondly, it

cannot distinguish the past exposure fi^om the present infection. The presence of

antigen in the circulation and urine of the infected individuals has shown to be a

better approach for diagnosing active filarial infection. A number of assays have

been developed previously by several investigators using polyclonal antibodies for

detection of circulating antigen in sera and urine of infected individuals and

experimental animals but the probelm of sensitivity and specificity of these assays

still remains. There is still a need to produce specific antibodies for developing

70

antigen detection test for filariasis. The excretory-secretory (E-S) products has

been known to be less complex and more specific as compared to the somatic

antigens. The present study was undertaken on the antigenic analysis of E-S

products ofS. cervi, a bovine filarial parasite.

The studies were initiated with the preparation of S. cervi E-S products, by

maintaining the adult parasites in asceptic, protein free medium Dulbecco's

modified eagle medium (DMEM) for 32 hrs at 37°C. These E-S products were

concentrated and dialysed. The prepared E-S products were used to immunized

the rabbits for raising hyperimmune polyvalent serum. Three rabbits (R1,,R2 &

R3) were immunized over a period of 5-6 months. The immune rabbit sera

collected after each immunization starting from the Iir'* injection and were tested

for the parasite specific antibodies by employing enzyme-linked immunosorbent

assay (ELISA). An increase in the titre value with increasing number of

immunization was observed and the titre value became constant after VII ' injection

in all the tliree rabbits (Rl, R2 and R3). The serum pool showed the reciprocal

antibody titre in the range of 100,000-250,000.

The protein and antigenic analysis of S. cervi E-S antigens were done by

employing the techniques of SDS-polyacrylamide gel electrophoresis (SDS-

PAGE), immunoelectrophoresis (lEP), and cross immunoelectrophoresis (CIE).

The SDS-PAGE resolved the .S". cervi E-S products into 14-15 protein bands in the

mol. wt. range of 10 to 200 Kd on 10% acrylamide gel. The number of protein

bands found in S. cervi somatic antigens was 30-35 which showed the more

complex nature than S. cervi E-S products.

The antigenic components of S. cervi E-S antigens were analysed initially by

employing the techniques of lEP using rabbit anti-5. cervi E-S semm. Immuno-

71

electrophoretic analysis showed an increase in the number of precipitin bands with

subsequent injections. The number of precipitin bands became constant with sera

obtained after the Vth injection from rabbits (Rl, R2 and R3). These results

suggest the presence of 6-8 parasite specific antigen in S. cervi E-S products. The

antigenic components of S. cervi E-S products were further analysed by crossed

immunoelectrophoresis using rabbit anti-5. cervi E-S serum and y-globulin

fi-action of pooled anti-E-S serum. The best resolution in CIE was obtained by using

5-10% concentration of anti-S. cervi E-S serum and it revealed the presence of 10-

12 precipitin peaks, whereas, in CIE using 2.5% concentration of y-globulin

fraction of pooled anti-E-S serum from the three rabbits, 13-14 precipitin peaks

were observed against S. cervi E-S products.

The S. cervi E-S products were also analysed for the presence of host serum

protein contamination by using rabbit anti-normal buffalo serum and it showed the

presence of 3-4 host proteins. Further, effort were made to identify these host

protein contaminants by using techniques such as crossed line immun­

oelectrophoresis (CLIE) and tandem crossed immunoelectrophoresis (tCIE). The

CLIE was done by adopting two ways, firstly 50 |xg of BSA was introduced in

the intermediate gel and secondly, by introducing 5% rabbit anti-BSA serum in the

intermediate gel and rabbit anit-E-S serum was used in both the cases. In the

former case, there were precipitin lines seen at the base of the two precipitin

peaks and reduction in the heights of these precipitin peaks were seen in the latter

case. In case of tCIE, BSA was run along with the E-S products in the first

dimension, against the rabbit anti-E-S serum and it showed considerable increase

in the heights of the same two corresponding precipitin peaks. The above results

72

with CLIE and tCIE indicates that the major components of host semm proteins is

found to be albumin.

The rabbit anti-E-S serum was evaluated for the detection of circulating

antigen in human filarial serum. Initially, the anti-E-S serum showed non-specific

reaction with the normal human serum. This may be due to the presence of host

serum albumin in the S. cervi E-S products. The efforts were made to absorb the

host serum proteins from the E-S products on anti-BSA sepharose beads. The

preabsorbed S. cervi E-S products were analysed using SDS-PAGE, CIE and

ELISA. The SDS-PAGE analysis of the preabsorbed E-S products showed the

removal of a major 66 Kd protein band, which is present in the unabsorbed S.

cervi E-S products.

The antigenic components of the absorbed E-S products were analysed in

CIE using rabbit anti-E-S serum. This revealed the presence of 10 precipitin

peaks in the preabsorbed E-S products. Further, the parasite specific antibodies

present in the anti E-S serum was tested in ELISA using the absorbed S. cervi E-S

product. The antibody titre was found to be 1:120,000. Similarly, the rabbit anti-

iS". cervi E-S serum was absorbed on BSA-sepharose beads inorder, to remove the

antibodies against the host protein components present in the hyperimmune serum.

The absorbed anti-E-S serum showed the removal of the antibodies against the

two major host protein contaminants of E-S products. This preabsorbed anti-E-S

serum was tested in ELISA for the presence of parasite specific antibodies using S.

cerv/E-S products. In this case also the antibody titre was found to be 1:120,000

as compared to the reciprocal antibody titre of 250,000 found for the unabsorbed

anti-E-S serum. Further the preabsorbed anti-E-S serum was evaluated for the

detection of circulating filarial antigen in patient sera. This absorbed rabbit anti-E-

73

S sera show no cross-reactivity with the normal human senim, as well as

detected the circulating antigen successfully in the filarial patient sera.

In conclusion, in the present study the E-S product were obtained by the in

vitro maintenance of the S. cervi adults worms. The hyperimmune sera was raised

in rabbits against the S. cervi E-S product. The rabbit anti-5. cervi E-S sera showed

high titres of parasite specific antibodies as detected by enzyme-linked immuno

sorbent assay (ELISA), i.e. in the range of 1:100,000 to 1:250,000. The SDS-

PAGE analysis revealed the less complex nature of 5". cervi E-S products

showing 12-14 protein bands in the molecular weight range of 10-200 Kd. The

crossed immunoelectrophoretic analysis of S. cervi E-S product showed the

presence of 10-12 antigenic components. Out of these 3-4 antigens were identified

as host protein present in the E-S products and one of them being albumin. The

host serum proteins were removed from the S. cervi E-S products by absorbing

on anti-BSA sepharose beads. After absorbtion S. cervi E-S products gave 10

precipitin peaks in CIE and also showed the absence of a major 66 Kd protein

bands in SDS-PAGE. The rabbit anXi-S. cervi E-S serum was also absorbed on

BSA-sepharose beads inorder to remove the antibodies against host serum

proteins. This absorbed anti-E-S serum gave reciprocal antibody titre of 120,000

when tested in ELISA. The rabbit immune sera could detect the filarial

circulating antigen in filarial patient sera. The absorbed anti-E-S serum was

successfully used to detect the filarial circulating antigen and also showed no non-

specificity with the normal human sera. Studies can be further directed to isolate the

cross-reactive anfigens between S. cervi E-S product and human filarial

parasites and antibodies raised against these common epitopes could be used for

developing specific immuno-diagnosfic reagents for the detection of human

filariasis.

74

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