Journal of Virological Methods, 12 (1985) 59-70

Elsevier

JVM 00439

59

USE OF CHICKEN AND RABBIT ANTIBODIES IN A SOLID PHASE

PROTEIN A RADIOIMMUNOASSAY FOR VIRUS DETECTION*

DAVID KATZ, SHOSHANA LEHRER and ALEXANDER KOHN

Israel Institute for Biological Research, P.O.B. 19, Ness-Ziona, Israel

(Accepted 17 June 1985)

A new rapid indirect solid phase radioimmunoassay was developed for the detection of Sindbis virus.

Chicken antibodies were adsorbed onto wells In microplates to serve as ‘capture antibodies* and rabbn

antlbodies were used as the second antibody. 12SI-labelled protein A that does not bind to chicken

antibodles, but binds firmly to rabbit antibodies was used as the tracer.

All the steps necessary for the development of the assay are described. The minimal amount of Sindbis

virus detectable was around 3 X lo5 PFU/ml and the interassay reproducibility was about f 30%.

solid phase radioimmunoassay Sindbis virus

INTRODUCTION

Protein A, a cell wall constituent of Staphylococcus UW~US (Cowan I), has a high

affinity for the Fc part of many mammalian immunoglobulins (Langone, 1982;

Lindmark et al., 1983; Richman et al., 1982; Richman, 1983). It can therefore replace

antispecies antibodies in indirect immunoassays, and serve as a broad reacting tracer

reagent. This feature of protein A was successfully applied for the detection of

antibodies in solid phase protein A radioimmunoassay (SPA-RIA) and enzyme immu-

noassay (Engvall, 1978; Jahrling et al., 1978; Anastasiu and Perrin, 1979; Colombati

and Hilgers, 1979; Marier et al., 1979; Mador et al., 1978; Potgieter et al., 1980; Sprouse

et al., 1981; Richman, 1983).

Detection of antigens using protein A, requires antigen adsorbtion directly onto a

solid phase (Goding, 1978; Enzman, 1978; Langone et al., 1977; Langone, 1978;

Langone, 1980; Richman et al., 1982; Richman, 1983; Butler et al., 1979). However,

such systems are not applicable to unpurified viruses or antigens in clincal specimens,

since irrelevant proteins and other materials present bind also, limiting the amount of

*In memory of the late Eva Gruber whose excellent technical assistance and contribution had a major

influence on this work.

0166.0934/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

60

specific antigen bound and reducing the sensitivity of the detection. The problem is

even worse with serum samples, because background values increase due to reactivity

of the immunoglobulins with the labelled protein A. There has been limited use of a

sandwich assay with protein A as tracer. Yolken and Stopa (1980) used goat antiserum

as ‘capture antibodies’, and Yolken and Leister (1981), used protein A enzyme

immunoglobulin conjugates and goat or burrow antisera as ‘capture antibodies’.

Chicken antibodies do not bind protein A (Kronval, 1974). Thus, we devised an

indirect sandwich type of immunoassay for the detection of antigens using chicken

antibodies which were absorbed on microwells to serve as ‘capture antibodies’. The

antigen, bound by the chicken antibodies was then detected by rabbit antibodies

followed by ‘2SI-labelled protein A. This assay, named solid phase protein A radioim-

munoassay (Sandwich) or SPA-RIA(S) was used in our laboratory to detect a variety

of antigens.

We describe here the steps involved in the development of SPA-RIA(S) for the

detection and titration of Sindbis virus as a model. Results are presented to demon-

strate the performance parameters of the assay, i.e., specificity, sensitivity and repro-

ducibility.

MATERIALS AND METHODS

Viruses

Sindbis virus was grown in baby hamster kidney (BHK-21) cells, cultivated in

Eagle’s medium supplemented with 5% fetal calf serum (FCS). Three types of prepara-

tions were used:

(a) Unpurified virus stocks derived from the infected cells and medium by repeated

freezing and thawing.

(b) Semi-purified Sindbis virus, prepared from unpurified virus stocks by centrifuga-

tion in Beckman L2-65 ultracentrifuge, SW 50.1 rotor, 45,000 rpm, for 75 min and

resuspension of the pellet to the original volume in phosphate-buffered saline

(PBS), pH 7.2.

(c) Purified Sindbis virus stocks, prepared from unpurified stocks by gradient centri-

fugation (Pfefferkorn, 1963).

In addition, the following unpurified virus stocks were prepared: Sindbis virus grown

in chick embryo fibroblasts (CEF) in Eagle’s medium supplemented with 5% normal

chick serum; Semliki forest virus (SFV), cultured in BHK-21 cells, as described for

Sindbis virus; West Nile virus (WNV) was grown in Vero-cells, in Eagle’s medium

supplemented with 5% FCS. All preparations were subsequently stored in -7O”Cuntil

used. Infectivity titers were determined by plaque assays.

All virus preparations were kindly provided by colleagues from our department:

Sindbis viruses by Dr. S. Lustig, Dr. A. Shapira and Dr. P. Fuchs; SFV by Dr. U.

Olshevski, and WNV by Dr. Y. Akov.

61

Protein A iodina tion

Protein A (Pharmacia, Sweden) was labelled by the chloramine T method (Hunter,

1973); 10 ul of a 1 mg/ml Protein A solution were mixed with 10 ul containing 1 mCi of

125I (Amersham, England) and 10 ul of chloramin T (0.5 mg/ml) for 60 s. The reaction

was stopped by adding 10 pl of sodium bisulphite (1 mg/ml) and 100 ul of potassium

iodide (10 mg/ml). L251-labelled protein A (‘251-PA) was separated from free iodine by

gel chromatography on a 0.8 X 20 cm Sephadex G-50 (Pharmacia) column. The

specific activity of this preparation was approximately 43 pCi/mg. The batch was then

diluted in PBS containing 40% FCS, to a concentration of 13 X 10 dpm/ml and stored

at -20°C for up to 4 mth, aliquoted in 4 ml volumes.

Preparation of anti-Sindbis antibodies in rabbits and chickens

Purified Sindbis virus (lOi PFU/ml) was inoculated twice intravenously, at 24

day intervals, into 6-mth-old, albino rabbits.

Chicken antibodies were prepared in two, 8- to lo-mth-old, laying hens, by 2

inoculations of approximately 5 X log PFU per ml of the CEF grown Sindbis virus

stock. The 2 inoculations were given intravenously at 40-day intervals. Blood was

taken at 0,8, 19,40,48, 55, and 78 days after the first inoculation. Eggs were collected

and kept at 4°C until used. For the determination of antibody content in eggs, the yolks

were separated from the whites, mixed with an equal volume of PBS containing 0.02%

sodium azide (PBS-AZ) and centrifuged 20 min at 10,OOOrpm in a Sorvall refrigerated

(4°C) centrifuge (Bar-Joseph and Malkinson, 1980). The supernatant thus obtained

was kept at -20°C until tested for antibodies.

SPA-RIA for rabbit antibodies The procedure was essentially the same as that described by Marier et al. (1979).

Microplates (polyvinyl chloride, 96 flat bottom wells, Dynatech Laboratories Inc.)

were coated with 0.2 ml per well of 10’ PFU per ml semi-purified Sindbis virus in 50

mM carbonate buffer and 0.02% AZ, pH 9.6 (coating buffer) at 4°C overnight. The

wells were then washed 3 times with 0.1% bovine serum albumin (BSA) in PBS-AZ

(PBS-BSA = blocking buffer). Duplicates (0.15 ml) of 5-fold rabbit serum dilutions

in PBS-AZ containing 0.05% Tween 20 (PBS-T) were added to the cells and incubated

at 37°C for 1 h. A known antibody-negative normal rabbit serum (NRS) was similarly

diluted and incubated in each test. After 3 cycles of washing with PBS-T the stock

solution of 1251-PA was diluted in PBS-T, so as to give 50,000 cpm (67,000 dpm) per

0.15 ml. This volume was added to the wells and incubated at 37°C for 1 h. After 4

cycles of washing with PBS-T, wells were numbered, cut from the plate and counted in

a gamma counter (Gammatic I, Kontron, Switzerland). Titration curves were then

drawn by plotting on semi-log paper the meancpmvaluesversus thereciprocalofserum

dilutions. From the NRS control a ‘cut-off curve was obtained by plotting the mean

cpm values to which 3 times the SD was added, versus the reciprocal of the NRS

dilutions. The titer of each serum was defined as the reciprocal of the serum dilution at

the intersection of the titration curve with the ‘cut-off curve.

62

SPA-RIA inhibition test for chicken antibodies

In the SPA-RIA inhibition test (SPA-RIA(IN)), chicken antibodies, first incubated

with the solid phase attached antigens, inhibit the binding of subsequently added,

rabbit antibodies that are directed to the same antigen. The inhibition is recognized by

the addition of “‘I-PA that binds only to rabbit antibodies. A decrease in the amount

of bound ‘251-PA, as compared to normal chick serum (NChS) or normal yolk (NY)

antibody-negative controls, is an indirect indication of the presence of chicken anti-

bodies.

The SPA-RIA(IN) test was performed as follows: Sindbis virus-coated microplate

wells prepared as for SPA-RIA, were incubated with 0.15 ml duplicate serial dilutions

(in PBS-T) of chicken sera or yolks, at 37°C for 1 h. As controls, a series of dilutions of

NChS or NY, negative for Sindbis virus antibodies, were used. The wells were then

washed 3 times with PBS-BSA. A predetermined rabbit anti-Sindbis virus serum

optimal dilution (1 : 1,000 in this study) was incubated (0.15 ml per well) at 37°C for 1

h. After 3 cycles of washes, ‘251-PA (50 000 cpm) was added and incubation continued > at 37°C for 1 h. After 4 cycles of washings, wells were prepared for the gamma counter

as decribed for SPA-RIA. From the data obtained, the percent of inhibition of bound

‘251-PA compared to controls (0% inhibition) was calculated and plotted on a semi-log

paper as a function of the sample dilution. The titer was defined as the reciprocal of the

sample dilution at the intersection of the curve with a 20% inhibition, arbitrarily

chosen ‘cut-off’ line.

SPA-RIA(S) for virus titration

The following procedure was used after defining optimal conditions: microplate

wells (as for SPA-RIA) were filled with 0.2 ml of a 1 : 1,000 dilution in coating buffer of

the pooled chicken anti-Sindbis virus antiserum (A-Sind-ChS) and incubated at room

temperature overnight. The cells were then washed 3 times with PBS-BSA. The coated

plates were dried and kept in sealed plastic bags at -70°C up to 1 yr.

For use, 0.15 ml of duplicate serial virus dilutions in PBS-T were incubated in wells

at 37°C for 1 h. As controls, 2 or more wells were incubated with PBS-T only ((-)virus

control). The wells were then washed 3 times with PBS-T and incubated at 37°C for 1 h

with 0.15 ml of a 1 : 1,000 dilution (in PBS-T) of rabbit anti-Sindbis virus antiserum

(A-Sind-RS). After additional 3 cycles of PBS-T washes, 0.15 ml of the conjugate

(50,000 cpm) were added to the wells and incubated at 37°C for 1 h. The wells were then

washed 4 times with PBS-T and counted in the gamma counter as described for

SPA-RIA.

The amount of bound lz51-PA was plotted on a semi-log (or log/log) paper as a

function of the reciprocal of the sample dilution. The titer was defined as the reciprocal

of sample dilution at the intersection with a cut-off line that equalled the mean cpm

values of the ‘(-)virus’ controls to which 3 times the SD was added. Alternatively and

preferably, the titer was determined using a TI-55-11 of TI-59 calculator (Texas

Instruments, Inc.) for computing a regression line from the log cpm bound 12’I-PA,

63

versus the log of the reciprocals of the sample dilutions. The titers were obtained from

the calculator program by introducing the cut-off value and finding the corresponding

reciprocal dilution value. Apart from titer values, the correlation coefficients, and the

slopes of the curves were computed from the same data. As a rule, the number of points

used for the regression line was never less than 3. Care was taken that the lowest

dilution used gave at least 2 times higher cpm than the ‘(-)virus’ controls.

RESULTS

Antibody response in sera and egg yolk of chickens inoculated with Sindbis virus

Chicken antibodies in sera or yolks were titrated by the SPA-RIA(IN) test. In typical

titration curves obtained with pooled sera and pooled egg yolks the titers obtained

with the 20% inhibition cut-off line were 16,000 for the serum and 10,000 for the yolk.

Maximal cpm mean values of the NChS and NY controls (0% inhibition) from which

the percent of inhibition values were calculated, were around 2,000 cpm.

Blood and eggs collected from the immunized chicken at different times after the

first inoculation were similarly titrated. The rise of SPA-RIA(IN) antibody titers in the

sera and egg yolks of the 2 chickens is shown at different times after incubation in Fig.

1. Relatively few eggs were laid during the follow-up period. The titers of sera and eggs

were similar, although as a rule the titer in the yolk was somewhat lower. Peak titers

were obtained at 8 days after the second inoculation. Six chicken sera which had titers

of 9,000 or higher were pooled (A-Sind-ChS); a similar yolk pool was prepared from

the 3 eggs with titers equal or higher than 9,000. Only the pooled chicken sera were

further used in this study.

Antibody response in rabbits inoculated with Sindbis virus One of the rabbits died of diarrhea within several days after the first injection. The

SPA-RIA antibody titers in sera of the surviving rabbit at 0, 24 and 34 days after the

first inoculation were 20, 10,000, and 530,000 respectively. The high titered serum,

obtained 34 days after the first inoculation, was the ‘A-Sind-RS’ preparation used

throughout the present study.

,Determination of the optimal chicken antiserum dilution for use in SPA-RIA(S) A series of titrations of Sindbis virus (stock 2 = 1 X lo9 PFU/ml) were performed in

microplates coated with a series of IO-fold chicken antiserum dilution from 10m2 to

10W6. The dilution of the rabbit antiserum was kept constant at 1: 1,000. From the

results (not shown) it was evident that with the 10m2, 10m3 and 10e4 dilutions of the

A-Sind-ChS similar titration curves were obtained. Significantly lower responses were

obtained with the lower A-Sind-ChS dilutions. Practically no response was obtained

with a 1: 1,000 dilution of a NRS control (used instead of the rabbit antiserum) in wells

coated with a 10e2 dilution of A-Sind-ChS. Some response, however, was obtained

when the rabbit antiserum was used in wells coated with a 10m2 dilution of randomly

pooled NChS.

Days after 1st inowlation

Fig. 1. Time course of antibody response in sera (black symbols) and egg yolks (open symbols) of 2 chickens

inoculated with Sindbis virus. Titers were determined by SPA-RIA(IN). Triangles represent chicken No. I, circles represent chicken No. 2. Arrows indicate time of inoculations.

Determination of the optimal rabbit antiserum dilution for use in SPA-RIA(S) Three series of Sindbis virus (Stock 2), 5-fold dilutions ( 10-2-10-4.8) were incubated

in wells coated with a 10e3 dilution of chicken antiserum. For each of the series a

different dilution of rabbit antiserum was used: 10w2, 10m3, and 10m4. As shown in Fig. 2,

the optimal dilution for SPA-RIA(S) was the low3 dilution. With this serum dilution

the highest titer, (3,900) namely, the lowest detection limit (2.6 X lo5 PFU/ml) was

obtained. With the 10m2 dilution of rabbit antiserum, the titer obtained was lowest (titer

= 2,400, detection limit = 4.2 X lo5 PFU/ml) due to the high cut-off values derived

from the ‘(-)virus’ controls (x -I- 3 SD = 420 + (3 X 58)~ 594). With the low4 dilution of

rabbit antiserum, an intermediate titer was obtained (titer = 3,200, detection limit =

3.1 X lo5 PFU/ml).

65

Fig. 2. Titrations of Sindbis virus (Stock 2) by SPA-RIA(S) in wells coated with a IO-’ dilution of

A-Sind-ChS. A-Sind-RS was used in 3 dilutions: 10-2(o--o), lo-’ (x---x), and 10e4 (o---o). Numbers indicate

cpm cut-off values calculated from PBS-T ((-) virus) controls (x + 3 SD); arrows point at the intersection of

the cut-off lines with the titration curves from which the titers were calculated.

Specificity of SPA-RIA(S) As observed in the preliminary experiment Sindbis virus adsorbed, to some extent,

nonspecifically to NChS coated wells. In the following experiment, we examined this

phenomenon more carefully, with another NChS preparation and a different stock of

Sindbis virus. The source of the NChS was a pool of preinoculation sera from the 2

hens immunized to produce the A-Sind-ChS. Sindbis virus used was ‘Stock 3’ with an

infectivity titer of 3 X IO9 PFU/ml. Six parallel titrations of Sindbis virus were

performed: 3 in wells coated with 1 : 1,000, 1: 4,000 and 1: 16,000 of A-Sind-ChS

dilutions and the other 3 in wells coated with identical dilutions of NChS. As shown in

Table 1, the NChS coated wells adsorbed the virus at any of the 3 dilutions tested. At

the highest virus dilution tested 90-100% of the virus was adsorbed to the NChS coated

wells as compared to the A-Sind-ChS coated wells. However, at the lower virus

dilution only 50% of the virus was adsorbed. Moreover, from the cut-off values

obtained from the ‘(-)virus’ controls, similar titers were calculated from the titrations

performed on NChS and A-Sind-ChS coated plates. The only difference noted was in

the slopes which were approximately -0.7 for the A-Sind-ChS coated plates and lower

(approximately -0.5) for the NChS coated plates.

,These results indicated that the normal chicken sera contained some virus binding

substance. Since the virus binding substance was not found in young chickens or adult

roosters, we concluded that it was present only in sera from adult laying hens (Katz and

Lehrer, 1983). In spite of the nonspecific binding of the chicken-derived catching

antibodies, the SPA-RIA(S) technique is on the whole specific. When heterologous

66

TABLE 1

Titrations of Sindbis virus (Stock 3) by SPA-RIA(S) in microplate wells coated with A-Sind-ChS compared

to titrations under similar conditions in wells coated with NChS. The rabbit antiserum (A-Sind-RS) was

diluted 1 : 1,000

-log,, virus

dilution

Mean cpm in wells coated with

A-Sind-ChS dilutions NchS dilutions

10-r 4 x 10-r 16 X lo-’ 10-s 4 x 10-j 16 X I--’

2 5,846 6,346 6,681 3,225 3,130 3,698

2.1 1,842 1,849 2,032 1,468 1,328 1,463

3.4 599 644 643 663 561 557

4.1 958 335 370 344 393 374

x’ 164 240 310 197 260 236

+ SDb 19 33 62 21 11 23

x + 3 SDC 221 339 496 260 293 304

Correlation

coefficientd

Sloped

Titerd

-0.999 -0.998 -0.999 -0.999 -0.999 -0.999

-0.70 -0.71 -0.72 -0.53 -0.53 -0.58

10,100 5,900 3,500 16,800 8,447 7,002

a Mean of (-)virus control.

b Standard deviation of controls.

’ Cut-off values.

d Values obtained from regression lines with a TI-59 calculator.

viruses (SFV and WNV) and a homologous virus (Sindbis virus - stock 4) were

compared in the SPA-RIA(S) (Fig. 3), as little as 2.5 X lo5 PFU/ml of Sindbis virus

were detected, while a lOO-fold higher concentration (2 X 10’ PFU/ml) of SFV and

WNV did not elicit any positive signal.

Sensitivity and reproducibility of SPA-RIA(S) The average sensitivity of SPA-RIA(S) was determined in a series of titrations of 7

Sindbis virus stocks with different infectivity titers. As summarized in Table 2, the

mean sensitivity in ‘detection limit’ values was 3.41 f 0.75 (X 105) PFU per ml.

Three of the Sindbis virus stocks (No. 2, 3, and 4) which were titrated in the

sensitivity experiment (Table 3), were also titrated at different times (see Figs. 2 and 3

and Table 1). The results are summarized (Table 3) and the average reproducibilities

shown as the percent coefficient of variation. From these data, an average reproduc-

ibility of f 27% was obtained.

61

1

* h x

z a ”

3 c

-_=-___ 4 _=_-__ .&&-kL_ - -*- 1 I 1 IO IO 01

PFU/ml I 10‘6

Ftg. 3. Spectfictty of SPA-RIA(S). Titration of homologous Sindbis virus - Stock 4(0--o) as compared to

titrattons of heterologous viruses, SFV (x---x) and WNV (o---c). Arrow Indicates the detection limit

obtained with the Sindbis virus at the intersection of the titration curve with the cut-off line (---) calculated

from the mean + 3 standard deviations (x + 3 SD).

TABLE 2

Determination of SPA-RIA(S) sensitivity (detection limit). Sindbis virus stocks 1-7 were simultaneously

titrated under identical conditions of reagents and incubations in the same microplates, as described in

Materials and Methods

Sindbis virus PFU/ml

stock no. x 109

SPA-RIA(S)

titer

Detection

limit

PFU/ml X IO’

1 5 15,600 3.2

2 I 2,100 4.8

3 3 7,500 4.0

4 2 6,200 3.2

5 0.65 2,300 2.8

6 1.5 4,600 3.3

7 0.5 1,900 2.6

68

TABLE 3

Reproducibility of SPA-RIA(S) detection limit determinations. Each one of the tests of experiments 1 and 2

were performed at different days under similar conditions, as described in Materials and Methods

Sindbis virus

stock no.

Infectivity

titer

(PFU/ml

x 109)

Detection limit

(PFU/ml X 105)

Exp. 1 Exp. 2d Mean

f SD

CV (%)

2 1 2.6a 4.8 3.7 f 1.6 43

3 3 3.0b 4.0 3.5 f 0.7 20

4 2 2.5’ 3.2 2.8 i 0.5 32

Mean reproducibility (CV%) zlz SD = 31.6 * Il.

a Data from Fig. 2.

b Data from Table 1.

’ Data from Fig. 3.

d Data from Table 2.

CV = coefficient of variation [(SD/mean) X 1001.

DISCUSSION

Indirect solid phase immunoassays have several advantages over direct assays (Yol-

ken and Stopa, 1980; Devergne et al., 1981; Halonen and Meurman, 1982; Yolken,

1982; Richman et al., 1984). The tracer, which is usually a labelled anti-species IgG can

be used for the detection of many antigens, obviating the need of labelling individual

antibodies for each antigen. The indirect assays are potentially more sensitive than

direct assays because there are more layers of antibodies in the indirect tests. Indirect

assays are easier and quicker to apply, since the titer of the second antibody used may

be lower than the one needed for a sensitive direct test.

The use of protein A as a general tracer instead of anti-species antibodies makes

construction and application of indirect assays even easier. Protein A is commercially

available as a purified, highly active powder; it is comparable in its activity to affinity

purified anti-species antibodies. The use of protein A, therefore, saves time and effort

required for affinity chromatography. Moreover, the labelled protein A can be used

for the detection of antibodies from many animal species (Langone, 1982; Richman,

1983).

Another improvement which we advocate, is the use of laying hens as a source of

viral antibodies. Chickens are good antibody producers and antibodies can be derived

either from blood or egg yolks or both (Patterson et al., 1962; Scherer and Pancake,

69

in indirect assays using a chicken antibody in one layer and a mammalian antibody (Al

Moudallal et al., 1984; Yolken, 1982;‘Devergne et al., 1981) for the second layer.

We have described here all the steps necessary for the development of an indirect

solid phase protein A, sandwich type of radioimmunoassay for Sindbis virus (SPA-

RIA(S)). Only chicken antisera were used, but the possibility of using yolk antibodies

was confirmed as well (unpubl. results).

The versatility of the ‘Z51-labelled protein A was examplified by its use, not only for

the estimation of viral antigens, but also for the estimation of viral antibodies. We have

also demonstrated a method for titrating chicken antibodies, which do not bind protein

A directly, by an inhibition assay (SPA-RIA(IN)).

The average minimal amount of Sindbis virus, detectable with SPA-RIA(S), was

(3.41 f 0.75) X IO5 PFU/ml. Assuming each PFU contains 20-30 virions 3 X lo5

PFU/ml were equivalent to 0.3-0.45 ng/ml viral protein. This sensitivity was within

the sensitivity range obtained by solid phase immunoassays (Halonen and Meurman,

1982; Yolken, 1982; Richman, 1984). Replacing the iZ51-protein A of SPA-RIA(S) by

an alkaline-phosphatase labelled protein A conjugate, colorogenic (SPA-EIA(S)) and

fluorogenic (SPA-ELFA( enzyme immunoassays resulted. In our hands, SPA-

RIA(S) was approximately 6 times more sensitive than the correspondent colorogenic

SPA-EIA(S) (Katz and Lehrer, 1982; Katz et al., 1982), and about 2 times more

sensitive than the fluorogenic SPA-ELFA (Katz et al., unpubl. data). SPA-RIA(S)

was recently used by us for the detection of a variety of viral and non-viral antigens,

e.g. rabies virus (Katz et al., 1984), West Nile virus (Akov and Katz, unpubl. data),

bovine serum albumin (Shneerson-Porath and Katz, 1983), and phytohemagglutinin

(Rosenberg, Bino and Katz, unpubl. data). The use of protein A as the only general

tracer for all those antigens was shown to be both efficacious and economic.

An interesting, serendipitous finding was that normal sera from laying hens, used as

a control ‘capture’ layer, bound Sindbis virus nonspecifically. Preliminary findings

(Katz and Lehrer, 1983), on the nature of the virus bindings substance in normal sera

from laying hens, indicated that this substance is most probably vitellogenin (Deeley et

al., 1975). However, SPA-RIA(S) as a whole was specific due to the specificity of the

second layer of rabbit antibodies.

In this work, we have dealt only with Sindbis virus from tissue cultures as a model.

Whether SPA-RIA(S) will perform equally for the detection of viruses in clinical

specimens, remains to be determined.

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