Vol. 13, No. 3JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1981, p. 449-4550095-1137/81/030449-07$02.00/0

Enzyme-Linked Immunosorbent Assay Compared withImmunoprecipitation for Serotyping Neisseria meningitidis

and Its Use in Demonstrating Serotype PolymorphismELEANOR B. MACKIE,'* LLOYD A. WHITE,2 KEITH N. BROWN,' AND LAWRENCE E. BRYAN'

Department ofMedical Microbiology and Infectious Diseases, Faculty ofMedicine, The University ofCalgary, Calgary, Alberta, Canada T2N 1N4,' and Defense Research Establishment Suffield, RaIston,

Alberta, Canada TOJ2No2

Rabbit antisera generated against 11 prototype strains of group B Neisseriameningitidis were titrated by using the enzyme-linked immunosorbent assay andthe Ouchterlony immunoprecipitation technique. The relative insensitivity of theimmunoprecipitation technique in detecting antibody compared with the enzyme-linked immunosorbent assay was demonstrated. Cross-reactions within the groupand among the 11 serotypes were characterized with respect to establishedserological techniques. Serotype polymorphism among the strains of N. menin-gitidis was demonstrated with the enzyme-linked immunosorbent assay.

To date, the enzyme-linked immunosorbentassay (ELISA) first introduced by Engvall andPerlmann (11) has had many applications (30).From viral work (9, 29, 30) through such diversesystems as aflatoxin B1 with haptenic inhibitors(21), serodiagnosis of toxoplasmosis (31), assayfor alpha-fetoprotein (5), measurement of serumanti-deoxyribonucleic acid antibody (24), and offactor-VIII-related antigen (4), superior sensitiv-ity, specificity, and ease of performance havebeen major attractive features of the assay. Ithas been used by Frasch and Robbins (17, 18) indetermining protective antibody in guinea pigswith implants into which were injected menin-gococci, and it has been used in other bacterialsystems (6-8, 20, 26). This study presents acharacterization of antisera produced in rabbitsagainst 11 serotype strains of group B Neisseriameningitidis. Cross-reactivity and serologicaldiversity were studied. The sensitivity of theELISA was compared with that obtained byOuchterlony immunoprecipitation, which hasbeen used extensively as a serotyping method(14, 16). Also, polymorphism detectable byELISA and not by immunoprecipitation amongmeningococcal strains is described.

MATERIALS AND METHODSBacterial strains. The prototype strains were

kindly provided by C. E. Frasch, Department ofHealth, Education and Welfare, Bethesda, Md. Theseincluded: M136(Tll), M990(T6), MlOll(T2,10),S3032(T12), M982(T9), M992(T5), M978(T8),M981(T4), M1080(T1), M986(T2,7), and B16B6(T2),all serotype strains of N. meningitidis group B.

Stock strains were maintained in the lyophilizedstate. No working cultures were used beyond the fifth

subculture after reconstitution of the lyophilizedstrains.

Antisera. Antisera were prepared in male BeltedDutch rabbits aged 2.5 months, with the exception ofsera prepared against strains M1080, M981, M992, andS3032. These were prepared in female San Juan rab-bits (28) aged 4 to 6 months. The immunization sched-ule was that of Frasch and Chapman (13), except that2.0 ml of Formalin-killed bacterial cells was substi-tuted for the last injection of 2 x 108 live organisms tocircumvent dealing with living meningococci in animalquarters (C. E. Frasch, personal communication). TheBelted Dutch rabbits were given one booster series ofinjections to obtain hyperimmune serum (14). Antiserawere absorbed as described by Frasch and Chapman(13), with the modification that organisms for absorp-tion were generally grown in 100- to 125-ml amounts.One gram of wet cells for each 2.0 ml of antiserum tobe absorbed was used. They were heat killed for 1 h at56°C while still in the growth medium, centrifuged,and washed once in phosphate-buffered saline (PBS),pH 7.4.

Preparation of serotype antigens. A modifica-tion of the saline extraction procedure (13) used alsoin serotyping of Neisseria gonorrhoeae (19) was used,whereby 2 ml of water was added per g of wet cells.The suspended cells were boiled for 20 min and cooledimmediately on ice. The remainder of the extractionprocedure was that supplied by Frasch (personal com-munication). The boiled suspension was pelleted at13,000 x g for 15 min. The supernatant "crude" extractwas centrifuged at 100,000 x g for 2 h, suspended indistilled water, and centrifuged for 2 h at 100,000 x g.The pellet containing the serotype antigen(s) was re-suspended, and sodium azide was added to 0.02%.After an initial favorable serological comparison withthe LiCI method of antigen preparation described byFrasch (above), this semipurified preparation was usedas the antigen in all cases.

Serological methods. (i) Ouchterlony immu-

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noprecipitation. Noble agar (1%; Difco Laboratories,Detroit, Mich.) in normal saline with 0.02% sodiumazide was layered on clean microscope slides, 3 mi perslide. Wells punched into the agar in groups were 3mm in diameter and 7 mm apart. For testing theantisera, the antigenic extracts were placed in thecentral well; antisera or dilutions thereof were ar-ranged in the peripheral wells. In the case of a knownantiserum used to check new extracts, the reversearrangement was used from time to time. The slideswere held at room temperature for 48 h, at which timeany line(s) of precipitation between antigen and anti-serum was recorded. This is the method used byFrasch (personal communication), a slight modifica-tion of which was described previously (14).

(ii) ELISA. The ELISA procedure was essentiallythat of Voller et al. (30), with modifications in volumes,times, and concentrations. Briefly, 200 ,ul of antigenicextracts in carbonate buffer (pH 9.6), at a concentra-tion of 1 ,ug per ml of protein as determined by themethod of Lowry et al. (23), were used to sensitize thewells of microtiter plates overnight at 4°C. The nextday the wells were washed three times with PBS (pH7.4) with 0.05% Tween 80 (PBS-Tween). They wereeither used immediately or stored at -70°C until used.Antiserum dilutions (200 Il) made in PBS-Tween wereadded, and the plates were incubated at room temper-ature for 2 h with agitation at intervals. The plateswere washed as above, 200 il of a 1:1000 dilution ofperoxidase-conjugated goat anti-rabbit immunoglob-ulin G (Miles Biochemicals, Elkhart, Ind.) in PBS-Tween was added, and the mixture was incubated for2 h at room temperature. The plates were againwashed, and 200,l of substrate, 5-aminosalicylic acid(8 mg in 10 ml of water, pH 6.0 ± 0.02) with 1 ml of0.05% H202 per 10 ml, was added. After 30 min ofincubation at room temperature the wells were scoredfor the intensity of the brown reaction product from 0to 4+. The negative controls were represented by thelight beige color of the substrate itself in a well inwhich saline or bovine serum albumin had been sub-stituted for the antigen and antiserum. The 4+ controlwas the color given by the reaction of 50,l of thediluted conjugate with the substrate.

(iii) Slide agglutination. Standard procedures forslide agglutination are given in several texts (12, 22).The procedure outlined by the manufacturer for com-mercially available (Wellcome Research Laboratories,Beckenham, England) grouping sera was extended tothe sera prepared above. A single colony or severalcolonies of a blood agar plate culture 18 to 24 h oldwere emulsified in a drop of saline on a glass micro-scope slide. Occasionally it was necessary to permitgrowth for 24 to 28 h. A drop of antiserum was addedand mixed. The slide was rocked gently and observedfor agglutination for 2 min. Commercial antiseraserved as positive controls, and saline was used as anegative control. In the case of inhibition of slideagglutination with group B purified capsular polysac-charide (a gift from H. J. Jennings, National ResearchCouncil, Ottawa, derived from strain 608B), antiserumand 1 mg of polysaccharide per ml of PBS in equalamounts were incubated at 37°C for 1 h first. Antise-

rum plus an equal amount of saline incubated in thesame way served as a control.

RESULTS

Sensitivity of ELISA versus immunopre-cipitation. The marked differences in the rangeof titers of antisera with homologous (immuniz-ing) antigens between the ELISA and the Ouch-terlony immunoprecipitation are emphasized inTable 1. Of particular note is that the highesttiters obtainable by Ouchterlony immunoprecip-itation were 1:40 in unabsorbed sera. Manydropped to the undiluted after absorption.ELISA titers were 10-4 and 10-5 in some cases.The figures for absorbed antisera represented

in Table 1 were those obtained after absorptionwith M1080(T1) in all except the instances in-dicated. M1080(T1) was chosen initially becauseof its frequent involvement in cross-reactionsusing the immunoprecipitation procedure. Sinceone absorption with M1080(T1) removed thecross-reactions (immunoprecipitation), this sug-gested that the group-specific capsular antigen,which was previously reported not to influenceimmunoprecipitation (14), was the common fea-ture among ail strains. Therefore, the cells weretested by slide agglutination.Characterization of the involvement of

the capsule (group specificity) in tests formembrane-located serotype antigens. Allhyperimmune sera from rabbits which receiveda booster immunization series agglutinated mostof the prototype strains (all were group B), insome instances equally as weil as, or morestrongly than, commercially available groupingsera. In most instances, the agglutinability wasalso removed with a single absorption with strainM1080(T1). Where agglutination remained, itwas with the homologous strain or with a straincarrying a common serotype antigen such asserotype 2. This suggests that in some instances(particularly in hyperimmune sera) the serotypeantigens are involved in the agglutination reac-tion.To test further the involvement of antibody

to capsular antigens, inhibition of agglutinationwith purified capsular polysaccharide was car-ried out on selected antisera which showed var-ious combinations of the reaction in homologousand heterologous systems. Table 2 shows thereduction in agglutination on addition of anequal volume of 1 mg of capsular polysaccharideper ml of PBS to the antiserum and compares itwith the absorbed antiserum (comparably di-luted 1:2 with PBS, in this case). Treatmentwith polysaccharide reduced or eliminated ag-glutination where absorption reduced or elimi-nated it. It had no effect where absorption had

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ELISA IN SEROTYPING OF N. MENINGITIDIS 451

TABLE 1. Comparison of reciprocal serum titers obtained with the ELISA and the Ouchterlonyimmunoprecipitation test with homologous (immunizing) antigens

Unabsorbed antiserum Absorbed antiserum'Antigen

ELISA (range) Ouchterlony (range) ELISA (range) Ouchterlony (range)

M136(Tll) 12,150-36,450 Undiluted to 5 3,645-10,935 Undiluted to 5M978(T8) 109,350-328,050 10-40 405-10,935 Undiluted to 4M982(T9) 36,450-109,350 20 10,935-98,415 Undiluted to 4M990(T6) 8,100-24,300 5-40 3,645-24,300 Undiluted to 2B16B6(T2) 12,150-328,050 Undiluted to 20 3,645-36,450 Undiluted onlyM986(T2,7) 4,050-12,150 10-20 1,600_3,200b Undiluted onlyM1O11(T2,10) 12,150-109,350 5-20 3,200-6,400b 0 to undilutedM981(T4) 4,050-12,000 10-20 4,050-12,000 Undiluted to 5M992(T5) 8,000-12,800 10-40 50-450 5 to 20S3032(T12) 8,100-24,300 Undiluted to 5 8,100-12,150 Undiluted to 4M1080(T1) 1,600-6,400 5-10 800-3,200C Undiluted to 10

a Absorbed with M1080 unless otherwise indicated.b Absorbed with B16B6(T2) to isolate the 7 and 10 antigenic components.C Absorbed with M978.

TABLE 2. Agglutination of antisera and the inhibition of agglutination through absorption and treatmentwith purified group B capsular polysaccharidea

Agglutination with strainAntiserum

M990(T6) M992(T5) Ml011(T2,10) M1080(T1)

Anti-M136(T11)Unabsorbed 4+ 3+Absorbed M1080(T1) Negative NegativePlus polysaccharide +/- Negative

Anti-M990(T6)Unabsorbed 2+ 1+Absorbed M136(T11) 2+ NegativePlus polysaccharide 2+ +/-

Anti-M986(T2,7)Unabsorbed 3+ 2+Absorbed B16B6(T2) Negative +/-Plus polysaccharide +/- +/-

a The limited quantity of this material prevented the performance of an inhibition test on other than selectedantigen-antibody combinations. One antiserum only from a group is represented. See text for scoring of data.+/-, Doubtful positive reaction.

no effect.However, Ouchterlony immunodiffusion tests

using the most reactive unabsorbed agglutinat-ing sera treated in the same way with 1.5, 2.5,4.5, or 6.5 mg of purified N. meningitidis groupB strain 608B polysaccharide per ml did notshow decreased cross-reactivity. Quantitativeprecipitation assays were also negative using 50p.l of antisera with 10, 25, 50, 75, 100, or 150 ,ug ofcapsular polysaccharide. Use of this polysaccha-ride preparation to coat wells for the ELISAresulted in no reaction when concentrations ofpolysaccharide from 1 to 15 ,ug/mI were testedwith strongly agglutinating antisera. By thesame token, pretreatment of the antiserum withpolysaccharide (which reduced agglutination)did not reduce ELISA titers when serotype an-

tigen extracts were used to coat plates (solidphase).Detection of additional antibody to dif-

ferent serotype antigens rendered specificin immunoprecipitation by absorption. Theextreme sensitivity of the ELISA prompted useof this assay for doser examination of additionalcross-reactions when it was observed that a sin-gle absorption did not remove reactions in thesame way in which their apparent removal wasachieved in the Ouchterlony immunoprecipita-tion.

Titrations performed with each absorbed an-tiserum against antigenic extracts from each pro-totype strain were compiled and subjected toanalysis of variance with subsequent F-testing.Methodology for this comprehensive form of

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statistical analysis is given in a number of stand-ard texts (2, 3, 25, 27). Table 3 shows the meanreciprocal ELISA titers with the number of an-tisera (n) in each group. A comparison is madewith titers obtained with unabsorbed antiseratitrated against homologous and absorbing an-tigens only. The mean titers ofabsorbed antiserareacting with homologous antigens are indicatedin boldface type. The mean titers remaining toabsorbing antigens are underlined.The following test criteria based on 1 and 5%

levels of the distribution ofF apply to the data.With the exception of anti-M990(T6), for whichunabsorbed antisera had equal titers with ho-mologous and absorbing antigens, titers weregreater with the homologous antigen, the degreeof significance being P« 0.01 or 0.01 <P < 0.05.The variance in mean titers remaining for all

heterologous antigens after absorption of theantisera was highly significant (P « 0.01) foreach group of antisera, as can be inferred frominspection of Table 3. For example, heterologoustiters of absorbed anti-T2 (B16B6) in the anal-ysis include those against Tl (M1080, absorb-ing), T4, T5, T6, T8, T9, Tii, and T12 (straindesignations as above), but not against T2 (ho-mologous), T2,7 or T2,10 (common antigen).Hence, for F(7,24) = 3.99, P« 0.01. Similarly, thereactions of different groups of antisera with anygiven heterologous antigen (columns) variedmarkedly. In short, absorption with a specificquantity of one serotype of bacterial cells af-fected the remaining titers to different antigensunequally in different groups of antisera. Thisindicates polymorphism in the bacterial cellsused to generate the antibody.A similar estimation of variance of remaining

titers which were equal to or greater than thatremaining for the absorbing antigen was alsodone. Significant variation of titers above theabsorbing in antisera against T2 and T4 (0.01 <P < 0.05), T9, T12, T2,7, and T2,10 (P « 0.01)was found. Other antisera showed either insig-nificant variation or had the highest titers re-maining against the absorbing antigen (e.g., anti-T8).However, anti-Tll sera showed a rather high

(1:1,333) titer against T2,10. Reciprocally, amean titer of 1:2700 against Tii in anti-T2,10sera was observed. Differential removal of theantibody was achieved by reabsorption of anti-M136(Tll) with M1011(T2,10) cells and reab-sorption of anti-M1O01(T2,10) with M136(Tll)cells, reducing the titers to these antigens mark-edly (1:179 and 1:115, respectively).

Figure 1 illustrates graphically the effect ofsequential absorptions of the group of anti-M1080(T1) on heterologous titers. Two absorp-

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tion protocols were used as illustrated. The sec-ond absorption in each case eliminated mostsignificant cross-reactions, although differentcombinations of antigens were affected depend-ing on the sequence of absorbing antigens[M978(T8) followed by M981(T4) followed byM1011(T2,10) or M1011 followed by M978 fol-lowed by M981]. Mean homologous antibodytiters remaining after these absorptions were 1:110 and 1:60, respectively, from 1:1,760 and 1:2,720, respectively, after the first absorption ineach series.

DISCUSSIONAlthough many cross-reactions are detectable

in undiluted sera by using the Ouchterlony im-munodiffusion technique, the insensitivity ofthis system precludes fine differentiation of thenature of the cross-reactions. Supplementary

tests are, therefore, required.The agglutination tests and agglutination in-

hibition (Table 2) suggest strongly that anti-bodies against capsular antigens are generatedin preparing the antisera by the describedmethod of immunization in some instances. Thisappears to be particularly true when immuniza-tions are repeated. The failure to precipitate thepurified capsular polysaccharide with the anti-sera is explainable on the premise that a solubleantigen-antibody reaction product was achievedwith this test. Failure of the polysaccharide toinhibit cross-reactions in the Ouchterlony im-

munoprecipitation is unexplained, except on thebasis that the test does not resolve serogroupand serotype reactions sufficiently well. Moreimportant, however, is the indication that thestrains are polymorphic with respect to theirmembrane-located serotype antigens as shown

VOL. 13, 1981

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in Table 3 and Fig. 1, where differential removalof serotype antibody with absorption is demon-strated by using the ELISA. Although the re-actions of antisera were much greater to thehomologous strain antigen in all except one case[Anti-M1080(T1) where antibody to T4 ap-proached the homologous], significant reactionsto one or more serotypes remained after absorp-tion (Table 3). Sequential absorption selectivelyremoved antibody reacting with some antigensand not others [Fig. 1, and the M136(T11)-M1011(T2,10) case]. Reciprocal relatedness isalso shown in Table 3 with other antigens inwhich heterologous titers were not as high, butthe variance was significant. Extensive cross-re-actions in serotyping in N. meningitidis are,therefore, a predictable characteristic. More-over, the reactions are detectable primarilythrough the use of a sensitive assay such as theELISA.

Additionally, the ELISA has shown thatstrain M1011(T2,10) contains the antigenic com-ponent of Tii carried by strain M136 which wasnot detected by the Ouchterlony immunodiffu-sion technique. This is supported by preliminarydata in the generation of monoclonal antibodyto strain M1011, in which 10 to 15% of the totalhybridoma clones generated antibody reactingwith strain M136. With these and other antigens,occasional clones reacted with heterologous an-tigens supporting the polymorphism demon-strated here (our unpublished data).This study adds to the information of Frasch

et al. on the serotyping system in group B N.meningitidis. The bactericidal assay (13) andthe bactericidal inhibition test (15) contributedstrongly in the characterization of the variousserotypes used here. Since death of the bacterialcell is the endpoint indicator of the bactericidalassay, it is not designed to measure antigenicconstituents present in lesser quantity or whichplay a subordinate role in protective immunity.Consequently, an assay system which measuresantigens in totality is an indicator of their diver-sity.We feel that the ELISA meets these require-

ments in sensitivity. It also permits some uniquedifferential analyses. Proteins, glycoproteins,glycolipids, denatured deoxyribonucleic acid, li-poproteins, and lipopolysaccharides absorb pas-sively to an inert polystyrene surface, but nativedeoxyribonucleic acid and neutral polysaccha-rides do not possess this property (10). Thecapsular polysaccharide used here would appearto fall in the latter category (others have, how-ever, reported coating plates with group B me-ningococcal polysaccharide [1]).

Frasch and Chapman (14) have documented

an antigen common to many group B meningo-coccal strains. The above discussion emphasizesthe use of a sensitive assay, but also addressesthe appropriateness of a choice of assays whichmay or may not detect contributing antigeniccomponents such as the relatively non-immu-nogenic capsule. The polymorphism among theserotype antigens has been examined statisti-cally. The overall diversity of all antigens maynot be dissimilar to those characterized in N.gonorrhoeae (32).

ACKNOWLEDGMENTSThis work was supported by Defense Research Establish-

ment Suffield, RaIston, Alberta, Canada, contract no. 8SU78-00112.

The technical assistance of Marilyn Brown and ChristineStefanowicz is gratefully acknowledged.

LITERATURE CITED1. Apicella, M. A. 1979. Lipopolysaccharide-derived sero-

type polysaccharides from Neisseria meningitidisGroup B. J. Infect. Dis. 140:62-72.

2. Bahn, A. K. 1975. Basic medical statistics. Grune andStratton, New York.

3. Balaam, L. N. 1974. Fundamentals of biometry. GeorgeAllen & Unwin Ltd., London.

4. Bartlett, A., K. M. Dormandy, C. M. Hawkey, P.Stableforth, and A. Voller. 1976. Factor-VIII-relatedantigen: measurement by enzyme immunoassay. Br.Med. J. 1:994-996.

5. Belanger, L., C. Sylvestre, and D. Dufour. 1973. En-zyme-linked immunoassay for alpha-fetoprotein bycompetitive and sandwich procedures. Clin. Chim. Acta48:15-18.

6. Brodeur, B. R., F. E. Ashton, and B. B. Diena. 1978.Enzyme-linked immunosorbent assays for the detectionof Neisseria gonorrhoeae specific antibodies. Can. J.Microbiol. 24:1300-1305.

7. Carlsson, H. E., A. A. Lindberg, and S. Hammar-strom. 1972. Titration of antibodies to salmonella Oantigens by enzyme-linked immunosorbent assay. In-fect. Immun. 6:703-708.

8. Carlsson, H. E., A. A. Lindberg, S. Hammarstrom,and A. Ljundren. 1975. Quantitation of Salmonella O-antibodies in human sera by enzyme-linked immuno-sorbent assay (ELISA). Int. Arch. Allergy Appl. Im-munol. 48:485-494.

9. Clark, M. F., and A. N. Adams. 1977. Characteristics ofthe microplate method of enzyme-linked immunosor-bent assay for the detection of plant viruses. J. Gen.Virol. 34:475-483.

10. Engvall, E., and H. E. Carlsson. 1976. Enzyme-linkedimmunosorbent assay, ELISA, p. 135-147. In G. Feld-man, P. Druet, J. Bignon, and S. Avrameas (ed.), FirstInternational Symposium on Immunoenzymatic Tech-niques INSERM Symposium No. 2. North HollandPublishing Company, Amsterdam.

11. Engvall, E., and P. Perlmann. 1972. Enzyme-linkedimmunosorbent assay, ELISA, III. Quantitation of spe-cific antibodies by enzyme-labelled anti-immunoglobu-lin in antigen-coated tubes. J. Immunol. 109:129-135.

12. Finegold, S. M., W. J. Martin, and E. G. Scott. 1978.Bailey and Scott's diagnostic microbiology. The C. V.Mosby Co., St. Louis.

13. Frasch, C. E., and S. S. Chapman. 1972. Classificationof Neisseria meningitidis group B into distinct sero-types. I. Serological typing by a microbactericidalmethod. Infect. Immun. 5:98-102.

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14. Frasch, C. E., and S. S. Chapman. 1972. Classificationof Neisseria meningitidis group B into distinct sero-types. II. Extraction of type-specific antigens for sero-typing by precipitin techniques. Infect. Immun. 6:127-133.

15. Frasch, C. E., and S. S. Chapman. 1973. Classificationof Neisseria meningitidis group B into distinct sero-types. III. Application of a new bactericidal-inhibitiontechnique to distribution of serotypes among cases andcarriers. J. Infect. Dis. 127:149-154.

16. Frasch, C. E., and S. S. Chapman. 1974. An outermembrane protein of Neisseria meningitidis group Bresponsible for serotype specificity. J. Exp. Med. 140:87-103.

17. Frasch, C. E., and J. D. Robbins. 1978. Protectionagainst Group B meningococcal disease. Il. Infectionand resulting immunity in a guinea pig model. J. Exp.Med. 147:619-628.

18. Frasch, C. E., and J. D. Robbins. 1978. Immunogenicityof serotype 2 vaccines and specificity of protection in aguinea pig model. J. Exp. Med. 147:629-644.

19. Geizer, E. 1972. Studies on serotyping of N. gonorrhoeae.WHO/VDT/RES/GON 72.67, p. 1-7.

20. Jodal, U., S. Ahistedt, B. Carlsson, L A. Hanson, U.Lindberg, and A. Sohl. 1974. Local antibodies inchildhood urinary tract infection. Int. Arch. AllergyAppl. Immunol. 47:537-546.

21. Lawellin, D. W., D. W. Grant, and B. K. Joyce. 1977.Enzyme-linked immunosorbent analysis for aflatoxinB,. Appl. Environ. Microbiol. 34:94-96.

22. Lennette, E. H., E. H. Spaulding, and J. P. Truant(ed.). 1974. Manual of clinical microbiology. AmericanSociety for Microbiology, Washington, D.C.

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