Enzyme-Linked Immunosorbent Assay Detection ... · 574 WETHERALL, HALLSWORTH, AND MCDONALD N.Y.). It was dissolved in phosphate-buffered saline (PBS,pH7.2) andstored at-20°C. Foruse

Vol. 11, No. 6JOURNAL OF CLINICAL MICROBIOLOGY, June 1980, p. 573-5800095-1 137/80/06-0573/08$02.00/0

Enzyme-Linked Immunosorbent Assay for Detection ofHaemophilus influenzae Type b Antigen

BRUCE L. WETHERALL,'* PETER G. HALLSWORTH,' AND PETER J. McDONALD2

Unit of Clinical Microbiology, School ofMedicine, Flinders University,2 and Flinders Medical Centre,1Bedford Park, South Australia, 5042

An enzyme-linked immunosorbent assay for the detection of Haemophilusinfluenzae type b antigen was developed. It was able to detect purified polyribosephosphate at concentrations of :1 ng/ml in cerebrospinal fluid. This was 50 timesmore sensitive than counterimmunoelectrophoresis with the same antiserum. Thesensitivity for polyribose phosphate in urine was similar, but that in serum was

about 10 times less. Nonspecific reactions were observed with blood-stainedcerebrospinal fluid and some sera. These were differentiated from true positivereactions by a blocking test with unconjugated immune serum. A wide range oforganisms was tested for cross-reactivity in the assay. With the exception of a

protein A-rich strain of Staphylococcus aureus, they gave absorbances of <8% ofthat of the homologous system. In a series of five cases of proven H. influenzaetype b meningitis, the sensitivity of the assay with cerebrospinal fluid was

confirmed to be at least 25 times greater than that of counterimmunoelectropho-resis. The results indicate that the enzyme-linked immunosorbent assay is highlysensitive and specific in detecting H. influenzae type b antigen. The necessity toperform the blocking assay on all sera limits its usefulness for the examination ofthese specimens. However, it should prove valuable for the detection of theantigen in cerebrospinal fluid and urine.

Enzyme-linked immunosorbent assay(ELISA) was first introduced in 1971-1972 byEngvall and Perlmann (13,14) and Van Weemenand Schuurs (30) for the detection of antibodiesand antigens in body fluids. This technique hasbeen used extensively for the detection of hor-mones, oncofetal proteins, serum proteins, andantibodies to a variety of bacteria, viruses, andparasites (31). The microbiological applicationsof ELISA have been directed mainly towardsthe measurement of antibodies, and relativelyfew assays for antigen have been developed.The detection of bacterial antigen in body

fluids has gained acceptance in recent years asa means of identifying the etiological agent ofcertain infectious diseases. Counterimmunoelec-trophoresis (CIE) has been the predominantmethod used (8, 9, 11, 16, 19, 27), but othersinclude latex agglutination (21, 25, 34), radioim-munoassay (22, 26), and staphylococcal coagglu-tination (12, 29). Of these, radioimmunoassay isundoubtedly the most sensitive and has beenreported to detect as little as 0.5 ng of antigenper ml in the case of polyribose phosphate(PRP). However, the reagents are labile, and thetechnique is hazardous and requires the use ofexpensive counting equipment, making it un-suitable for widespread use. ELISA has none ofthese disadvantages, and in those cases where a

direct comparison has been made (hepatitis Bsurface antigen [17, 36] and Escherichia colienterotoxin [37]) it appears to be as sensitive asradioimmunoassay. It seemed appropriate,therefore, to explore the possibility of usingELISA to detect bacterial antigen. Haemophilusinfluenzae type b was the test organism selectedbecause (i) it has a well-defmed capsular antigenwhich can be obtained in purified form, (ii) good-quality antiserum is available commercially, and(iii) all of the assays mentioned above have beentested with this antigen, making comparison eas-ier. Crosson et al. (10) published a short paperon the same subject recently but did not examinethe behavior of negative specimens in ELISA orattempt to confirm positive results in a blockingassay. The present paper describes the results ofpreliminary studies in these aspects and exam-ines the sensitivity of the assay in cerebrospinalfluid (CSF), urine, and serum.

MATERIALS AND METHODSAntigens. The haemophilus was isolated from the

CSF of a child with meningitis and cultured in brainheart infusion broth with supplements (2). For prep-aration of antigen, the cells were grown to a concen-tration of approximately 109 organisms per ml andsonicated for 5 min at 40 W (Branson Sonifier B-12).Purified PRP was the generous gift of Porter Anderson(University of Rochester Medical Center, Rochester,

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574 WETHERALL, HALLSWORTH, AND MCDONALD

N.Y.). It was dissolved in phosphate-buffered saline(PBS, pH 7.2) and stored at -20°C. For use in assays,

the antigens were diluted in saline, CSF, urine, or

serum.

Test samples. CSF and urine specimens were ob-tained from those submitted to the Microbiology De-partment for routine examination. They were storedat -20°C before testing or pooling. Normal humanserum was obtained from specimens submitted forroutine antenatal serology.Antiserum. Rabbit antisera to H. influenzae type

b from various manufacturers (Wellcome ReagentsLtd., Beckenham, Kent, U.K.; Hyland Laboratories,Costa Mesa, Calif.; and Difco Laboratories, Detroit,Mich.) were fractionated by sodium sulfate precipita-tion and dissolved in PBS after exhaustive dialysis.Normal rabbit serum (Commonwealth Serum Labo-ratories, Parkville, Victoria, Australia) was treatedsimilarly.

Conjugation. Immunoglobulin was conjugatedwith alkaline phosphatase (Boehringer Mannheim,grade 1) by the method of Engvall and Perlmann (14).A 0.3-ml volume of enzyme suspension (1.5 mg ofprotein) was centrifuged, and the pellet was mixedwith 0.5 mg of immunoglobulin. After dialysis againstPBS, glutaraldehyde was added to a final concentra-tion of 0.2%. After 2 h at room temperature, thesolution was diluted to 1 ml and dialyzed against PBS.An additional dialysis against 0.05 M tris(hydroxy-methyl)aminomethane-hydrochloride (pH 8.0) was

performed, and the conjugate was diluted to a finalvolume of 5 ml with 0.05 M tris(hydroxymethyl)-aminomethane-hydrochloride (pH 8.0) containing1.0% bovine serum albumin, fraction V (Common-wealth Serum Laboratories), 1 mM MgCl2, and 0.02%NaN;i. The conjugate was stored in the dark at 4°C,without loss of activity over a 12-month period.ELISA procedure. The basic procedure was an

adaptation of that described by Voller et al. (32). A200-dl sample of fractionated antiserum in 0.05 Mcarbonate buffer (pH 9.6) was added to each well of a

polystyrene microtiter plate (Kayline Plastics, Ade-laide, S. Australia) and incubated at 37°C for 4 h andthen overnight at 4°C. The plate was washed threetimes with PBS containing 0.05% Tween-20 (PBS-Tween). A 200-fl portion of test sample was added toeach of duplicate wells and incubated at room tem-perature (22°C) for 2 h in a humid chamber. Afterfurther washing with PBS-Tween, 200 ul of conjugatein PBS-Tween containing 4% bovine serum albuminwas added, and the plate was incubated at room tem-perature for another 2 h. It was washed again, and 200[l of substrate (1 mg of p-nitrophenyl phosphate perml in 10% diethanolamine buffer, pH 9.8) was added.After 60 min at room temperature, the reaction was

stopped by the addition of 50 [ld of 3 N NaOH, and theoptical density (OD) of the solution was measured ina spectrophotometer at 400 nm.

Blocking test. The test sample was mixed with an

equal volume of either immune globulin, normal rabbitglobulin (both at 200 jig/ml in PBS), or saline andincubated at 37°C for 1 h. Each was submitted to thenormal ELISA procedure to determine the effect ofthe pretreatment. Samples showing >50% reduction inOD with immune globulin relative to that with normal

rabbit globulin were considered to be positive. Whenantigen was present in excess, the sample was dilutedin saline before pretreatment.

CIE. CIE was performed as described by Coonrodand Rytel (9). Glass slides were coated with 1% agarosein 0.05 M Veronal buffer (pH 8.2) to a depth of 1.5 to2 mm. The wells (3 mm in diameter, 2 mm apart) werefilled with 10 ld of antigen or antiserum, the latterbeing from the same batch as that used in the ELISA.The reservoirs contained 0.05 M Veronal acetatebuffer (pH 8.6). Electrophoresis was performed at 15mA for 45 min. The slides were examined for precipitinlines immediately, and if negative, they were examinedagain after 1 h and 18 h of storage at 4°C.

RESULTSDetermination of optimal conditions for

ELISA. Each step of the basic procedure wasexamined systematically with two aims in mind:(i) to obtain maximum differentiation betweenpositive and negative specimens and (ii) to com-plete the assay in the shortest time possible. Theantigen used in this initial work was a salinesuspension of whole cells (heat killed at 56°C for30 min) at a concentration of approximately 5x 106/ml. Saline was the negative control.Three brands of antisera were tested for po-

tency. The Difco product gave significantlylower readings than the other two. The Well-come antiserum was selected for subsequentwork because of ease of supply.A variety of microtiter plates were tested,

including a polyvinyl flat surface (Cooke Micro-titer, Dynatech Corp., Cambridge, Mass.) andseveral brands of polystyrene. These were theCooke Micro-Elisa flat- and U-bottom plates,Linbro flat tissue culture and nonsterile plates(Flow Laboratories, Rockville, Md.), and twolocal brands (Disposable Products flat and Kay-line Plastics U-bottom plates). The polyvinyland flat-bottom polystyrene plates were clearlyinferior to the others. There was little to choosebetween the various brands of U-bottom plates,so the one of local manufacture was selected forreasons of economy and supply.The optimal amounts of antiserum (for coat-

ing the plates) and conjugate were determinedby checkerboard titration. The antiserum pro-duced optimal coating at a concentration of 2,ug per well. Batches of conjugate were found tovary in their activity, but the optimum responseoccurred usually with dilutions of 1:40 or 1:80 inPBS-Tween + bovine serum albumin.

Incubation times of less than 2 h were testedin conjunction with temperatures above ambient(22°C) for the binding of antigen to coat andconjugate to antigen in an attempt to reduce thetotal assay time below 5 to 6 h. The elevatedtemperatures caused a significant increase inbackground readings (i.e., the negative control)

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ANTIGEN DETECTION BY ELISA 575

without a comparable increase from the positivecontrol. Similarly, the differentiation betweenthe two was decreased when the time was re-duced and the temperature was kept at ambient.The rate of the alkaline phosphatase reactionwas not affected significantly by increasing thetemperature, so this also was maintained at22°C. In summary, incubations of 2 h at 220Cfor the antigen-antibody binding steps and 1 hat 22°C for the enzyme reaction were found togive the greatest differentiation between positiveand negative specimens.

Sensitivity. The sensitivity of ELISA wascompared to that of CIE in terms of both puri-fied antigen (PRP) and sonicated cells, dilutedin pooled CSF. The cells were from a log-phaseculture subjected to ultrasonic treatment as de-scribed. The ELISA was considered positive ifthe test sample gave an OD more than doublethat of the CSF after correcting for background(due to unreacted substrate + alkali). TheELISA was able to detect as little as 1 ng ofPRP per ml and 3.0 x 105 cells per ml, whereas50 ng of PRP per ml and 1.2 x 107 cells per mlwere the minimum concentrations detectable byCIE. The limit of sensitivity of the test for PRPin pooled urine was also 1 ng/ml using the samecriterion for positivity as above.The OD is expressed graphically as a function

of antigen concentration in Fig. 1. Saturation ofthe antibody coat occurred at concentrations ofapproximately 30 ng of PRP per ml and 108 cellsper ml. The difference in OD between the twoantigens at saturation is significant (approxi-mately fourfold) and may be due to steric fac-tors.

Specificity. The specificity of ELISA wasexamined by testing saline suspensions of a widevariety of organisms in the standard assay forcross-reaction to H. influenzae type b, as fol-lows: H. influenzae types a, c, d, e, f; non-encap-sulated H. influenzae; Haemophilus canis, H.haemolyticus, H. parainfluenzae, and H. para-phrohaemolyticus; Enterobacter aerogenes andE. cloacae; E. coli Bi 7509/41 and Easter; beta-hemolytic streptococci groups A, B, C, G; Kleb-siella pneumoniae; Neisseria meningitidisgroups A, B, C, X, Y; Staphylococcus aureusCowan 1 (protein A-rich) and miscellaneousstrains; Staphylococcus epidermidis; Strepto-coccus pneumoniae types 6, 15, 29, 35; Candidaalbicans; Citrobacter freundii; Proteus mirabi-lis; Pseudomonas aeruginosa; and viridansstreptococcus. Organisms were obtained eitherby isolation from clinical specimens or from typeculture collections. Single strains were grown onchocolate or blood agar, and the colonies wereemulsified in saline to give a concentration ofapproximately 108 cells per ml. A suspension of

ORGANISMS/ml

Ec

00I.-

C')zUJa0

0-0~

1 20 60 100 140CONCENTRATION OF PRP ng/ml

FIG. 1. Dose-response curve for PRP and soni-cated H. influenzae type b in the ELISA showing ODat 400 nm as a function of the concentration of anti-gen (nanograms per milliliter and organisms permilliliter, respectively) in CSF.

H. influenzae type b, prepared similarly, wasused as the positive control. This plate growth-derived control gave an OD of 1.02, which islower than the OD of 2.0 recorded for the broth-grown culture shown above (Fig. 1). This wasprobably due to the organism shedding excesssurface antigen during growth in the liquid cul-ture. The saline diluent gave an OD of 0.04.Except for the protein A-rich staphylococcus,which gave an OD of 3.1, the other organismsgave negligible readings, the greatest being 0.07(beta-hemolytic streptococcus group B).Determination of the normal range for

negative CSF. A total of 100 samples of CSFthat were bacteriologically negative by micros-copy and culture were tested by ELISA to de-termine the normal range for such material (Fig.2). Of the 100, 80 gave readings of less than 0.1,11 gave readings of from 0.1 to 0.2, and 9 weregreater than 0.2. All of the third group weremacroscopically blood stained to varying de-grees. Statistical analysis of the non-blood-stained specimens produced a mean of 0.06 witha standard deviation of 0.05.Attempts to determine the cause of the high

backgrounds in the blood-stained specimens

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n=91F] Normal CSF 2=0 06

(S.D.= 00520E 20 Blood-stained CSF

£ 16 +2 S.D.>_ ~~~+3 S.D.

Z 12 0

w 8-IL

4-

-04 -08 -12 -16 -20 *24 *28 -32 *36 -40 -44 *48 >-50

OPTICAL DENSITY (400nm)FIG. 2. Histogram of the OD values of bacteriologically negative CSFs. Blood-stained specimens were

excluded from statistical analysis.

were unsuccessful. Normal CSF spiked withwhole or lysed erythrocytes with or withouthomologous serum failed to show a significantrise in OD, even when the reconstituted speci-men was allowed to stand at room temperatureovernight before being tested.Urine samples. Thirty specimens of urine

were examined to determine the range of nega-tive values. Of the 30 urines, 16 were sterile, 11contained low levels of nonpathogenic orga-nisms, and 3 contained significant numbers of E.coli, K. pneumoniae, and Streptococcus faecalis,respectively. All specimens were clarified by cen-trifugation before testing. The OD values ob-tained were in the range of 0.01 to 0.09, with amean of 0.05 and standard deviation of 0.02.Serum samples. Serum was found to behave

in an erratic manner in the ELISA. Some sam-ples gave low backgrounds, whereas others gen-erated high readings for no apparent reason.This unpredictable behavior meant that serumcould not be tested confidently for the presenceof antigen in the normal ELISA. However, theuse of the blocking assay appears to overcomethe problem (see below). Using this technique,PRP was detectable in serum at a concentrationof 13 ng/ml. This compares with a CIE detect-ability of 250 ng/ml.Blocking assay. In view of the high back-

ground readings obtained with blood-stainedCSFs and some sera, a blocking assay was de-veloped to differentiate these from positive spec-imens. The samples were pretreated with im-mune globulin before addition to the standardELISA, and the readings were compared withthose obtained after pretreatment with salineand with normal rabbit globulin (Table 1). TheODs obtained with positive specimens were notaffected much by normal rabbit globulin but

were reduced significantly by the immune glob-ulin (specimens 2 and 5), except when the anti-gen was present in excess (specimen 6). In con-trast, negative specimens either were not af-fected by either globulin (specimens 1 and 3) orwere reduced by both (specimens 7 and 8). Inthe latter case, the ODs were reduced more bythe immune globulin than by the normal rabbitglobulin. However, the effect is not great, andthe percentage of reduction with immune versusnormal rabbit globulin is well below the nominal50% for a positive result. When antigen waspresent in excess (specimen 6), the specimenbehaved as though it were negative. This wasdifferentiated from a true negative by diluting iteither in a low background pool (specimen 5) orin saline (result not shown). In either case, theimmune globulin then caused the OD to dropsignificantly. Similar treatment of a negativespecimen did not have the same effect (speci-mens 7 and 8). The blocking assay clearly failedto work when antigen was present in excess. Itwas therefore of interest to determine the con-centration range of antigen over which the assaywould work. Dilutions of PRP were prepared inserum and urine and tested as before. The max-imum concentration of PRP permitting morethan 50% reduction in OD with immune globulinpretreatment was found to be 250 ng/ml in bothmedia. The minimum concentration detectablein serum was 13 ng/ml, whereas that in urinewas less than 5 ng/ml.

Clinical specimens. During the course ofthese studies, CSF was collected from five chil-dren with H. influenzae type b meningitis. Di-lutions of each were prepared in pooled CSF andtested by CIE and ELISA. Results of the latterwere considered positive if the readings weremore than double that of the diluent. In all cases

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TABLE 1. Effects of the blocking assay on positive and negative specimensOD at 400 nm

Specimen +Saline +Normal rabbit +Immune globu- % Reduction"

globulin lin

1. Normal CSF 0.11 0.11 0.10 92. Positive CSFb 0.35 0.39 0.08 803. Blood-stained CSF 1.57 1.61 1.78 -

4. Low-background serum 0.02 NDc ND -

5. PRP (13 ng/ml) in specimen 4 0.07 0.08 0.02 756. PRP (2.5 yg/ml) in specimen 4 0.46 0.41 0.36 127. High-background serum 0.22 0.12 0.09 258. Specimen 7 diluted 1:4 in saline 0.09 0.07 0.05 29

a Percent reduction in OD after treatment with immune globulin compared to that with normal rabbitglobulin. -, Not applicable.

b The CSF was seeded with organisms at a concentration of approximately 5 x 106 celis per ml.'ND, Not done.

the specimen was positive by ELISA at concen-trations 40 to 320 times less than that detectableby CIE. One of them was negative by CIE, yetin the ELISA it gave an OD of 0.49 for theundiluted fluid. This figure is clearly positive onthe basis of the normal range for negative CSFs(Fig. 2).

DISCUSSIONThe present investigation shows that ELISA

can be used to detect the capsular antigen of H.influenzae type b in a highly sensitive and spe-cific manner. The conditions found to give max-imum sensitivity were similar to those recom-mended by Voller et al. (32). The assay timecould not be reduced below 5 to 6 h without asignificant loss in sensitivity. This is appreciablylonger than the time normally required for CIE,though even that may take up to 18 h to producemaximum sensitivity (18, 28).The assay was able to detect 1 ng of PRP per

ml and 3 x 105 cells per ml in CSF. This is,respectively, 50 and 40 times less than the min-imum detectable by CIE. A few authors havereported the sensitivity of CIE to be in the 1- to5-ng/ml range for PRP (28, 33), but the majorityare unable to detect less than 10 to 50 ng/ml (8,11, 16, 19, 24, 27). This may be significant interms of accurate detection of PRP in meningi-tis, since the CSF of 50% of such cases has beenfound to contain less than 10 ng of the antigenper ml (26). The level of detectability is deter-mined to a large extent by the potency of theantiserum. For this reason, care was taken in thepresent study to ensure that the same batch wasused in both tests, so that comparison of theirsensitivities would be valid. The minimum con-centration of PRP detectable in urine was of thesame order as that detected in CSF, whereas inserum, the minimum detectable was 13 ng/mlby the ELISA blocking assay and 250 ng/ml by

CIE. The drop in sensitivity with ELISA wasdue to the higher background readings obtainedwith serum, necessitating the use of the blockingassay. The drop in sensitivity with CIE was ofan extent similar to that observed by Ward etal. (33) and was presumably due to a reductionin the concentration of free migrating PRP.

Increasing the concentration of antigen abovethe minimum detectable gave increases in ODuntil saturation of the antibody coat occurred.This was at approximately 30 ng of PRP per mland 108 cells per ml in pooled CSF. The ODobtained at this level was fourfold greater withthe cellular antigen than with the PRP. Thereason is unclear but may be due to the largersurface area of the cell having more binding sitesavailable to the conjugate. The dose-responsecurve for the PRP is similar to that obtained byCrosson et al. (10), except that in their handssaturation occurred at about 100 ng/ml. Thismay reflect differences in the polystyrene platesor in the nature of the anti-PRP antiserum.The assay was shown to be specific for H.

influenzae type b. Organisms reported to cross-react with this antigen (E. coli Easter [6] andpneumococcus types 6, 15, 29, and 35 [1]) showedlittle activity, although a high concentration ofantigen was tested. Crosson et al. (10) also noteda lack of cross-reactivity with E. coli Easter byELISA despite a strong reaction with the sameantiserum by CIE. The explanation for this maylie in the fact that the enzyme assay uses theantiserum at approximately 1/1,000 the concen-tration used in the electrophoretic assay. Thislow level may be insufficient to permit theweaker (i.e., heterologous) reactions to occur ata significant level. The Cowan 1 strain of S.aureus produced a strong reaction, which wasexpected; the affinity of the protein A compo-nent for the Fc region of immunoglobulin G iswell documented (see reference 23). However,

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the reaction is unlikely to cause problems in theclinical situation, since S. aureus is an uncom-mon cause of meningitis and urinary tract infec-tion.When specimens of bacteriologically negative

CSF were tested in the assay, they generated arelatively uniform set of readings. This is per-haps surprising in view of the fact that thespecimens were examined over a 9-month periodduring which two batches of antiserum and twopreparations of conjugate were employed. Onlyblood-stained specimens gave readings above0.2. This uniformity allows the ELISA to beused directly in examining non-blood-stainedCSF for the presence of antigen. Three standarddeviations from the mean cover >99% of a nor-mally distributed population (4), and in thisseries all normal CSFs fell within that limit. Itis reasonable, therefore, to call specimens posi-tive (P < 0.01) if they give readings greater than3 standard deviations from the mean, which inthis case is a reading of 0.21. The factors respon-sible for the high readings with blood-stainedCSF could not be identified in this study. How-ever, rheumatoid and some nonspecific factorsin serum (heat-labile components and antibodyto plasma proteins) have been reported to gen-erate false-positive reactions in latex agglutina-tion testing (21, 25, 34) and are likely to producethe same effect in ELISA. It appears not to bea significant problem if the blocking assay isused on these specimens.Examination of 30 urine samples known to be

free of H. influenzae type b antigen producedresults similar to those obtained with normalCSFs. The above statistical considerations aretherefore applicable. Three standard deviationsfrom the mean in this case is equivalent to areading of 0.11. ELISA appears to work partic-ularly well with urine, and in view of the knownexcretion of bacterial antigen in this fluid inpatients with bacterial meningitis (15), it maywell find its greatest value with this type ofspecimen.Serum was not examined to any depth in the

assay because of its erratic behavior. Berdal etal. (5) noted the same phenomenon with serumin a recent study to detect Legionella pneumo-phila antigen by ELISA. The factors mentionedabove for blood-stained CSF may have beenresponsible; sera positive for rheumatoid factorcertainly gave elevated readings in the presentstudy (results not shown), though these speci-mens were excluded from the group tested ini-tially. A further explanation may lie in the na-ture of the polystyrene of the microtiter plates.Chessum and Denmark (7) found that antigenbound unevenly to the surface, causing unpre-

dictable variations in assay results. Uneven coat-ing of the plate with immunoglobulin may havecaused similar effects in the present study.The blocking assay added an hour to an in-

cubation time which was already 5 h long. Theextra time is considered justified because it enablesthe routine assay to differentiate nonspecificreactions obtained with blood-stained CSF andsome sera from true positive reactions by theuse of a preincubation step with immune serum.In theory, this step can be applied either beforeor after the specimen is added to the plate, andin practice both methods have been used inconfirming hepatitis B positives by ELISA (18,33). In our hands, the latter method gave unsat-isfactory results (not shown) despite the theo-retical advantage that excess antigen is washedoff before the blocking antibody is added. Theformer method gave clear differentiation be-tween positive and blood-stained CSFs. In thecase of high-background sera, the blocking assayis more difficult to interpret because of the dropin OD caused by the normal rabbit globulin. Therabbit globulin reduces the nonspecific compo-nent of the binding by competition, whereas theimmune globulin also reduces that binding dueto the specific antigen when it is present. Com-parison of the reduction in OD with immuneversus normal rabbit globulin is therefore still avalid test for the presence of antigen. The block-ing assay did not work when antigen (whenpresent) was in excess of the blocking antibody.This "excess" level was reached at a PRP con-centration of 250 ng/ml. The need to dilutespecimens containing antigen at concentrationsabove this level limits the usefulness of the testwith serum. In the case of urine, however, theblocking assay would be necessary only to con-firm a result that was positive by the standardassay.

Wolters et al. (36) found it necessary to per-form a confirmatory test (blocking assay) on allsera positive for hepatitis B surface antigen bythe routine ELISA. Our experience with seraagrees with that, and ideally the same shouldapply to CSF. However, limitations in specimenvolume usually preclude this, so it is fortunatethat the CSF behaves so predictably in theassay. The five clinical specimens from childrenwith proven H. influenzae type b meningitisgave positive results at substantial dilutions.This suggests that in routine testing it may bepossible to perform the blocking assay on dilutedmaterial to confirm the screening result obtainedwith undiluted CSF. The five specimens alsoconfirmed that ELISA was at least 32 timesmore sensitive than CIE in detecting.the hae-mophilus antigen in CSF. Of particular interest

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was the specimen that was negative by CIE. Notonly was it positive to a dilution of 1:40 whencompared to the reading of the CSF used asdiluent, but also the undiluted fluid gave a read-ing more than double the maximum of the neg-ative range, strongly implying a positive result.The work presented here shows that ELISA

can be used as a highly sensitive and specificassay for H. influenzae type b antigen in CSFand urine. The requirement to perform theblocking assay on dilutions of serum limits itsuse with these specimens. The applicability ofthe test for the detection of antigen in bodyfluids will only be determined after further workon clinical specimens, in conjunction with com-

parisons with other antigen detection methods.

ACKNOWLEDGMENTSWe thank D. Hansman and Sandra Duncan of the Micro-

biology Department, Adelaide Children's Hospital, for provid-ing us with three of the positive clinical specimens of CSF andstrains of pneumococci, meningococci, and Haemophilus. Wealso thank R. Matters of the Institute of Medical and Veteri-nary Science for providing the E. coli strains and several otherHaemophilus strains.

LITERATURE CITED

1. Alexander, H. E. 1965. The hemophilus group, p. 724-741. In R. J. Dubos (ed.), Bacterial and mycotic infec-tions of man, 4th ed. J. B. Lippincott Co., Philadelphia.

2. Anderson, P., R. B. Johnston, and D. H. Smith. 1972.Human serum activities against Hemophilus influenzaetype b. J. Clin. Invest. 51:31-38.

3. Anderson, P., G. Peter, R. B. Johnston, L. H. Wetter-low, and D. H. Smith. 1972. Immunization of humanswith polyribophosphate, the capsular antigen of He-mophilus influenzae type b. J. Clin. Invest. 51:39-44.

4. Armitage, P. 1973. Statistical methods in medical re-search, p. 44-81. Blackwell Scientific Publications, Lon-don.

5. Berdal, B. P., C. E. Farshy, and J. C. Feeley. 1979.Detection of Legionella pneumophila antigen in urineby enzyme-linked immunospecific assay. J. Clin. Micro-biol. 9:575-578.

6. Bradshaw, M. W., R. Schneerson, J. C. Parke, and J.B. Robbins. 1971. Bacterial antigens cross-reactivewith the capsular polysaccharide of Haemophilus influ-enzae type b. Lancet i:1095-1097.

7. Chessum, B. S., and J. D. Denmark. 1978. InconstantELISA. Lancet i: 161.

8. Coonrod, J. D., and M. W. Rytel. 1972. Determinationof aetiology of bacterial meningitis by counterimmu-noelectrophoresis. Lancet i: 1154-1157.

9. Coonrod, J. D., and M. W. Rytel. 1973. Detection oftype-specific pneumococcal antigens by counterimmu-noelectrophoresis. I. Methodology and immunologicproperties of pneumococcal antigens. J. Lab. Clin. Med.81:770-777.

10. Crosson, F. J., J. A. Winkelstein, and E. R. Moxon.1978. Enzyme-linked immunosorbent assay for detec-tion and quantitation of capsular antigen of Haemophi-lus influenzae type b. Infect. Immun. 22:617-619.

11. Edwards, E. A., P. M. Muehl, and R. 0. Peckinpaugh.1972. Diagnosis of bacterial meningitis by counterim-munoelectrophoresis. J. Lab. Clin. Med. 80:449-454.

12. Eldridge, J., E. M. Sutcliffe, J. D. Abbott, and D. M.Jones. 1978. Serological grouping of meningococci anddetection of antigen in cerebrospinal fluid by coagglu-

tination. Med. Lab. Sci. 35:63-66.13. Engvall, E., and P. Perlmann. 1971. Enzyme-linked

immunosorbent assay (ELISA). Quantitative assay ofimmunoglobulin G. Immunochemistry 8:871-874.

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Enzyme-Linked Immunosorbent Assay Detection ... · 574 WETHERALL, HALLSWORTH, AND MCDONALD N.Y.). It was dissolved in phosphate-buffered saline (PBS,pH7.2) andstored at-20°C. Foruse