Vol. 20, No. 2JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1984, p. 259-2650095-1137/84/080259-07$02.00/0Copyright C) 1984, American Society for Microbiology

Detection of Neisseria meningitidis Group A, Haemophilusinfluenzae Type b, and Streptococcus pneumoniae Antigens in

Cerebrospinal Fluid Specimens by Antigen Capture Enzyme-LinkedImmunosorbent Assays

JOHN E. SIPPEL,1* CATHERINE M. PRATO,' NABIL I. GIRGIS,2 AND EARL A. EDWARDS3Naval Biosciences Laboratory, Naval Supply Center, Oakland, California 946251; Naval Medical Research Unit No. 3,

Cairo, Egypt2; and Naval Health Research Center, San Diego, California 921383

Received 27 January 1984/Accepted 9 May 1984

Antigen capture enzyme-linked immunosorbent assay was compared to coagglutination and counterimmuno-electrophoresis for the detection of meningococcal, Haemophilus, and pneumococcal antigens. Enzyme-linkedimmunosorbent assay detected 1 ng of purified meningococcal and Haemophilus polysaccharides per ml and 5ng of pneumococcal polysaccharide per ml; coagglutination detected 20, 25, and 30 ng/ml, respectively, of thesepolysaccharides; and counterimmunoelectrophoresis detected 10, 50, and 60 ng/ml. Double-antibody sandwich-antiglobulin enzyme-linked immunosorbent assays, which employed antibodies produced in two animal species,differentiated 100% of the cerebrospinal fluid (CSF) specimens from meningococcal meningitis patients and95% of the CSFs from Haemophilus patients from heterologous control CSFs. Double-antibody sandwichprocedures, which use the same antiserum preparation for coating the wells of microtiter plates and for alkalinephosphatase-conjugated immunoglobulin, differentiated meningococcal CSFs from control specimens but wereunable to effectively differentiate the Haemophilus or pneumococcal specimens from control CSFs. Coaggluti-nation detected specific antigen in 92% of the meningococcal CSFs, 80% of the Haemophilus CSFs, and 92% ofthe pneumococcal specimens. The comparable percentages for counterimmunoelectrophoresis were 76, 95, and71%.

Detection of bacterial antigens in cerebrospinal fluid(CSF) was shown to be feasible in the early part of thiscentury (1, 13). However, this approach to the diagnosis ofacute bacterial meningitis was rarely employed until coun-terimmunoelectrophoresis (CIE) was evaluated in the 1970s(5, 10, 12). When high-quality antisera were used, theefficacy of this procedure, which could provide a diagnosisin less than 1 h, usually approached that of culture (16). Themore recent development of coagglutination (COAG) andlatex agglutination, simple immunoagglutination procedureswhich make it possible to identify specific antigens in CSFsin minutes, has increased the interest in immunoassays forthe diagnosis of meningitis (4, 15, 20). Because of itssensitivity, another immunological method which has beenused for this purpose is antigen capture enzyme-linkedimmunosorbent assay (ELISA) (3, 6-8, 11, 14, 18, 19).Two antigen capture ELISA procedures which might be

employed are (i) a double-antibody sandwich (DAS) ELISAwhich uses the same antiserum for the coating of the solidphase and for the enzyme-conjugated antibody and (ii) aDAS-antiglobulin (DAS-A) ELISA which uses antiserumproduced in one animal species for antibody coating, antiser-um raised in a second species for antigen recognition (secondantibody), and a conjugated antiglobulin to the secondantibody. Previous studies involving small numbers of speci-mens have shown DAS-A ELISA procedures to be effectivefor the detection of meningococcal (18) and Haemophilus (7,14) antigens in CSFs obtained from meningitis patients.Results of other studies suggest that DAS ELISA may alsobe effective for the detection of bacterial antigens in CSFs (3,6, 11).

* Corresponding author.

This investigation was performed to compare the efficacyand sensitivities of the DAS with DAS-A ELISAs and theELISAs with COAG and CIE for the detection of meningo-coccal, Haemophilus, and pneumococcal antigens eitherpurified or in CSF specimens obtained from bacterial menin-gitis patients.

MATERIALS AND METHODS

CSF specimens. Samples were obtained from patientsadmitted to the Abbassia Fever Hospital, Cairo, Egypt, from1979 to 1982 with signs and symptoms of acute bacterialmeningitis. The specimens were from 25 group A meningo-coccal patients, 20 Haemophilus influenzae type b patients,and 24 pneumococcal patients. All of the meningococcal andpneumococcal specimens and 17 of the Haemophilus CSFswere culture positive at the time they were obtained. Theremaining three Haemophilus specimens were subsequentlydiagnosed by CIE or COAG or both. The CSFs were storedat -70°C until tested.

Purified antigens. Meningococcal group A polysaccharidewas extracted from the supernatant of a Mueller-Hintonbroth culture as described by Apicella (2); H. influenzaepolyribophosphate was generously provided by Hynson,Westcott and Dunning (Baltimore, Md.); and Pneumovaxpolysaccharide pneumococcal vaccine was purchased fromMerck Sharp & Dohme (West Point, Pa.). Outer membraneprotein was obtained from a group A meningococcal cultureby lithium chloride extraction and treatment with Triton X-100 (17).

Antisera. Horse anti-meningococcus group A serum andburro anti-H. influenzae type b serum were provided by JohnRobbins, National Institute of Allergy and Infectious Dis-eases (Bethesda, Md.); rabbit anti-H. influenzae type bserum was obtained from Hyland Diagnostics (Deerfield,

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260 SIPPEL ET AL.

Ill.); rabbit anti-Streptococcus pneumoniae omni-serum wasobtained from Statens Seruminstitut (Copenhagen, Den-mark); and the immunoglobulin G fraction of anti-rabbitglobulin prepared in goat was purchased from Cappel Labo-ratories (West Chester, Pa.). Rabbit anti-meningococcusgroup A serum preparations 33 and 2H7 were prepared byrepeated immunizations with killed cells, followed by injec-tion with live organisms (17).Immunoassays. CIE was carried out as previously de-

scribed with barbital buffer (pH 8.6) and 1% agarose (12),using both rabbit and equine sera. COAG was performed bymixing a drop of COAG reagent with a drop of CSF on amicroscope slide and rotating the slide for up to 1 min. TheCOAG reagents were prepared by adsorbing antibodies ontoprotein A-containing staphylococcal cells (Cowan strain) bythe procedures of Edwards et al. (9). Since equine immuno-globulins do not adsorb well to the protein A on staphylococ-cal cells, only rabbit sera were used to prepare the COAGreagents. The meningococcal reagent was prepared withrabbit serum 2H7.Antigen capture ELISA was performed with Immulon I

microtitration plates (Dynatech Laboratories, Inc., Alexan-dria, Va.). The globulin fractions of antisera were preparedby ammonium sulfate precipitation (21), the washing buffer

HARA - R HAH

was phosphate-buffered saline containing 0.05% polyoxyeth-ylene sorbitan monolaurate, alkaline phosphatase conjugateswere prepared by glutaraldehyde fixation (21), and absor-bances were read in a Titertek Multiskan spectrophotometer(Flow Laboratories, Inc., McLean, Va.) after ca. 30 min ofsubstrate incubation at room temperature. Because antiseraprepared in two animal species were available, DAS-AELISAs, as well as DAS assays, were employed for thedetection of meningococcal and Haemophilus antigens (Fig.1 and 2). Only the DAS ELISA was used for pneumococcaldetection (Fig. 3) since a suitable second antiserum was notavailable. To decrease high background activity due tononspecific binding in the pneumococcal system, plateswere adsorbed overnight with phosphate-buffered salinecontaining 3% fetal calf serum. Checkerboard titrations (21)were performed with polysaccharide antigens to determinethe optimal concentrations of all of the antibody prepara-tions; the antisera used for coating the microtiter plates werediluted from 1:1,000 to 1:10,000, antisera used for antigenrecognition in the meningococcal and Haemophilus DAS-AELISAs were diluted 1:1,000, and the conjugated immuno-globulins were diluted 1:500. The assay for purified polysac-charide was considered positive when it had an absorbance1.5 times that obtained with a phosphate-buffered saline

RAR

Horse Anti-meningococcusgrp.A globulin, pH 9.6

overnight40C

Wash

Antigen (CSF)

I nc u bateI hr 37°C

Wash

Rabbit Anti-meningococcusgrp. A globulin

I ncubateI h r 370C

Wash

Alkaline Phosphatase conjugatedgoat anti-rabbit globulin

I nc ubateI hr 37°C

Wash

Substrate

Horse Anti-meningococcusgrp. A globulin, pH 9.6

overnight4°C

Wash

Antigen (CSF)

I ncubateI hr 370C

Wash

Alkaline Phosphatase conjugatedhorse anti-meningococcus

grp. A globulinIncubateI hr 37°C

Wash

Substrate

Read Absorbance405 nm

Rabbit Anti-meningococcusgrp. A globulin, pH9.6

overnight40C

Wash

Antigen (CSF)

I ncubateI hr 370C

Wash

Alkaline Phosphatase conjugatedrabbit anti- meningococcus

grp A globulin

I ncubateI hr 370C

Wash

Substrate

Read Absorbance405 nm

Read Absorbance405 nm

FIG. 1. Protocols for the HARA-R DAS-A ELISA, HAH DAS ELISA, and RAR DAS ELISA. Rabbit serum preparation 2H7 was thesecond antibody in the HARA-R ELISA. Rabbit serum 2H7 was used for both antigen capture and recognition in one RAR ELISA (RAR2H7),and rabbit serum 33 was used for both antigen capture and recognition in another ELISA (RAR33).

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ANTIGEN CAPTURE ELISA FOR MENINGITIS 261

BARA-R

Burro Anti - H. influenzaetype b globulin, pH 9.6

overnight4°C

Wash

Antigen (CSF)

I ncubateI h r 37°C

Wash

Rabbit Anti - H. influenzaetype b globulin

I ncubateI h r 370C

Wash

Alkaline Phosphatase conjugatedgoat anti-rabbit globulin

I ncubateI h r 37°C

Wash

Substrate

BAB

Burro Anti - H. influenzaetype b globulin, pH 9.6

overnight40C

Wash

Antigen (CSF)

I nc u bateI hr 370C

Wash

Alkaline Phosphatase conjugatedBurro Anti - H. influenzae

type b globulin

I nc ubateI hr 370C

Wash

Substrate

Read Absorbance405 nm

RAR

Rabbit Anti - H. influenzaetype b globulin, pH9.6

overnight40C

Wash

Antigen (CSF)

I ncubateI hr 370C

Wash

Alkaline Phosphatase conjugatedRabbit Anti - H. influenzae

type b globulin

I ncubateI hr 370C

Wash

Substrate

Read Absorbance405 nm

Read Absorbance405 nm

FIG. 2. Protocols for the Haemophilus BARA-R DAS-A ELISA, BAB DAS ELISA, and RAR DAS ELISA.

(i.e., no antigen) control. Absorbance values with CSFspecimens were calculated by subtracting the absorbancewith phosphate-buffered saline from that obtained with thespecimens and were considered positive when they werehigher than that obtained with any of the heterologouscontrol CSF specimens tested.

RESULTS

Relative sensitivity of the immunoassays. The three Haemo-philus ELISAs and the meningococcal horse globulin, anti-gen, rabbit globulin, anti-rabbit conjugate (HARA-R), horseglobulin, antigen, horse globulin conjugate (HAH), andrabbit globulin, antigen, rabbit globulin conjugate (RAR)with rabbit serum 33 (RAR33) ELISAs detected 1 ng ofspecific polysaccharide per ml, whereas the meningococcalRAR with rabbit serum 2H7 (RAR2H7) and the pneumococ-cal ELISAs detected ca. 5 ng of antigen per ml (Fig. 4). Theconcentrations of polysaccharide detected by the meningo-coccal, Haemophilus, and pneumococcal COAG assayswere 20, 25, and 30 ng/ml, respectively. The concentrationsdetected by CIE with rabbit sera were 40, 100, and 60 ng/ml.The sensitivity of the meningococcal CIE increased fourfoldwhen horse serum was used, and that of the HaemophilusCIE increased twofold when burro serum was employed.The meningococcal HARA-R ELISA detected ca. 10 ng of

outer membrane protein per ml, whereas COAG detected300 ng and CIE detected 3 Vxg of this antigen per ml.

Detection of meningococcal antigen in CSF by ELISAs. Allof the meningococcal CSFs were differentiated from controlCSFs by the HARA-R, HAH, and RAR33 ELISAs, and 24 ofthe 25 meningococcal CSFs were differentiated by theRAR2H7 ELISA (Fig. 5); The mean ELISA values with thehomologous CSFs were 0.912, 0.793, 0.902, and 0.493,respectively, whereas the values with the heterologous con-trol specimens were 0.052 or less.

Detection of Haemophilus antigen in CSF by ELISAs. Of 20Haemophilus CSF specimens, 19 were differentiated fromcontrol CSFs by the Haemophilus burro globulin, antigen,rabbit globulin, anti-rabbit conjugate (BARA-R) system (Fig.6). However, only seven of the Haemophilus specimensproduced ELISA values greater than those obtained with thecontrol CSFs with the RAR system, and, because the highestELISA reading with the burro globulin, antigen, burroglobulin conjugate (BAB) system was obtained with a menin-gococcal control specimen, none of the Haemophilus speci-mens were considered to be positive with this protocol. Themean ELISA values with the homologous CSFs were 0.447with BARA-R ELISA, 0.415 with BAB ELISA, and 0.219with RAR ELISA. The corresponding mean values withheterologous controls were 0.009, 0.115, and 0.032.

Detection of pneumococcal antigen in CSF by ELISA. The'

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PNEUMOCOCCAL ASSAY

Rabbit "omni" Anti-pneumococcuSglobulin, pH 9.6

1

over ni gh t40C

Wash

3% Fetal calf serum in PBS

overnight4°C

Wash

Antigen (C SF)

i n c u bateI h r 370C

Wash

Alkaline Phosphatase conjugatedRabbit "omni" anti-pneumococcusglobulin

i nc u bI hr 3

Wash

iate

370C

S u b st ra te

Read Absorbance405 nm

FIG. 3. Protocol for the pneumococcal DAS ELISA.

DAS pneumococcal ELISA system was able to differentiate14 of 24 pneumococcal CSF specimens from meningococcalcontrol samples (Fig. 7). The mean ELISA value with thepneumococcal specimens was 0.179, whereas that with thecontrol CSFs was 0.016.

Detection of antigens in CSF by COAG and CIE. Thecomparative efficacy of ELISA, COAG, and CIE for thedetection of antigen in the CSF specimens is shown in Fig. 5,6, and 7. Specific antigen was detected in 23 of the 25meningococcal specimens by COAG, in 19 of the specimensby CIE with horse serum, and in 13 of the specimens by CIEwith rabbit serum preparation 33 or 2H7. Of the 20 Haemo-philus specimens, 16 were positive by COAG, 19 were

positive by CIE with burro serum, and 14 were positive byCIE with rabbit serum. Pneumococcal antigen was detectedin 22 of the 24 CSFs by COAG and in 17 by CIE. There wereno false-positive results with COAG and CIE when theheterologous CSF samples were tested.

DISCUSSIONThe DAS-A ELISAs should be more sensitive than the

DAS assays, as the former has one additional step andtherefore additional amplification. However, although theDAS-A systems tested here were highly sensitive, they were

not superior to the DAS ELISAs with horse or burroantiserum for the detection of purified polysaccharide (Fig.4). Presumably, the equine sera have higher affinity than therabbit antisera for these antigens, and using the superiorequine-conjugated immunoglobulin compensates for the the-oretical advantages of the DAS-A assays. The RAR33ELISA was also as effective as the meningococcal DAS-Aassay for the detection of antigen in CSFs (Fig. 5). SinceELISA can detect noncapsular meningococcal antigens inthese specimens (19), the excellent results with the RAR33system with clinical material may be due to the high affinitythat rabbit serum 33 has for antigens other than surfacepolysaccharide.The failure of the Haemophilus and pneumococcal DAS

ELISAs to differentiate homologous CSFs from controlspecimens was surprising in light of reports (3, 6, 11) ofsuccess when similar ELISA systems were used andthe sensitivity of our assays for purified polysaccharideantigens. The poor performance with the Haemophilus BABELISA resulted from elevated ELISA values obtained withthree of the meningococcal control specimens; apparentlythe burro serum contains antibodies that cross-react withsome meningococcal determinants. The burro serum did notdetect meningococcal antigens in the control CSFs by CIE,presumably because the sensitivity of this assay is less,relative to ELISA, for the determinants involved. SinceELISA appears to be considerably more sensitive than theother assays for the detection of outer membrane compo-nents, the cross-reacting antigens are most likely noncapsu-lar. There was no evidence for cross-reacting antibodies with.the Haemophilus RAR ELISA as none of the control speci-mens produced elevated ELISA values. However, the val-ues with the Haemophilus specimens were also low. Thus,the RAR ELISA system that detected 1 ng of purifiedHaemophilus polysaccharide per ml had limited sensitivityfor Haemophilus antigen in CSF. Even with the fetal calfserum blocking step there was some background activitywith the pneumococcal DAS ELISA, making it difficult todetermine if cross-reactions or lack of sensitivity contributedto the poor results with this assay. Superior performancewith the meningococcal and Haemophilus DAS-A ELISAswas probably due in part to the excellent specificity obtainedwith such systems; nonspecific antigens must react with thetwo different antiserum preparations to be detected. In thisregard, the data of Drow and Manning (8), which show that aDAS-A ELISA can differentiate pneumococci from poten-tially cross-reacting gram-negative bacteria, suggest that suchan assay would be as effective as the analogous meningococ-cal and Haemophilus systems.The DAS ELISAs with rabbit sera were more sensitive

than COAG and CIE with rabbit sera for detecting purifiedmeningococcal, Haemophilus, and pneumococcal polysac-charides in saline. Since the same preparations of anti-meningococcal, anti-Haemophilus, and anti-pneumococcalrabbit sera were used in these assays, the superior resultswith ELISAs must be due to the inherent properties of theassays themselves (since they cannot be due to differences insera). These results also demonstrate that it is essential touse clinical material in evaluating immunoassay techniquessince the Haemophilus DAS ELISAs and the pneumococcalELISA which had greater sensitivity than COAG and CIEfor purified antigens were less successful than these assaysfor detecting specific antigens in CSF specimens.The importance of the quality of the antiserum used in a

particular immunoassay was also demonstrated in this study.The meningococcal RAR ELISA with rabbit serum 33 was

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POLYSACCHARIDE (n g/ml)FIG. 4. Sensitivity of the ELISA protocols for purified polysaccharide antigens. HARA-R, HAH, RAR33, RAR,H7 are meningococcal

assays, BARA-R, BAB, and RARH are Haemophilus assays, and RARpn is the pneumococcal assay.

HAR A-R

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Meningococcal Meningococcal Meningococcal MeningococcalCSFs Pneumococcal CSFs Pneumococcal CSFs Pneumococcal CSFs Pneumococcal

CSFs CSFs CSFs CSFsFIG. 5. Comparison of ELISA, COAG, and CIE for the detection of meningococcal antigens in CSF (positive CIE results with horse or

rabbit serum or both). Each CSF specimen is positive by COAG and CIE (0); positive by COAG and negative by CIE (A); negative by COAGand positive by CIE (x); or negative by both COAG and CIE (0). The mean absorbance values with homologous and heterologous specimensand the values 3 standard deviations from the mean of the heterologous specimens are indicated.

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264 SIPPEL ET AL.

BAR A-R BAB RAR0

1.6

4,

a,.0

-D

044on

Meningococcal Meningococcal MeningococcalHaemophilus CSFs Haemophilus CSFs Haemophilus CSFs

CSFs CSFs CSFs

FIG. 6. Comparison of ELISA, COAG, and CIE for the detection of Haemophilus in CSF (positive CIE results with burro or rabbit serumor both). Each CSF specimen is positive by COAG and CIE (0); negative by COAG and positive by CIE (x); or negative by both COAG andCIE (0). The mean absorbance values with homologous and heterologous specimens and values 3 standard deviations from the mean of theheterologous specimens are indicated.

superior to that with serum 2H7, and the sensitivity ofmeningococcal and Haemophilus CIEs increased substan-tially when the horse and burro sera were used rather thanrabbit sera.

In preliminary experiments, the DAS-A meningococcalELISAs detected antigen in supernatants of group A brothcultures but not in supernatants from cultures of othermeningococcal serogroups (unpublished data). Presumably,these ELISAs are essentially serogroup specific. However,since some meningococcal surface components are commonto different serogroups (22), it would be unwise to considerthe meningococcal ELISAs group specific unless polyclonalsera raised against polysaccharide or monoclonal antibodiesagainst the capsular antigens are employed.A question which was not addressed in this study was the

ELISA value which must be achieved with a specimenbefore it is considered positive. Since such a determinationwould be arbitrary until a large number of clinical specimenswere tested, it was felt that the best criterion for this studywas simply the ability of the ELISA to differentiate a samplefrom a reasonable number of control specimens. Anothercriterion for evaluating specimens would be to consider thempositive when they produce absorbance values which are 3standard deviations greater than the mean of the heterolo-

gous CSFs. As indicated in Fig. 5, 6, and 7, the resultsobtained with the two methods are similar.The efficacy found in this study with COAG was impres-

sive. It detected antigen in almost 90% of the 69 CSFs andwas more effective than CIE (P < 0.02). Considering itssensitivity and simplicity, COAG should be consideredhighly appropriate for the diagnosis of bacterial meningitisfrom CSF, especially when only a few samples are to beassayed.The results reported here suggest that ELISA is superior

to CIE and COAG (P < 0.05) for the detection of specificantigens in CSF from meningococcal and Haemophilusmeningitis patients and might have been superior for thedetection of pneumococcal antigens if appropriate sera wereemployed. The least-sensitive method, CIE, still detectedspecific antigen in 80% of the 69 CSF specimens in this studyand in 76% of the 25 meningococcal specimens which werecollected 1 to 3 years before they were tested. A previousstudy in which CSFs were assayed for meningococcal groupA antigen as they were collected from 259 meningococcalmeningitis patients demonstrated that CIE is as effective asculture for the diagnosis of this disease (16). The datareported here therefore suggest that antigen capture ELISAcan be more effective than culture. ELISA, with its high

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ANTIGEN CAPTURE ELISA FOR MENINGITIS 265

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Pneumococcal MeningococcalCSFs CSFs

FIG. 7. Comparison of ELISA, COAG, and CIE for the detec-tion of pneumococcal antigens in CSF. Each CSF specimen ispositive by COAG and CIE (0); positive by COAG and negative byCIE (A); or negative by both COAG and CIE (0). The mean

absorbance values with homologous and heterologous specimensand the values 3 standard deviations from the mean of the heterolo-gous specimens are indicated.

sensitivity, may be particularly useful for diagnosing bacteri-al meningitis, especially when the assay becomes standard-ized and automated.

ACKNOWLEDGMENTSThis work was supported by contract no. N00014-81-C-0570,

proposal no. 110-83 from the Office of Naval Research, and workunit no. M0095-PN-002-5020 of the Naval Medical Research andDevelopment Command.We express our appreciation to R. Giard for technical assistance

and to M. L. Takacs for preparation of this manuscript. We alsothank R. J. Sugasawara for her thoughtful suggestions.

LITERATURE CITED1. Alexander, H. G., and G. Rake. 1937. Studies on meningococcus

infection X. A further note on the presence of meningococcusprecipitinogens in the cerebrospinal fluid. J. Exp. Med. 65:317-321.

2. Apicella, M. A. 1976. Immunological and biochemical studies ofmeningococcal C polysaccharides isolated by diethylaminoethylchromatography. Infect. Immun. 14:106-113.

3. Beuvery, E. C., F. van Rossum, S. Lauwers, and H. Coignan.1979. Comparison of counterimmunoelectrophoresis andELISA for diagnosis of bacterial meningitis. Lancet i:208.

4. Collins, J. K., and M. T. Kelly. 1983. Comparison of Phadebactcoagglutination, Bactogen latex agglutination, and counterim-munoelectrophoresis for detection of Haemophilus influenzaetype b antigens in cerebrospinal fluid. J. Clin. Microbiol.17:1005-1008.

5. Coonrod, J. D., and M. W. Rytel. 1972. Determination ofaetiology of bacterial meningitis by counterimmunoelectrophor-esis. Lancet i:1154-1157.

6. Crosson, F. J., Jr., J. A. Winkelstein, and E. Richard Moxon.1978. Enzyme-linked immunosorbent assay for detection andquantitation of capsular antigen of Haemophilus influenzae typeb. Infect. Immun. 22:617-619.

7. Drow, D. L., D. G. Maki, and D. D. Manning. 1979. Indirectsandwich enzyme-linked immunosorbent assay for rapid detec-tion of Haemophilus influenzae type b infection. J. Clin. Micro-biol. 10:442-450.

8. Drow, D. L., and D. D. Manning. 1980. Indirect sandwichenzyme-linked immunosorbent assay for rapid detection ofStreptococcus pneumoniae type 3 antigen. J. Clin. Microbiol.11:641-645.

9. Edwards, E. A., M. E. Kilpatrick, and D. Hooper. 1980. Rapiddetection of pneumococcal antigens in sputum and blood serumusing a coagglutination test. Mil. Med. 145:256-258.

10. Fossieck, B., Jr., R. Craig, and P. Y. Paterson. 1973. Counterim-munoelectrophoresis for rapid diagnosis of meningitis due toDiplococcus pneumoniae. J. Infect. Dis. 127:106-109.

11. Harding, S. A., W. M. Scheld, M. D. McGowan, and M. A.Sande. 1979. Enzyme-linked immunosorbent assay for detectionof Streptococcus pneumoniae antigen. J. Clin. Microbiol.10:339-342.

12. Higashi, G. E., J. E. Sippel, N. I. Girgis, and A. Hassan. 1974.Counterimmunoelectrophoresis: an adjunct to bacterial culturein the diagnosis of meningococcal meningitis. Scand. J. Infect.Dis. 6:233-235.

13. Maegraith, B. G., and M. B. Adelaide. 1935. The rapid diagnosisof cerebrospinal fever. Lancet i:545-546.

14. Pepple, J., E. R. Moxon, and R. H. Yolken. 1980. Indirectenzyme-linked immunosorbent assay for the quantitation of thetype-specific antigen of Haemophilus influenzae b: a prelimi-nary report. J. Pediatr. 97:233-237.

15. Severin, W. P. J. 1972. Latex agglutination in the diagnosis ofmeningococcal meningitis. J. Clin. Pathol. 25:1079-1082.

16. Sippel, J. E., and N. I. Girgis. 1978. Meningococcal infection inEgypt: laboratory findings in meningitis patients and the preva-lence of pharyngeal infection in patients and contacts. Am. J.Trop. Med. Hyg. 27:980-985.

17. Sippel, J. E., and A. Quan. 1977. Homogeneity of proteinserotype antigens in Neisseria meningitidis group A. Infect.Immun. 16:623-627.

18. Sippel, J. E., and A. Voller. 1980. Detection of Neisseriameningitidis cell envelope antigen by enzyme-linked immuno-sorbent assay in patients with meningococcal disease. Trans. R.Soc. Trop. Med. Hyg. 74:644-648.

19. Sugasawara, R. J., C. M. Prato, and J. E. Sippel. 1984. Enzyme-linked immunosorbent assay with a monoclonal antibody fordetecting group A meningococcal antigens in cerebrospinalfluid. J. Clin. Microbiol. 19:230-234.

20. Thirumoorthi, M. C., and A. S. Dajani. 1979. Comparison ofstaphylococcal coagglutination, latex agglutination, and coun-terimmunoelectrophoresis for bacterial antigen detection. J.Clin. Microbiol. 9:28-32.

21. Voller, A., D. Bidwell, and A. Bartlett. 1980. Enzyme-linkedimmunosorbent assay, p. 359-371. In N. R. Rose and H.Friedman (ed.), Manual of clinical immunology, 2nd ed. Ameri-can Society for Microbiology, Washington, D.C.

22. Zollinger, W. D., C. L. Pennington, and M. S. Artenstein. 1974.Human antibody response to three meningococcal outer mem-brane antigens: comparison by specific hemagglutination as-says. Infect. Immun. 10:975-984.

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