This investigation was performed to compare the efficacy and sensitivities of the DAS with DAS-A ELISAs and the

JOURNAL OF CLINICAL MICROBIOLOGY, Aug. 1984, p. 259-265 95-1137/84/8259-7$2./ Copyright C) 1984, American Society for Microbiology Vol. 2, No. 2 Detection of Neisseria meningitidis Group A, Haemophilus influenzae Type b, and Streptococcus pneumoniae Antigens in Cerebrospinal Fluid Specimens by Antigen Capture Enzyme-Linked Immunosorbent Assays JOHN E. SIPPEL,1* CATHERINE M. PRATO,' NABIL I. GIRGIS,2 AND EARL A. EDWARDS3 Naval 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 counterimmunoelectrophoresis for the detection of meningococcal, Haemophilus, and pneumococcal antigens. Enzyme-linked immunosorbent assay detected 1 ng of purified meningococcal and Haemophilus polysaccharides per ml and 5 ng of pneumococcal polysaccharide per ml; coagglutination detected 2, 25, and 3 ng/ml, respectively, of these polysaccharides; and counterimmunoelectrophoresis detected 1, 5, and 6 ng/ml. Double-antibody sandwichantiglobulin enzyme-linked immunosorbent assays, which employed antibodies produced in two animal species, differentiated 1% of the cerebrospinal fluid (CSF) specimens from meningococcal meningitis patients and 95% of the CSFs from Haemophilus patients from heterologous control CSFs. Double-antibody sandwich procedures, which use the same antiserum preparation for coating the wells of microtiter plates and for alkaline phosphatase-conjugated immunoglobulin, differentiated meningococcal CSFs from control specimens but were unable to effectively differentiate the Haemophilus or pneumococcal specimens from control CSFs. Coagglutination detected specific antigen in 92% of the meningococcal CSFs, 8% of the Haemophilus CSFs, and 92% of the pneumococcal specimens. The comparable percentages for counterimmunoelectrophoresis were 76, 95, and 71%. Detection of bacterial antigens in cerebrospinal fluid (CSF) was shown to be feasible in the early part of this century (1, 13). However, this approach to the diagnosis of acute bacterial meningitis was rarely employed until counterimmunoelectrophoresis (CIE) was evaluated in the 197s (5, 1, 12). When high-quality antisera were used, the efficacy of this procedure, which could provide a diagnosis in less than 1 h, usually approached that of culture (16). The more recent development of coagglutination (COAG) and latex agglutination, simple immunoagglutination procedures which make it possible to identify specific antigens in CSFs in minutes, has increased the interest in immunoassays for the diagnosis of meningitis (4, 15, 2). Because of its sensitivity, another immunological method which has been used for this purpose is antigen capture enzyme-linked immunosorbent 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) ELISA which uses the same antiserum for the coating of the solid phase and for the enzyme-conjugated antibody and (ii) a DAS-antiglobulin (DAS-A) ELISA which uses antiserum produced in one animal species for antibody coating, antiserum raised in a second species for antigen recognition (second antibody), and a conjugated antiglobulin to the second antibody. Previous studies involving small numbers of specimens have shown DAS-A ELISA procedures to be effective for 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 also be effective for the detection of bacterial antigens in CSFs (3, 6, 11). * Corresponding author. 259 This investigation was performed to compare the efficacy and sensitivities of the DAS with DAS-A ELISAs and the ELISAs with COAG and CIE for the detection of meningococcal, Haemophilus, and pneumococcal antigens either purified or in CSF specimens obtained from bacterial meningitis patients. MATERIALS AND METHODS CSF specimens. Samples were obtained from patients admitted to the Abbassia Fever Hospital, Cairo, Egypt, from 1979 to 1982 with signs and symptoms of acute bacterial meningitis. The specimens were from 25 group A meningococcal patients, 2 Haemophilus influenzae type b patients, and 24 pneumococcal patients. All of the meningococcal and pneumococcal specimens and 17 of the Haemophilus CSFs were culture positive at the time they were obtained. The remaining three Haemophilus specimens were subsequently diagnosed by CIE or COAG or both. The CSFs were stored at -7 C until tested. Purified antigens. Meningococcal group A polysaccharide was extracted from the supernatant of a Mueller-Hinton broth culture as described by Apicella (2); H. influenzae polyribophosphate was generously provided by Hynson, Westcott and Dunning (Baltimore, Md.); and Pneumovax polysaccharide pneumococcal vaccine was purchased from Merck Sharp & Dohme (West Point, Pa.). Outer membrane protein was obtained from a group A meningococcal culture by lithium chloride extraction and treatment with Triton X- 1 (17). Antisera. Horse anti-meningococcus group A serum and burro anti-h. influenzae type b serum were provided by John Robbins, National Institute of Allergy and Infectious Diseases (Bethesda, Md.); rabbit anti-h. influenzae type b serum was obtained from Hyland Diagnostics (Deerfield,

26 SIPPEL ET AL. Ill.); rabbit anti-streptococcus pneumoniae omni-serum was obtained from Statens Seruminstitut (Copenhagen, Denmark); and the immunoglobulin G fraction of anti-rabbit globulin prepared in goat was purchased from Cappel Laboratories (West Chester, Pa.). Rabbit anti-meningococcus group A serum preparations 33 and 2H7 were prepared by repeated immunizations with killed cells, followed by injection with live organisms (17). Immunoassays. CIE was carried out as previously described with barbital buffer (ph 8.6) and 1% agarose (12), using both rabbit and equine sera. COAG was performed by mixing a drop of COAG reagent with a drop of CSF on a microscope slide and rotating the slide for up to 1 min. The COAG reagents were prepared by adsorbing antibodies onto protein A-containing staphylococcal cells (Cowan strain) by the procedures of Edwards et al. (9). Since equine immunoglobulins do not adsorb well to the protein A on staphylococcal cells, only rabbit sera were used to prepare the COAG reagents. The meningococcal reagent was prepared with rabbit serum 2H7. Antigen capture ELISA was performed with Immulon I microtitration plates (Dynatech Laboratories, Inc., Alexandria, Va.). The globulin fractions of antisera were prepared by ammonium sulfate precipitation (21), the washing buffer J. CLIN. MICROBIOL. was phosphate-buffered saline containing.5% polyoxyethylene sorbitan monolaurate, alkaline phosphatase conjugates were prepared by glutaraldehyde fixation (21), and absorbances were read in a Titertek Multiskan spectrophotometer (Flow Laboratories, Inc., McLean, Va.) after ca. 3 min of substrate incubation at room temperature. Because antisera prepared in two animal species were available, DAS-A ELISAs, as well as DAS assays, were employed for the detection of meningococcal and Haemophilus antigens (Fig. 1 and 2). Only the DAS ELISA was used for pneumococcal detection (Fig. 3) since a suitable second antiserum was not available. To decrease high background activity due to nonspecific binding in the pneumococcal system, plates were adsorbed with phosphate-buffered saline containing 3% fetal calf serum. Checkerboard titrations (21) were performed with polysaccharide antigens to determine the optimal concentrations of all of the antibody preparations; the antisera used for coating the microtiter plates were diluted from 1:1, to 1:1,, antisera used for antigen recognition in the meningococcal and Haemophilus DAS-A ELISAs were diluted 1:1,, and the conjugated immunoglobulins were diluted 1:5. The assay for purified polysaccharide was considered positive when it had an absorbance 1.5 times that obtained with a phosphate-buffered saline HARA - R Horse Anti-meningococcus grp.a globulin, ph 9.6 4C I nc u bate I hr 37 C Rabbit Anti-meningococcus grp. A globulin I h r 37C goat anti-rabbit globulin I nc ubate I hr 37 C HAH Horse Anti-meningococcus grp. A globulin, ph 9.6 4 C I hr 37C horse anti-meningococcus grp. A globulin Incubate I hr 37 C 45 nm RAR Rabbit Anti-meningococcus grp. A globulin, ph9.6 4C I hr 37C rabbit anti- meningococcus grp A globulin I hr 37C 45 nm 45 nm FIG. 1. Protocols for the HARA-R DAS-A ELISA, HAH DAS ELISA, and RAR DAS ELISA. Rabbit serum preparation 2H7 was the second 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).

VOL. 2. 1984 BARA-R BAB ANTIGEN CAPTURE ELISA FOR MENINGITIS 261 RAR Burro Anti - H. influenzae type b globulin, ph 9.6 Antigen 4 C (CSF) I h r 37 C Rabbit Anti - H. influenzae type b globulin I h r 37C goat anti-rabbit globulin I h r 37 C Burro Anti - H. influenzae type b globulin, ph 9.6 4C I nc u bate I hr 37C Burro Anti - H. influenzae type b globulin I nc ubate I hr 37C 45 nm Rabbit Anti - H. influenzae type b globulin, ph9.6 4C I hr 37C Rabbit Anti - H. influenzae type b globulin I hr 37C 45 nm 45 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 CSF specimens were calculated by subtracting the absorbance with phosphate-buffered saline from that obtained with the specimens and were considered positive when they were higher than that obtained with any of the heterologous control CSF specimens tested. RESULTS Relative sensitivity of the immunoassays. The three Haemophilus ELISAs and the meningococcal horse globulin, antigen, rabbit globulin, anti-rabbit conjugate (HARA-R), horse globulin, antigen, horse globulin conjugate (HAH), and rabbit globulin, antigen, rabbit globulin conjugate (RAR) with rabbit serum 33 (RAR33) ELISAs detected 1 ng of specific polysaccharide per ml, whereas the meningococcal RAR with rabbit serum 2H7 (RAR2H7) and the pneumococcal ELISAs detected ca. 5 ng of antigen per ml (Fig. 4). The concentrations of polysaccharide detected by the meningococcal, Haemophilus, and pneumococcal COAG assays were 2, 25, and 3 ng/ml, respectively. The concentrations detected by CIE with rabbit sera were 4, 1, and 6 ng/ml. The sensitivity of the meningococcal CIE increased fourfold when horse serum was used, and that of the Haemophilus CIE increased twofold when burro serum was employed. The meningococcal HARA-R ELISA detected ca. 1 ng of outer membrane protein per ml, whereas COAG detected 3 ng and CIE detected 3 Vxg of this antigen per ml. Detection of meningococcal antigen in CSF by ELISAs. All of the meningococcal CSFs were differentiated from control CSFs by the HARA-R, HAH, and RAR33 ELISAs, and 24 of the 25 meningococcal CSFs were differentiated by the RAR2H7 ELISA (Fig. 5); The mean ELISA values with the homologous CSFs were.912,.793,.92, and.493, respectively, whereas the values with the heterologous control specimens were.52 or less. Detection of Haemophilus antigen in CSF by ELISAs. Of 2 Haemophilus CSF specimens, 19 were differentiated from control CSFs by the Haemophilus burro globulin, antigen, rabbit globulin, anti-rabbit conjugate (BARA-R) system (Fig. 6). However, only seven of the Haemophilus specimens produced ELISA values greater than those obtained with the control CSFs with the RAR system, and, because the highest ELISA reading with the burro globulin, antigen, burro globulin conjugate (BAB) system was obtained with a meningococcal control specimen, none of the Haemophilus specimens were considered to be positive with this protocol. The mean ELISA values with the homologous CSFs were.447 with BARA-R ELISA,.415 with BAB ELISA, and.219 with RAR ELISA. The corresponding mean values with heterologous controls were.9,.115, and.32. Detection of pneumococcal antigen in CSF by ELISA. The'

262 SIPPEL ET AL. Rabbit PNEUMOCOCCAL ASSAY "omni" Anti-pneumococcuS globulin, ph 9.6 1 over ni gh t 4C 3% Fetal calf serum in PBS 4 C Antigen (C SF) i n c u bate I h r 37C Rabbit "omni" anti-pneumococcus globulin i nc u b iate I hr 3 S u b st ra te 45 nm 37C FIG. 3. Protocol for the pneumococcal DAS ELISA. DAS pneumococcal ELISA system was able to differentiate 14 of 24 pneumococcal CSF specimens from meningococcal control samples (Fig. 7). The mean ELISA value with the pneumococcal specimens was.179, whereas that with the control CSFs was.16. Detection of antigens in CSF by COAG and CIE. The comparative efficacy of ELISA, COAG, and CIE for the detection of antigen in the CSF specimens is shown in Fig. 5, 6, and 7. Specific antigen was detected in 23 of the 25 meningococcal specimens by COAG, in 19 of the specimens by CIE with horse serum, and in 13 of the specimens by CIE with rabbit serum preparation 33 or 2H7. Of the 2 Haemophilus specimens, 16 were positive by COAG, 19 were positive by CIE with burro serum, and 14 were positive by CIE with rabbit serum. Pneumococcal antigen was detected in 22 of the 24 CSFs by COAG and in 17 by CIE. There were no false-positive results with COAG and CIE when the heterologous CSF samples were tested. DISCUSSION The DAS-A ELISAs should be more sensitive than the DAS assays, as the former has one additional step and therefore additional amplification. However, although the DAS-A systems tested here were highly sensitive, they were J. CLIN. MICROBIOL. not superior to the DAS ELISAs with horse or burro antiserum for the detection of purified polysaccharide (Fig. 4). Presumably, the equine sera have higher affinity than the rabbit antisera for these antigens, and using the superior equine-conjugated immunoglobulin compensates for the theoretical advantages of the DAS-A assays. The RAR33 ELISA was also as effective as the meningococcal DAS-A assay for the detection of antigen in CSFs (Fig. 5). Since ELISA can detect noncapsular meningococcal antigens in these specimens (19), the excellent results with the RAR33 system with clinical material may be due to the high affinity that rabbit serum 33 has for antigens other than surface polysaccharide. The failure of the Haemophilus and pneumococcal DAS ELISAs to differentiate homologous CSFs from control specimens was surprising in light of reports (3, 6, 11) of success when similar ELISA systems were used and the sensitivity of our assays for purified polysaccharide antigens. The poor performance with the Haemophilus BAB ELISA resulted from elevated ELISA values obtained with three of the meningococcal control specimens; apparently the burro serum contains antibodies that cross-react with some meningococcal determinants. The burro serum did not detect meningococcal antigens in the control CSFs by CIE, presumably because the sensitivity of this assay is less, relative to ELISA, for the determinants involved. Since ELISA appears to be considerably more sensitive than the other assays for the detection of outer membrane components, the cross-reacting antigens are most likely noncapsular. There was no evidence for cross-reacting antibodies with. the Haemophilus RAR ELISA as none of the control specimens produced elevated ELISA values. However, the values with the Haemophilus specimens were also low. Thus, the RAR ELISA system that detected 1 ng of purified Haemophilus polysaccharide per ml had limited sensitivity for Haemophilus antigen in CSF. Even with the fetal calf serum blocking step there was some background activity with the pneumococcal DAS ELISA, making it difficult to determine if cross-reactions or lack of sensitivity contributed to the poor results with this assay. Superior performance with the meningococcal and Haemophilus DAS-A ELISAs was probably due in part to the excellent specificity obtained with such systems; nonspecific antigens must react with the two different antiserum preparations to be detected. In this regard, the data of Drow and Manning (8), which show that a DAS-A ELISA can differentiate pneumococci from potentially cross-reacting gram-negative bacteria, suggest that such an assay would be as effective as the analogous meningococcal and Haemophilus systems. The DAS ELISAs with rabbit sera were more sensitive than COAG and CIE with rabbit sera for detecting purified meningococcal, Haemophilus, and pneumococcal polysaccharides in saline. Since the same preparations of antimeningococcal, anti-haemophilus, and anti-pneumococcal rabbit sera were used in these assays, the superior results with ELISAs must be due to the inherent properties of the assays themselves (since they cannot be due to differences in sera). These results also demonstrate that it is essential to use clinical material in evaluating immunoassay techniques since the Haemophilus DAS ELISAs and the pneumococcal ELISA which had greater sensitivity than COAG and CIE for purified antigens were less successful than these assays for 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

to w z m (I) 2 1, 1, 1 1.1 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 HAH RA R2H7 RAR33 a) a). Co. 1.6 7 I.4-1.2-1.-.6-.4- a. * O 16 x A S S. * * a i 1 A a A.2 - I. T 8 R-g-o HO o ml i 'm I- I. T _,An, I 1 fl 1 co I 86 f -.2 Meningococcal Meningococcal Meningococcal Meningococcal CSFs Pneumococcal CSFs Pneumococcal CSFs Pneumococcal CSFs Pneumococcal CSFs CSFs CSFs CSFs FIG. 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 (); positive by COAG and negative by CIE (A); negative by COAG and positive by CIE (x); or negative by both COAG and CIE (). The mean absorbance values with homologous and heterologous specimens and the values 3 standard deviations from the mean of the heterologous specimens are indicated. 263 S 1 a I - A A I ax C II AA A A

264 SIPPEL ET AL. J. CLIN. MICROBIOL. BAR A-R BAB RAR 1.6 4, -D a,. on 44 Meningococcal Meningococcal Meningococcal Haemophilus 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 serum or both). Each CSF specimen is positive by COAG and CIE (); negative by COAG and positive by CIE (x); or negative by both COAG and CIE (). The mean absorbance values with homologous and heterologous specimens and values 3 standard deviations from the mean of the heterologous specimens are indicated. superior to that with serum 2H7, and the sensitivity of meningococcal and Haemophilus CIEs increased substantially when the horse and burro sera were used rather than rabbit sera. In preliminary experiments, the DAS-A meningococcal ELISAs detected antigen in supernatants of group A broth cultures but not in supernatants from cultures of other meningococcal serogroups (unpublished data). Presumably, these ELISAs are essentially serogroup specific. However, since some meningococcal surface components are common to different serogroups (22), it would be unwise to consider the meningococcal ELISAs group specific unless polyclonal sera raised against polysaccharide or monoclonal antibodies against the capsular antigens are employed. A question which was not addressed in this study was the ELISA value which must be achieved with a specimen before it is considered positive. Since such a determination would be arbitrary until a large number of clinical specimens were tested, it was felt that the best criterion for this study was simply the ability of the ELISA to differentiate a sample from a reasonable number of control specimens. Another criterion for evaluating specimens would be to consider them positive when they produce absorbance values which are 3 standard deviations greater than the mean of the heterologous CSFs. As indicated in Fig. 5, 6, and 7, the results obtained with the two methods are similar. The efficacy found in this study with COAG was impressive. It detected antigen in almost 9% of the 69 CSFs and was more effective than CIE (P <.2). Considering its sensitivity and simplicity, COAG should be considered highly appropriate for the diagnosis of bacterial meningitis from CSF, especially when only a few samples are to be assayed. The results reported here suggest that ELISA is superior to CIE and COAG (P <.5) for the detection of specific antigens in CSF from meningococcal and Haemophilus meningitis patients and might have been superior for the detection of pneumococcal antigens if appropriate sera were employed. The least-sensitive method, CIE, still detected specific antigen in 8% of the 69 CSF specimens in this study and in 76% of the 25 meningococcal specimens which were collected 1 to 3 years before they were tested. A previous study in which CSFs were assayed for meningococcal group A antigen as they were collected from 259 meningococcal meningitis patients demonstrated that CIE is as effective as culture for the diagnosis of this disease (16). The data reported here therefore suggest that antigen capture ELISA can be more effective than culture. ELISA, with its high

VOL. 2, 1984 ANTIGEN CAPTURE ELISA FOR MENINGITIS 265 I a. -. Pneumococcal CSFs Meningococcal CSFs FIG. 7. Comparison of ELISA, COAG, and CIE for the detection of pneumococcal antigens in CSF. Each CSF specimen is positive by COAG and CIE (); positive by COAG and negative by CIE (A); or negative by both COAG and CIE (). The mean absorbance values with homologous and heterologous specimens and the values 3 standard deviations from the mean of the heterologous specimens are indicated. sensitivity, may be particularly useful for diagnosing bacterial meningitis, especially when the assay becomes standardized and automated. ACKNOWLEDGMENTS This work was supported by contract no. N14-81-C-57, proposal no. 11-83 from the Office of Naval Research, and work unit no. M95-PN-2-52 of the Naval Medical Research and Development Command. We express our appreciation to R. Giard for technical assistance and to M. L. Takacs for preparation of this manuscript. We also thank R. J. Sugasawara for her thoughtful suggestions. LITERATURE CITED 1. Alexander, H. G., and G. Rake. 1937. Studies on meningococcus infection X. A further note on the presence of meningococcus precipitinogens in the cerebrospinal fluid. J. Exp. Med. 65:317-321. 2. Apicella, M. A. 1976. Immunological and biochemical studies of meningococcal C polysaccharides isolated by diethylaminoethyl chromatography. Infect. Immun. 14:16-113. 3. Beuvery, E. C., F. van Rossum, S. Lauwers, and H. Coignan. 1979. Comparison of counterimmunoelectrophoresis and ELISA for diagnosis of bacterial meningitis. Lancet i:28. 4. Collins, J. K., and M. T. Kelly. 1983. Comparison of Phadebact coagglutination, Bactogen latex agglutination, and counterimmunoelectrophoresis for detection of Haemophilus influenzae type b antigens in cerebrospinal fluid. J. Clin. Microbiol. 17:15-18. 5. Coonrod, J. D., and M. W. Rytel. 1972. Determination of aetiology of bacterial meningitis by counterimmunoelectrophoresis. Lancet i:1154-1157. 6. Crosson, F. J., Jr., J. A. Winkelstein, and E. Richard Moxon. 1978. Enzyme-linked immunosorbent assay for detection and quantitation of capsular antigen of Haemophilus influenzae type b. Infect. Immun. 22:617-619. 7. Drow, D. L., D. G. Maki, and D. D. Manning. 1979. Indirect sandwich enzyme-linked immunosorbent assay for rapid detection of Haemophilus influenzae type b infection. J. Clin. Microbiol. 1:442-45. 8. Drow, D. L., and D. D. Manning. 198. Indirect sandwich enzyme-linked immunosorbent assay for rapid detection of Streptococcus pneumoniae type 3 antigen. J. Clin. Microbiol. 11:641-645. 9. Edwards, E. A., M. E. Kilpatrick, and D. Hooper. 198. Rapid detection of pneumococcal antigens in sputum and blood serum using a coagglutination test. Mil. Med. 145:256-258. 1. Fossieck, B., Jr., R. Craig, and P. Y. Paterson. 1973. Counterimmunoelectrophoresis for rapid diagnosis of meningitis due to Diplococcus pneumoniae. J. Infect. Dis. 127:16-19. 11. Harding, S. A., W. M. Scheld, M. D. McGowan, and M. A. Sande. 1979. Enzyme-linked immunosorbent assay for detection of Streptococcus pneumoniae antigen. J. Clin. Microbiol. 1:339-342. 12. Higashi, G. E., J. E. Sippel, N. I. Girgis, and A. Hassan. 1974. Counterimmunoelectrophoresis: an adjunct to bacterial culture in the diagnosis of meningococcal meningitis. Scand. J. Infect. Dis. 6:233-235. 13. Maegraith, B. G., and M. B. Adelaide. 1935. The rapid diagnosis of cerebrospinal fever. Lancet i:545-546. 14. Pepple, J., E. R. Moxon, and R. H. Yolken. 198. Indirect enzyme-linked immunosorbent assay for the quantitation of the type-specific antigen of Haemophilus influenzae b: a preliminary report. J. Pediatr. 97:233-237. 15. Severin, W. P. J. 1972. Latex agglutination in the diagnosis of meningococcal meningitis. J. Clin. Pathol. 25:179-182. 16. Sippel, J. E., and N. I. Girgis. 1978. Meningococcal infection in Egypt: laboratory findings in meningitis patients and the prevalence of pharyngeal infection in patients and contacts. Am. J. Trop. Med. Hyg. 27:98-985. 17. Sippel, J. E., and A. Quan. 1977. Homogeneity of protein serotype antigens in Neisseria meningitidis group A. Infect. Immun. 16:623-627. 18. Sippel, J. E., and A. Voller. 198. Detection of Neisseria meningitidis cell envelope antigen by enzyme-linked immunosorbent 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. Enzymelinked immunosorbent assay with a monoclonal antibody for detecting group A meningococcal antigens in cerebrospinal fluid. J. Clin. Microbiol. 19:23-234. 2. Thirumoorthi, M. C., and A. S. Dajani. 1979. Comparison of staphylococcal coagglutination, latex agglutination, and counterimmunoelectrophoresis for bacterial antigen detection. J. Clin. Microbiol. 9:28-32. 21. Voller, A., D. Bidwell, and A. Bartlett. 198. Enzyme-linked immunosorbent assay, p. 359-371. In N. R. Rose and H. Friedman (ed.), Manual of clinical immunology, 2nd ed. American Society for Microbiology, ington, D.C. 22. Zollinger, W. D., C. L. Pennington, and M. S. Artenstein. 1974. Human antibody response to three meningococcal outer membrane antigens: comparison by specific hemagglutination assays. Infect. Immun. 1:975-984.