INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Apr. 1988, p. 201-206 Vol. 38. No. 2 0020-7713/88/040201-06$02.OO/O Specificity of a Monoclonal Antibody for Alkaline Phosphatase in Escherichia coli and Shigella Species M. 0. HUSSON,1*2*P. A. TRINELY2C. MIELCAREK,2 F. GAVINI,2 C. CARON,lq2D. IZARD,l AND H. LECLERC' Faculte' de Me'decine, Laboratoire de Bacte'riologie A, 59045 Lille Cedex, France' and Unite' Institut National de la Sante' et de la Recherche Me'dicale 146, Domaine dir CERTIA, 59650 Villeneuve-d'Ascq Cedex, France2 The specificity of monoclonal antibody 2E5 for the alkaline phosphatase of Escherichia coli was studied against the alkaline phosphatases of 251 other bacterial strains. The organisms used included members of the six species of the genus Escherichia (E. coli, E. fergusonii, E. hermannii, E. blattae, E. vulneris, E. adecarboxylata), 41 species representing the family Enterobacteriaceae, and, in addition, Pseudomonas aeruginosa, Aeromonas spp., Plesiomonas shigelloides, Acinetobacter calcoaceticus, and Vibrio cholerae non-01. Three methods were used. An enzyme-linked immunosorbent assay was performed against 21U of alkaline phosphatase per ml; immunofluorescence against bacterial cells and Western blotting against periplasmic proteins were also used. All of our experiments demonstrated the high specificity of monoclonal antibody 2E5. This antibody recognized only E. coli (118 strains tested) and the four species of the genus Shigella (S. sonnei, S. flexneri, S. boydii, S. dysenteriae; 12 strains tested). Since the description of hybridoma production by Kohler mouse immunized with purified Escherichia coli ATCC and Milstein in 1975 (14), many monoclonal antibodies 10536 alkaline phosphatase as described elsewhere (12). (MoAbs) have been produced against microorganisms. Some Specificity testing of MoAb 2E5. (i) ELISA. Activated of these are now used for the detection of bacteria, such as polyvinyl chloride microtitration plates (Nunc or Flow Lab- Chlamydia trachomatis (29), Legionella pneumophila (9), oratories) were coated with 100 pl of alkaline phosphatase (2 and group B Streptococcus (19). The success of such anti- IU/ml) in phosphate-buffered saline (PBS; 0.05 M, pH 9.6) bodies is correlated with their high antigenic affinity and by incubating the preparation at 4°C for 18 h. The plates especially with their specificity, as established by enzyme- were washed three times with 200 pl of this buffer and were linked immunosorbent assay (ELISA), immunofluores- saturated by using 2% bovine serum albumin in PBS in the cence, or immunoblotting against a great number of various wells at 37°C for 1 h. After three washes with 200 pl of PBS bacterial species. containing 0.05% Tween 20, the plates were covered and Escherichia coli alkaline phosphatase was chosen to im- stored at 4°C. The immune reaction was performed by munize BALB/c mice and to obtain specific antibodies. This adding 100 pl of MoAb 2E5 for 1 h at 37°C. Subsequently, enzyme protein is a dimer composed of two identical sub- the wells were washed three times with PBS containing units. It is localized in the periplasmic space and at the cell 0.05% Tween 20 and then incubated with 100 r~.lof horse- surface of the gram-negative cell envelope in association radish peroxidase-conjugated goat anti-mouse immunoglob- with lipopolysaccharides (8, 17). Studies of its immunologi- ulin (Institut Pasteur Production Laboratories), Peroxidase cal properties by immunoprecipitation or quantitative micro- activity was detected colorimetrically by adding 0.04% 0- complement fixation (6, 8) have demonstrated a protein phenylenediamine in citrate-phosphate buffer (pH 5) supple- sequence divergence between Escherichia coli and some mented with 0.006% hydrogen peroxide. The reaction was other species of Enterobacteriaceae. Recently, we produced developed in the dark for 15 min at room temperature and MoAbs against alkaline phosphatase of Escherichia coli (12). stopped by adding 30 pl of 1.3 M H2S0,. Absorbance at 490 In this paper, we describe the specificity of MoAb 2E5, nm was measured with a micro-ELISA plate photometer which was studied by using the three techniques described (Autoreader MR600; Dynatech Laboratories, Inc.). Assays above. were carried out in duplicate. Negative controls (serum of nonimmunized BALB/c mice) and positive controls (serum MATERIALS AND METHODS of immunized BALB/c mice) were included in each test. Reactions were considered positive when the mean of repli- Bacterial strains and alkaline phosphatase extraction. The cate titrations was two times higher than the mean of 251 bacterial strains included in this study are listed in Table negative controls. For some species (Escherichia hermannii, 1. Cells were grown at 30°C for 18 h in phosphate limiting Escherichia vulneris, Enterobacter cloacae, Proteus vul- medium to derepress alkaline phosphatase synthesis, as garis, Proteus penneri, Providencia rustigianii, Pvovidencia described by Garen and Levinthal (11). Bacteria were har- alcalifaciens, Yersinia pseudutuberculosis, Buttiauxella vested, and alkaline phosphatase was released with other agrestis) no enzymatic activity was found after heat treat- periplasmic proteins by heat treatment at 56°C in tris(hy- ment or sonication. In these cases, the ELISA was per- droxymethy1)aminomethane (Tris) hydrochloride buffer (pH formed by using periplasmic proteins (15 pg/ml). 8) as described by Tsuchido et al. (26). Alkaline phosphatase (ii) Immunofluorescence assay. Indirect immunofluores- activity was assayed with 0.2 mg of the substrate p-nitro- cence was performed with MoAb 2E5 and fluorescein iso- phenyl phosphate per ml in 1 M Tris buffer (pH 8) at 400 nm. thiocyanate-conjugated rabbit anti-mouse immunoglobulin MoAb 2E5 production. MoAb 2E5 was obtained from the (Institut Pasteur Production). The working dilutions of the fusion of SP20/Ag-14 myeloma cells and spleen cells from a MoAbs and the conjugate were determined by checkerboard titration. PBS (pH 7.6) was used to dilute the antibody and * Corresponding author. the conjugate. The MoAb and the conjugate were diluted 201 :!02 HUSSON ET AL. INT. J. SYST.BACTERIOL. TABLE 1. Strains tested and cross-reactivities of MoAb 2E5 specific for Escherichia coli alkaline phosphatase Cross-reactivity as determined by: Bacterial strain(s)" ELISA against Immuno- enzymatic fluorescence of Western extract bacterial cells Escherichia spp. strains E. coli ATCC 10536 + + E. coli (38 environmental strains) + E. coli (59 medical strains) + E. coli (20 fecal strains) + E. fergusonii CDC 3296-73, CDC 3014-74 - E. fergusonii CDC 3458-74, CDC 1016-74, CDC 568-73 - E. hermannii ATCC 33650, CDC 899-73 E. hermannii CDC 2808-70, CDC 1933-74, CDC 2472-72 E. vulneris ATCC 33821, CDC 2898-73, CDC 3763-72, CDC 2954-71 E. blattae ATCC 29907 E. adecarboxylata ATCC 23216 Shigella spp. strains S. sonnei ATCC 9290, CUETM 77-73 + S. sonnei CFML 969 S. dysenteriae WRAIR 1617, CUETM 79-327 S.flexneri WRAIR 24570, CFML 970, ATCC 12661, CFML 17 S. boydii CDC 2406-51, WRAIR C10, CFML 398 Salmonella spp. strains S. typhimurium ATCC 23565 S. typhimurium ATCC 7823, CFML 1010 S. paratyphi CUETM 79-368 S. derby CFML 303, CFML 302 S. sohio CFML 305, CFML 317 S. enteritidis CFML 308 S. arizonae ATCC 7823 Levinea spp. strains L. rnalonatica CDC 25408 L. malonatica CUETM 77-12, CFML 359, CFML 416, CFML 875 L. amalonatica CDC 25406, CUETM 77-9 ICitrobacter freundii ATCC 8090 ICitrobacterfreundii CDC 460-61, CUETM 78-263, CUETM 78-280, CUETM 78-273, CFML 615, CFML 495, CFML 624 (Citrobacrerfreundii CU ETM 77-166, CUETM 77-152 Klebsiella spp. strains K. pneumoniae ATCC 13882 K. pneumoniae CUETM 77-130, CUETM 77-174, CUETM 78-146, CUETM 78-258, CUETM 77-180, CFML 509, CFML 220 K. oxytoca ATCC 13182, ATCC 77-146, CUETM 78-177, CUETM 78-182, CUETM 78-181, CUETM 78-178, CFML 685, CFML 173, CFML 48 Enterobacter spp. strains E. cloacae ATCC 13047 E. cloacae CUETM 77-121, CUETM 77-120, CUETM 77-126, CUETM 77-116, CUETM 77-125 E. amnigenus ATCC 33072 E. aerogenes ATCC 13048, CUETM 67-71 E. gergoviae ATCC 33028 E. sakazakii ATCC 29544 E. taylorae CDC 697-81 E. agglomerans ATCC 27155 Hafnia alvei CFML 877, CFML 880, CFML 884, CFML 883, CFML 165 Serratia spp. strains S. marcescens ATCC 16880 S. marcescens CUETM 78-210, CUETM 78-212, CUETM 78-248, CUETM 78-164, CFML 485, CFML 757 S. liquefaciens CUETM 78-243, CUETM 78-172, CUETM 78-244 S. liquefaciens CUETM 78-174, CUETM 78-228 S. fonticola ATCC 29844, CUETM 78-10 S. odorifera CUETM 83-88 Proteus spp. strains P. rnirabilis ATCC 29906 P. mirabilis CFML 699, CFML 1004 P. vulgaris ATCC 13315 P. penneri ATCC 33519 Continued on following page VOL. 38, 1988 SPECIFICITY OF MoAb 203 TABLE 1-Continued Cross-reactivity as determined by: Bacterial straints)" ELISA against Immuno- enzymatic fluorescence of extract bacterial cells blotting Morganella morganii ATCC 25830 Providencia spp. strains P. rustigianii ATCC 33673 P. stuartii ATCC 29914 P. alcalfaciens ATCC 9886 Yersinia spp. strains Y. enterocolitica CDC 135 Y. pseudotuberculosis ATCC 29833 Rahnella aquatilis ATCC 33071 Ewingella americana ATCC 33852 Buttiauxella agrestis ATCC 33320 Kluyvera spp. strains K. ascorbata ATCC 33434 K. cryocrescens ATCC 33435 Edwarsiella tarda CUETM 78-301 Cedecea spp. strains C. lapagei ATCC 33432 C. davisae ATCC 33431 Leminorella spp. strains L. richardii ATCC 33998 L. grimondii ATCC 33999 Pseudomonas aeruginosa ATCC 10145 Aeromonas spp. strains A. sobria CDC 9538-76 A. punctata ATCC 15468 A. hydrophila ATCC 7966 Acinetobacter lwofi ATCC 17986 Plesiomonas shigelloides ATCC 14029 Vibrio cholerae non-01 CUETM 77-100 ATCC, American Type Culture Collection, Rockville. Md.; CDC, Centers for Disease Control, Atlanta, Ga.; WRAIR, Walter Reed Army Institute of Research, Washington, D.C.; CUETM, Collection de I'unite Ecotoxicologie Microbienne, Villeneuve d' Ascq, France; CFML, Collection Faculte de Mkdecine de Lille, Lille, France. +, Positive; -, negative; (-), no alkaline phosphatase activity found, ELISA performed against periplasmic proteins extracted by heat treatment (26).