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Identification of Streptococcus Sobrinus with Monoclonal Antibodies

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Identification of Streptococcus Sobrinus with Monoclonal Antibodies JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1987, p. 2285-2288 Vol. 25, No. 12 0095-1137/87/122285-04$02.00/0 Copyright © 1987, American Society for Microbiology Identification of Streptococcus sobrinus with Monoclonal Antibodies JOHANNESJ. DE SOET,l* PHILIPPUS J. VAN DALEN,' BEN J. APPELMELK,2 AND JOHANNES DE GRAAFF' Department of Oral Microbiology, ACTA,' and Department of Medical Microbiology, Research Group for Commensal Infections,2 Vrije Universiteit, 1081 BT Amsterdam, The Netherlands Received 17 April 1987/Accepted 13 August 1987 Identification of Streptococcus sobrinus is often difficult to perform because of the great resemblance of the organism to other oral streptococcal species. Therefore, monoclonal antibodies were prepared which were shown to be highly specific for S. sobrinus. Cross-reactivity with other oral microorganisms has not been observed in an enzyme-linked immunosorbent assay and an immunofluorescence assay. These monoclonal antibodies belonged to the subclass immunoglobulin G2b. To be certain that the strains used in cross-reactivity tests were S. sobrinus, their DNA base composition was measured as a golden standard. Additional tests like colony morphology and sugar fermentation with the API 20 Strep system (Analytab Products, Montalieu- Vercieu, France) were performed. These additional tests turned out to be necessary because 100% correct identification could not be obtained by separate tests. Immunological characterization with the clones OMVU10 and OMVU11 proved to be discriminative between S. sobrinus and other streptococcal species. Streptococcus sobrinus, previously called Streptococcus immunization schedule consisted of four injections of 0.1 ml mutans serotype d/g, is known to be cariogenic in animals of bacterial suspension given at 2-week intervals. The first (18). It has been found in higher numbers in dental plaque injection was given intravenously, the second intraperitone- from humans with active caries than from caries-free indi- ally with complete Freund adjuvant, and the third intraperi- viduals (11-13). These results suggest that S. sobrinus may toneally with incomplete Freund adjuvant. The last injection be more cariogenic than other members of the S. mutans (intravenous) was given 4 days before fusion. group. Hybridomas were made by fusing the splenocytes with It is evident that for an evaluation of the role of S. sobrinus P3-X63-Ag8.653 myeloma cells at a 1:5 ratio. The fusion in dental caries, a reliable method for identification and procedure was that described by De Haan et al. (7). Hybrids quantification is required (26). Mitis-salivarius bacitracin were screened for the production of immunoglobulin by agar (MSB) is often used for the isolation of streptococci of testing the hybridoma culture supernatant (HCS) in an the S. mutans group. However, this selective medium is not enzyme-linked immunosorbent assay (ELISA). suitable for direct identification of S. sobrinus and, more- ELISA. Bacterial cell suspension (75 pil) was incubated at over, strongly inhibits its growth (8, 14, 15, 21, 25, 27, 29). 4°C for 16 h in 96-well microdilution plates (655001; Greiner). Therefore, S. sobrinus may be easily missed when this Free binding places were blocked by incubation with 1% medium is used. A recently developed selective Trypticase- bovine serum albumin and 0.05% (wt/vol) Tween 80 in PBS yeast-cystine-sucrose-bacitracin agar (TYCSB) has been re- at room temperature for 1 h. Between every incubation, the ported to be more suitable for the isolation and identification plates were washed three times with PBS supplemented with of S. sobrinus colonies which exhibit a "halo" on it (21, 27). 0.05% Tween 80 (PBST). HCS (75 ,ul) diluted in PBST was On the other hand, antisera have proved to be useful for the identification of pathogenic bacteria in dental plaque samples added. The plates were incubated for 2 h at room tempera- (2, 9, 19, 20). Monoclonal antibodies (MAbs) have also been ture. After washing, 75 ,ul of peroxidase-conjugated goat used for this purpose (3, 23). anti-mouse immunoglobulin was added (1:1,000; affinity- The aims of this study were (i) to produce MAbs against S. purified; Bio Makor, Israel) and incubated for 1 h at room sobrinus that were highly specific and therefore suitable for temperature. Peroxidase activity was measured by adding 75 identification purposes, and (ii) to compare the results with ,ul of o-phenylenediamine (8 mg) with 8 ,ul of H202 in 20 ml commonly used identification methods, such as sugar fer- of citrate buffer (0.01 M, pH 9.5). The reaction was stopped mentation, colony morphology on selective media, and with 50 p.l of H2SO4 (10%) after 15 min. The OD of each well guanine plus cytosine content determination of the DNA. was measured with a Micro plate reader (MR 600; Dynatech Industries, Inc., Torrance, Calif.) at 490 nm. MAbs against Escherichia coli lipopolysaccharide served as a negative MATERIALS AND METHODS control (B. J. Appelmelk, A. M. J. J. Verweij-van Vught, Bacterial strains. The microorganisms used in this study J. J. Maaskant, W. F. Schouten, A. J. R. De Jonge, L. G. are listed in Table 1. Bacterial cell suspensions were pre- Thijs, and D. M. Maclaren, J. Med. Microbiol., in press). pared by growing the cells anaerobically (85% N2, 5% C02, The titer of HCS was defined as the dilution by which the and 10% H2) overnight in Todd-Hewitt broth at 37°C. The OD of the ELISA was two times the OD of the negative cells were centrifuged, washed, and diluted in phosphate- control. Clones which were specific for S. sobrinus were buffered saline (PBS) to an optical density (OD) at 650 nm of detected by their positive reactions in ELISA with S. 1.0. sobrinus and by their negative reactions with other strepto- Hybridoma production. The bacterial cell suspension of cocci. strain HG459 was used to immunize BALB/c mice. The Screening of the microorganisms was performed by ELISA. A positive ELISA was defined as an OD of 0.5 or * Corresponding author. greater at an HCS dilution of 1:1,000. 2285 2286 DE SOET ET AL. J. CLIN. MICROBIOL. TABLE 1. Microorganisms used for cross-reactivity studies and their determination Acid production from": Species and strain (origin') G + C%b ELISA' Halo" Fluorescent cells Inulin Raffinose Streptococcus sobrinus HG456 43.5 +l- v + HG459 6715DP (6) 44.3 + + v + HG594 (7) 45.0 + v + HG596 OMZ176 (4) 44.4 +l- + HG598 50B4 (4) 44.7 + + HG599 OMZ65 (4) 44.4 + +l1- V v + HG688 OMZ65 (3) 43.9 + HG689 B276 (3) 44.6 + HG690 B13 (3) 43.7 + +l- + HG702 (7) 44.2 + + HG707 ATCC 33478 44.3 + HG713 OlHl (1) 43.7 + HG716 5OB4SS (1) 44.9 + HG718 50B4 (1) 44.1 + HG732 (7) 44.2 + HG738 OMZ176 (2) 44.2 + HG739 KIR (2) 43.4 + HG743 6715 (2) 43.0 + Streptococcus mutans HG724 SE1l (1) ND + HG210 4540 (5) 38.8 + HG221 999 (5) 38.0 + HG222 1000 (5) 38.2 + + HG236 (7) ND + HG237 (7) 38.3 + HG240 (7) 38.4 + HG801 JC2 (4) 37.0 HG712 16Ba (1) ND + HG714 LM7 (1) 38.0 + HG730 AT10 (1) ND + Streptococcus cricetus + HG48 E-49 N 42.0 ND v +D HG709 370 (1) ND ND + HG773 E-49 (2) ND ND V HG744 HS6 (2) 42.1 ND ND ND Streptococcus rattus HG725 Z-BHT (1) ND HG726 JH145 (1) ND ND ND HG751 FAl (3) ND + + HG745 BHT (3) 41.7 Various microorganisms ND ND ND a Origins were as follow: 1, C. Van Looveren, The Netherlands; 2, A. L. Coykendall, United States; 3, R. Whiley, United Kingdom; 4, W. H. Van Palenstein Helderman, The Netherlands; 5, J. S. Van Der Hoeven, The Netherlands; 6, S Hamada, Japan; 7, our isolate. b Guanine and cytosine content of DNA. ND, Not determined. C An OD of 0.5 or higher in an ELISA with clone OMVU10 (diluted 1:1,000). d Existence of a halo around the colony on TYCSB and TSY20B agar plates. When differences between the two media were observed, both results are given as TYCSB/TSY20B. +, a halo of at least 1 mm was observed around the colony; -, no halo was observed; ND, not determined. e Acid production from inulin and raffinose by the API 20 Strep system. V, Variable; ND, not determined. f Fluorescent cells by immunofluorescence technique. " The various oral microorganisms tested were S. ferus 8S1 (3) and HD3 (2); S. macacae H1569 (3); Streptococcus sanguis I and II HG168, HG169, HG310, HG311, HG533, HG536, HG857, and HG865 (7); Streptococcus mitis HG170, HG178, and HG467 (7); Streptococcus milleri HG293, HG294, and HG587 (7); Streptococcus sdlivarius HG668 (7); Streptococcus pyogenes HG228 and HG336 (7); Streptococcus morbillorum HG858 and HG874 (7); Actinomyces viscosus HG484 (NY334; 5); Lactobacillus case HG498 (7); Bacteroides gingivalis HG762 (7); Bacteroides melaninogenicus HiG753 (7); Bacteroides intermed us HG763 (7); Capnocytophaga ochracea HG347 (7); and Candida albicans HG428 (7). These strains were tested for reactivity on ELISA and immunofluorescence only. Immunofluorescence assay. The immunofluorescence as- slides were incubated for 1 h at room temperature with 40 ,ul say used in this study was performed with pure and mixed of an antibody dilution (clone OMVUiO; 1:500 in PBST). bacterial cultures. The method used was a modification of After the slides were washed, 40 ,uI of fluorescein isothiocy- that described by Mouton et al. (19). Briefly, bacterial anate-conjugated, affinity-purified goat anti-mouse immuno- suspensions were diluted to an OD at 650 nm of 0.1. The globulin G (IgG; Bio Makor) was added and incubated for 1 suspensions wère placed on objective slides (XBM-302- h at room temperature. The slides were examined with a 89912; Bio Merieux) and dried at 70'C for 1 h.
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