Immunology Letters, 33 (1992) 201-206 0165 - 2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved IMLET 01819
Monoclonal antibodies against Salmonella porins: generation and characterization V.R. M u t h u k k a r u p p a n , K.S. N a n d a k u m a r and V. Palanivel Department of Immunology, School of Biological Sciences, Madurai Kamaraj University, Madurai, India (Received 11 May 1992; accepted 25 May 1992)
Monoclonal antibodies (mAbs) were generated against porins, one of the major outer membrane proteins of Salmonella typhi. Six clones, designated MP1, MP2, MP3 (IgG2ak), MPN4, MPN6 (IgGlk) and MPN5 (IgG2bk) were characterized by enzyme immunoassay (ELISA) for their reactivity to porins from S. typhi, Salmonella paratyphi A, S. paratyphi B, S. paratyphi C, Salmonella choleraesuis, Salmonella enteritidis, Salmonella krefeld, Salmonella panama, Salmonella typhimurium, Escherichia coli B, Shigella flexneri lb and Pseudomonas aeruginosa. All the clones positive for S. typhi porins showed varying reactivity towards several Salmonella species. However, none of them was positive for porins from other Gram-negative bacteria or for lipopolysaccharide (LPS). The affinity constant of these mAbs, except MPN4, was found to be in the higher range. Dot ELISA revealed that the mAbs recognized porins only in their native form. The results of inhibition ELISA using horseradish peroxidase (HRP)-conjugated MP1 suggest that the clones MPI, MP2, MP3, MPN5 and MPN6 secreted antibodies to identical epitope(s) of a 36-kDa peptide and MPN4 to a different epitope of a 35-
kDa peptide. The possible applications of these mAbs were discussed. 2.
Porins are the pore-forming proteins present in the outer membrane of Gram-negative bacteria, and are 34-36 kDa in size . Several studies have shown that this protein could mount both cell-mediated and humoral immune responses . Also, the importance of porins as a suitable eliciting antigen for the delayed type hypersensitivity reaction in murine salmonellosis has been demonstrated . Either the native form or the cleaved products of porins could act as a potent mitogen for human peripheral blood tymphocytes and as a polyclonal activator for normal B cells . Further, porins have been shown to be highly immunogenic proteins with the ability to induce good protective immunity in mice . Calderan et al. (1986) demonstrated a high level of anti-porin antibodies in patients with typhoid infection . With the understanding of such immunological properties of porins, the present study was aimed to generate and characterize mouse mAbs against porins of S. typhi 3.
Materials and Methods
Key words: Porin; Monoclonal antibody; Specificity; Sub-type; Affinity constant
Correspondence to: Dr. V.R. Muthukkaruppan, Department of Immunology, School of Biological Sciences, Madurai Kamaraj University, Madurai 65 021, India.
The bacterial strains were obtained as follows: S. typhi (Ty21a) from E. Furer, Swiss Serum and 201
Vaccine Institute, Switzerland; S. typhi (Ty2), S. paratyphi A, B and C, S. choleraesuis, S. enteritidis, S. krefeld and S. panama from the Central Research Institute, Kasauli, India; S. typhimurium (C5) from C.E. Hormaeche, University of Cambridge, UK; P. aeruginosa from R. Sambasiva Rao, Department of Microbiology, JIPMER, India; E. coli B and Sh. flexneri lb from K. Dharmalingam, Genetic Engineering Research Unit of this University. The bacteria were cultured in Luria-Bertani broth containing peptone-Beef extract (HI Media, India)-NaC1 for 18 h at 37°C, harvested, washed with saline followed by Tris-HC1 (pH 7.2) and suspended in Tris-HCl. 3.2.
Porins from the outer membrane of different bacterial strains were purified as described by Tokunaga et al. . Porins extracted from other Gram-negative bacteria, using the above method, also resolve in the same molecular weight range (34-36 kDa). The purity of our porin preparation was reported earlier . 3.3.
Media and chemicals
Dulbecco's modified Eagle's medium (DMEM), 2-mercaptoethanol (2-ME), hypoxanthine-thymidine (HT)-aminopterin (HAT), polyethylene glycol (PEG) 1300-1600, dimethyl sulfoxide (DMSO), 2-6-10-14 tetramethyl pentadecane (Pristane), bovine serum albumin fr. V (BSA), Ophenylene diamine (OPD) diaminobenzidine (DAB), polyoxyethylenesorbitan monolaurate (Tween-20) and Gelatine ty. I were purchased from Sigma Chemical Co., St Louis, MO, USA. Fetal calf serum (FCS), sodium bicarbonate solution, L-glutamine, sodium pyruvate, penicillin and streptomycin (Pen-Strep) Freund's complete adjuvant (FCA), gentamycin sulfate and heparin sulfate were from Gibco Laboratories (Grand Island, NY, USA). Hybridoma cloning factor (HCF) was from Origen Inc., (Maryland, USA). 3.4.
in complete DMEM containing 10% FCS, 2 mM L-glutamine, 0.05 mM 2-ME, 100 U/ml PenStrep. BALB/c mice were immunized subcutaneously (s.c) either with 75 #g of Ty21a porins mixed with equal volume (100/fl) of FCA or 75 ~g of Ty2 porins in polyacrylamide gel (200 pl). After three weeks, a similar injection schedule was adopted for the respective groups. Subsequently, three weeks later, booster injection of porins alone (125/~g in 200 ktl) was given intraperitoneally (i.p.) to both groups. Somatic cell hybridization was performed three days after the last immunization following the method of Lerner . Either the SP2/O-Ag 14 or the P3X63Ag8.653 cell line was used for fusion in the presence of 40% (v/v) PEG in serum free DMEM. The hybrid cell clones were selected in DMEM supplemented with HAT. The homologous erythrocytes were used as the feeder layer. The hybridomas were screened by indirect ELISA and cloned twice by limiting dilution technique  in the presence of HCF. 3.5.
Screening for anti-porin activity in the hybridoma culture supernatants was performed by ELISA . The polystyrene plates were pulsed with porins (5 #g/ml) in carbonate-bicarbonate buffer pH 9.6. PBS-Tween 20 with or without 1% BSA was used in blocking and in washing steps. Subsequently incubated with culture supernatants and then with HRP-conjugated goat anti-mouse IgG (Cappel, PA, USA) at 1:4000 dilution. The color developed with the substrate (OPD plus hydrogen peroxide) was measured at 490 nm. The cross- reactivity of the mAbs was analyzed as described above, except that the plates were coated with porins from different Gram-negative bacteria or 16 /~g/ml of phenol/chloroform/petroleum ether extract of S. typhimurium LPS from Sigma. The mouse polyclonal anti-porin serum was included as standard positive. Ascites to unrelated antigen and no antigen pulsing served as negative control and blank respectively.
Generation of monoclonal antibodies 3.6.
BALB/c myeloma cell lines, SP2/O-Ag 14 and P3X63-Ag8.653 (non-secretors) were maintained 202
Indirect enzyme-linked immunosorbent assay
Measurement of affinity constant
The affinity constant of mAbs was determined
by following the method of Friguet et al. . Briefly, twice the concentration of mAb giving 50% binding in ELISA was incubated with different concentrations of porins. The unbound mAbs were detected against porins by ELISA.
3.7. lsotype characterization The subtypes of mAbs from hybridoma supernatants were identified by ELISA. The HRP-conjugated goat anti-mouse subclass-specific antibodies (Serotec, Oxford, UK) were used.
3.8. Production of ascites Hybridomas were grown as ascites in pristaneprimed (0.5 ml i.p.) male BALB/c mice with some modifications in the standard procedure . Ten days after pristane, the mice received i.p. 1 mg of gentamycin sulfate. They were inoculated i.p. with 107 hybridoma cells in 0.5 ml saline. Ascitic fluid
was collected after 7-10 days and stored at -
3.9. Enzyme labeling of mAbs The affinity purified MP1 mAbs were labeled with HRP by the glutaraldehyde method . The labeled MP1 was tested for its anti-porin activity by ELISA.
3.10. Inhibition enzyme-linked immunosorbent assay The porin-coated wells were incubated with various dilutions of ascites (MP2, MP3, MPN4, MPN5 or MPN6). Following this, the wells were treated with HRP-conjugated M P 1 mAbs (1:1000). The rest of the procedure was as described above.
TABLE 1 Characterization of clones secreting anti-porin antibodies. Source of porins
S. typhi S. typhimurium S. paratyphi A S. paratyphi B S. paratyphi C S. choleraesuis S. enteritidis S. krefeld S. panama E. coli B Sh.flexneri lb P. aeruginosa LPS d Isotypee
Anti-porin activitya'b MP1
40 40 30 12.5 0.02 2.5 0.02 55 25 0.02 0.02 0.1
35 30 30 12.5 0.02 2.5 0.02 25 25 0.02 0.02 0.1
1250 1250 1250 625 62.5 600 25 1250 1250 0.02 0.02 2.5
625 0.5 2.5 0.1 125 600 125 62.5 62.5 0.02 0.02 0.02
312 312 312 62.5 62.5 100 12.5 312 312 0.02 0.02 0.1
2400 1250 1250 312 1250 2400 625 1250 1250 0.02 0.02 0.1
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0. !
0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02
Affinity const. ( x 10 -9 M) 0.80
aAscitic fluid, precipitated with ammonium sulphate and reconstituted to half the original volume was used. ~Dilution ( X 104) giving 0.15 A units ELISA. CReactiveto unrelated antigen, aReactivity with LPS-eoated wells. CAllwere found to be x-fight chain. MPI, MP2, MP3, by Ty21a porins immunization. MPN4, MPN5, MPN6, by Ty2 porins immunization. 203
Dot immunoenzymatie assay
To characterize the specificity of mAbs, the purified porins (50 ng) from Salmonella (native and heat-denatured forms) and E. coli were spotted on nitrocellulose discs. The discs were incubated with ascites and stained using HRP-conjugated goat anti-mouse IgG. DAB-hydrogen peroxide was used as substrate . 4.
Results and Discussion
In order to generate mAbs, the porin-sensitized splenocytes were fused with myeloma cells. Six hybrid cell clones were selected and characterized for their reactivity to porins from several Gramnegative bacteria. The clones were injected into pristane-primed BALB/c mice. We have found i.p. injection of gentamycin was required to have the consistant development and better yield of ascites. As shown in Table 1, all six clones reacted with porins of S. typhi. Also, these clones showed varying binding with porins of other Salmonella species. However, none of the clones were positive for porins of other Gram-negative bacteria and LPS. The isotypes and the affinity constant of these mAbs are presented in Table 1. For the determination of reactivity of these
mAbs with native and denatured forms of porins, the dot ELISA was performed (Fig. 1). All the mAbs recognized the porins in their native form, thus indicating that they were directed to the conformational epitope(s). With respect to various polypeptides of porins, all mAbs showed binding to 36-kDa peptide except MPN4 which identified 35-kDa peptide, as revealed by ELISA using purified preparations (data not shown). The affinity constant measurements revealed that all the clones except MPN4 secreted antibodies at high affinity range. To further characterize the mAbs for their epitope specificity and for the development of typhoid diagnostic tests, we have selected a mAb (MP1) reacting with porins of both S. typhi and S. typhimurium for HRP conjugation. The protein-A purified MP1 was labeled with HRP and tested for its anti-porin activity. As shown in the titration curve (Fig. 2), it was evident that the MP1-HRP conjugates had strong reaction towards porins of S. typhi. Reactivity with E. coli porins was negligible even at the dilution of 1:200. The data reveal that after conjugation MP1 retained its reactivity as well as its specificity. Using this M P I - H R P conjugate, the inhibition ELISA was carried out to determine the epitope specificity of different mAbs. Among the mAbs
Fig. 1. Anti-porin activityof mAbs in dot ELISA.Heat denaturationwas done in boilingwater for 5 min. Lanes(1-3) MPI-MP3; (46), MPN4-MPN6; (7) NMS; (8) control ascites; (9) standard positivecontrol (polyclonal). 204
E c 1.2
Fig. 2. Level of anti-porin activity in HRP-conjugated MP1 mAbs. The ELISA plates were coated with porins of either S. typhi ( 0 ) or E. coli (O). BSA-coated wells served as blank. Each point represents an average of five tests.
tested, MP2, MP3, MPN5 and MPN6 inhibited the binding of MP1-HRP, whereas the MPN4 did not (Fig. 3). The results indicate that the former set of mAbs were directed towards an identical epitope as that of MP1 and the latter one to a different epitope. From the above findings, it can be concluded that the mAbs generated are specific 1.2
to porins of Salmonella and also directed towards different epitopes. The primary idea of generating mAbs and giving importance for porins is to use these mAbs as a tool for the diagnosis of human typhoid infection. Porins are one of the major constituents among the outer membrane proteins , found to be secreted directly by bacteria or derived from the lysis of bacterial cells . They are resistant to the action of proteolytic enzymes of the host . These properties make the porins a candidate antigen to be detected (using the mAbs) in the Salmonella-infected mice  as well as in humans. As an alternative, porin-specific antibodies can be detected by inhibition enzyme immunoassays. We have observed that sera collected from typhoid patients as early as three days after onset of fever inhibited the binding of MP1-HRP mAb to porins. Whereas sera from persons infected with other Gram-negative bacteria or healthy controls did not (manuscript in preparation). Experiments are in progress along these lines to develop a method for typhoid diagnosis. Different classes of mAbs to porins could be useful in studying their protective ability against Salmonella infection. Such mAbs would be important for developing passive therapy. In this context, the clones MP1 and MPN5 were capable of complement-mediated killing of S. typhi in vitro (Lakshmi Mundkur, unpublished observation). This also indicates that these mAbs bind to the porin epitopes exposed on the bacterial surface. These mAbs could also be used in studying immunobiological properties of porins, in distinguishing certain Salmonella species and in the identification of recombinant clones of E. coli expressing S. typhi porins.
Acknowledgements KSN is a recipient of research fellowship from CSIR, India. I DILUTIONS
Fig. 3. Inhibition ELISA with HRP-conjugated MPI mAbs. Each point represents an average of two tests. The top most horizontal line represents the absorbance value for the MP1HRP.MP (O), MP3 (O), MPN4 (*), MPN5 (A) and MPN6
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