FEMS Microbiology Letters 78 (1991) 227-230 © 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50

ADONIS 037810979100132S

227

FEMSLE 04329

Production of monoclonal antibodies against a hemagglutinin/protease of Vibrio cholerae non-01

Takeshi Honda, Atsuko Hata-Naka, Kanchalee Lertpocasombat and Toshio Miwatani

Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka, Japan

Received 8 October 1990 Accepted 2 November 1990

Key words: Monoclonal antibodies; Hemagglutinin; Protease; Vibrio cholerae; Vibrioparahaernolyticus

1. SUMMARY

Two hybridoma cell lines producing mono- clonal antibodies (MAbs) against a hemag- glutinin/protease (HA/P) from Vibrio cholerae non-01 were produced and characterized. The two MAbs contained the x light chain and were IgG1 type. They similarly neutralized HA/P protease activity derived from both V. cholerae non-01 and V. cholerae 01, whereas they were unable to neu- tralize the hemagglutinating activity of HA/P, suggesting that the epitopes for protease and hemagglutination activities are different. Western blotting analysis and the cross-neutralization test with the two MAbs confirmed the identity of HA/P produced by V. cholerae non-01 and 01. This study also suggests that HA/P of V. cholerae

Correspondence to: T. Honda, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita, Osaka 565, Japan.

and a protease of V. parahaemolyticus are im- munologically unrelated.

2. INTRODUCTION

Vibrio cholerae non-01 strains, enteropathogens closely related to V. cholerae 01, have been identi- fied as the causative agents of small outbreaks and sporadic cases of gastroenteritis [1-3]. The pro- duction of various toxins and toxic substances [4-7], including a hemagglutinin/protease (HA/ P) [8,9], by strains of V. cholerae non-01 have been reported. Several investigators [11-15] have reported that the H A / P of V. cholerae 01 may serve as a mediator of attachment of bacteria onto intestinal epithelium and may enhance cholera toxin toxicity by nicking. We recently purified and characterized H A / P of V. cholerae non-01 and suggested the identity of H A / P of V. cholerae non-01 and 01 [10].

In this study, to investigate in more detail the H A / P of V. cholerae non-01 and 01, we describe, for the first time, the establishment and char-

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acterization of two monoclonal antibodies (MAbs) against HA/P of V. cholerae non-01.

3. MATERIALS AND METHODS

3.1. Purification of H A / P of V. cholerae non-O1 HA/P of V. cholerae non-01 was purified from

cultures of a clinical isolate of K cholerae non-01 TH81 by published procedures [10].

3.2. Preparation of monoclonal antibody Immunization was essentially performed as de-

scribed elsewhere [16]. In brief, BALB/c mice were intraperitoneally immunized with 20 /~g of purified HA/P emulsified in Freund complete adjuvant. An additional booster dose of 20/~g of HA/P together with Freund incomplete adjuvant was given intraperitoneally 4 weeks later. Three days after the last intravenous booster injection of 20 /lg of HA/P, spleen cells from immunized mouse were fused with X6-Ag8.653 myeloma cells as described previously [16]. After selecting hybrid cells by HAT selection, clones producing MAbs were screened using an enzyme-linked immuno- sorbent assay (ELISA) using purified HA/P- coated plates [16].

The positive hybridomas were finally recloned at least twice by the limiting dilution method. Ascites fluids induced by the hybridoma in BALB/c mice were used in the following experi- ments.

3.3. Protease of V. cholerae 01 and V. parahaemo- lyticus

A HA/P of V. cholerae 01 was obtained from cultures of V. cholerae 01 as described [9,10]. A V. parahaemolyticus protease was obtained from the V. parahaemolyticus strain TH25m as previously described [17].

3.4. Determination of immunoglobulin isotype and concentration

Immunoglobulin isotype determination of MAbs was performed using a MonoAB-ID EIA kit (Zymed Laboratories Inc.) according to the procedure recommended by the manufacturer. Im- munoglobulin concentrations of MAbs were de-

termined by radial immunodiffusion test as de- scribed previously [16].

3.5. Polyclonal antibody Polyclonal antiserum against purified HA/P

was prepared in rabbit as described previously [10]. Immunoglobulins were purified from the polyclonal antiserum by a protein A-conjugated Sepharose 4B affinity column [17].

3.6. Neutralization Neutralization of protease activity of H A / P by

MAbs and polyclonal antibodies was performed in an azocasein assay as described previously [10]. After incubating MAbs and protease preparations at 37 °C for 15 rain, the remaining protease activ- ity was assayed with azocasein as a substrate.

Neutralization of hemagglutinating activity of HA/P by antibodies was examined by a micro- titer method by mixing chicken erythrocytes sus- pended in Krebs-Ringer buffer and serially di- luted antibodies as described [9,10].

3. 7. Western blot analysis Immunoblot (Western blot) analysis using crude

and purified H A / P preparations obtained from V. cholerae non-01 and 01 was performed as previ- ously described [16].

4. RESULTS

Among 480 hybridomas obtained, 24 hy- bridomas developed a positive reaction in ELISA. After two to four rounds of subcloning, two stable clones producing antibodies against H A / P of V. cholerae non-01 were established. The properties of the two MAbs are summarized (Table 1). The two MAbs belonged to the IgG class having x light chains (Table 1).

The reactivity of the two MAbs to H A / P de- rived from both V. cholerae non-01 and 01 was examined by Western blot analysis (Fig. 1). The two MAbs reacted similarly to HA/P derived from both V. cholerae non-01 and 01 at an identi- cal position, indicating the identity of HA/P of the two organisms. On the other hand, the two MAbs did not react with a protease preparation of

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43 k

30 k

Fig. 1. An example of Western blot analysis with MAbs. Lanes: 1, 2, purified HA/P of V. cholerae 01 and non-01, respectively; 3, crude protease preparation of 1I. parahaemo- lyticus. (A and B) Results with 2.4.1 and 2.4.2 MAbs, respec-

tively.

V. parahaemolyticus (Fig. 1), suggesting the im- munological unrelatedness of proteases of V. cholerae and V. parahaemolyticus.

Although polyclonal antibodies neutralized both the protease and hemagglutinating activities of HA/P , the two MAbs similarly neutralized the protease activity (Fig. 2), but not the hemag- glutinating activity of H A / P , indicating that the epitopes related to these activities are different. The two MAbs did not neutralize the protease activity of V. parahaemolyticus (Fig. 2).

E ~" 0.3 O O4

0.2 c 0

o.~ .<

10 -1 10 -2 1'0 3

Dilution of monoclonal ant ibody

E c

O ¢q

=o

O

2~ <

0 , 3 -

J 0.2 y 0.1

t0-' lb -2 lO-a

Oilution of monoclonal antibody

Fig. 2. Neutralization of protease activity of V. cholerae non-01, V. cholerae 01 and V. parahaemolyticus. (A and B) Results with 2.4.1. and 2.4.2 MAbs, respectively. • m, HA/P of V. cholerae 01; • • , H A / P of V. cholerae non-01; • e, II. parahaemolyticus protease. The concentration of the two MAbs was adjusted to 100 /tg/ml and diluted as

indicated.

5. DISCUSSION

The pathogenic importance of H A / P of V. cholerae has been documented by various investi-

Table 1

Summary of the features of two MAbs against HA/P

gators [11-15]. H A / P is an interesting molecule with bifunctional protease and hemagglutination activity [14]. The similarity between H A / P pro- duced by both V. cholerae non-01 and 01 has been

Clone Isotype and light chain

Neutralization of protease (hemagglutination) activity of H A / P derived from

V. cholerae non-01 V. cholerae 01

Reactivity with HA/P by Western blotting

MAbs 2.4.1 IgG1, MAb 2.4.2 IgG1,

Polyclonal antibody unidentified

+ (_ a) + (_) + + ( - ) + ( - ) +

+ ( - ) + (+) +

a Parentheses contain neutrafization of hemagglutination.

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suggested by physicochemical comparative studies [8-101.

In this study, to investigate HA/P in more detail, we established, for the first time, two clones producing MAbs against the HA/P derived from 1~. cholerae non-01. The results of neutralization and Western blot analyses reveal the similarity of HA/P of both E cholerae non- /and 01. Interest- ingly, our results also suggest that the epitope(s) involved in protease and hemagglutinating activi- ties are present at different sites on the bifunc- tional HA/P molecule.

Moreover, data obtained by neutralization ex- periments and Western blotting analysis in this study suggest an immunological difference be- tween HA/P of V. cholerae and a protease pro- duced by ld. parahaemolyticus, an enteropathogen similar to V. cholerae [1-3].

ACKNOWLEDGEMENTS

This work was supported by a Grant-in-Aid for Scientific Research and for Special Project Re- search from the Ministry of Education, Science and Culture of Japan.

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