Journal oflmmunological Methods, 156 (1992) 267-269 © 1992 Elsevier Science Publishers B.V. All rights reserved 0022-1759/92/$05.00



Short communication

Use of passive immunization for the production of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 1,6 and 12 E. Stenb~ek National Veterinary Laboratory, DK-1790 Copenhagen II, Denmark (Received 30 April 1992, revised received 8 June 1992; accepted 11 September 1992)

Hyperimmune sera from BALB/c mice immunized intraperitoneally (IP) with Actinobacillus pleuropneumoniae serotype 2 were used for passive intraperitoneal (i.p.) immunization of BALB/c mice. The immunized mice were subsequently immunized i.p. with a mixture of A. pleuropneumoniae serotypes 1, 6 and 12. Numerous monoclonal antibodies specific for serotypes 1, 6 and 12 were obtained. Using this immunization scheme antibodies can be obtained against specific antigens from closely related bacteria. Key words: Actinobacillus pleuropneumoniae; Passive immunization; Monoclonal antibody

Actinobacillus pleuropneumoniae (App) is a gram-negative bacterium which causes severe pleuropneumonia in pigs. This disease is often fatal and therefore causes severe economic losses to the pig industry (Nicolet et al., 1981). To date 12 App serotypes have been defined on the basis of differences in the carbohydrate composition of their lipopolysaccharide (LPS) and their capsular polysaccharide (CPS) (Altman et al., 1990). The variation in carbohydrate composition is very small for certain serotypes. In contrast the protein composition between App serotypes does not differ substantially (Rapp et al., 1986; M~ller et al., 1992), although this has not been thoroughly investigated. It can therefore be difficult to obtain monoclonal antibodies towards serotypespecific App epitopes, especially to epitopes which are not very immunogenic.

Correspondence to: E. Stenbaek, National Veterinary Laboratory, Biilowsvej 27, 1790 Copenhagen V, Denmark.

Limited antibody production against individual antigens in crude antigen mixtures may be due to inhibition of the immune response to antigenic epitopes caused by the presence of other antigenic epitopes. Attempts to avoid such mechanisms and to increase the polyclonal antibody production against individual antigenic epitopes in crude extracts of bacterial antigens have been overcome by passive immunization (Brody et al., 1967; Gershon et al., 1971; Thalhamer et al., 1985). The effects of these passively administrated antibodies, it was assumed, was to 'cover up' the dominant antigens or epitopes thereby limiting the immune response against them. The present work demonstrates the influence of passive immunization on the production of monoclonal antibodies (MAb) against App serotype specific epitopes using a crude extract of App antigens. App strains (kindly provided by Dr. R. Nielsen) were grown on PPLO agar overnight at 37°C. The plates were harvested in phosphate-buffered


saline (PBS) pH 7.2 (antigen I) and frozen at - 2 0 ° C (antigen II). Antigen II was sonicated on ice three times for 15 s with intervals of 1 rain, centrifuged at 3000 X g for 10 min and the supernatant recovered (antigen III). B A L B / c mice were immunized twice intraperitoneally (i.p.) with App-2, Danish strain 4226 (antigen II, 5 x 108 cells/mouse), in Freund's incomplete adjuvant (FICA) (1 : 1), and once with App-2 (antigen I, 5 x 108 cells/mouse) in FICA (1:1) with intervals of 2 weeks. The antibody response was investigated in an indirect enzyme immunosorbent assay (EIA) as described previously by Engvall et al. (1972) using antigen III for coating the microtiter plates. Sera that showed a n 0 D 4 9 0 > 1.000 when diluted 1/30,000 in PBS were considered positive. Responding mice were boosted 4 weeks later with App-2 (antigen I, 5 x 108 cells/mouse) in FICA (1:1) and bled after 1 week. Sera from the App-2 mice were used for passive i.p. immunization of B A L B / c mice (250 /xl/mouse). After 1 h the mice were immunized i.p. with a mixture of App-1, -6 and -12 (5 X 108 cells/mouse) (ref. strains Shope 4074, Femo and 8329, respectively) in FICA (1 : 1). This procedure was repeated three times at 2 week intervals using App-1, -6 and -12 (antigen II, 5 x 108 cells/mouse) in FICA (1:1) for the first immunization and App-1, -6 and -12 (antigen I, 5 x 10 ~ cells/mouse) in FICA (1 : 1) for the last two immunizations. Mice sera were tested in indirect EIA for the presence of antibodies against App-1, -6 and -12. Responding mice were immunized i.p. with App-2 mouse sera (250 izl/mouse) and boosted

after 1 h with App-1, -6 and -12 (antigen I, 5 x 108 cells/mouse) in FICA (1 : 1). After 3 days one mouse spleen was recovered and fused with the myeloma cell line P3-X63-Ag 8.653, using PEG 1500 as a fusogen, for production of monoclonal antibodies (MAb) (K6hler et ai., 1975). Two fusions were performed and hybridoma supernatants were screened in indirect EIA for the presence of serotype-specific antibodies against App-1, -6 or -12. Haemophilus parasuis (Hp), a closely related gram-negative bacterium, was used as a negative control in indirect EIA. Two additional fusions were performed in which B A L B / c mice were immunized i.p. with App-6 (5 x 108 cells/mouse) in FICA (1:1) as described above but omitting the passive immunization (standard immunization). The indirect EIA procedure was performed as follows. Microtiter plates were coated with App-1, App-6, App-12 or Hp antigens (antigen III, 10 6 cells/ml) overnight at 4°C. The plates were washed three times in PBS, 0.05% Tween 20, pH 7.2, incubated with 25 tzl hybridoma supernatant plus 75 /.~l PBS for 1 h, washed and incubated with horseradish peroxidase-conjugated sheep anti-mouse Ig (Na 931, Amersham) for 1 h. After washing, binding of MAb was detected with 0.01% H 2 0 2, 0.06% orthophenylene-diamine (OPD) in 0.1 M citrate acid pH 5.0 for 30 min. The reaction was stopped with 0.5 M H2SO 4. Reactivity was measured at 490 nm. An enhancement in the production of serotype-specific MAbs was noted following passive immunization (Table I). The App serotype specificities of the hybridomas were determined as follows. Taking the reactivity against one App




Spleen cells

Clones producing MAb








App-x a

IA 2A 3A 4A

6 6 1, 6, 12 1, 6, 12

Standard Standard Passive im. Passive ira.

7x 2x 7x 6x

42 38

4 16 13 26

23 35

6 121 115 114

107 107 107 107

Clones producing MAb reacting with one or several App serotypes.


serotype (OD490 value) as 100%, the reactivity against the other App serotypes and the Hp control strain should be less than 10%. In fusion 3A, 42, 13 and 23 clones producing MAb against App-1, -6 and -12, respectively, were detected. In fusion 4A, 38, 26 and 35 clones producing MAb against App-1, -6 and -12, respectively, were detected. This shows a significant increase in the number of clones producing App serotype-specific antibodies compared to fusion 1A and 2A where only four and 16 clones, respectively, were detected. Production of MAbs by standard immunization procedures using mixtures of App serotypes was not performed. A mixture of App serotypes will decrease the concentration of the serotypespecific antigens. This may therefore decrease the antibody response against serotype-specific antigens and enhance the antibody response against common epitopes. The enhancement of monoclonal antibodies may be due to the exclusion of common App epitopes which are highly immunogenic, the reduction of the antigenic repertoire and eliciting an antibody response against epitopes which are otherwise not immunogenic. Thalhamer et al. (1985) have demonstrated the presence of antigens which only elicit an antibody response after passive immunization. The antibody repertoire against App-specific epitopes can therefore be successfully increased. The administration of passive antibodies against antigens common to App2, -1, -6 and -12, prior to immunization demonstrates an effective method for enhancement of the antibody response towards specific epitopes in crude antigenic extracts. The major benefits of passive immunization for the production of monoclonal antibodies can thus be summarized as: (a) the use of crude antigenic mixtures for immuniza-

tion, (b) enhancement of the antibody response against epitopes which are weakly immunogenic, (c) production of antibodies against extremely small epitope differences in closely related bacteria, (d) a significant increase in the number of antibodies produced and thereby an increase in the antibody repertoire, and (e) a reduction in the cost of antibody production due to the increased numbers of antibody producing clones per fusion.

References Altman, E., Brisson, J.-R. and Perry, M.B. (1990) Structural characteristics of the antigenic capsular polysaccharides and lipopolysaccharides involved in the serological classification of Actinobacillus (Haemophilus) pleuropneumoniae. Serodiagn. Immunother. Infect. Dis. 4, 299. Brody, N.I., Walker, J.G. and Siskind, G.W. (1967) Studies on the control of antibody synthesis. J. Exp. Med. 126, 81. Engvall, E. and Perlmann, P. (1972) Enzyme-linked immunosorbent assay, ELISA. J. Immunol. 109, 129. Gershon, R.K. and Kondo, K. (1971) Antigenic competition between heterologous erythocytes. J. Immunol. 106, 1532. K6hler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 227, 495. Moiler, K., Nielsen, R., Vraa Andersen, L. and Kilian, M (1982) Clonal analysis of the Actinobacillus pleuropneumoniae population in a geographically restricted area by multilocus enzyme electrophoresis. J. Clin. Microbiol. 30, 623. Nicolet, J. and Scholl, E. (1981) Haemophilus infections. In: A.D. Leman, R.D. Glock, W.L. Mengeling, R.H.C. Penny, E. Scholl and B. Straw (Eds.), Diseases of Swine. Iowa State University Press, Ames, IA. Rapp, V., Munson, R.S. and Ross, R.F. (1986) Outer Membrane Protein Profiles of Haemophilus pleuropneumoniae. Infect. Immun. 52, 414. Thalhamer, J. and Freund, J. (1985) Passive immunization: a method of enhancing the immune response against antigen mixtures. J. Immunol. Methods 80, 7.

Use of passive immunization for the production of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 1,6 and 12.

Hyperimmune sera from BALB/c mice immunized intraperitoneally (IP) with Actinobacillus pleuropneumoniae serotype 2 were used for passive intraperitone...
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