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On-line version ISSN 1414-431X
Braz J Med Biol Res vol.34 no.8 Ribeirão Preto Aug. 2001
Production and characterization of a monoclonal antibody against an Ascaris suum allergenic component
R.R. Pires, T.M. Oshiro, D.M. Itami, I. Fernandes and M.F. Macedo-Soares
Laboratório de Immunopatologia, Instituto Butantan, São Paulo, SP, Brasil
Ascaris suum allergenic components (PIII) separated by gel filtration chromatography of an adult worm extract were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells using polyethylene glycol (MW 1450) as fusogen. The hybridomas were cultured in HAT-containing medium and cloned at limiting dilutions. Supernatants from the growing hybrids were screened by ELISA using plates coated with PIII or the A. suum crude extract. The monoclonal antibody obtained, named MAC-3 (mouse anti-A. suum allergenic component), is an IgG1 kappa mouse immunoglobulin that specifically recognizes a 29,000 molecular weight protein (called allergenic protein) with an affinity constant of 1.7 x 109 M-1. The A. suum components recognized by MAC-3 induce specific IgE antibody production in immunized BALB/c mice. Ascitic fluid induced in Swiss mice by injecting ip the hybridoma cells and incomplete Freund's adjuvant was purified by affinity chromatography using a protein A-Sepharose column. The purified monoclonal antibody was then coupled to activated Sepharose beads in order to isolate the A. suum allergenic component from the whole extract by affinity chromatography.
Key words: Ascaris suum, allergen, IgE, monoclonal antibodies
Helminth infections are associated with production of substantial amounts of both parasite-specific and nonspecific IgE, occurrence of immediate hypersensitivity reactions and proliferation of hypersensitivity-associated cells, such as mast cells and eosinophils. The ascaroid nematodes are particularly active in inducing atopic reactions (1,2). Regarding Ascaris suum, extracts in various stages of purification have been widely used for allergen characterization in different animal species (3-5). Several allergens have been isolated from worm extracts, body fluid or culture supernatant. A potent allergen was identified in the body fluid of adult A. suum and characterized as a 14,000 molecular weight protein that induces an IgE antibody response in a rat model (6). It was also demonstrated that A. suum potentiates the IgE response in different species, although attempts made using the mouse model were unsuccessful (7). Working with A. suum, we have previously shown that whole extract obtained from adult worms suppresses the production of antibodies to an unrelated antigen in the mouse system (8). Moreover, we have demonstrated that the suppressive components present in the A. suum extract could be isolated from a 29,000 molecular weight component which induces high levels of IgE antibody (9). In order to further characterize this allergenic component, the aim of the present study was to produce a specific monoclonal antibody (mAb) which provides an important tool for a better identification of the allergen.
BALB/c mice weighing 18-22 g (provided by the animal house of Butantan Institute) were injected into the footpad with an emulsion of complete Freund's adjuvant containing 40 µg of A. suum allergenic component purified as previously described (8). Two weeks later, the animals received 40 µg of the antigen in incomplete Freund's adjuvant by the same route and the popliteal lymph nodes were excised 3 days later for fusion with a nonsecreting SP2/O cell line using 50% polyethylene glycol (MW 1450). The hybridomas were cultured in HAT (hypoxanthine-aminopterin-thymidine)-containing medium and cloned under limiting dilution conditions. Supernatants from growing hybrids were screened by ELISA using plates coated with A. suum allergenic components purified by gel filtration (PIII) and visualized with 0.1 µg of peroxidase-labeled rat mAb against different mouse Ig isotypes (kindly provided by Dr. H. Bazin, Experimental Immunology Unit, University of Louvain, Brussels, Belgium).
The cloned hybrid cells produced an IgG1 kappa mAb named MAC-3 - mouse anti-A. suum allergenic component. The specificity of MAC-3 mAb was determined by ELISA using plates coated with each fraction eluted from a Sephacryl S-300 column to which 30 mg of A. suum extract was applied. At each step, the plates were incubated for 1 h at 37oC and washed five times with PBS, pH 7.2. They were then saturated with 5% defatted milk in PBS and 1 µg/well of MAC-3 was added followed by peroxidase-labeled rat anti-mouse IgG1 mAb and 100 µl/well of o-phenylenediamine diluted in sodium citrate buffer, pH 5.0, at a concentration of 0.4 mg/ml. The mAb was visualized by the addition of 0.012% H2O2. Plates were read in a Titerteck Multiskan apparatus at 492 nm. The MAC-3 mAb recognized only fractions eluted in the third peak (fractions 37 to 50), represented by the low molecular weight proteins of the A. suum extract (Figure 1).
The allergenic properties of A. suum components were determined by their ability to induce IgE measured by passive cutaneous anaphylaxis (PCA) in rats according to the method of Mota and Wong (10). Several dilutions of the serum from mice immunized with Al(OH)3 (7.5 mg) plus 250 µl of each fraction of A. suum crude extract eluted on a Sephacryl S-300 column were injected intradermally into the shaved back of three rats. The animals were challenged intravenously with 0.5 mg of A. suum crude extract in 0.25% Evans blue solution after a sensitization period of 18-24 h. The PCA titer was expressed as the reciprocal of the highest dilution that produced a lesion of more than 5 mm in diameter. The results shown in Figure 1 demonstrate that the same fractions recognized by MAC-3 induced specific IgE antibody production.
A large amount of MAC-3 was purified from ascitic fluid of Swiss mice injected with the secreting hybridoma and incomplete Freund's adjuvant by affinity chromatography using a protein A-Sepharose column. Purified MAC-3 was conjugated with activated Sepharose and the adult worm crude extract was applied to the column. The allergenic protein was then eluted using citrate buffer, pH 2.8. Allergenic protein purity was evaluated by SDS-PAGE and by immunoblotting. The protein banding pattern of the whole extract was quite complex, as revealed by SDS-PAGE performed on a 7.5% acrylamide gel at 20-mA constant current and visualized with silver staining (Figure 2). On the other hand, the material eluted from the MAC-3-Sepharose column (allergenic protein) showed a single band corresponding to a 29,000 molecular weight protein. The different components of the whole extract separated by SDS-PAGE were transferred to a nitrocellulose membrane that was then treated with MAC-3, followed by the addition of anti-mouse IgG1 mAb labeled with peroxidase plus H2O2 and 4-chloro-1-naphthol. As shown in Figure 2, the mAb MAC-3 recognized only the 29,000 molecular weight protein present in the whole extract.
MAC-3 mAb binds to allergenic protein with an affinity constant of 1.7 x 109 M-1, as determined by ELISA according to Van Heymingen et al. (11).
We produced a mouse mAb against the A. suum allergen that is an IgG1 kappa immunoglobulin with an affinity constant of 1.7 x 109 M-1 and which recognizes specifically a 29,000 molecular weight protein from the adult worm extract. This mAb will be used for the identification of allergenic protein in different stages of the A. suum life cycle as compared to immunosuppressive components.
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Address for correspondence: M.F. Macedo-Soares, Laboratório de Imunopatologia, Instituto Butantan, Av. Vital Brasil, 1500, 05503-900 São Paulo, SP, Brasil. E-mail: email@example.com
Presented at the XVI Annual Meeting of the Federação de Sociedades de Biologia Experimental, Caxambu, MG, Brazil, August 22-25, 2001. R.R. Pires, T.M. Oshiro and D.M. Itami are recipients of FAPESP fellowships. Received April 12, 2001. Accepted June 12, 2001.