Preparation Of Monoclonal Antibodies Against Porcine SIgA, IgM And The Conjuates With Horseradish Peroxidase (HRP)

With the development of scale swine industy, some new infectious diseases have appeared in China, include porcine reproductive and respiratory syndrome (PRRS), porcine circovirus II type (PCVII) infection, Haemophilusparasuis (HPS) infection etc, which caused a considerable financial loss. It is a great need to prepare monoclonal antibodies (Mabs) against porcine immunoglobulin, in order to understand the basic immune response to these pathogens and study on the corresponding diagnostic and protective methods.In this study, porcine Secretory immunoglobulin A (SIgA) and immunoglobulin M (IgM) were isolated and purified by a procedure of precipitation with saturated ammonium sulfate, chromatography with Sephadex G-200 and ion-exchange chromatography with DEAE52 from colostrums and serum, respectively, and they were identified by SDS-PAGE electrophoresis. The BALB/c mice were immunized four times with the purified SIgA and IgM, respectively, then the spleen cells of the hyperimmunized mouse and SP2/0 cells were fused with PEG-1500. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect monoclonal antibodies (Mabs) secreted by the hybridoma cell lines. To prepare Mabs against porcine SIgA, cell fusion was made twice. The cell fusion rates were 79.7% and 60.3%, and the positive rates were 4.1 % and 16.8 %, respectively. Two hybridoma cell lines secreting monoclonal antibodies , named CA6 and 5D4, were developed by twice subclones. After repeated frozen storage % thawed revival and generation, CA6 cells were steadily producing high titer of Mabs to SIgA. The results of latex agglutination test showed that the antibody secreted by CA6 cells belongs to IgM, K isotype. The specificity of antibody against SIgA was identified by indirect ELISA and Western-blot. The liters of its culture and ascitic fluids were 1:128 and 1:6400-12800, respectively. The antibody protein in the ascitic fluids was well purified by PEG6000, with its concentration of 4.26mg/ml. To prepare Mabs against porcine IgM, cell fusion was made once, and the cell fusion rate of 80%, and the positive rate of 100% were obtained , respectively. Five hybridoma cell lines secreting monoclonalantibodies, named M1, M2, C1, D1 and H2 were developed by twice subclones. After repeated frozen storage, thawed revival and generation, they were steadily producing high titer of Mabs to porcine IgM.. And all five Mabs belong to mouse IgGl, K isotype. In both indirect ELISA and western-blot, all five Mabs reacted with the mu-chains of porcine IgM, but did not react with porcine IgG, IgA, or the light chains of IgM. The liters of its culture and ascitic fluids were 1:256 and 1:12800-1:25600, respectively. The Mabs protein against porcine IgM from ascitic fluids prepared by caprylic acid/ ammonium sulfate was high purified, with its concentration of 4.84mg/ml.The purified Mabs were both labeled with horseradish peroxidase (HRP) by the method of Sodium periodate. To determine the optimal concentrations of Ig-HRP, a direct ELISA was used. The results indicated that the optimal dilutions of Mabs Ig-HRP against SIgA, IgM were 1: 200 and 1: 300, respectively. For the detection and quantification of porcine IgM, a double antibody sandwich (DAS) assay was developed and optimized. The results implied that the dilutions of 1: 300 appeared to be appropriate for McAb Ig-HRP. The DAS-ELISA could be used to detect porcine IgM specifically at the level of at least 2ug/ml.It is believed that the Mabs against SIgA should be helpful in detecting the level of mucosal immune and purifying SIgA, and the Mabs to IgM should be useful hi quantitative analysis to surface membrane IgM-bearing cells in peripheral blood and early-diagnosis for porcine disease.