| Objective: To prepare and identify monoclonal antibodies against purified extracts of egg white.Methods: BALB/c mice were immunized subcutaneously with purified extracts of egg white on it back. By use of the chessboard titration method,the working concentrations of coated antigen was determined. Indirect ELISA was used to detect antibody titers in the serum of immunized mice, and the mouse with high antibody titer was selected used for monoclonal antibody preparation. The mouse spleen was removed under the sterile condition, and the spleen cells were dispersed through the strainer to get a single cell suspension. Hybridoma cell lines were obtained by fusing immunized mouse spleen cells with myeloma NS-1 cells. 10 positive hybridoma with the capability of secreting McAb against extracts of egg white were screened by indirect ELISA, and were subjected to clone and subclone by limiting dilution method. Incrementation culture was used to preparation of McAbs. Ig classes and subclasses of McAbs were detected by ImmunoType kit. The titers of mAbs secreted by hybridoma before or after freeze and thaw were tested by indirect ELISA. The specificities of McAbs were determined by Western blot analysis.Result: 100 ng·mL-1 was selected as the working concentrations of coated antigen. Two hybridoma cells,named 5E12 and 4F1, were obtained after 3～4 rounds of cloning which can stably secrete the McAbs against extracts of egg white. Both McAbs were IgG1. Western blot analysis showed that 4F1 could recognize a target molecule with relative molecular mass of 44kD. Results of indirect ELISA showed that the titers of two McAbs , 5E12 and 4F1 secreted by two strains of hybridoma cells before or after freeze and thaw were 1:5000 and 1:300, respectively.Conclusion: Two McAbs ,5E12 and 4F1,against extracts of egg white are prepared successfully, which provides valuable experiment foundation for quickly measuring allergen in foods, and for isolation, purification and identification allergens.
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