| Porcine adipose tissues are turned into fat cells after being incubated in medium containing 1mg/ml of collagenese and centrifuged. Adipocytes which are homogenized in membrane extraction medium are comminuted by ultrasonic cellular disintergrator and recentrifuged to separate the triacylalycerols from other suspended components. Adipocyte plasma membranes are purified by the method of sucrose gradient centrifugence. The BALB/c mice were immunized with proteins of adipocytes plasma membranes in pigs plus CFA/IFA, B lymphocytes hybrized with the myeloma cell strain SP2/0 by PEG (MW4000) and selected with HAT medium. Hybridoma clones secreting monoclonal antibodies against adipocytes plasma membranes in pigs were screened by indirect ELISA, and positive clones were subcloned two times by limited dilution method. Two clones which can stably secrete McAb were obtained and designated cell strain 3B2 and 3F3. The cell strains are identified by measuring the ascites titer, analyzing the numbers of chromosomes, stability to heat, acid and alkali, regimentation and sub-regimentation of immnoglobulin, peculiarity and affinity. The concentration of adipocyte plasma membranes obtained is 4.97mg/ml. The proteins are devided into five proteins that is, there are two proteins between 116.0kDa-66.2 kDa, two proteins between 66.2 kDa-45.0 kDa and one below 14.4kDa by SDS-PAGE electrophoresis. The astite titer of antiserum in three immune mice is much higher than that of positive serum. The McAb obtained has high specificity and was IgG1 subtype and IgG2b subtype. Its astite titer could reach to 1:105.Through identification, the chromosome number of hybridoma cell strains is from 80 to 100. Its restraint rate to hybridoma cell strains is higher than 50%.Its affinity constant to adipocytes plasma membranes in pigs were 4.63×109 (mol/L)-1 , at the same time, the McAb secreted is stable to environmental factors such as temperature, acid and alkali etc. |
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