Preparation Of McAb Against γ Chain Of Human IgG And Application In The Quantitative Detection Of VEGF Huaman Monoclonal Antibodies

Objective: To preparate the hybridoma cells and McAb against γ chain of human IgG . To research and appraise it. To build a quantitative detection method of huaman monoclonal antibodies in the processes of large scale culture and purification and biologicals by appling it which have many advantages in the easiness, speediness,specifity and repetitiveness against other ways.Methods: The Balb/c mice were immunized intraperitoneally with the pure human IgG isolated from human serum, it' s B lymphocytes hybrized with myelomacells SP2/0 by PEG(MW4000). The antibody to human IgG were screened out by indirected ELISA. Using limited dilution four times cloning and subcloning, one strain of hybridoma cells(MKIG12) which secrete antibody(McAb G12) against human IgG.. In the Sandwich Enzyme-Linked Immunosorbent Assay (Sandwich Elisa), MccAb G12 was coated to surface of the wells of amicrotiter plate, the HRP- MKIG12 detected the quantitative sample after an unknown test human IgG was added .Results: The eluate from the column were analyzed by SDS-PAGE. It is indicated that the human IgG was consisted of two subunits, heavy chain(52. 73 KD) 和 and light chain (26. 44 KD). MKIG12 was characterized by better stability and more chromosome numbers(100 ± 5).There were four characteristics of McAb G12 : (1) McAb G12 belonged class IgG, subclass IgG2; (2) McAb G12 had higher specificity.no cross-reaction occurred to other huaman Ig (IgA, IgD, IgE, IgM ) expected IgG and other species' s Ig (mice, chiken, cattle, duck, goal); (3) McAb G12 possed of highter titer, titers of McAb G12 from both supernant and ascite was l:106; (4) McAb G12 which is a antibody against heay chain of human IgG ( Y chain) appraised by WESTON-BLOT. The Sandwich Elisa builed by McAb G12 is specific and repetitive.With the assumption that antigen minimum of this method results was 2. 5ng/ml for VEGF huaman monoclonal antibodies. The CV of the results of repetitive test in same time were below 5% and in continuous three times below 6%.Conclution: The hybridoma cells (MKIG12) and McAb(McAb G12) against γ chain of human IgG were preparated andcharacterized, a specific link occurred between McAb G12 with constant regions of y chain of human IgG. (CH) by conclution based on the results. This kind of monoclonal antibodies, were ont reported in china. The Sandwich Elisa builed by McAb G12 has many advantages in the easiness, speediness, specifity and repetitiveness against other ways. .This method were not reported in china.