Gossypol Artificial Antigen Synthesis And Preparation And Identification Of Anti Gossypol Monoclonal Antibody

Gossypol is a kind of small molecule,fat-soluble compounds without immunogenicity,which has obvious accumulation within the animal body.It will lead to the chronic poisoning with a long-term consumption and damage to the liver cells,blood vessels,and the nervous system,causing heart failure,tissue edema and poisoning symptoms.At present,in the analysis of the traditional detection methods,the samples which are under test need to be more trouble before processing.The analysis of gossypol poisoning is by using HPLC method.Immune analysis has the characteristics of strong specificity,high sensitivity,simple operation etc.It has many advantages in the treatment of detection of gossypol.Artificial antigens for immunization(Gossypol-BSA)were sybthesized though EDC and glutaraldehyde method.The coupling results are identified by the characteristic absorption peak detection and SDS-PAGE.We have taken Balb/C female mice as the immune animals.After the five immunizations,we measured the blood serum of the Balb/C mice.The titers were determined by indirect ELISA and the specificity was detemnined by the complete indirect ELISA.The spleen cells from the immunized mice and the myeloma cells were fused together by the PEG.Used HAT selective medium to screen the monoclonal cell lines and identify the cell subtype of the clones which was obtained.Finally,the hybrid tumor cells which stably secreted Ig G antibodies were obtained.These cell lines were injected into mice's abdominal cavity to induce Mabs.The purity and affinity constants of the purified ascites were purified by protein G column.The results showed that gossypol and carrier proteins were condensated successfully.The titer of the antiserum obtained from immunized mice was up to1:6400.We obtained two Ig G1? kappa subclasses of monoclonal cell lines strainsafter the screening.The purity of antibody reached more than 90%,affinity constant range 62.73~115.Therefore,the present results established the foundation of binding ELISA kit for the free gossypol for the next study.