Establishment Of Quantitative Human HPPCn Sandwich ELISA With Newly-developed Monoclonal Antibodies

Objective:HPPCn （Hepatopoietin Cn）, a hepatic stimulator factor isolated from hepatic stimulator substance, was confirmed to be related with the development of hepatocellular carcinoma. In order to further identity the biological function of HPPCn, analyze its dynamic changes in the occurrence process of hepatitis, cirrhosis and liver cancer and other liver diseases and the correlation with the disease, we need more anti-HPPCn antibody. So the present study’s purpose is to establish the double antibody sandwich ELISA method of quantitively detecting the HPPCn.Method:The female BALB/c mice were mimunized with the recombinant HPPCn proteins and then collected antiserum. Splenocytes and Sp2/0cells were fused with PEG-1500. The positive clone was identified through indirect ELISA and then subcloned by lmited dilution. The property of depurative anti-HPPCn Monoclonal antibodies （mAb） were identified through immunohistochemistry and Western blot. Then we pair bond detected the eight strains of antibodies by gliding chip method, determined the optimal working concentration of coating antibodies and enzyme labelled antibody by square matrix titrimetry. We used the depurative HPPCn antigen as the standard substance to establish the standard curve.Result: we obtained eight strains hybridoma cell which stably secretes anti-HPPCn antibodies and they are Ab₃-3-3, Ab₆5-29-6, Ab₉-34-1, Ab₇-8-10, Ab₄-8-12, Ab₂-30-23, Ab₁2-27-23（W2-D5） and Ab₁1-11-12. The best pair bond is Ab₄-8-12and Ab₂-30-23verified by the anti-bodies pair bond detection. The optimal working concentration of recognition anti-body Ab₇-8-12and coating anti-body Ab₆5-29-6are respectively1:2000and2.5μg/ml.Conclusion:We produced one monoclonal antibodies that could specially detects human HPPCn protein and established double antibody sandwich ELISA method which could be used for detecting HPPCn.