Anti-human April Preparation Of Monoclonal Antibodies And The Sensitivity Comparison Of Three Immunoassay Methods

A proliferation-inducing ligand (APRIL), a member of TNF superfamily, is a typeⅡtransmembrane protein with full length of 250 amino acids. It is expressed very highly in many tumors including human cancers of colon, thyroid and lymphoid tissues. Since 1998, APRIL has been identified to stimulate growth of tumor cells in vitro and in vivo. In recent research, it has been found that this molecular plays an important role in regulating humoral immunity and stimulates the mature and activation of T and B lymphocytes.B cell activating factor belonging to the TNF family (BAFF), also a member of the TNF superfamily, is a fundamental survival factor for mature B cells. APRIL shares the same receptors, BCMA and TACI, with BAFF. But it appears to play a different biological role. We prepared anti-human APRIL monoclonal antibodies to assess the expression of APRIL and its correlation with BAFF in patients with autoimmune disease, such as SLE and RA. And then, we will check if these two cytokines can be used as useful markers to judge the effect of treatment and for the prognosis of disease.The present study consists of two parts as follows. In the first part, we extracted total RNA from human PBMCs to amplify the whole cDNA of APRIL by RT-PCR. The cDNA fragments were cloned into NdeⅠand HindⅢsites of pET-30a(+), an E.coli vector with a His-tag that allows heterologous protein expression. The expressed and purified fusion APRIL protein maintained the original immunogenicity. And then, the anti-hAPRIL monoclonal antibodies were prepared. ELISA and Western blot analysis showed that the McAbs had a good antigen specificity. These works may provide evidence for the further study of the APRIL protein.In the second part, we attempted several ways to improve the sensitivity of the immunoassay. We detected a cytokine by sandwich ELISA, BA-ELISA and immuno-PCR. BA-ELISA is much more sensitive than sandwich ELISA mostly. But it is less sensitive sometimes. As for the immuno-PCR assay, it is more sensitive than sandwich ELISA, too. But it is less stable.