Measurement of scFv antibody affinity using non-competitive enzyme-linked immunosorbent assay

Objective: To measure scFv antibody af finity using non-competitive enzyme-linked immunosorbent assay (ELISA). Methods:Using serial dilutions of antigen (, ) and antibody (anti-MAGE-A1 scFv antibody), the affinity constant (K aff) was measured by non-competitive ELISA. Two sigmoid curves of optical density (OD) v ersus logarithms of antibody concentrations were estimated by SPSS 10.0 software . The maximum value of OD (OD-100) of each curve was computed respectively and OD-50, half of OD-100, was obtained. Then the concentrations of antibody corre sponding to OD-50 on the curves, named t and t were calculated . For =1/2 , K aff=1/{2 t- t}. Results: The affinity constant of scFv antibody was about 1.432×1 06 L/mol. Conclusion: Based on the Law of Mass Action, n on-competitive ELISA method for measurement of antibody-antigen affinity const ant is simple, rapid and reliable.

The effect of MAGE-A1 gene on NIH3T3 cells

Objective: Melanoma antigen genes(MAGE) genes have been found in many kinds of tumor tissue, but not in normal tissue except testis and placentas. The Ags encoded by MAGE genes therefore are strictly tumor-specific. The most current researches associated with these genes focus on the tumor vaccination using these Ags. Few reports are concerning these genes' functions. In this study, we investigated the role of MAGE-A1 gene on NIH3T3 cells after transferring with it. Methods: Clone the MAGE-A1 into the plasmids pEGFP-C3 and pcDNA3.1, then transfer the reconstructed plasmids and primary plasmids into the NIH3T3 cells using a new transfer reagent FuGENE 6. Selecting the positively transferred cells by G418. Identified by RT-PCR, Western blot, Immunocytochemistry, Laser Scanning Confocal Microscope and Fluoroscope. The cells mobile ability was measured with Millicell-PCF. The cell cycle and apoptosis were measured with Flow Cytometry. Results:The apoptosis rate of NIH3T3 cells that transferred with control plasmid pcDNA3.1 was 13.4% and the ratios that stay in S phase and G2-M phase were 5.68% and 1.04% respectively. The apoptosis rate of NIH3T3 cells that transferred with pcDNA3.1-A1 was 0.90% and the ratios that stayed in S phase and G2-M phase were 19.31% and 13.47% respectively. The apoptosis rate of the cells that transferred with control plasmid pEGFP-C3 was 1.87%, a little higher than 1.47% of those transferred with pEGFP-C3-A1. Conclusion:The MAGE-A1 gene may enhance the cell cycle, inhibit the apoptosis and raise the mobile (ability) of NIH3T3 cells.