An optimal blocking strategy for non-specific staining in immuno-infiltration assay / 西安交通大学学报(医学版)

ABSTRACT

Objective To select an optimal non-specific antigen blocking method by using immuno-infiltration assay so as to suit protein chip preparation. Methods Human papillomavirus type 16 L1 protein expressed by insect-baculovirus espressin system was incubated with skimmed milk powder, calf serum, bovine serum albumin (BSA) combinations of five kinds of methods to block the non-specific antigen. PBS was used as control. The effect of eliminating non-specific stain was detected by immuno-infiltration assay. Results After repeated tests, the results showed that the stability and repeatability of blocking effects were poor for the fixing up antigen first and then blocking method, and the blank control was prone to false positive. The infiltration rate of NC membrane would be affected by using skimmed milk powder as a blocking agent because the pore of NC membrane was easily plugged by milk powder particles. The use of calf serum as a blocking agent made it very difficult to determine the result because the calf serum absorbed by NC membrane produced the background; however, when 20g/L BSA was used to blocking before fixing up antibody, the results became satisfactory. Conclusion Fixing up antibody after blocking in immuno-infiltration assay showed that the blocking effect against non-specific antigen was satisfactory, stable and repeatable, indicating this method is a novel optimal blocking method compared with others.