Primary Study In Preparation Of TORCH Antigen Microarray Using Fluorescence Detection In Perinatal Period

Objective: To establish an optimized method for preparing protein microarray using agarose film coated glass slides and compare its protein loading capacity with three different substrates coated glass slide. To simultaneously detect TORCH infectious, A new method was developed and tested for printing protein microarrays on agarose film coated glass slides by using fluorescence as signal detection.Methods: The protein loading capacity of the protein microarry spotted on poly-L-lysine coated, agarose film glass slide and amino glass slide, three different substrates were investigated and compared. Clean glass slides are coated with different concentration of agarose film, the optimal agarose concentration was determined by average spot signal intensities. The TORCH antigen (Toxoplasma gondii,rubella virus,cytomegalovirus,and herpes simplex virus type2 )were printed on glass slide modified with the agarose films. Human sera were applied to study the ability of the TORCH microarray and detect the present or absence of IgM directed against the diferent TORCH antigens by using fluorescently labeled secondary antibodies.Results: When the concentration of agarose was1.0%, the agarose coated glass slides based protein microarray performed the best. And it had a 1.4 fold and 4.6 fold higher protein loading capacity than poly-L-lysine glass slides and amino glass slides based microarrays. TORCH protein microarray can detect minim antibodies against the TORCH antigen. The cross-reactivities of the anti-IgM goat were negligible. when 30 positive serum and 30 negative serum were detected, The sensitivity and specificity was 100% and 93.3%.Conclusion: Agarose film coated glass slides not only have a big protein loading capacity but can minimize the denaturation of the immobilized protein, the agarose film coated glass slides are more suitable for protein microarray preparation. TORCH antigen microarray can simultaneously determine infection of TORCH by detecting antibodies against Toxoplasma gondii,rubellavirus,cytomegalovirus,herpes virus type 2.The TORCH assay performs equivalently to ELISA and may have potentially .The TORCH protein microarray for the detection of TORCH infections may possess advantages in high throughput, convenience, micromation, low cost and will be used in clinic in the future.