Study Of Construction And Optimization Of The Detection System For TORCH Infections Based On Protein Chip

TORCH infections in pregnancy are the most common congenital infection , which include five microorganisms such as toxoplasma gondii , rubella virus, cytomegalovirus, chlamydia trachomatis and herpes simplex virus type 2 and so on . TORCH group infection may result in long-term neurodevelopmental disabilities and are the most serious infectious diseases during pregnancy due to the seriousness of possible embryo-fetal lesions. However, it is very hard to discriminate between primary and recurrent infections in pregnancy, notwithstanding the possible implications. Since at present, neither effective vaccines nor resolutive therapies are available against viral infections, the main means against infection of the foetus still remains the prevention of infection in pregnant woman .We have developed and tested a method for printing protein microarray on netrocellulose membrane and use these microarrays in a comparativeimmunogold probe to detect TORCH infection . This method is based on specific recognition between proteins and show high-throughput, simple, low-cost forma . We purchase five antigens from Microbix incorporation in Canada. First, we got five antigens' concentrations by means of Cummas G250 method. Second, we evaluated five antigens' values through enzyme linked immunosorbent assay(ELISA). Third, we detected proteins' specificity via absorption-suppression experiment. Then, we optimized preparation crafts of colloid gold and immunogold , besides increasing the amount of staphylococal protein A (SPA) which was labelled to the colloid gold and improving methods of purifying immunogold , to develop a more uniform and more stability nanogold particle. The only printing concentration of each antigen was confirmed by concentration-grade assay. Nitrocellulose (NC) membrane from Pharmacia company was selected for its high quality and 0.45um aperture. We generated TORCH protein microarrays by printing five antigens(Toxoplasma gondii , rubella virus , cytomegalovirus , chlamydia trachomatis and herpes simplex virus type 2 ) on NC membranes. The results wrer demonstrated as red spot on naked eyes and can be easily detected with commercial CCD camera as gray spot. Human sera were applied to study the ability of the TORCH microarray and detect the present or absence of IgG directed against the different TORCH antigens . Standard sera were all tested for the presence or absence of antibodies directed against TORCH antigens by the TORCH ELISA detection kits made in ZUES company in USA. The results of our microarray were compared with the results of ELISA methods .All results proved antigens purchased from Microbix incorporation can meet expectations of both research and production. Immunogold can detect IgG as less as 0.0125 mg/mL in solution in this assay , and no cross-reactivity...