The Preparation And Optimization Of Transfusion Related Disease Pathogens Antibodies Protein Chip And The Preliminary Testing

Protein chip,also known as protein arrays or protein microarrays(protein microarray),refers to the protein molecules(such as enzymes,antigen,antibodies, receptosr,ligands,cytokines,etc.)as a ligand,it is orderly fixed on solid-state carrier; capture the specific combination of protein(found in serum,plasma,lympHatic, interstitial fluid,urine,effusion,dissolved in liquid,fluid,etc.).Nowadays,the detection method of many infectious diseases is enzyme linked immunosorbent assay(ELISA),comparing to the protein chip technology,some shortcomings of this traditional way exist obviously in such five factors,from Total time,Amount of sample,Total steps,Number of samples in a determination and Limit of detection.Therefore in the purpose of saving time,having high-throughput and high sensitivity,in this paper,several blood-related diseases protein antigen were printed on solid-pHase slides,and by testing serum specimens collected form some hospital, to build a new protein chip approach to diagnostic tests.Firstly we established a preliminary model with indirect way of ELISA;Secondly, with purification of human IgG and human IgM,this paper got these conditions for successful protein chip;Finally,with these identified optimum conditions,all kinds of genetic engineering purified protein were diluted into the specific antigen concentration,and then were printed with ProSys 5510(U.S.Cartesian),one kind of chip printing machine,the slides were scanned by Express(USA Packard Company) biochip scanner scans Antibody combination of circumstances,the results of antigen-antibody combination were analyzed by Quant Analysis software.The results are follwing:1.ELISA indirect method was determined as the basic method of testing the corresponding antibody in serum.This system and ELISA test results confirmed the positive,negative results were in line with the rate reached 82.5%through the optimization process,and by the indirect method successfully detected antibodies related factors were as follows:antigen diluted multiples for the 500×,seized an anti-serum to be diluted multiples for the 10×,the tag-concentration of 1000×,the cleansing frequency for the 5×3(5min,3).2.With successful purified human IgG and IgM,we got the conditions and standard curve,Point system temperature:22℃;Humidity of:(RH)65%;Carrier: optical epoxy substrate(EPX);Printing buffer:EPX corresponding B solution; Antigen protein dilution:Carbonate buffer(CB);Blocking buffer:1%bovine serum albumin(BSA)of pHospHate buffer(PBS);The time for blocking:2 h(37℃); Washing buffer for:containing 0.05%Tween-20 and the PBST-PBS;Washing time: 5 min,3,(5×3);One of incubation time:30 rain(37℃);2th antibody incubation time for:30 min(37℃);Standard curve chip was successfully prepared,antibody can be found in serum detection threshold of 0.05 ng/nl.3.Pathogens associated antigen(purified HBcAg,purified HSVⅠ,purified HSVⅡ,purification CMV,purification HIV1-2,purified TP,purification of HCV, purification TOX)antibody detection chip successful preparation.Negative and Positive of the rate reached 75.74%,chip-chip average rate of 84.05%,to repeat the average sensitivity:an anti-dilution 100×,reached 0.1 ng / nl;average specificity 84.5%,stability up to 12 weeks.The chip system,bloodtransfusion-related infectious disease pathogens antibody detection chip,has been initially established.but the index value low enough stability, the promotion is also available from the clinical application of greater distance, replace the traditional method of ELISA kit also need to improve the indicators strongly in-depth.The study of the various factors affecting the development and improvement.