Development Of Colloidal Gold Immunochromatographic Strip Of Human Pregnancy Specific β1Glycoprotein

Pregnancy-specific β1glycoprotein is a protein which is synthesized by theplacental trophoblast cells during pregnancy. Itwas secreted into the maternalcirculation after synthesis.Whenconcentration is lower than normal, it often causesome adverse effects, such as poor fetal development and abortion. Underpathological conditions， PSG1can also be detected in many tumor tissues.As apregnancy-specific glycoprotein,PSG1have many functions, such as:monitoringplacental function, detection of fetal growth restriction, detection of tumor cells,involved in immune regulation and maintaining the mother’s immune tolerance duringpregnancy environment.Presently considered that the levels of PSG1in maternalserum can be used as a reliable reference in such filed as diagnosis of early pregnancy,monitoring placental function, monitoring high-risk pregnancy and determine fetaloutcome.Therefore, the study of PSG1also more and more attention. In previouswork,the laboratory has been able to build a stable cell lines expressing PSG1mAband its prokaryotic expression system.On this basis, three monoclonal antibodyhybridisma cells which were used to choose two of them with smaller impact onepitopes.Combined with GICA technology, so we could establish a detection methodof PSG1by using the double antibody sandwich method. The detection method issensitive, specific, rapid and simple.1, Extraction and purification of recombinant proteins PSG1We exact the inclusion bodyprotein byrecover and expand training the DE3strainswith recombinant prokaryotic expression system. Refolding and purification for thesubsequent detection.The concentration of purified protein is0.6mg/ml.2, Screen the PSG1monoclonal cell lineWe haverecover three monoclonal cell lines and test its stability.Screening thePSG1monoclonal cell line is using the superimposedELISA test to pick out twomonoclonal with little impact ofantigenic epitopes,9G6and2D10.3,Preparation and purification of monoclonal antibodiesThe hybridoma cells was injected into the intraperitoneal of BALB/c mice toprepare antibody.The preliminary purification was centrifugation to remove impurities,and furtherpurified by protein G affinity chromatography. The purification effect was detected bySDS-PAGE electrophoresis and WB.Protein concentration was determined by BCA.1.69mg/ml and1.37mg/ml. Titer of antibody is greater than1:2.48×106.4, Preparation of colloidal gold, labeled antibody and determinethe assemblyconditions of test trip.Preparation of colloidal gold and detect its quality,about30nm.Double antibodysandwich method,9G6as a labeled antibody,2D10as the capture antibody which iscoated on the nitrocellulose membrane.Quality control line coated rabbit anti-mouseIgG antibody.The protein labeling conditions and strip assembly conditionswasdetermined.5,Performance evaluation of colloidal gold stripAfter the test strip assembly, its sensitivity is betterby the detection of recombinantantibodies. Its sensitivity could be seen to reduce only in the reaction with the nativeprotein. The test strip is with a better specificity when compare the reactions of otherproteins. After detected the trip which was stored at different temperatures, thestability is good.In this study,a good activity, high purity PSG1recombinant proteins andmonoclonal antibodies was obtain. We also prepared the colloidal gold test strip ofPSG1withgood sensitivity, stability and specificity. The next step needs to be done isto develop the quantitative test strip, and with further optimize the conditions so thatthere is more obvious response with natural PSG1protein.And meet the needs ofclinical testing.