The Application Of Clustered Acetylcholine Receptors In The Detection Of Myasthenia Gravis Antibody

Objectives1. To test acetylcholine receptor antibody in24MG patients by enzyme-linkedimmunosorbent assay, and to screen patients for the positive or negative acetylcholinereceptor antibody, in order to prepare for the following test.2. To establish acetylcholine receptor antibody detection model through the celltransfection technique by which the adult nicotinic acetylcholine receptor and RAPSN mayco-express on the surface of human embryonic kidney cells (HEK293T). Then the24MGpatients were detectde acetylcholine receptor antibody by the model.At last, compared theresults of the two methods.Methods1．To collect sera from24MG patients and to detect serum acetylcholine receptorantibody by ELISA, then to distinguish the patients with or with not the serum antibody.2．To extract plasmid of the target genes from bacteria liquid containing five plasmids:AchR subunits alpha, beta, delta, epsilon and Rapsn, respectively; To conduct transfectionwith plasmid of AchR alpha, beta, and delta, epsilon and Rapsn, proportionally, and tomake them co-express on the surface of cells,and to detect AchR-Ab by the cell model inimmunofluorescence next,then to compare the results between immunofluorescence andELISA.3. Main Outcome Measures(1) To test the sample absorbance value.(2) To test the gene fragment size by agarose electrophoresis and gene sequencing.(3) To test the target protein’s expression inevery transfected models by Western blot.(4) To test the expression and colocalization of these fluorescent proteins byfluorescent microscope after transfections.(5) Compare the test results the positive rate of two methods.Results1.ELISA method: According to the results of30cases of adult volunteers withoutMG,theOD range of the suspicious positiveof MG was0.249-0.272and the positive ODrange was more than0.272.3positive,4suspicious positive and17negative patients werefound in24MG patients. 2.Agarose electrophoresis confirmed that the gene fragments of plasmid RAPSN-EGFP was consistent with that of the target genes. Gene sequencing results of Western blothad showed the size of the target stripe.3.Fluorescent microscope showed that the aggregate and granular green fluorescentproteins could be seen in the cell which had transfected with subunits of AchR andRPASN.4.24patients were test for detecting the acetylcholine receptor antibody by the assayof immunofluorescence and Immunofluorescenct colocalization could be detected withinpositive serum.5.The total positive rate of acetylcholine receptor antibody in24MG cases by theassay of immunofluorescence was70.83%.The result was significantly higher than theELISA(P<0.05). The positive rate of acetylcholine receptor antibody in17SNMG was58.82%.Conclusions1.Though ELISA test was currently the major method in detecting the acetylcholinereceptor antibody within MG patients, but it is not sensitive enough.2.Clustered acetylcholine receptor was successfully transfected and expressed onHEK293T cells After the primary test, it was believed that the model wasbetter thanELISA in detecting the acetylcholine receptor antibody assay.