The Establishment And Evaluation Of Testing Methodology Of Magnetic Chemiluminescence Enzyme Immunoassay Based On Alpha Enolase For Anti-Endothelial Cell Antibody

Objective Establishment and initial evaluation of testing methodology of magnetic chemiluminescence enzyme immunoassay based on ENO1for anti-endothelial cell antibody (AECA).Methods The cDNA was synthesized using Human Umbilical Vein Endothelial Cell (HUVEC) total RNA as the template. ENO1gene was amplified from cDNA using specific forward and reverse primers by polymerase chain reaction (PCR). The recombinant expression vector was obtained by ligating ENO1gene and pET28(a) using T4ligase. ENO1protein was produced by transfecting expression vector pET28(a)-ENO1into BL21(DE3) E.Coli. and subsequent IPTG induction. The His-tagged ENO1protein was purification using Ni-NTA column. HRP was coupled to ENO1protein by sodium periodate oxidation method. The biotin labeled AECA monoclonal antibody was coated onto the magnetic beads. After adding serum, the light quantum yield was detected. We assayed ACEA in23patients with SLE and19patients with RA, as well as20healthy controls, using this detection system and ELISA, and compare the positive detection rate of the two methods. Results The cloned ENO1into pET28(a) was correct and accordant with the sequence in GeneBank. We obtained recombinant ENO1protein with high purity, the molecular weight was about47,000. Initial establishment of testing methodology of magnetic chemiluminescence enzyme immunoassay based on ENO1for anti-endothelial cell antibody (AECA).2times of the mean values of light quantum from20healthy control as positive cutoff value, the specificity was92%, and the positive rate in SLE and RA were69.56%and52.63%, exceeding ELISA.Conclusion We obtained recombinant ENO1protein using a prokaryotic expression system, and further applied recombinant ENO1protein to a competent magnetic chemiluminescence immunoassay for detection of AECA.