Development Of Sandwich ELISA Systems For Evaluating Human Soluble BTLA And Study Its Applications

The regulatory expression and interaction of cosignaling molecules onimmunocytes modulate and maintain the optimal and effective immune response. Thecosignaling molecules consist of two superfamilies based on their structure, theimmunoglobulin (Ig) superfamily and the tumor necrosis family (TNF)/TNF receptor(TNFR) super-family. Further, the cosignaling molecules can also be classified ascostimulatory or coinhibitory molecules based on their functional effect upon hostimmune responses. The crosslinking and aggregation of TCR and cosignaling receptorsby MHC/peptide and its cosignaling ligands leads to full activation of T cells. Incontrast, TCR engagement without costimulation leads to suboptimal T cell andactivation and unresponsiveness to antigen during secondary stimulation, a phenomenoncalled T cell anergy. As a coinhibitory receptor, human B and T lymphocyte attenuator(BTLA) plays an important role on inhibiting T cell activation and proliferation,maintaining peripheral tolerance and preventing organisms from autoimmunity.Interestingly, as a member of CD28superfamily, BTLA interacts with the unique ligandherpesvirus entry mediator (HVEM), a member of TNF superfamily. The BTLA/HVEMpathway provides inhibitory signal that blocked the cell cycle and inhibited IL-2production and CD4~+and CD8~+T cell proliferation. However, emerging evidenceindicated that there were two modes of action between BTLA and HVEM, andBTLA/HVEM pathway forms a bidirectional signaling system. The trans-interactionbetween BTLA and HVEM can directly activates the HVEM-dependent NF-κB RelAtranscriptional complex. The cis-interaction in na ve T cells, however, limits receptivityto signals from cells in the surrounding microenvironment and provides a mechanismmaintaining T cells in a resting state. Therefore the regulatory co-expression ofBTLA/HVEM on T cells plays a crucial role on immune response.Mounting data demonstrated that many cosignaling molecules assume two forms of expression. Except membrane-bound forms, soluble forms have been found forseveral members of B7family including OX40L, CD40L, B7-H3and PD-1. The solubleprotein is either shed from the membrane or produced by the special mRNA. Manysoluble proteins are valuable for clinical diagnosis. However, the existence of sBTLA inphysiological and pathologic condition is still unexposed, the functions and biologicalsignificance of soluble BTLA (sBTLA) remain unknown.In this study, a cell line CHO/BTLA-Fc stably expressing the human BTLA-Fcfusion protein has been obtained, and human BTLA-Fc fusion protein was generatedsuccessfully. At the same time, BTLA-Fc is a alternative forms of sBTLA to study thebiological role of T cells in vitro. Then the fusion protein and mAbs which recognizedifferent epitopes were used to develope sandwich enzyme-linked immunosorbent assay(ELISA) systems for the detection and quantification of sBTLA. Then we evaluate thelevels of sBTLA in human sera from healthy donors and RA patients.Part Preparation of Human BTLA-Fc Fusion Protein and Study of ItsFunctionObjective: To establish transfected CHO cells expressing human BTLA-Fc fusionprotein.Method: The gene encoding human IgG1(Fc) was amplified by PCR fromrecombinant vector pEGZ-Term/B7-H1-Fc, and then the target fragment was insertedinto eukaryotic expression vector pIRES2-EGFP after being digested with XhoI, BamHI.Then the extracellular gene of human BTLA was amplified by PCR from recombinantvector pEGZ-Term/BTLA, inserted into recombinant vector pIRES2-EGFP/Fc afterbeing digested with NheI, XhoI. The recombinant vector was transfected into CHO cellswith LipofectAMINETM2000, and the cells were further selected with G418. Analysethe expression of BTLA-Fc fusion protein by flow cytometry analysis. BTLA-Fc fusionprotein was purified by affinity chromatography and analyzed qualitatively via Westernblot. Using CCK-8to detect its influence on the process of T lymphocyte proliferation.Results: The stable secretion of human BTLA-Fc fusion protein from transfectedcell line was identified by flow cytometry analysis. Western blot shows the fusionprotein can combine with mouse-anti-human BTLA monoclonal antibody. BTLA-Fcfusion protein inhibits proliferation of T cells in vitro.Conclusion: A cell line CHO/BTLA-Fc stably expressing the human BTLA-Fc fusion protein has been obtained. BTLA-Fc fusion protein inhibits proliferation of Tcells in vitro.Part П Development of sandwich ELISA systems for evaluating humansoluble BTLAObjective: To establish specific ELISA systems with high sensitivity to detecthuman soluble BTLA proteins respectively. Then detect the levels of sBTLA in the seraof healthy donors and patients with rheumatoid arthritis by the sBTLA ELISA.Method: Four Mouse anti-human BTLA mAbs (1F11,3B6,7D7,8H9) obtained byus and a commercial monoclonal antibody(330104) were used as coating antibodies,were precoated in the ELISA plate with the carbonate buffer solution (CBS). Anotherfour biotin-anti-BTLA mAbs (1F11-bio,3B6-bio,7D7-bio,8H9-bio) were used asdetecting antibodies to recognize the coating antibodies-bound soluble BTLA protein.Then the Streptavidin-HRP was added to the reaction system. Finally, TMB substratewas added for colorable reaction. The absorbance was measured by the microplatereader. Then choosed a couple of antibodies with a good linear relationship to develop asandwich ELISA systems for evaluating human sBTLA. We tested the soluble sBTLAin the sera of78healthy donors and60patients with rheumatoid arthritis, and tested thesoluble sBTLA in the sera of148healthy donors with different age ranges.Results: The results showed that the8H9and7D7-bio had a good linearrelationship and the ELISA systems for detecting human sBTLA was establishedsuccessfully. It could be used to detect human sBTLA protein specially without anycross-reaction with other proteins. Standard curve of the system displayed favorablelinear correlation when the concentration of sBTLA was0.31~20ng/ml. The ELISAsystem had favorable stability, precision and specificity. The result showed that levels ofsBTLA were significantly increased in patients with rheumatoid arthritis. The sBTLAconcentration climbed along with the age and took on the statistical significance ofdifferences between investigated age ranges.Conclusion: The ELISA systems with high specificity and sensitivity provideuseful methods to detect human sBTLA. And levels of sBTLA were significantlyincreased in serum of patients with rheumatoid arthritis, the sBTLA concentrationclimbed along with the age and took on the statistical significance of differencesbetween investigated age ranges. In conclusion, human BTLA-Fc fusion protein was generated successfully, anELISA assay for the detection of soluble BTLA with high stability, veracity andspecificity was developed. Then detect the levels of sBTLA in the sera of healthydonors and patients with rheumatoid arthritis by the sBTLA ELISA.