| Objective: Inflammation,angiogenesis and bone remodeling are three important biological processes in the bone marrow microenvironment of multiple myeloma,which play important roles in the pathogenesis and disease progression of multiple myeloma.Based on the clinical demand for multi-analytes parallel detection,it is of great significance to develop quantitative detection reagents of multiple protein biomarkers for clinical application.In this study,a robust,sensitive,and user-friendly platform,multiplex beadbased immunoassay by flow cytometry,was used to develop quantitative detection reagents of cytokines,angiogenic factors,and bone remodeling molecules.Application was carried out to analyze the expression levels of cytokines,angiogenesis and bone remodeling proteins in relapse/refractory multiple myeloma(RRMM).Methods: The discrimination ability and the homogeneity of carboxyl modification level of the double fluorescence-coded microspheres used in flow cytometry-based immunoassay were evaluated.Based on the double-antibody sandwich method,quantitative detection reagents of multiple protein markers were developed,and optimization of immunoassays was carried out,including coupling amount of capture antibody,detection antibody concentration,cross-reactivity test and diluents screening.Then immunoassay validation was performed,and the methodological evaluation consisted of sensitivity,accuracy,linearity-of-dilution and precision.It was applied to analyze the expression levels of cytokines(IL-1β,IL-2,IL-4,IL-6,IL-8,IL-10,IL-12 p70,IL-17,TNF-α,IFN-γ),inflammatory markers(Serum amyloid A1 [SAA1]),angiogenic factors(Vascular endothelial growth factor [VEGF] and Tissue inhibitor of metalloproteinases 1[TIMP1])and bone remodeling molecules(Dickkopf-related protein 1 [DKK1],Receptor activator of nuclear factor kappa-B ligand [RANKL] and Osteoprotegerin [OPG])in 55 patients with RRMM and 22 healthy controls.And the correlation between these proteins and clinical characteristics such as DS,ISS stage and the level of M protein was analyzed.Results: The analytical sensitivity of ten cytokines(IL-β,IL-2,IL-4,IL-6,IL-8,IL-10,IL-12 p70,IL-17,TNF-α,IFN-γ),VEGF,DKK1,RANKL and OPG ranged from 0.2 to 25.3pg/ml,with the average sensitivity of 6.8 pg/m L.The sensitivities of SAA1 and TIMP1 were 2700.0 pg/m L and 46.0 pg/m L,respectively.The recovery rate of the 16 analytes developed was in the range of 67.1% to 125.8%,with the average recovery rate of 98.6%.Except for IL-2,IL-4,IL-8,and DKK1,the recovery rates of other analytes were within the acceptable range of 80%～120%.The average linearity of the 16 analytes was 127.8%,and the linearity of IL-1β,IL-2,IL-6,IL-8,SAA1,TIMP1,and VEGF was good,ranging from90.3% to 115.8%.The intra-assay coefficients of variation of the 16 analytes were lower than 8.3%,and the average intra-assay coefficient of variation was 4.2%.The inter-assay coefficients of variation were lower than 15.1%,and the average inter-assay coefficient of variation was 10.2%.All of the above analytes had good precision.Compared with healthy controls,the levels of IL-1β,IL-2,IL-4,IFN-γ,SAA1,DKK1,RANKL,OPG and RANKL/OPG increased in RRMM,while the levels of IL-6,IL-8,IL-10,IL-17 and VEGF decreased.There was no significant difference in levels of IL-12、TNF-α and TIMP1.The level of VEGF was positively correlated with the levels of IL-1β,IL-4,IL-6,IL-8,IL-10,IL-12,and TNF-α(r values were 0.39,0.36,0.50,0.57,0.47,0.28,0.49,respectively).The levels of IL-2,IL-17,DKK1,RANKL and OPG in patients were positively correlated with Ig G monoclonal protein(r values were 0.41,0.54,0.55,0.50,and 0.53,respectively),and the level of TIMP1 was positively correlated with Ig G and Ig A monoclonal protein(r values were 0.59,0.61,respectively).Conclusion: In this study,16 kinds of quantitative detection reagents for protein markers with high sensitivity and specificity were developed.The development process of multiplex detection reagents established has important reference significance for expanding the development and application of multiplex detection reagents for protein markers in the future.In addition,we found that the levels of IL-2,IL-17,DKK1,RANKL and OPG were well correlated with the level of M protein in the Ig G type MM,and the level of TIMP1 was well correlated with the levels of M protein in the Ig G type MM and Ig A type MM.The above proteins have potential value in tracking the disease progression of MM. |
