Establishment Of A Novel QDs-encoded Microbead-based Flow Cytometric Method For The Quantification Of Soluble FcεRⅠα And Immunoglobulin E In Serum

Objective:The aim of this research is to prepare anti-FcεRⅠαmonoclonal antibody（m Ab）conjugated magnetic CuInS₂/Zn S core-shell quantum dots（QDs）microbeads,and to establish microbead-based flow cytometric method for the quantification of soluble FcεRⅠαand IgE in serum.To evaluate the performance of the method by its sensitivity,specificity,linear range,precision and stability.Analyze the efficiency of quantifying MCs activation-related biomarkers in autoimmune diseases.Methods:（1）The human IgE Cε3-Cε4 gene was obtained by RT-PCR.The gene was cloned and expressed in E.coli.The immunological activity of the protein was identified by western blot and flow cytometry.（2）The BALB/c mice were immunized with recombinant FcεRⅠαprotein to prepare monoclonal antibody against human FcεRⅠαin hybridoma cells.The m Abs were screened and identified by ELISA and flow cytometry.（3）A membrane emulsification technique was utilized to incorporate CuInS₂/Zn S QDs into microbeads.The beads were carboxylated and coated with capture antibody monoclonal anti-human FcεRⅠα.The influence of the amount of coating m Ab,the number of microbeads,incubation temperature and incubation time were analyzed.The limit of detection,precision and stability were determined according to the CLSI guidelines.（4）89 cases of SLE patients,40 cases of AILD patients and 64 healthy controls were collected to analyze sFcεRⅠαand IgE levels.Results:（1）The IgE Cε3-Cε4 prokaryotic expression system was successfully constructed.The prokaryotic protein obtained retained IgE antigenicity.（2）A pair of matched antibody A8H1 and D7C1 was obtained,which could be used for sandwich ELISA.B5A11 was a good antibody for flow cytometry analysis,which binds to CHO3D10 cells and KU812 cells expressing human FcεRⅠαwith high efficiency.（3）The method exhibited good linearity（R²>0.99）and sensitivity with wider dynamic range.The precision of the assay validated by intra-and inter-assay variability met the acceptance criteria with the mean recovery falling within 80%-110%of the theoretical concentration and a corresponding CV<20%.（4）The levels of serum sFcεRⅠαand IgE in SLE patients were significantly higher than those in healthy controls（P<0.05）.The level of sFcεRⅠαin serum of AILD patients was increased（P<0.05）.There was no significant difference between sFcεRⅠαand IgE levels in SLE patients and AILD patients（P>0.05）.Conclusion:These results indicated that anti-FcεRⅠαm Ab-conjugated QDs-encoded microbeads were successfully synthesized to establish a method for the quantification of soluble FcεRⅠαand IgE in serum.The method had a wider detection range,and was more sensitive,more convenient and stable.This method achieved the aim of comparing sFcεRⅠαand IgE levels in the same detection system to avoid bias.In addition,MCs activation-related biomarkers can be detected in sera of patients with SLE and AILD.These results suggested that the new method may be a efficient method of quantifying MCs activation-related biomarkers in clinical application.The prokaryotic IgE expression vector and anti-human FcεRⅠαmonoclonal antibody provided a guarantee for future study.