Application Of Monoclonal Antibodies To Brucella OMPs For Antigen Detection In PBMCs Of Brucellosis Patients

BackgroundBrucellosis is a serious infectious zoonosis caused by Brucella.spp,which easily develops chronic infection and causes multiple organ failure.At present,laboratory of brucellosis is largely depended on serologic detection.However,there are some cross-reactions and weakness in reflecting the patients’ situation of carrying bacteria.On the other hand,bacterial culture is the gold standard for pathogenic detection of brucellosis,but it has a low isolation rate and a high bio-safety risk.PCR as a hot research topic of brucellosis diagnosis,it’s operation simplicity has not yet reached the requirements.It is great significance that the intracellular pathogens or brucella of brucellosis patients can be detected directly for diagnosis and evaluate therapeutic efficacy of brucellosis.However,there are few reports in this field.ObjectiveThis study intends to develop a flow cytometry(FCM)assay for detecting intracellular Brucella pathogens in peripheral blood mononuclear cells(PBMCs)of brucellosis patients to evaluate the clinical therapeutic effect of brucellosis.This way is based on identifying and screening monoclonal antibodies(mAbs)against outer membrane proteins(OMPs)of Brucella.Materials and Methods1.MaterialsRecombinant OMPs of Brucella Omp31,Bp26 and Omp25;mAbs and hybridoma cell to Omp31,Bp26 and Omp25 of Brucella;three kinds of inactivated Brucella.spp strains(B.melitensis M5-90,B.suis S2 and B.abortus 2308,104M and S19 strains);fluorescein isothiocyanate(FITC),phycoerythrin(r-PE).2.ObjectsThe whole blood samples from 52 brucellosis patients and 55 healthy blood donors were used as the experimental group and the control group,respectively.PBMCs were freshly separated from whole blood of brucellosis patients and blood donors.3.Methods3.1 Purification and preparation of mAbs:Hybridoma cells were re-cloned by limiting dilution,and then ascites fluid was prepared by injecting subcutaneously mouse with collected mAbs’ cells and purified by Protein G NUPharose FF.The concentration and purity of mAbs were determined by Nanodrop2000 and SDS-PAGE,respectively.3.2 Identification and screening of mAbs:The titers and affinity of purity mAbs were identified ELISA.Antigenic epitopes of Brucella Omp25 recognized by mAbs to Omp25 were identified by Peptide-ELISA and cross-matching mAbs were screened by the double-antibody sandwich ELISA(DAS-ELISA).3.3 Reactivity of mAbs with three common strains of Brucella:Western-blot and ELISA were used to identify B.melitensis,B.suis and B.abortus.3.4 Flow cytometry(FCM)for detecting intracellular Brucella antigen:The transduced 293 FT cells and PBMCs from blood donors and brucellosis patients were detected by FCM with mAbs labeled FITC or r-PE,which application value in clinical practice was analyzed.Results1.The mAbs to Omp31,Bp26 could be used to detected different types of Brucella antigen by various immunological assays,such as ELISA,Western-blot and IFS,which titers were about 1:200,000 and purity were more than 90%.2.By mapping of Omp25 epitopes,mAb 6C12 reacted with a semi-conformational epitope,and other four mAbs(4A12 and 4F10,8F3,2B10)recognized linear epitope P2,P4 and P7,respectively.The paired mAbs(8F3/6C12-HRP and 8F3/4A12-HRP)were selected for detecting Brucella species by ELISA,indicating that 8F3 was suitable for solid phase capture and 6C12 or 4A12 was suitable for conjugation with HRP to detect Brucella Omp25 by ELISA3.MAb 6C12 labeled with r-PE was used for detecting the frequency of carrying Brucella in brucellosis patients’ PBMCs.The results showed that there were 42.3%(22/52)brucellosis patients with Brucella positive frequency>1%(Cutoff)of PBMCs,which was detected by FCM and was positively correlated with the number of carrying BrucellaConclusionIn this study,we screened and identified mAbs for the immunoassay of Brucella OMPs and developed a FCM assay based on mAb 6C12 for detecting intracellular Brucella pathogens in brucellosis patients’ PBMCs,which provided a new useful method for diagnosis and efficacy evaluation of brucellosis.InnovationIt’s the first time that FCM for detecting brucellosis PBMCs’ Brucella pathogens was developed and applied to evaluate the efficacy of antimicrobial therapy of brucellosis.The research results have been published in Frontiers in Cellular and Infection Microbiology(2020).