Research On Suspension Array Reagents Used For Detection Of Human Serum Tumor Markers

Tumor markers(TMs) have an important place in clinical diagnosis andtreatment of tumors.They are not only useful in tumor screening,earlydiagnosis,assistant diagosis,discrimination of benign and malignant diseases,andclinical staging,but also significant in efficacy monitoring,prognosis,prediction ofrelapse and metastasis.To improve diagnostic sensitivity and specificity oftumors,clinicians usually adopt the detection of multiple TMs.At present,thetraditional methods of detecting serum TMs include RIA,ELISA,CLIA,TRFIA,andetc,which have a common limitation,ie, only one TM can be measured at atime.Undoubtedly, multiple TMs detection will increase both economic burden onpatients and cost of time,energy and reagents.Suspension array is a novel bio-chip platform based on xMAP(flexibleMulti-Analyte Profiling)technology developed by Luminex Corporation, USA.Assays of antigen-antibody, enzyme-substrate,receptor-ligand and nucleic acidhybridization are completed on different fluorescence-encoded microspheres whichhave specific spectral addresses.When the microsphere pass by two separate lasers inthe Luminex 100 analyzer,it is classified based on its spectral address and the reactionon the surface is quantified based on the reporter fluorochrome bound to themicrosphere surface, xMAP technology allows multiplexing of up to 100 analytes in a singl reaction vessel.Thus suspension array is a new generation of high-throughputmolecular diagnosis tool.Compared with other immunoassays,in addition tospeed,sensitivity, flexibility and broad detection ranges,the most remarkable benefit ofsuspension array is its unique capability of qualitative and quantitative analysis ofdifferent analytes in one sample simultaneously.Because of the clinical needs for combined analysis of multiple TMs and thelimitation of present detection methods,our aim is to develop a new immunologicaldiagnostic reagent for detecting multiple serum TMs by using suspension arraytechnology.In the present study, we chose four TMs—carcinoembryonicantigen(CEA),alpha-fetoprotein(AFP),total prostate specific antigen(tPSA) andneuron-specific enolase(NSE) as the analytes.Based on the principle of doubleantibody sandwich immunoassay, we developed reagents which could determine 3 or4 analytes simultaneously(ie.CEA+AFP+NSE,CEA+AFP+tPSA andCEA+AFP+tPSA+NSE).Analytical performance such as dynamic range,lower limitof detection,precision,normal range,stability were investigated for each reagent.Wealso compared the concentration of each analyte in the human serum samples withthose obtained by CLIA and TRFIA.The results indicated that:(1) CEA+AFP+NSE: Dynamic ranges were 0.078-200 ng/ml,0.030-30.3ng/ml,0.146-75 ng/ml,and lower limits of detection were 39.1 pg/ml,19.7 pg/ml,73.2pg/ml for CEA,AFP and NSE,respectively.Intra-assay variances were less than 9％and inter-assay variances were less than 15％.Kappa coefficients were0.937,0.965,0.906(P＜0.001,n=64)compared with CLIA for CEA, AFP andNSE, respectively. Correlation coefficients(r) were0.989, 0.966, 0.947(P＜0. 001, n=64)compared with CLIA for CEA, AFP and NSE,respectively.(2) CEA+AFP+tPSA: Dynamic range were 0.031-400 ng/ml,0.026-166.4ng/ml,0.008-40 ng/ml,and lower limits of detection were 31.3 pg/ml,13.0 pg/ml,5.2pg/ml for CEA,AFP and tPSA,respectively.Intra-assay variances were less than10%,and inter-assay variances were less than 14%. Kappa coefficients were0.940,0.959(P＜0.001,n=100),0.945(P＜0.001,n=109)compared with CLIA forCEA,AFP and NSE,respectively. Correlation coefficients(r) were0.961,0.972(P＜0.001, n=100),0.962(P＜0.001, n=109) compared with CLIA,and0.958,0.980(n=100),0.955(n=109)compared with TRFIA for CEA,AFP andtPSA,respectively.(3) CEA+AFP+tPSA+NSE: Dynamic range were 0.039-400 ng/ml,0.030-121ng/ml,0.007-30 ng/ml,0.146-300 ng/ml,and lower limits of detection were 26.0pg/ml,19.7pg/ml,4.9pg/ml,73.2pg/ml for CEA,AFP, tPSA andNSE,respectively.Intra-assay variances were less than 9%,and inter-assay varianceswere less than 14%.Correlation coefficients(r) were0.986,0.979(P＜0.001, n=64),0.964(P＜0.001, n=60),0.958(P＜0.001, n=64) comparedwith CLIA for CEA,AFP, tPSA and NSE,respectively.(4)Stability:After the major components of suspension assay reagent,ie,antibodycoupled microspheres,antigen standards and biotinylated detection antibody for CEAwere placed under 37℃for 7 days,the fluorescence intensity of standards had somedecrease,however, the detection range did not change and intra-and interassayvariance were still less than 10%.The results of our study demonstrated that suspension array reagents couldsimultaneously determine CEA,AFP, NSE and tPSA levels from 1 to 2 microliter ofhuman serum in 2 to 3 hours.This techique wassimple, fast,flexible,reproducible(coefficient of variance less than 14%),highly sensitive(pg level) and wide-ranging(3 to 5 orders of magnitude).The measurementsof clinical serum samples correlated well with those obtained by CLIA andTRFIA.Furthermore,it was much more labor-saving andcost-effective.Therefore,suspension array proved potential to become a newdiagnostic and therapy.Our study has laid a foundation for future research anddevelopment of mature clinically-used suspension array reagents for multiple TMsdetection.