Method For Determination Of Tumor Marker By The Horseradish Peroxidase And Immonomanetic Beads

Objective:1. To establish an analytical method for determination the horseradishperoxidase （HRP） activity by electrochemistry.2. To establish an analytical method for determination of tumormarker immobilized magnetic beads by electrochemistry.3. To investgate a correlation of methods for determination of tumormarker between immobilized magnetic beads by electrochemistry andenzyme linked immunoassay （ELISA）.Method:1. Method for determination of HRP catalyzed the3,3,5,5-tetramethyl benzidine （TMB） by H2O2.Working electrode was analysed by screen printed carbon electrode（SPCE）. Detection conditions: substrate solution contained with0.8mmol/L TMB15L and15mmol/L H2O225L, which reacted with4L5.0×10-10g/mL HRP for5min at37. The HRP activity was characterized by Cyclic voltammetry and quantitied by Differential pulsevoltammetry （DPV） at the range of-0.1V0.4V respectively.2. Determination of tumor marker modified of epoxy-functionalizedsuperparamagnetic immobilized beads by electrochemistry.Three strategies using different binding agents were prepared for theimmobilization of monoclonal anti-CA199solution on magnetic beads.Firstly, the immobilization of Streptavidin to the magnetic beads wascommonly accomplished through the active epoxy group existing on theirsurface, and biotin anti-CA199was modified the surface of magnetic beadsthrough the specificity of biotin and Streptavidin. Lastly, the activitysettings were blocked by BSA （1%） to prevent non-specific binding. Whenthe treated beads were connected with the antigen of CA199labelled withHRP, and then washed to remove the physically adsorbed antibodies. Theworking concentration of HRP-labeled anti-CA199modified theimmobilized magnetic beads was investigated after adding into the TMBand H2O2substrate solutions by the method of electrochemistry.Results:1. HRP activity by electrochemistry: The linearity was from1.1×10-13to5.0×10^-11g/mL for HRP. The detection limit was8.7×10-13g/mL.2. Under the optimal conditions, the current intensity increasedlinearly with the increasing CA199antigen concentration from3⁶0U/mL（R2=0.9779）. No obvious change of the modified immunomagnetic beads was observed after storing for7days. The within-day precision range forCA199antigen concentration was0.70¹.36%the between-day precisionranged from1.32⁶.92%. The detection limit was1.6U/mL for CA199antigen concentration.Conclude:1. The proposed electrochemical enzyme-linked immunoassay methodwas developed for detecting HRP and labeled-HRP, which wasreproducible, sensitive and showed promising for in the clinical diagnosis.2. A novel, simple enzyme immunoassay for CA199based on epoxymodified microbeads as immobilization support is described. This low costand flexible electrochemical immunosensor, combined with a low costreagent system, is a simple, sensitive and rapid noncompetitiveimmunoassay for CA199.3. The proposed method was found significant correlation withEnzyme-linked immunosorbent assay （ELISA）.