Enzyme-linked Immune Method Based On Chemical Oscillation For Detection Of Insulin

Insulin can combine alkaline phosphatase-labeled antibody in immune complexes,because its immunological specificity. Alkaline phosphatase is a hydrolase, It can catalyze thehydrolysis of phosphoric acid mono esters, and product of phosphate monoester hydrolysiscan be detected through the chemical oscillations system. The design ideas of this paper is tofind a material which itself has no effect on the chemical oscillation, but the alkalinephosphatase-catalyzed hydrolysis products of it can direct disturbance on chemicaloscillations. Alkaline phosphatase can happen in enzyme immunoassay, so it can be used intne enzyme immunoassay to detect product of chemical oscillation.This paper proposes to establish a new type of trace amounts of B-Z oscillation system,and use this new type of trace to detect the concentration of insulin in Enzyme-linkedimmunoassay. Different concentrations of insulin combine alkaline phosphatase-labeledantibody in enzyme immunoassay. The use of alkaline phosphatase this product on theimmune is to catalyze the certain concentration of vitamin c sodium phosphate hydrolysisreaction, generated vitamin c can be direct measure by the new B-Z oscillating system. Inthe particular situation, different concentrations varying amounts of alkaline phosphatasecatalyzed the different generation of vitamin c, thereby the amount of changes on amplitudeor cycle system in different trace amounts B-Z oscillation.According to this changes, theamount of chemical oscillation parameters can be linked to the concentrations of insulin inimmunosorbent reactions.Create a new trace chemical oscillating of malic acid system, and optimize theconcentration of each component by its trace experiment system. The results show, when atrace component concentrations B-Z chemistry of malic acid system oscillation are:[H2SO4]=1.05mol/L,[NaBrO3]=0.60mol/L,[[CuL](ClO4)2]=2.21*10-3mol/L,[DL-malic acid]=0.6mol/L, Vitamin c cause the maximum disturbance on the system. When the concentrationof vitamin c in the0.5*10-9~1*10-7mol/L range, the variables of this oscillation amplitudeand log[VC] show a good linear relationship, the detection limit is3.15*10-10mol/L(3δ), theMinimum detectable amount is7.875*10-17mol; After adding vitamin c, the first electrode potentialmaximum value and log[VC] show a good linear relationship, the detection limit is3.95*10-10mol/L(3δ), the Minimum detectable amount is9.875*10-17mol. The traces B-Z oscillating chemicalof malic acid system to measure the concentration of alkaline phosphatase: When alkalinephosphatase activity is in the1~16U/L range, he variable of the oscillation amplitude and thelog[ALP] show a good linear relationship, the detection limit is0.59U/L(3δ), the Minimumdetectable amount is1.475*10-6U; the variable of oscillation cycle and log[ALP] also show alinear relationship, and the detection limit is0.56U/L(3δ), the Minimum detectable amount is 1.40*10-6U.Create a new trace chemical oscillating of glucose-acetone system, and optimize theconcentration of each component by its trace experiment system. The results show, when atrace component concentrations B-Z chemistry of glucose-acetone system oscillation is:[H2SO4]=1.05mol/L,[NaBrO3]=0.06mol/L,[[CuL](ClO4)2]=0.05mol/L,[glucose]=0.06mol/L,[acetone]=0.06mol/L, Vitamin c cause the maximum disturbance on the system.When the concentration of vitamin c in the0.5*10-9~1*10-7mol/L range, the variables of thisoscillation amplitude and log [VC] show a good linear relationship, the detection limiti2.6*10-10mol/L(3δ), the Minimum detectable amount is6.5*10-17mol; the variables of this oscillationcycle and log[Vc] show a good linear relationship, the detection limit is2.70*10-10mol/L(3δ),the Minimum detectable amount is6.75*10-17mol. The traces B-Z oscillating chemical ofglucose-acetone system to measure the concentration of alkaline phosphatase: When alkalinephosphatase activity is in the1~32U/L range, the variable of the oscillation amplitude andthe log [ALP] show a good linear relationship, the detection limit is0.57U/L(3δ) the Minimumdetectable amount is14.5*10-6U.Different concentrations of insulin and alkaline phosphatase-labeled antibody are directlyenzyme immunoassay, using the two traces of B-Z oscillating system to quantitative analysisits protect, in order to achieve the determination of insulin. The results show, When theconcentration of insulin in the0.5*10-9~1*10-7g/L range, the variables of this oscillationamplitude and log[INS] show a good linear relationship, the detection limit is2.35*10-10g/L(3δ), the Minimum detectable amount is5.875*10-17g; the traces B-Z oscillating chemical ofglucose-acetone system to measure the concentration of insulin: the variables of thisoscillation amplitude and log [INS] show a good linear relationship,the detection limit is1.85*10-10g/L(3δ), the Minimum detectable amount is4.625*10-17g.Different concentrations of insulin is performed with alkaline phosphatase-labeledantibody by avidin-biotin enzyme immunoassay, their biological immunity is measured byboth the chemical trace B-Z oscillating, to achieve the quantitative detection of insulin. Theresults show, When the concentration of insulin in the0.5*10-9~1*10-7g/L range, thevariables of this oscillation amplitude and log[INS] show a good linear relationship,thedetection limiti is2.00*10-10g/L(3δ), the Minimum detectable amount is5.1*10-17g; the traces B-Zoscillating chemical of glucose-acetone system to measure the concentration of insulin: thevariables of this oscillation amplitude and log[INS] show a good linear relationship, thedetection limit is1.90*10-10g/L(3δ), the Minimum detectable amount is4.75*10-17g.The significance of this thesis is: to establish a system of two different trace (totalvolume of1ml) B-Z oscillating chemical firstly, as a result its detection sensitivity isincreased40-fold ratioing constant constants (total volume of40ml) of B-Z oscillation. Thismethod expands the scope of for the detection of trace substances; Secondly, this thesis emplythe chemical traces of B-Z oscillating to obtain the quantitative analysis of insulin in enzymeimmunoassay, so a new method for detecting the of immune products is setted up. This willinjecte a new force to ELISA technique; finally, this new ELISA method can not only detec the concentration of insulin, but aslo extends the research field of methods for thedetermination of other antibodies or antigens.