广州医科大学《临床免疫学检验》9.理论课-酶联免疫技术.pptVIP

一、pinciple of enzyme immunoassay Characteristic Antigen-antibody reaction: 1、Reversibility: the process?of antigen antibody reaction?is?a dynamic balant reaction: Ag + Ab? Ag · Ab The?equilibrium constant K reflect the antigen-antibody affinity. K = [Ag ·?Ab]?/ [Ag][Ab] Dissociation degree of Ag · Ab?is associated with K?value. After dissociation, Antigen?and antibody?can keep?the original?structure and?activity. 2、specificity： The?antigen determinant?can combine with antibody binding sites.?This reaction has a high degree of specificity because of the complementary?relationship between chemical?structure and the?spatial structure. when we test a particular?substance,?we don’t need separate the?samples first. 3、optimal ratio ： 4、sensitivity ： Sensitivity of Colorimetric assay?can reach mg/ml level. Sensitivity of enzyme?assay??is about 5-10μg/ml. Sensitivity of enzyme-labelled?immunoassay?can be up to ng/ml?level. For example,?HBsAg,?the sensitivity?can reach 0.1ng/ml. characteristics of enzyme: High efficiency: it has good catalytic ability to promote the antigen and antibody reaction in the process. Specificity: the catalytic role to substrate is specific and enzyme is not affected by other reagent in the solution. Stable: easy to react with antigen or antibody and the immunocompetence of enzyme、antigen or antibody in complex is not affected. Non-toxic, no harm Horseradish peroxidase(HRP) HRP is a kind of compound enzyme, which is consists of glycoprotein and ferroheme. PH range: 5-9 Substrate: DH2+H2O2====D+H20 donor receptor Common substrate for HRP TMB liquid A: contain 0.5‰ H2O2 liquid B：contain 0.26‰ TMB easy to decompose and store in the dark bottle Mix two liquid together before using. Other enzyme Alkaline Phospharase(AP) substrate: PNP c