Preparation Of Goat-Anti-Chicken Enzyme Label Secondary Antibody

Enzyme-labeled secondary antibody is a reagent that was born with immunoassay.It not only has high specificity and sensitivity as an antibody,but also has the ability to efficiently catalyze enzyme substrates.Compared with the enzyme-labeled primary antibody that directly performs immunoassay,it has the effects of improving efficiency,reducing cost,reducing false positives and signal cascade amplification.Compared with similar products,goat anti-chicken enzyme secondary antibody has many advantages.Immune host using chicken as antigen has a wider range of antigen determination,and using goat antibody as secondary antibody has great market prospects.There are four key technologies for the preparation of goat anti-chicken enzyme-labeled secondary antibodies,namely the purification of chicken yolk antibody,sheep immune reaction,purification of sheep serum(plasma)antibody and enzyme-linked reaction.In this study,based on the synthesis of previous research work,it is proposed to establish a method for preparing enzyme-labeled secondary antibodies.The experimental results of the paper are as follows:(1)Prepare chicken egg yolk antibody IgY by multiple methods and compare the methods.The protein purity obtained by water dilution-ethanol fractionation precipitation,chloroform-ethanol fractionation precipitation,chloroform-DEAE column chromatography and chloroform-ammonium sulfate secondary precipitation method were respectively 54%,55%,80% and 91%.Water dilution-ethanol fractionation precipitation method IgY yield is 11.99 mg per egg,chloroform-ethanol fractionation precipitation protein yield is 16.50 mg per egg,the highest yield of IgY in the four methods is chloroform-ammonium sulfate secondary precipitation Method,17.26 mg IgY can be extracted from each egg.Both chloroform-DEAE column chromatography and chloroform-ammonium sulfate secondary precipitation method can obtain high-purity IgY,but after comprehensive efficiency and cost and other factors,choose chloroform-ammonium sulfate secondary precipitation method as the purification method of primary antibody.(2)Only the first and second immunizations use adjuvanted IgY as the antigen during the sheep immune reaction.The amount of the first,second and thirdimmunization antigens is 200 mg,the amount of the antigen used in the fourth immunization is 300 mg,and the fifth immunity passes the neck Intravenous injection of 300 mg of IgY after heating and coagulation.None of the two experimental sheep had serious allergic reactions.Blood was taken from the jugular vein after the immune response was completed.(3)Purify IgG in sheep plasma using the octanoic acid-ammonium sulfate method.The purity of the obtained antibody is 80%,and the yield is 0.9 mg per ml of plasma.(4)Use sodium periodate oxidation method to make covalent cross-linking reaction between goat IgG and HRP.In order to test the binding capacity of the enzyme-labeled sheep secondary antibody to the chicken yolk primary antibody and the effect of the cross-linking reaction,we transferred the primary antibody IgY to the NC membrane by SDS-PAGE electrophoresis,and then incubated with the enzyme-labeled sheep secondary antibody.Exposure after chemiluminescence reaction.The results showed that no positive exposure band appeared in the IgY sample.It is unclear whether IgG and HRP have failed to cross-link effectively,or that the chicken yolk primary antibody antigen has failed to elicit the sheep's immune response to produce a sufficient amount of secondary antibody IgG.