【摘要】 本文报道了HRP的二种标记抗体的简易方法。操作简便,易于掌握。可用国产酶(RZ≥2)标记。效果良好。更多还原

【Abstract】 A method for the preparation of peroxidase conjugate for ELESA with glutaraldehyde in two steps was introduced by Avrameas and Ternynci. The method has been modified and simplified as follows:1. 5 mg of HRP is put in a test tube. Add 0.4ml of 0.05M carbonate buffer, pH9.6 to dissolve the enzyme, then add 0.1ml of 25% glutaraldehyde (A₂₃₅/A₂₈₀ -10), mix well and incubate at 37℃ for 2 hours. Then add 0.1ml of 22% NaCl solution. Cool the solution. Introduce 2.4 ml of cold absolute ethyl alcohol（approximately —5℃） into the test tube, mix thoroughly. Centrifuge at 1000 rpm for 10—15 minutes and remove the supernatant. Wash the precipitate with 80% cold ethanol, centrifuge as above. Pour out alcohol as much as possible. Add 0.5ml of 0.05M carbonate buffer, pH9.6 to dissolve the precipitate. Mix it with 0.5ml of antibody solution containing 5—10 mg IgG. Keep in refrigerator over night. The preparation is ready for use.2. Another method is a modification of the periodate method established by Nakane and Kawaoi, using formaldehyde （final concentration 0.6%） or glutaraldehyde （final concentration 5%） instead of 1-fluoro-2, 4-dinitrobenzene, to avoid the production of HF, which is an inhibitor of HRP. The carbohydrate portion of the enzyme is oxidized by 0.03M periodate solution （final concentration） at 0—5℃ for 30 minutes. The oxidized enzyme is precipitated by cold absolute ethanol. The precipitate is washed with 80% alcohol, dissolved in 0.05M carbonate buffer, and then coupled with antibody （10—15mg）.更多还原