Preparation of ELISA (EIA) enzyme conjugate

Immunoenzymatic technique (immunoenzymatic technique), also known as enzyme immunoassay (EIA), is another immunolabeling technique developed after immunofluorescence and radioimmunoassay. It is established by combining the specific reaction of antigen and antibody with the efficient catalytic action of enzyme. This technology combines enzymes with antibodies or anti-antibodies through chemical methods to form enzyme labels. These enzyme labels still maintain immunological activity and enzyme activity, and then react it with corresponding antibodies or antigens to form enzyme-labeled complexes When the enzyme bound to the immune complex meets the corresponding substrate, it catalyzes the substrate to produce hydrolysis, oxidation, or reduction reactions, and produces soluble or insoluble colored substances. Then reflect the content of the antigen or antibody in the sample to be tested according to the depth of color development. If the colored substance produced is soluble, the optical density (OD) can be determined qualitatively or quantitatively by visual colorimetric or microplate reader; Colored substances are insoluble precipitates, but also electron-dense substances, which can be identified and positioned by optical microscope or electron microscope. Therefore, immunoenzyme technology is a comprehensive technology of positioning, qualitative and quantitative (sensitivity can reach ng ~ pg level per ml). Commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AKP).
Enzyme-labeled antibody or anti-antibody (anti-immunoglobulin) conjugate can be prepared by glutaraldehyde method or periodate oxidation method. The modified sodium periodate method of horseradish peroxidase (HRP) labeling antibody established by Guo Chunxiang is simple and easy to perform, and the labeling effect is good. It is especially suitable for small batch preparation in the laboratory. The operation method is now introduced as follows:
(1) Marking of the conjugate Dissolve 5mg HRP in 05ml distilled water, add 05ml of newly prepared 006mol / L NaIO4 aqueous solution, mix well, put in the refrigerator for 30min, take out and add 016mol / L ethanol aqueous solution 05ml, after standing at room temperature for 30min, 5mg of purified antibody in 1ml of aqueous solution (or PBS), mix and pack in a dialysis bag, dialyze with 005mol / L, pH 95 carbonate buffer for 6h (or overnight) and stir to bind, then aspirate and add NaBH4 solution / ml) 02ml, set in the refrigerator for 2h. The above conjugate mixture was added to an equal volume of saturated ammonium sulfate, placed in a refrigerator for 30 minutes, centrifuged, the resulting precipitate was dissolved in a little 002mol / L, pH 74 PBS, and dialyzed against it overnight. The next day, the insoluble material was removed by centrifugation again to obtain the enzyme-antibody conjugate. Add 5ml with 002mol / L, pH 74 PBS, and then freeze-dry or store at low temperature.
(2) Determination of the amount of HRP and antibody in the conjugate The enzyme-labeled conjugate was measured for its optical density (OD) at a wavelength of 280 and 403 nm on a 751 spectrophotometer, and the ratio of OD403 to OD280 and the ratio of HRP to antibody mol / L ratio.
Concentration of HRP in conjugate (mg / ml) = OD403 × 04
Concentration of IgG in conjugate (mg / ml) = (OD280-OD403 × 03) × 062
Enzyme / antibody mol / L ratio = HRP concentration IgG concentration × 4
(3) Determination of the dilution of the conjugate
The polystyrene reaction plate was directly coated with the antigen corresponding to the antibody in the enzyme conjugate, and the titer of the enzyme conjugate was measured by ELISA direct method. Generally, when the enzyme conjugate prepared by this method is diluted 1:10 000, its OD403 is still above 01.

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