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Notes on the double antibody sandwich method

Views : 1380
Update time : 2023-03-01 15:29:00
Hello everyone, today we will talk about the precautions for the double antibody sandwich method.
The following are precautions.
1. Soluble antigen or antibody is adsorbed on the solid phase carrier and becomes insoluble form, which is the basic condition for enzyme labeling assay. Many substances can be used as solid-phase carriers, such as cellulose, cross-linked dextrose, agarose beads, polypropylene, polystyrene, polyethylene and polyvinyl chloride, etc.. However, the most commonly used in ELISA are polystyrene or polyvinyl chloride micro reaction plates and plastic tubes.

2. The antigen used for encapsulation must be soluble and requires a high quality and stable preparation with high purity and immunogenicity. If it is impure, it will compete for a limited position on the solid phase carrier due to impurities contained in the antigen, reducing sensitivity and specificity. It should also be considered that the preparation of encapsulated antigens should not destroy their immunological activity.

3. For some antigens must be purified, such as viral tissue cultures and chicken embryo cultures and virus-inoculated animal organs, etc., which contain many non-antigenic proteins, which must be removed, commonly purified by gradient ultracentrifugation or affinity chromatography, which cannot be used without purification.



4. Using the most appropriate dilution of antigen, coating the solid phase carrier is not only economical, but also can overcome the pre-banding phenomenon. It can be measured by tessellation titration, but it is simpler to use single titration method, which is to wrap the antigen in a series of dilutions in micro reaction plate, then wash after incubation with positive reference serum and negative reference serum at a certain dilution, then add enzyme conjugate for reaction, after incubation and washing, add substrate solution for color development, terminate the reaction and measure the OD value respectively, and select the one with OD value ≥ 1.0 Dilution is the most suitable concentration for coating. Generally always choose that OD value slightly greater than 1.0, but not the concentration of antigen less than 1.0. Negative reference sera require an OD value of <0.1-0.2, which means that the OD values of positive and negative reference sera are significantly different.

5. In addition to the concentration of antigen encapsulated solid phase carrier, it has a relationship with time, temperature and PH. High temperature (such as 37℃, 45℃, or 56℃) can shorten the coating time, and low temperature can prolong the coating time. However, for the sake of convenience, overnight coating at 4°C is usually used to make the antigen adsorption more complete and uniform. When using polystyrene or polyethylene micro reaction plates or plastic tubes as solid phase carriers, overnight coating at 4°C under alkaline conditions (e.g. 0.1 mol/L, PH9.6 carbonate buffer diluted antigen) is more appropriate. However, in some experiments, such as when encapsulating with LPS or toxin proteins, dilution with PH 7.2-7.4 PBS is satisfactory. The concentration of the specific protein to be encapsulated is 1-10 ug/ml, while for some pathogenic microbial antigens 5-20 ug/ml is more appropriate, but all should be titrated.

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