First, the relevant product solid phase carrier
There are many substances which can be used as an enzyme in the enzyme-linked immunosorbent assay, such as polystyrene, polyvinyl chloride, magnetic particles containing iron, and microporous membranes such as nitrocellulose membranes, nylon membranes and the like.
The most commonly used is polystyrene. Polystyrene has a strong ability to adsorb proteins, and the antibody or protein antigen retains its original immunological activity after being adsorbed thereon. Polystyrene is plastic and can be made in various forms. In the enzyme-linked immunosorbent assay, it is widely used as a carrier and a container, and does not participate in chemical reactions.
There are three main types of enzyme-linked immunosorbent assays for the determination of carriers: small tubes, beads, and microplates. The most commonly used carrier is a microreaction plate. A good enzyme-linked immunosorbent assay plate should have good adsorption performance, low blank value, high transparency at the bottom of the well, and similar performance between the plates and the holes in the same plate.
Second, antigens and antibodies
The quality of antigens and antibodies during the implementation of the enzyme-linked immunosorbent assay is a key factor in the success of the experiment.
There are three sources of antigens used in the immunosorbent assay: natural antigens, recombinant antigens, and synthetic polypeptide antigens. The natural antigen is obtained from animal tissues or body fluids, microbial cultures, etc., and generally contains a plurality of antigen components, which are purified and extracted to extract specific antigen components, and are therefore also referred to as purified antigens. The recombinant antigen and the polypeptide antigen are artificial synthetic products, which are safe to use, high in purity, and low in interference substances.
Antibodies for enzyme-linked immunosorbent assays are available in polyclonal and monoclonal.
Third, the immunosorbent
The solid phase antigen or antibody is called an immunosorbent. The process of immobilizing an antigen or antibody is called coating. The method of coating differs depending on the carrier. The coated enzyme-linked immunosorbent assay plate can be placed at low temperatures for a period of time without losing its immunological activity.
Fourth, enzymes and substrates
The enzyme used in the enzyme-linked immunosorbent assay requires high purity, high conversion rate of catalytic reaction, strong specificity, stable nature, abundant source, inexpensive price, stable enzyme-labeled antibody or antigen, and remains. Its active part and catalytic ability. The most commonly used enzyme in the enzyme-linked immunosorbent assay is horseradish peroxidase (HRP).
Five, the combination
An enzyme-labeled antigen or antibody is referred to as a conjugate. Due to the different chemical structures, antigens can be combined with enzymes in different ways. For protein antigens, reference is basically made to the method of abzyme labeling.
1. Glutaraldehyde Crosslinking Process Glutaraldehyde is a bifunctional group reagent that allows an enzyme to be coupled to the amino group of a protein or other antigen. The glutaraldehyde cross-linking can be carried out in one step (such as connecting AP) or in a two-step method (such as connecting HRP).
2. Periodate Oxidation Method This method is only used for cross-linking of HRP. The enzyme contains 18% carbohydrates, and the periodate oxidizes the polysaccharide on its molecular surface to an aldehyde group. The excess periodate was neutralized with sodium borohydride (NaBH4). The aldehyde radical on the enzyme is very active and can be combined with the protein to form a molar ratio of the enzyme-bound conjugate. Some people think that this method is the most effective method for horseradish peroxidase cross-linking. However, it is also believed that the results of each batch of experiments are not easy to repeat due to the violent use of the reagents used.
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