Hapten-carrier connection method
1. When connecting the hapten to the carrier, an appropriate method should be selected, and the selection of the binding method should consider the following factors:
1) The solubility and stability of hapten: it should not cause the change of hapten activity in the binding reaction, and at the same time, it should not denature the carrier to the degree of insolubility.
2) The position of the binding bond: the antibody has the best specificity to the part of the hapten away from the protein connection point, so the connection bond should be away from the determinant of the hapten.
3) Select the appropriate coupling reagent: when different haptens are coupled, the appropriate coupling reagent should be selected according to the chemical structure of the hapten and the reaction mode. For example, small-molecule peptides have a certain tertiary structure, and rely on amino acid residues in solution to maintain their structural stability. Therefore, the use of bifunctional imidates not only selects amino acids, but also replaces the positive charge of each ε-amino group being substituted. After the protein is contacted with the coupling agent, the functional groups of different protein molecules are cross-linked and coagulated. The solubility of widely cross-linked proteins often decreases, but this poorly soluble protein is an effective source of immunity.
2. Ligation method The hapten and the carrier can usually be connected by physical and chemical methods. Carriers that are physically adsorbed include carboxymethyl cellulose, polyvinylpyrrolidone, etc. They adsorb haptens by electric charges and micropores. The chemical rule is to use certain functional groups to connect the hapten to the carrier. These carriers include serum albumin, thyroglobulin, ceruloplasmin, egg protein, and synthetic polylysine. The hapten with free amino group or free carboxyl group and both groups can be directly connected to the carrier. Other haptens that do not carry amino groups or carboxyl groups must be properly modified to be converted into derivatives with free amino groups or free carboxyl groups before they can be connected to the carrier.
1 There are free amino groups or free carboxyl groups and the connection method of hapten and carrier with both groups
(1) Carbodiimide method: Carbodiimide is a bifunctional reagent with very active chemical properties. They can be condensed with the carboxyl group on the hapten and the amino group on the hapten. This method is very simple, as long as the hapten and carrier protein are mixed in an appropriate solution according to a certain molecular ratio, then carbodiimide is added, stirred for 1-2 hours, and left at room temperature for 24 hours, and finally dialysis to remove unreacted hapten Artificial immunogens are available.
(2) Glutaraldehyde method: Glyoxal is a bifunctional linker with two active groups. It is connected to the carrier and the amino group of the hapten by a covalent bond via the aldehyde groups at both ends. The reaction is as follows:
(3) Mixed acid anhydride method: Mixed acid anhydride method is also called chloromethyl isobutyl ester method, which is mainly used to couple steroid hormones and proteins. With alkyl chloromethyl as the coupling agent, the most commonly used is chloromethyl isobutyl ester. The hapten containing a carboxyl group reacts with chloromethyl isobutyl ester to form a mixed acid anhydride, and then reacts with an amino group on a protein carrier to form a peptide bond. The reaction is as follows:
(4) Periodic acid oxidation method: Periodic acid oxidizes the sugar epoxide to the dialdehyde group, and then couples with the amino group on the protein. This method is commonly used for the coupling of glycoside drugs and proteins.
2 The hapten with amino or carboxyl groups can be modified by the following method, and then connected by the above method
(1) Succinic anhydride method: This method is used for the modification of hapten with hydroxyl group. Succinic anhydride is converted into succinic acid by adding water. If the hydroxyl-bearing hapten and succinic anhydride are reacted in anhydrous pyridine, the carboxyl-bearing hapten succinic acid derivative can be obtained. The reaction formula is as follows:
(2) Carboxymethyl hydroxylamine method: the hapten with ketone group (such as progesterone, testosterone) reacts with O- (carboxymethyl) hydroxylamine to transform into hapten derivative with carboxyl group The reaction formula is as follows:
(3) Diazotized p-aminobenzoic acid method: This method is suitable for the transformation of hapten with phenol group (such as certain drugs). First, p-aminobenzoic acid and sodium nitrite are reacted, and the reaction product is then applied to a hapten with a phenol group, thereby preparing a hapten derivative with a carboxyl group.
(4) Sodium monochloroacetate: This method is suitable for transformation with phenolic hapten. By reacting a drug with a phenol group with sodium monochloroacetate, a hapten derivative with a carboxyl group can be obtained.
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