Coupling of the carriers and small molecule haptens
In last special subject we talked about the structural modification of small molecule haptenes, this part we will start discussing the choice of carrier proteins and coupling methods.
Carrier protein is a kind of large molecule that can initiate immune reaction by itself, and it would be a totally artificial antigen after coupling of the carriers and small molecules. Carrier proteins commonly used include bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), ovalbumin(OVA), bovine thyroid globulin(BTG),etc.
For selecting carrier protein, there are some main factors to be considered, such as its solubility, size, coupling groups, immunogenicity and cost price. At present bovine serum albumin (BSA) is the most widely used as the carrier on the market(the three dimensional structure as Fig.1), mainly because of its stable chemical properties, not easy to be denatured, with many kinds of amino groups in its molecule, the high content of lysine, and cheap price. However, BSA is limited used in some immune assay experiment as blocking agent. The next choice of the carrier more is keyhole limpet hemoeyanin (KLH) and ovalbumin(OVA).
The following is some brief introduction about the coupling method of haptenes and carriers:
1.Compounds with carboxyl or amino groups are suggested to use the carbodiimide method and mixed anhydride method ;
2.Compounds with mercapt groups are suggested with the method by using maleimide;
3.For the compounds with amino groups in the molecular structure, it is suggested to use glutaraldehyde method, and it is the most commonly used method on the market.
In general haptenes and carrier proteins, as the method above, it is recommend to have the reaction with an appropriate coupling rate at room temperature or 4 degree overnight, then dialyse the product for 2 days through separator column or dialysis bag, finally totally artificial antigen could be obtained.
Then, we can compare the changes of UV absorption between products coupled and uncoupled by the ultraviolet scanning method. If the UV absorption characters of artificial antigen both have or is different from that of primary hapten and carrier protein, coupling is successful. Then the final coupling rate will be got through calculation.
After a lot of research and development, Cloud-Clone Corp. company can provide various kinds of coupled small molecule compounds and ploypeptide for researchers to choose and use.
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