We need to raise an antibody against Progesterone in the rabbit. For that, we want to conjugate it with KLH. Please share a standardized protocol to do it.
Please be aware that the design of a proper immunoconjugate is a science. It is not sufficient to conjugate something to a carrier protein. The structure of the hapten conjugate highly influences the cross-reactivity pattern and the affinity of the serum. This is particularly important in the field of steroids, where many similar compounds may be present in a sample. I fear that you need to read many papers.
Try carbodiimide (EDC) method (Hermanson, GT, 2008). EDC reacts with available carboxyl groups on either the protein carrier or peptide hapten to form an active O-acylisourea intermediate which then reacts with a primary amine to form an amide bond and a soluble urea by-product. EDC conjugations usually
result in a certain amount of polymerization which may enhance the immunogenicity of the peptide and produce a greater antibody response.
Progesterone does not have readily available active group to link it to KLH. Sigma/Aldrich sell two progesterone products that already have a carboxylate group attached to the molecule. You can use these to carry out standards EDC/NHS linkage procedure of progesterone (via the carboxylate group) to NH2 groups on KLH. The two products are : progesterone-11-alpha glucuronide (product no. UC184) and progesterone-3-(O-carboxymethyl)oxime (product no. P3277)
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