Hapten—carrier conjugation using

This chapter describes the design, preparation, and use of hapten-carrier conjugates used to elicit an immune response toward a coupled hapten. The chemical reactions discussed for these conjugations are useful for coupling peptides, proteins, carbohydrates, oligonucleotides, and other small organic molecules to various carrier macromolecules. The resultant conjugates are important in antibody production, immune response research, and in the creation of vaccines. 

As reported by Seif (S6), the /3-type antibody can be replaced with a macro-moiecuie muitiply iabeied with the target hapten, such as hapten-carrier conjugates used for immunogen. The noncompetitive assay for cortisoi (Gl), which was discussed in Section 3.3, is based on this principle, but employs a different method for selective detection of hapten-occupied antibody. 

The following is a generalized protocol for the activation of a protein with sulfo-SMCC with subsequent conjugation to a sulfhydryl-containing second molecule or protein. Specific examples of the use of this crosslinker to make antibody-enzyme or hapten-carrier conjugates may be found in Chapter 20, Section 1.1 and Chapter 19, Section 5, respectively. 

Figure 19.13 Conjugation to KLH often can cause precipitation due to the high-molecular weight of the carrier protein. The conjugation of [Met5]-enkephalin to KLH yields a soluble immunogen if the level of EDC addition is about 0.1 times that typically used with BSA as a carrier. This figure shows the gel filtration pattern on Sephadex G-25 after completion of the crosslinking reaction. The first peak is KLH and the second peak is excess peptide. Depletion of the peptide peak correlates to hapten-carrier conjugation.

The hapten-carrier conjugate now may be mixed with adjuvant and used for injection purposes without further purification. 

Positive clones A phrase usually used to describe those hybridoma clones which bind reasonably to their respective hapten in an enzyme-linked immunosorbent assay, thereby eliminating non-specific antibodies raised to different epitopes of the hapten/carrier conjugate. 

Another method of NHS ester mediated hapten—carrier conjugation is to create reactive sulfo-NHS esters directly on the carboxylates of the carrier protein using the EDC/sulfo-NHS reaction described in Chapter 3, Section 1.2. A carbodiimide reaction in the presence of sulfo-NHS activates the carboxylate groups on the carrier protein to form amine-reactive sulfo-NHS esters. The activation reaction is done at pH... 

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