Cationized BSA

Raising the pi of macromolecules also can significantly alter the immune response toward them upon in vivo administration. Cationized proteins (those modified with diamines to increase their net charge or pi) are known to generate an increased immune response compared to their native forms (Muckerheide et al., 1987a, b Apple et al., 1988 Domen et al., 1987 Domen and Hermanson, 1992). The use of cationized BSA as a carrier protein for hapten conjugation can result in a dramatically higher antibody response toward a coupled hapten (Chapter 19). 

The most common carrier proteins in use today are keyhole limpet hemocyanin (KLH MW 4.5 X 105 to 1.3 X 107), BSA (MW 67,000), aminoethylated (or cationized) BSA (cBSA), thyroglobulin (MW 660,000), ovalbumin (OVA MW 43,000), and various toxoid proteins, including tetanus toxoid and diphtheria toxoid. Other proteins occasionally used include myoglobin, rabbit serum albumin, immunoglobulin molecules (particularly IgG) from bovine or mouse sera, tuberculin purified protein derivative, and synthetic polypeptides such as poly-L-lysine and poly-L-glutamic acid. 

Cationized BSA containing blocked carboxylates with additional positively charged amines... 

Figure 272 The effectiveness of cBSA as an immunogen can be seen by the comparison of specific antibody response in mice to arginine vasopressin coupled to both native and cationized BSA. The quantity injected was standardized according to the amount of arginine vasopressin present. The cationized carrier results in higher concentrations of antibody produced against the peptide than the immunogen made with native BSA.

Figure 273 Cationized BSA even can increase the specific antibody response to large proteins coupled to it. This graph shows a comparison of the relative antibody response in mice to injections of ovalbumin, either in an unconjugated form or conjugated to native or cationized BSA. The quantity injected was standardized according to the amount of ovalbumin present. The highly basic cBSA molecule modulates the immune response to enhance the production of antibodies toward even proteins conjugated with it.

Carriers having molecular weights similar to that of BSA or OVA may be activated at the same level with good success. Cationized BSA requires less cross-linker addition due to its greater quantity of amines present. 

To increase the yield of conjugated hapten using this procedure, cationized BSA is used as the carrier protein in the method described below (Section 2.1). The greater density of amine groups on cBSA available for participation in the Mannich reaction over that available on native proteins provides better results in coupling active hydrogen-containing haptens. 

Figure 293 The conjugation of phenol red to cationized BSA using the Mannich reaction.

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