Thermo Scientific Imject Cationized Bovine Serum Albumin (cBSA) functions as an easy-to-use and highly immunogenic carrier protein for preparing antigens for use in antibody production.
Imject cBSA is prepared by modifying native BSA with excess ethylenediamine, essentially capping all negatively-charged carboxyl groups with positively-charged primary amines. The result is a highly positively-charged protein (pI > 11) that has significantly increased immunogenicity compared to native BSA. In addition, the increased number of primary amines provides for a greater number of antigen molecules to be conjugated with typical crosslinking methods. Imject cBSA is an excellent choice for immunogen preparation when greater immunogenicity is needed relative to BSA but conditions are not amenable to using keyhole limpet hemocyanin (KLH) or Blue Carrier* Protein.
- Purified bovine serum albumin (BSA) that is cationized and supplied lyophilized in PBS or MES buffer
- Single polypeptide protein with molecular mass > 67,000 Daltons
- Contains 59 lysine residues, plus numerous other primary amines added by cationization, most of which are capable of reacting with a conjugation reagent to attach antigens
- Net positive charge (pI >11), conferring solubility in conditions not suitable for the negatively-charged native BSA (pI 5.1)
- Significantly greater immunogenicity than native BSA because the positively charged protein is processed differently by the immune-response system (see references).
Carrier proteins are large, complex molecules capable of stimulating an immune response upon injection. Successful production of antibodies specific to small antigens (i.e., peptides or drug compounds) requires that these haptens be covalently conjugated to a larger, more complex molecule (usually a protein) to make them immunogenic. Carrier proteins are chosen based on immunogenicity, solubility, and whether adequate conjugation with the carrier can be achieved. Imject Cationized BSA (cBSA), which was previously called "SuperCarrier Protein" is very soluble in aqueous buffers and contains numerous primary amines that can be targeted for conjugation with glutaraldehyde, EDC and other crosslinking reagents. For sulfhydryl-target conjugation (e.g., cysteine-containing peptide antigens), use Imject Maleimide-Activated cBSA.
|Higher and longer-sustained immune response with Thermo Scientific Imject Cationized BSA. Mice were immunized with a mildly immunogenic hapten (ovalbumin protein) alone or as conjugates with BSA or cationized BSA (cBSA). Hapten-specific antibody production was measured using an ELISA procedure and the values expressed as a percent of the highest data point (cBSA conjugate at 49 days).
Panel A. Immunizations with biweekly boosts.
Panel B. Immunization without any boosts.
- Muckerheide, et al. (1987). Cationization of protein antigens. I. Alteration of immunogenic properties. J. Immunol. 138:833-837.
- Muckerheide, et al. (1987). Cationization of protein antigens. II. Alteration of regulatory properties. J. Immunol. 138:2800-2804.
- Domen, P.L., et al. (1987). Cationization of protein antigens. III. Abrogation of oral tolerance. J. Immunol. 139:3195-3198.
- Apple, R.J., et al. (1988). Cationization of protein antigens. IV. Increased antigen uptake by antigen-presenting cells. J. Immunol. 140:3290-3295.
- Peterson, E.C., et al. (2007) Using hapten design to discover therapeutic monoclonal antibodies for treating methamphetamine abuse. J. Pharmacol. Exp. Ther. 322:30-39.
- Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. Chapter 5 discusses the use of carrier proteins.
- Hermanson, G.T. (2008). Bioconjugate Techniques. 2nd edition, Academic Press, New York. (Part No. 20036). Chapter 19 discusses carrier protein uses and immunogen preparation.
Review of antibody production and immunogen preparation methods
Review of carbodiimide (EDC) crosslinker chemistry
Carrier protein activation and conjugation data for immunogen preparation
EDC Crosslinker – carbodiimide crosslinker for conjugating via carboxyl and amine groups
CarboxyLink Immobilization Kit – for preparing an affinity column with a carboxylate peptide or hapten
BS(PEG)5 – homobifunctional NHS-ester crosslinker for conjugating amine to amine groups
Maleimide-Activated cBSA – for conjugating cysteine peptides via sulfhydryl groups
Adjuvants and other Carrier Proteins