Thermo Scientific Imject BSA and the accompanying EDC Conjugation Kit are packages of purified bovine serum albumin carrier protein that enable simple preparation of effective immunogens with amine- or carboxyl-peptide antigens.
BSA is a popular immunogenic carrier protein used for preparation of peptide antigens for immunization and antibody production. BSA preparations in phosphate buffer are ready for hapten-carrier conjugation via amine-reactive NHS-ester or glutaraldehyde crosslinking. Preparations in MES buffer are optimized for peptide-carrier conjugation via the carboxyl-reactive carbodiimide crosslinker called EDC (or EDAC). The Imject EDC Spin Kit includes ready-to-use units of BSA carrier protein, optimized reaction buffers and trouble-free spin desalting columns for immunogen preparation using the EDC method.
- Convenient kit – contains lyophilized BSA (in MES buffer), EDC crosslinker and accessories components to easily prepare ready-to-use immunogens for injection
- High-yield conjugation – each molecule of 59 lysine residues, 30 to 35 of which have primary amines that are capable of reacting with a conjugation reagent
- Validated quality – purified BSA (Fraction V) is available lyophilized and stabilized in PBS or MES buffer, ready for use in glutaraldehyde, NHS-ester or EDC crosslinking methods
- Compatible solubility – numerous carboxyl groups account for the net negative charge (pI 5.1) of BSA and its high solubility, allowing its use in a broad range of conjugation conditions
- Immunogenic – at 67 kDa, BSA is sufficiently large to function as the primary immunogen or it can be used as an irrelevant protein carrier for antibody screening and immunoassays after using KLH as the carrier protein to generate the immune response against the hapten
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 hapten-carrier conjugation can be achieved. Imject BSA is very soluble in aqueous buffers and contains numerous primary amines and carboxyl groups that can be targeted for conjugation with glutaraldehyde, NHS esters, EDC and other crosslinking reagents. Imject BSA is purified and lyophilized in buffers that optimize its stability and solubility for hapten conjugation.
||Glutaraldehyde hapten-carrier conjugation. A variety of amine-based and carboxyl-based crosslinking methods can be used to conjugate haptens (e.g., peptides) and carrier proteins such as bovine serum albumin (BSA) for antibody production.
Bovine serum albumin (BSA) is a convenient protein for a variety of uses in the laboratory because, like most abundant plasma proteins, it is very stable and soluble. In addition, the 67 kDa protein is sufficiently large and complex to be fully immunogenic. Consequently, BSA is a popular carrier protein for conjugation to haptens and other weak antigens to make them more immunogenic for the purpose of antibody production. It also provides an independent hapten-carrier protein for microplate and other assay techniques required to screen antibodies produced using KLH or another more immunogenic carrier.
The carbodiimide crosslinker EDC conjugates carboxyl-containing haptens (e.g., C-terminus of peptide antigens) to BSA carrier protein. This method of immunogen preparation is ideal for peptide antigens with few or no aspartic and glutamic acid residues (carboxylates) and lysine residues (primary amines) within the central portion of the primary sequence. Because peptides contain both carboxylate and amines, EDC conjugation results in their becoming variously polymerized and randomly oriented in their linkage to the carrier protein. Typically, this results in a high level of antigen loading on the carrier protein as well as presentation in all possible orientations for antibody production. However, important (desired) epitopes within the antigen peptide sequence may be blocked by EDC-mediated conjugation if those regions contain primary amines (lysine residues) or carboxylates (aspartic and glutamic acid residues). In these cases, either use a homobifunctional amine-reactive crosslinker with the purified BSA in phosphate buffer or synthesize the peptide with a unique terminal cysteine and use a kit with Malemide-Activated BSA to prepare the carrier protein conjugate.
- 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 carboxyl groups to 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 BSA and Conjugation Kit – for conjugating cysteine peptides
Adjuvants and other Carrier Proteins