Thermo Scientific Imject OVA is purified ovalbumin protein conveniently formulated for preparation of hapten-carrier conjugates.
Because ovalbumin is only weakly immunogenic, its primary use in antibody production workflows is as a secondary (independent) carrier protein to make antigens more amenable to antibody screening procedures. For example, peptide antigens are more easily coated in polystyrene microplates when conjugated to ovalbumin, thereby facilitating the screening of antisera that were produced using immunogen conjugates with KLH or other more immunogenic carrier protein.
- Purified from chicken egg whites and lyophilized in phosphate buffered saline
- Single polypeptide protein with molecular mass of 45,000 Daltons
- Contains 20 lysine residues, most of which have primary amines that are capable of reacting with a conjugation reagent
- Contains 14 aspartic acid and 33 glutamic acid residues whose carboxyl groups account for the net negative charge (pI 4.63) of OVA
- Not as immunogenic as keyhole limpet hemocyanin (KLH) but more soluble in DMSO (dimethyl sulfoxide) for applications that require this solvent
- Useful 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 conjugation with the carrier can be achieved.
Ovalbumin (OVA), the most abundant protein in chicken egg whites, is a convenient protein for a variety of uses in the laboratory. The 45 kDa protein is not as immunogenic as other proteins, such as keyhole limpet hemocyanin (KLH). However, OVA's independent structure make it especially useful as a carrier for peptides and other small antigens that by themselves are difficult to manipulate in typical screening assays such as ELISA. Although it is more hydrophobic and less soluble in aqueous buffers than BSA, ovalbumin remains soluble in up to 70% DMSO, making it useful for conjugation with haptens that require DMSO for solubility. Ovalbumin contains numerous primary amines and carboxyl groups that can be targeted for conjugation with glutaraldehyde, NHS-ester, EDC and other crosslinking reagents.
- 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 Ovalbumin – for conjugating peptides and haptens via cysteine or sulfhydryl groups
Adjuvants and other Carrier Proteins