Thermo Scientific Imject Maleimide-Activated Ovalbumin is sulfhydryl-reactive ovalbumin (OVA) for hapten-carrier conjugation with cysteine-containing peptides and other thiolated antigens.
This product consists of purified, carrier-grade ovalbumin that has been modified with SMCC crosslinker to attach maleimide groups capable of forming covalent crosslinks with sulfhydryl (-SH) moieties on cysteine residues of peptides and other molecules. Immunogens are easily prepared from peptide antigens that have been engineered with a terminal cysteine residue as a precise conjugation point. Several antigen molecules can be attached per carrier protein molecule without blocking intended epitopes by crosslinking to primary amines or carboxylates within the peptide sequence.
- Maleimide-Activated Ovalbumin, supplied lyophilized in PBS (pH 7.2) with stabilizer
- Activated with 5 to 15 moles of maleimide per mole of ovalbumin
- Ideal for conjugating cysteine-containing peptide antigens to make immunogen or screening target
- Conjugation reaction complete in 2 hours; results in stable, covalent thioether bonds
- Single polypeptide protein with molecular mass of 45,000 Daltons
- 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
|Summary of OVA carrier protein activation and conjugation to peptide antigen. Maleimide-Activated Ovalbumin is supplied lyophilized in the form shown at the center of the diagram. Addition of peptides or other molecules containing reduced sulfhydryl groups results in efficient and specific reaction to form stable thioether bonds. Each carrier protein molecule contains several maleimide activations.
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 45kDa protein is sufficiently large and complex to be mildly immunogenic. Consequently, OVA can be used as a carrier protein for conjugation to haptens and other antigens to make them more immunogenic for immunization. If not commonly used to prepare the primary immunogen, OVA is a popular carrier protein to make antigens more amenable to analysis (e.g., screening assays involving plate coating) of antibodies that were produced using KLH or other more immunogenic carrier protein.
Imject Ovalbumin 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, some of which have been modified to make Maleimide-Activated Ovalbumin.
- 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 the maleimide-activation chemistry.
- Schofield, L., et al. (2002). Synthetic GPI as a candidate anti-toxic vaccine in a model of malaria. Nature 418:785-789.
- Mahnke, K., et al. (2003). Induction of CD4+/CD25+ regulatory T cells by targeting of antigens to immature dendritic cells. Blood 101:4862-4869.
Review of antibody production and immunogen preparation methods
Review of sulfhydryl-reactive (maleimide) crosslinker chemistry
Carrier protein activation and conjugation data for immunogen preparation
Sulfo-SMCC – crosslinker for activating ovalbumin amines to make them sulfhydryl-reactive
SulfoLink Immobilization Kit for Peptides – for preparing an affinity column with a cysteine peptide
Ovalbumin (non-activated OVA) – for hapten conjugation through amines or carboxylates
Keyhole Limpet Hemocyanin (KLH) and other Carrier Proteins