Thermo Scientific Imject Maleimide-Activated Blue Carrier products enable conjugation of sulfhydryl-containing peptide antigens to this soluble immunogenic carrier protein for immunization and effective antibody production.
Blue Carrier* Protein is a large hemocyanin that is highly immunogenic like keyhole limpet hemocyanin (KLH) yet is more soluble. The carrier is rendered sulfhydryl-reactive by chemical modification with Sulfo-SMCC crosslinker, and the resulting maleimide-activated protein is then stabilized and lyophilized in phosphate buffer. Maleimide groups form covalent crosslinks with sulfhydryl (-SH) moieties on cysteine residues of peptides and other thiol-containing haptens. Numerous antigen molecules can be conjugated to each Blue Carrier Protein molecule to make them highly immunogenic and increase the likelihood of eliciting immunized animals to produce high-titer antisera to the epitopes of interest.
- Optimized activation – specifications require that each lot is activated with more than 400 moles of sulhydryl-reactive maleimide per mole of carrier, ensuring consistently high levels of conjugation
- Sulfhydryl-reactive – engineered for conjugating cysteine-containing peptide antigens to make effective immunogens for high-titer antibody production
- Simple, rapid procedure – complete the hapten-carrier conjugation reaction in two hours to yield sufficient immunogen for several injections and booster immunizations of host animals
- High-quality carrier protein – Blue Carrier Protein is ultrapure Concholepas concholepas hemocyanin (CCH) prepared from molluscan hemolymph
- Highly immunogenic and soluble – CCH has a high molecular mass that elicits a stronger immune response than BSA or ovalbumin
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 an adequate level of hapten-carrier conjugation can be achieved.
Blue Carrier* Protein is specially purified hemocyanin from the mollusk Concholepas concholepas. The CCH protein is composed of two very large polypeptide subunits (404/351 kDa) that form an extremely stable heterodidecameric structure even in the absence of divalent cations. (By contrast, KLH has a less stable and soluble homodidecameric structure). The complex molecular arrangement of CCH subunits contains diverse repeated antigenic structures that elicit a strong immune reaction mediated by T and B lymphocytes.
Because of their large size and molecular complexity, KLH and CCH hemocyanins are carrier proteins of choice for use as immunogens to produce antibodies against haptens and peptides. Moreover, studies suggest that the strong DTH immune response elicited by hemocyanins in animals and in humans has beneficial therapeutic effects in certain types of cancer. New developments in the immunotherapy of cancer have taken advantage of the unique immunogenic properties of hemocyanins in the development of novel conjugate vaccines for treatment of emerging diseases.
|Structure and conjugation reaction scheme of Thermo Scientific Imject Maleimide-Activated Blue Carrier Protein. High-quality protein is modified with Sulfo-SMCC crosslinker to produce the maleimide-activated carrier protein, which is supplied lyophilized. 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 hundred maleimide activations, allowing conjugation of many peptide hapten molecules per immunogenic Blue Carrier* Protein molecule.
Efficient peptide conjugation and high immunogenicity. Comparisons of conjugation efficiency (left) and immunogenicity (right) among three maleimide-activated carrier protein formulations: Thermo Scientific Imject mcKLH, PEGylated mcKLH and Blue Carrier* Protein. Conjugation data are graphed as the percent of mcKLH, where error bars are the standard deviations for the two or three replicates of each condition. Immunization data are the means of 2 rabbits with each condition.
- 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.
Review of antibody production and immunogen preparation methods
Review of sulfhydryl-reactive (maleimide) crosslinker chemistry
Carrier protein activation and conjugation data for immunogen preparation
Maleimide-Activated KLH and Conjugation Kit ===== Looking for KLH or a convenient kit?
Sulfo-SMCC – crosslinker for activating BSA amines to make them sulfhydryl-reactive
SulfoLink Immobilization Kit for Peptides – for preparing an affinity column with a cysteine peptide
Blue Carrier Protein (non-activated) – for hapten conjugation through amines or carboxylates
Other Carrier Proteins