Blue Carrier* Protein and accompanying Thermo Scientific EDC Conjugation Kit are packages of the highly-soluble, mollusk-derived hemocyanin that enable simple preparation of effective immunogens with amine- or carboxyl-peptide antigens.
Blue Carrier Protein is a purified preparation of Concholepas concholepas hemocyanin (CCH) that confers high immunogenicity to conjugated antigens for injection and immunization in antibody production procedures. The large protein exhibits most of the same immunogenic properties as the popular carrier protein, keyhole limpet hemocyanin (KLH). However, its signficantly better solubility provides greater flexibility in immunogen preparation protocols by allowing a broader range of buffer and pH conditions for coupling peptides, proteins and other haptens using classical crosslinkers such as glutaraldehyde, Sulfo-SMCC and EDC. The accompanying Thermo Scientific Imject EDC Blue Carrier Spin Kit provides for easy immunogen preparation.
- Convenient kit – contains lyophilized Blue Carrier Protein (in MES buffer), EDC crosslinker and accessories components to easily prepare ready-to-use immunogens for injection
- High-yield conjugation – each molecule of CCH contains hundreds of primary amines available for coupling haptens via EDC or NHS ester crosslinkers
- Validated quality – the immunogenic carrier protein is specially purified to ensure uniform chemical structure and solubility for consistent performance
- Superior solubility – increased solubility compared to KLH allows Blue Carrier Protein to be used in a broader range of conjugation protocols to accommodate unusual or poorly soluble haptens
- Highly immunogenic – provides essentially the same level of immunogenicity and phylogenetic distance as keyhole limpet hemocyanin (KLH)
Properties of Blue Carrier Protein:
- Alternative names: Concholepas concholepas hemocyanin, CCH
- Molecular weight: Whole molecule: ~ 8,000,000; Subunits: ~ 400,000 and ~ 350,000
- Absorption: maxima 280nm, 350nm and 558nm
- Extinction Coefficient (A 1mg/mL solution): 1.4 @ 280nm; 0.35 @ 346nm
- A280/A350 Ratio: ~ 4.0
- Copper/protein ratio: ~ 16 Cu2+ per protein molecule
- Isoelectric point: 6.0
- Solubility: Soluble in aqueous buffers: pH 7.0-8.0
- Stability: Stable in aqueous buffers: ~ pH 7.2 without Ca2+ or Mg2+
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.
Immune response comparison to mariculture KLH carrier protein. Rabbits (n=2) were immunized with a synthetic peptide that had been coupled with Thermo Scientific Imject mcKLH or Blue Carrier* Protein using glutaraldehyde or EDC. Serum samples were collected immediately before immunization and 35 days post-immunization and then screened in a peptide-specific, direct ELISA procedure.
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 Blue Carrier in phosphate buffer or synthesize the peptide with a unique terminal cysteine and use a kit with Malemide-Activated Blue Carrier to prepare the carrier protein conjugate.
EDC-mediated conjugation of peptides and carrier proteins. Carrier proteins (C) and peptides (P) have both carboxyls and amines, so conjugation occurs in both orientations. Carrier proteins are very large in comparison to typical peptide haptens; therefore, numerous conjugation sites exist on each carrier protein molecule.
- De Ioannes, P., et al. (2004) Hemocyanin of the molluscan Concholepas concholepas exhibits an unusual heterodecameric array of subunits. Journal of Biological Chemistry. 279:26134-26142.
- Manosalva H., et al. (2004) Development of monoclonal antibodies bearing the internal image of the Gizzerosine epitope and application in a competitive ELISA for fish meal. Hybridoma and Hybridomics. 23:45-54.
- Oliva H., et al. (2002) Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits. Hybridoma and Hybridomics. 21:365-373.
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 Blue Carrier and Conjugation Kit – for conjugating cysteine peptides
Adjuvants and other Carrier Proteins