Keyhole limpet hemocyanin (KLH) immunogenic carrier protein and easy conjugation kit for amine- and carboxyl-containing peptide antigens.

Thermo Scientific Imject Mariculture KLH and the accompanying EDC Conjugation Kit are convenient packages of purified keyhole limpet hemocyanin (KLH) carrier protein that enable simple preparation of highly effective immunogens with peptides and other haptens for immunization and antibody production.

Mariculture KLH (mcKLH) is purified and lyophilized in buffers that optimize its stability and solubility for hapten conjugation. The large, complex protein contains hundreds of sites per molecule for effective conjugation of peptides and other antigens using amine-reactive or carboxyl-reactive crosslinkers.

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 KLH carrier protein, optimized reaction buffers and trouble-free spin desalting columns for immunogen preparation using the EDC method.

Highlights:

• Convenient kit – contains lyophilized mcKLH (in MES buffer), EDC crosslinker and accessories components to easily prepare ready-to-use immunogens for injection
• High-yield conjugation – each molecule of KLH contains hundreds of primary amines available for coupling haptens via EDC or NHS ester crosslinkers
• Validated quality – purified and stabilized mariculture KLH maintains solubility in aqueous solutions, unlike traditional sources of the carrier protein
• Sustainable source – KLH is harvested from select populations of the mollusk Megathura crenulata (keyhole limpet) that are grown in mariculture, not extracted from wild populations
• Highly immunogenic – KLH has a high molecular mass (4.5 x 10⁵ - 1.3 x 10⁷ Daltons; aggregates of 350 and 390 kDa subunits) and elicits a stronger immune response than BSA or ovalbumin

Product Details:

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.

Keyhole limpet hemocyanin (KLH) is widely used as a carrier protein for conjugation to haptens and other antigens to make them more immunogenic for the purpose of antibody production. Because of its large mass and complexity, KLH elicits a stronger immune response than other carrier proteins. Because the protein is derived from a mollusk, it is phylogenetically distant from mammalian species and less likely to produce antibodies that will cross-react with typical target samples in assays.

Imject Mariculture KLH (mcKLH) is KLH that has been harvested from limpets grown in mariculture rather than captured from the wild. Imject mcKLH provides the highest immunogenicity and good solubility. As a large protein, KLH has hundreds of primary amines and carboxyl groups that can be targeted for conjugation with NHS-ester, EDC, and other crosslinking reagents.

High-solubility KLH carrier protein. Thermo Scientific Imject mcKLH is purified and lyophilized in buffers that optimize its stability and solubility. After reconstitution, the suspension-solution is an opalescent blue, which is characteristic of highly purified, nondenatured protein with intact heme groups.

The carbodiimide crosslinker EDC conjugates carboxyl-containing haptens (e.g., C-terminus of peptide antigens) to the highly immunogenic KLH carrier protein for immunization and antibody production. 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 mcKLH in phosphate buffer or synthesize the peptide with a unique terminal cysteine and use a kit with Malemide-Activated mcKLH 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.

References:

1. Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. (Product #15050). Chapter 5 discusses the use of carrier proteins.
2. Hermanson, G.T. (2008). Bioconjugate Techniques. 2nd edition, Academic Press, New York. (Product #20036). Chapter 19 discusses carrier protein uses and immunogen preparation.
3. Wajih, N., et al. (2004). The inhibitory effect of calumenin on the vitamin K-dependent-carboxylation system. J. Biol. Chem. 279:25276-25283.
4. Mitrofanova, E., et al. (2004). Rat sodium iodide symporter for radioiodide therapy of cancer. Clin. Cancer Res. 10:6969-6976.
5. Shibata, T., et al. (2002). 15-deoxy-delta12,14-prostaglandin J2, a prostaglandin D2 metabolite generated during inflammatory processes. J. Biol. Chem. 277:10459-10466.
6. Barrett, J.W., et al. (2007). M135R is a novel cell surface virulence factor of myxoma virus. J. Virol. 81:106-114.
7. Jerry, D.J. (1993). Monitoring coupling of peptides to carrier proteins using biotinylated peptide. Biotechniques 14(3):464-469.
8. Comer, F.I. (2001). Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Anal. Biochem. 293:169-177. (Glutaraldehyde-conjugation)
9. Gembitsky, D.S., et al. (2004). A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation. Mol. Cell. Proteomics 3:1102-1118. (Glutaraldehyde-conjugation)
10. Tarp, M.A. (2007). Identification of a novel cancer-specific immunodominant glycopeptide epitope in the MUC1 tandem repeat. Glycobiology. 17:197-209. (Glutaraldehyde-conjugation)
11. Shibata, T., et al. (2002). 15-deoxy-delta12,14-prostaglandin J2, a prostaglandin D2 metabolite generated during inflammatory processes. J. Biol. Chem. 277:10459-10466. (EDC conjugation)
12. Kawai, Y., et al. (2006). Formation of N-(succinyl)lysine in vivo: a novel marker for docosahexaenoic acid-derived protein modification. J. Lipid Res. 47:1386-1398. (EDC-modification method).

Related Products and Resources:

Protein Methods Library article – Antibody production and immunogen preparation
Example Product Data – Activation, conjugation and immune-response data for Imject Carrier Proteins

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 KLH and Conjugation Kit – for conjugating cysteine peptides
Adjuvants and other Carrier Proteins