Keyhole limpet hemocyanin (KLH) is a potent, safe immunostimulating protein with an extensive history of use in vaccine development and immunological research.
This vitally important protein can only be derived from a limited natural resource – the Giant Keyhole Limpet (Megathura crenulata) which naturally lives only in the Pacific coastal waters, from Monterey Bay, California to Isla Asuncion off Baja California, Mexico.
KLH is well known for its potent and safe immune-stimulating properties, with an extensive (40+ years) history of use in immunology....as a vaccine carrier molecule, as a standard assay for measuring humoral and cellular immune function, and as an essential tool for immunotoxicology screening of new drugs.
KLH is a very large, high molecular weight, oxygen-carrying metalloprotein from the hemolymph of the marine mollusc Megathura crenulata.
The KLH protein is expressed in two subunit isoforms (KLH1 and KLH2) of approximately 360,000 and 400,000 monomeric molecular weight. The KLH monomers are each composed of 7 or 8 functional unit domains; each functional unit contains an oxygen binding site containing two copper atoms.
Both KLH isoforms assemble into native homo-decamers and -didecamers of 4,000,000 to 8,000,000 molecular weight in hemolymph.
KLH's immunogenicity is partly attributed to the presence of unusual carbohydrate moieties, including unique Fuc(alpha1-3)GalNAc, Gal-(beta1-6)Man-, Gal(beta1-4)Fuc-, and Gal(beta1-4)Gal(beta(1-4)Fuc- structural motifs on N-glycans.(1,2,3) Terminal mannose residues are mannose receptor ligands and fucose residues are DC-SIGN ligands, suggesting that endocytosis of KLH via C-lectin receptors for antigen presentation in KLH-based conjugate vaccines. KLH has been reported to stimulate dendritic cell maturation from human monocytes in processes possibly involving the mannose receptor.(4)
Size (and Source) Matters
Due to its exceptional size and unusual glycosylation, KLH has not been reproduced synthetically, and is more efficiently and cost-effectively prepared by purification from the hemolymph of the source animal.
Studies have shown that different forms of KLH have distinctly different structural properties and, importantly, may have different inductive effects on the immune system.(5,6,7,8,9,10,11,12,13,20)
Further, studies have reported that the culture, handling and processing of KLH can impact isoform structure and immunogenicity.(14,15,16)
Different forms and supply sources of KLH have been associated with differing magnitudes and variability in IgM and IgG responses.(17,18,19)
 Kantelhardt SR, et al. Fuc(α1à3)GalNAc-: the major antigenic motif of Schistosoma mansoni glycolipids in infection sera and keyhole-limpet haemocyanin cross-reactivity.
 Kurokawa T, et al. Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N-glycans with Gal(β1-6)Man-motifs. Euro J Biochem 269(22):5459-5473; 2002.
 Wuhrer M, et al. A novel Gal(beta1-4)Gal(beta1-4Fuc(alpha1-6)-core modification attached to the proximal N-acetylglucosamine of keyhole limpet haemocyanin (KLH) N-glycans. Biochem J 378(2):625-632; 2004.
 Presicce P, et al. Keyhole limpet hemocyanin induces the activation and maturation of human dendritic cells through the involvement of mannose receptor. Molecular Immun 45(4):1136-1145; 2008.
 Swerdlow RD, et al. Keyhole limpet hemocyanin: Structural and functional characterization of two different subunits and multimers. Comp Biochem Physiol 113B(3):537-548; 1996.
 Amkraut AA, et al. Immunogenicity of hemocyanins and their subunits. J Immunol 103:1301-1310; 1969.
 Bartel AH, et al. Some immunochemical differences between associated and dissociated hemocyanin. Arch Biochem Biophys 82:232-234; 1959.
 Green AA, et al. In vitro response of human leukocytes to associated and dissociated hemocyanin. J Immunol 107:293-297; 1971.
 Herscowitcz HB, et al. Immunochemical and immunogenic properties of a purified keyhole limpet hemocyanin. Immunology 22:51-61; 1972.
 Markl J, et al. The role of two distinct subunit types in the architecture of keyhole limpet hemocyanin (KLH). Naturwiss 78:512-514; 1991.
 Vandenbark AA, et al. All KLH preparations are not created equal. Cell Immunol 60:240-243; 1981.
 Harris JR, et al. Keyhole limpet hemocyanin (KLH): a biomedical review. Micron 30:597-623; 1999.
 Miller JS, et al. Diminished neo-antigen response to keyhole limpet hemocyanin (KLH) vaccines in patients after treatment with chemotherapy or hematopoietic cell transplantation. Clin Immun 117:144-151; 2005.
 Oakes FR, et al. The effect of captivity and diet on KLH isoform ratios in Megathura crenulata. Comp Biochem Physiol 138A:169-173; 2004.
 Harris JR, et al. Keyhole limpet hemocyanin: negative staining in the presence of trehalose. Micron 26:25-33;1995.
 Gerbauer W, et al. Quaternary structure, subunits and domain patterns of two discrete forms of keyhole limpet hemocyanin: KLH1 and KLH2. Zool 98:51-68; 1994.
 Herscowitz, Harold, and Stavitsky. Immunochemical and immunogenic properties of a purified keyhole limpet haemocyanin. Immunol. 22: 51-61; 1972.
 Miller, Curtsinger, Berthold, Malvey, Bliss, Le, Fautsch, Dudek, Blazar, and Panoskaltsis-Mortari. Diminished neo-antigen response to keyhole limpet hemocyanin (KLH) vaccines in patients after treatment with chemotherapy or hematopoietic cell transplantation. Clin. Immunol. 117: 144-151; 2005.
 Lebrec, Hock, Sundsmo, Mytych, Chow, Carlock, Joubert, Reindel, Zhou, and Bussiere. T-cell Dependent Antibody Responses in the rat: Forms and sources of keyhole limpet hemocyanin matter. J of Immunology 11 (3): 213-221; July-Sep 2014.
 Milgrom, Kesler, Byron, Harbeck, Holliday, and Leung. Response to cutaneous immunization with low-molecular-weight subunit keyhole limpet hemocyanin. Int Arch Allergy Immunol. 157 (3):269-274; Feb 2012.