Keyhole limpet hemocyanin,

Dissolve the protein to be modified at a concentration of 1-10 mg/ml in 0.1 M sodium phosphate, pH 7.4. NaCl may be added to this buffer if desired. For the modification of keyhole limpet hemocyanin (KLH Thermo Fisher) as described by Staros et al., 1986, include 0.9 M NaCl to maintain the solubility of this high-molecular-weight protein. If lower or higher concentrations of the protein are used, adjust the amounts of the other reactants as necessary to maintain the correct molar ratios. 

The most common carrier proteins in use today are keyhole limpet hemocyanin (KLH MW 4.5 X 105 to 1.3 X 107), BSA (MW 67,000), aminoethylated (or cationized) BSA (cBSA), thyroglobulin (MW 660,000), ovalbumin (OVA MW 43,000), and various toxoid proteins, including tetanus toxoid and diphtheria toxoid. Other proteins occasionally used include myoglobin, rabbit serum albumin, immunoglobulin molecules (particularly IgG) from bovine or mouse sera, tuberculin purified protein derivative, and synthetic polypeptides such as poly-L-lysine and poly-L-glutamic acid. 

In rodents, sheep red blood cells (SRBC) are routinely used for immunization. The antibody response is determined using the plaque forming cells assay (PFC) or by plasma SRBC-specific antibody titer [21, 22], As an alternative to SRBC, other T-cell-dependent antigen may be used, including keyhole limpet hemocyanin (KLH), tetanus toxoid (TT), or pneumococcal antigen. 

Serum antibody responses of B lymphocytes can be assessed either by vaccination with a potent primary antigen such as keyhole limpet hemocyanin (KLH), or by boosting with a recall antigen such as tetanus toxoid. This results in either a primary or secondary antibody response in normal individuals, which is often reduced or absent in AIDS patients. 

Since small haptens are not immunogenic -i.e. do not usually stimulate the formation of antibodies-, they must be coupled to a carrier protein. For instance, Schultz [22b] described the coupling of the hapten to the carrier proteins bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), in order to generate antibodies for his specific studies. 

Lymphocytes Streptokinase Phytohemoagglutin Tuberculin (PPD) Keyhole limpet Hemocyanine Human y-globulin Tuberculin... 

For keyhole limpet hemocyanine (KLH) both antibody responses and delayed type hypersensitivity (DTH) reactions can be determined [43—45]. In addition several infectious models, including bacterial, viral and parasitic infections may be used to challenge the immune system [18,46]. As survival and eradication of the infections is the primary function of the immune system, these models provide direct information on the functional status of the immune system. Direct immunotoxic compounds will induce immunosuppression and thus an increase in infection rate and/or severity of the infection. The number of infectious agents (bacteria, parasites, or viral colonyforming units), increased morbidity and mortality are indications for an immunotoxic effect. Also a reduction in specific antibody levels in animals treated with the test compound compared to nontreated controls indicates immunosuppression. 

Figure 13.12 A synthetic vaccine bearing multiple carbohydrate antigens KLH, keyhole limpet hemocyanin.

Immunocyanin is a stable modification of keyhole limpet hemocyanin, a non-heme, oxygencarrying copper protein found in arthropods and mollusca. It is an aspecific activator of both cellular and humoral reactions. Immunocyanin is used for the local treatment of bladder cancer. Its systemic adverse effects are usually limited to some mild fever. 

Riggs DR, Jackson B, Vona-Davis L, McFadden D. In vitro anticancer effects of a novel immunos-timulant keyhole limpet hemocyanin. J Surg Res 2002 108(2) 279-84. 

Therapeutic vaccines were tested in BALB/c mice bearing TA3-Ha mammary carcinoma. The treatment consisted of 4 subcutaneous injections, at 3-6 days intervals, of Detox [a commercial preparation of cell wall skeletons from Mycobacterium phlei and non-toxic monophosphoryl lipid A from Salmonella minnesota (S. minnesota) in squalane oil and Tween 80 from Ribi Immunochemical research, Montana, USA] mixed with Thomsen-Friedenreich (TF) antigen coupled with KLH (Keyhole Limpet Hemocyanin) performed 5 days after the tumor cell injection. This vaccination achieved the survival of 25 % of the mice. Pretreatment of mice with cyclophosphamide in order to inhibit any suppressive response, increased survival to 50 % when the treatment began 5 days after tumor cell injection, and to 90 % when the treatment began 2 days after tumor cell injection. Both antibody as well as delayed-... 

Keyhole limpet hemocyanin (KLH) is an immune stimulant and a hapten carrier, derived from a circulating glycoprotein of the marine mollusk Megathura crenulata. KLH has significant antiproliferative effects in vitro against several types of cancers (23). It has also been conjugated to a variety of immunogens and used as a vaccine adjuvant (24). 

McFadden DW, Riggs DR, Jackson BJ, et al. Keyhole limpet hemocyanin, a novel immune stimulant with promising anticancer activity in Barrett s esophageal adenocarcinoma. Am J Surg 2003 186(5) 552-555. 

Geyer, H., Wuhrer, M., Kurokawa, T. and Geyer, R. (2004) Characterization of keyhole limpet hemocyanin (KLH) glycans sharing a carbohydrate epitope with Schistosoma mansoni glycoconjugates. Micron 35, 105-106. 

Kantelhardt, S.R., Wuhrer, M., Dennis, R.D., Doenhoff, M.J., Bickle, Q. and Geyer, R. (2002) Fuc(alpha1-3) GalNAc- major antigenic motif of Schistosoma mansoni glycolipids implicated in infection sera and keyhole limpet hemocyanin cross-reactivity. Biochemical Journal 366, 21 7-223. 

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