Keyhole limpets

Ki Antagonist binding affinity Ki67 Nuclear membrane antigen KLH Keyhole limpet haemocyanin K , Michaelis constant KOS KOS strain of herpes simplex... 

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. 

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

In this method the keyhole limpet haemoglobin conjugate was prepared as follows Keyhole limpet haemocyanin (KLH, Calbiochem, La Jolia, CA) and bovine serum albumin (BSA, BDH Chemicals) were coupled to the adduct (2), derived from 6-bromohexanoic acid and monoquat (3), via a carbodiimide reaction, as reported previously by Niewola et al. [184], The resulting conjugates contained 662mol of Paraquat per mole of KLH and 15mol of Paraquat per mole of 6-bromohexanoic acid. The amount of Paraquat bound to the protein was determined by spectrophotometric dithionite assay for Paraquat and the protein concentration was established by a standard Lowry test. 

KLH Keyhole limpet haemocyanin, used for its excellent antigenic properties. It is used as a carrier protein in order to bestow immunogenicity in small haptens. 

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. 

Maeda, S., Morikawa, A., Kato, M., Motegi, Y., Shigeta, M., Tokuyama, K., Kuroume, T., Naritomi, Y., Suehiro, K., Kusaba, K., Minoshima, M., and Iwata, S. (1991). Eleven cases of anaphylaxis caused by grand keyhole limpet (abalone-Uke shellfish). Arerugi 40, 1415-1420. 

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. 

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