Hapten direct reaction with protein

Skin sensitization is believed to occur as a result of nickel binding to proteins (particularly on the cell surface) and hapten formation. Essentially, the body perceives the nickel-protein complex as foreign and mounts an immune reaction to it. For example, sweat may react with the nickel in plated jewelry that comes in direct contact with skin dissolved metal may penetrate and react with proteins in the skin and lead to immune sensitization. Nickel may substitute for certain other metals (especially zinc) in metal-dependent enzymes, leading to altered protein function. High nickel content in serum and tissue may interfere with both copper and zinc metabolism. It also readily crosses the cell membrane via calcium channels and competes with calcium for specific receptors. 

The synthesis of the protein conjugates is shown in Figure 3. Commercially available carboxylic acids were converted to activated esters by direct DCC (N,N-dicyclohexylcarbodiimide) coupling with N-hydroxysuccinimide or by conversion to the corresponding acid chloride derivative followed by reaction with N-hydroxysuccinimide. Reactiion of the resulting N-hydroxysuccinimide esters with either BSA or OVA led to the desired lysine bonded nitroaromatic hapten-protein conjugates. 

In the two-site assay, the antigens bind to the immobilized antibodies and in the second step labeled antibodies bind to second binding sites of the antigen. This approach is applicable only to large molecules such as proteins (Scheme IB). In another variation the hapten is immobilized and competes with the sample hapten for the reaction with the labeled antibodies (Scheme 1C). For the determination of antibodies a so-called sandwich or double antibody assay can be used. The hapten to which the antibodies bind is immobilized and in the second step second labeled antibodies (anti-immunoglobulin), which are directed against the antibodies to be determined, are added (Scheme ID). In another variation, labeled haptens are added after binding of the sample antibody to the immobilized hapten (Scheme IE). 

Carrier protein Macromolecule to which a hapten is conjugated, thereby enabling the hapten to stimulate the immune response. catELISA Similar to an ELISA, except that the assay detects catalysis as opposed to simple binding between hapten and antibody. The substrate for a reaction is bound to the surface of the microtitre plate, and putative catalytic antibodies are applied. Any product molecules formed are then detected by the addition of anti-product antibodies, usually in the form of a polyclonal mixture raised in rabbits. The ELISA is then completed in the usual way, with an anti-rabbit second antibody conjugated to an enzyme, and the formation of coloured product upon addition of the substrate for this enzyme. The intensity of this colour is then indicative of the amount of product formed, and thus catalytic antibodies are selected directly. 

Dissolve up to 4 mg of the peptide or hapten to be coupled in 1 ml of the reaction buffer chosen in step 1. If the peptide to be coupled is already in solution, it may be used directly if it is in a buffer containing no other amines or carboxylic acids and is at a pH between 4.7 and 7.2. Note If an assessment of the degree of peptide coupling is desired, measure the absorbance at 280 nm of the 1 ml peptide solution before proceeding to step 3. In some cases, a dilution of the peptide solution may be necessary to keep the absorbance on scale for the spectrophotometer. If the peptide is sparingly soluble in aqueous solution, it may be dissolved in DMSO and an aliquot added to the carrier solution. See the previous discussion on carrier proteins to determine the levels of DMSO compatible with carrier protein solubility. 

With photoallergic reactions, cytopathologic events are believed to be even more complex than with direct phototoxicity. Although many mechanistic features remain obscure, fundamental concepts include the photoactivation of endogenous or xeno-biotic haptens so that they combine with cellular proteins and form a complete antigen. Subsequent immunologic reactions, especially cell-mediated hypersensitivity, complete the sensitivity process. 

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