Hapten carboxyl

Table 3 Conjugation of a carboxyl-containing hapten to a protein...

Alternatively, competitive ELISA can be used to estimate the hapten density if an antibody that specitically recognizes the hapten is available. At first observation this approach seems circular because the immunoassay developed is used to determine hapten density on proteins used for immunization. However, if a small molecule mimic of the protein conjugate is used as a standard, the method can be accurate. For example, a hapten containing a carboxylic acid can be coupled to phenethylamine or tyramine, its structure confirmed and the material used to generate a calibratron curve to estimate hapten density. 

NOTE The carboxylic acid group of the hapten was coupled to bovine serum albumin... 

The reactions involved in an EDC-mediated conjugation are discussed in Chapter 3, Section 1.1 (Note EDC is l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride MW 191.7 and is sometimes referred to as EDAC). The carbodiimide first reacts with available carboxylic groups on either the carrier or hapten to form a highly reactive o-acylisourea intermediate. The activated carboxylic group then can react with a primary amine to form an amide bond, with release of the EDC mediator as a soluble isourea derivative. The reaction is quite efficient with no more than 2 hours required for it to go to completion and form a conjugated immunogen. 

Conjugation chemistry for the coupling of haptens to carrier molecules is fairly well defined for compounds having common functional groups to facilitate such attachment. The types of functional groups generally useful for this operation include easily reactive components such as primary amines, carboxylic acids, aldehydes, or sulfhydryls. 

The X-ray crystal strudures of antibody 5C8 in complex with piperidine-N-oxide hapten 4 and piperidinium hapten 5 were determined at 2.0 A resolution.1151 In the 5C8 5 complex, two carboxylate residues (AspH95 and AspHlOl) flank the cationic... 

An example of esterase behaviour is provided by a catalytic antibody developed by Tramontano et al. (1988), using a phosphonate transition state analogue [53] as the hapten. The antibody cleaves the carboxylic ester [54, R = Me] with enzyme-like efficiency (kc/ku = 6.25 X 106 = 1.5 mM ... 

Fig. 7 Using the bait and switch principle, hapten [7] elicited an antibody, 43D4-3D12, which catalysed the -elimination of [8] to a trans-ene-one [9], The carboxyl function in [7] is necessary for its attachment to the carrier protein.

In order to generate antibodies which catalyse the hydrolysis of carbonates (6, 10), carboxylic esters (9) and amides with a certain degree of specificity, the phosphates (7a. lOai and phosphonates 9a were used as haptens that mimic the tetrahedral negatively charged transition state of the spontaneous hydrolysis reaction (see Scheme 11.3) [27] [29]. 

Phosphonates have been widely used as analogues of carboxylic acids. They have been particularly effective as analogues of tetrahedral transition states that occur in the course of enzyme-catalyzed reactions such as hydrolysis of the amide (peptide) bond. As such, they may be used as inhibitors of enzymes (e.g., 82, 83) or as haptens for producing antibodies that are catalytic (e.g., 84). A notable example is H203P— CH2—CH2—CH(—NH2)—COOH, which has effects that are likely to be due to its interference with glutamate as a neurotransmitter (85). 

Functional groups on haptens that are mostly used for linkage are the amino and carboxyl groups. As a result, the most common linkages are peptide bonds between a carboxyl and an amino group. Other groups that can be also used for... 

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