Antibody-hapten complexes

CD has been considered as a sensitive method for comparison of combining sites of antibodies with nitrophenylated haptens (345). With antinitrophenyl antibody fragments of rabbit y-immunoglobins, no significant differences in the far ultraviolet CD spectra with the antibody-hapten complex have been observed (346). 

The rate of the reverse reaction is (AL), where (AL) is the concentration of the antibody-hapten complex. The units of k2i are reciprocal seconds. If initial forward rates are measured with sufficient rapidity... 

Fig. 6 Binding site details for antibody 48G7 complexed with hapten p-nitrophenyl 4-carboxybutanephosphonate (Patten et al., 1996). N.B. Amino acid residues in antibodies are identified by their presence in the light (L) or heavy (H) chains with a number denoting their sequence position from the N-terminus of the chain.

A prominent advantage of this assay procedure is the feature that the complex of hapten and labeled antibody was captured on a solid phase (PMP) and separated from the reaction medium before signal determination. This additional step not only reduces interference due to biological specimens but also eliminates the tedious transfer of supernatant, which is essential in conventional immunometric assays. This immunometric assay provided somewhat improved specificity in terms of the cross-reactivities with T2 and reverse T3 (3,3, 5 -L-triiodothyronine). The authors speculated that the dissociation rate of the antibody-cross-reactant complex would be faster than that of an antibody-analyte complex thus the former binding would be preferentially substimted by T2 immobilized on CPG. 

Extracellular antigens are detected by APCs, such as lymphocytes, macrophages, and dendritic cells in interstitial fluid and blood. These detect the hapten and engulf the whole antigenic complex. Then, when inside the APC, the complex is partly dismantled and peptides attached to proteins similar to immunoglobulins, known as MHC II. The modified peptide-hapten complex is moved to the surface of the APC and presented as a complex with MHC to T-helper cells (CD4+), which activates and instructs the APC to make antibodies to the hapten and also B cells (memory cells with "memory" of the hapten) to proliferate. These events lead to types I to III responses. 

The MHC complex and antigen can then be detected such as by CD8 cytotoxic T cells and the cells destroyed. However, the fragments of the hapten complex can also be detected by other cells such as APC, and with the collaboration of T-helper cells, B cells would become activated and antibodies produced. These could then bind to the surface antigen as described for a type II reaction. 

The three-dimensional crystal structure was solved for the AZ-28 antibody Fab complexed with hapten (Ulrich et al., 1997 Ulrich and Schultz,... 

DeSilva, B. S., Orosz, G., Egodage, K. L., Carlson, R. G., Schowen, R. L., Wilson, G. S. (2000) Catalytic antibodies for complex reactions hapten design and the importance of screening for catalysis in the generation of catalytic antibodies for the NDA/CN reaction, Appl. Biochem. andBiotechn. 83, 195-208. 

Diffusion in agar performed by the conventional method shows a strong reaction between the antibody and Man-BSA, but not with BSA (Fig. 12, plates A). However, both compounds give precipitin complex with anti-BSA serum. Oxidation of the antigens with periodate no longer gives a precipitin reaction with the anti-mannose antibodies, but has no effect on the anti-BSA antibodies. Hapten-inhibition results are also shown in Fig. 12, plates C and D. These tests are conducted with the purified antibody... 

Antibody esterase 48G7 was elicited against hapten 1 and effectively catalyzed the hydrolysis of the corresponding activated ester 2 (27). The X-ray crystal structure of this catalytic antibody Fab complexed with 1 revealed the corresponding stabilization of the oxyanion by a nearby cationic Arg residue (27, 38). Hydrogen bonds from the side chains of the adjacent amino acids His and Tyr serve to stabilize the polarized phosphoryl bonds of hapten 1 that would assist in forming the transition state of ester 2. Main-chain amide bonds from Tyr and Tyr also provide additional hydrogen-bond stabilization forces. 

This analysis can be applied to enzymatic as well as to simple chemical transformations [9-11], for uni- and multi-substrate [12] reactions according to Eqs. (1) and (2). nNKM denotes the product of Michealis-Menten constants for all substrates. In this analysis one assumes that kinetics follow the Michaelis-Menten model, which is the case for most antibody-catalyzed processes discussed below. The kcat denotes the rate constant for reaction of the antibody-substrate complex, Km its dissociation constant, and kuncat the rate constant for reaction in the medium without catalytic antibody or when the antibody is quantitatively inhibited by addition of its hapten. In several examples given below there is virtually no uncatalyzed reaction. This of course represents the best case. 

Fig. 4. Conformational diversity in an antibody esterase. Whether the amino acid belongs to a heavy or light chain is indicated with the letter H or L before the sequence number, a) Superposition of the germ-line Fab-hapten complex (Fab=antigen-binding fragment of an antibody purple) and the affinity-matured Fab-hapten complex (red). Somatically mutated residues (X Y) are shown in green, b) Superposition of the structures of the germ-line Fab domain...

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