Recombination of H and L chains

This chapter describes methods of separating and recombining H and L chains and considers some of the physical and biochemical properties of the individual chains. The contribution of each chain to the antigen-binding capacity and to the formation of an active site will be discussed. Other topics included are the preferential recombination of H and L chains derived from the same molecule, such as a myeloma protein or antibody from an individual animal, the separation and recombination of univalent half-molecules, each comprising a complete L and H chain, and the formation of hybrid antibody molecules through recombination of half-molecules of different specificity. Interactions of chain segments, produced by proteolysis, are also considered. 

Recovery of Antigen-Binding Activity of Specifically Purified Rabbit Anti-/7-Azobenzoate IgG Antibody after Recombination of H and L Chains Isolated from the Antibody of an Individual Rabbit"... 

In view of the heterogeneity of ordinary rabbit antihapten antibodies, it seems quite remarkable that as much as half of the activity was restored one might expect that random recombination of H and L chains, from molecules with different structure but having the same specificity, might yield inactive recombinants. The answer is that recombination does not occur at random there is preferential pairing of autologous chains (see below). 

Recombination of H and L Chains from Antibodies of THE Same Specificity but from Different Animals... 

A. Capacity of H and L Chains to Recombine through Noncovalent Interaction... 

Data described so far were based on qualitative precipitin tests in agar gel. Quantitative studies were carried out to assess the capacity of isolated H and L chains to react with rabbit anti-D antibodies directed to a human IgG myeloma protein having L chains of the k type (76). Substantial inhibitory activity was observed in an autologous recombinant, comprising H and L chains of the immunogen, but not in isolated L chains or in recombinants in which only one of the chains was derived from the immunogen. Very similar results were obtained with rabbit anti-D antibodies to two other monotypic proteins of the k type, one IgG and one IgM (99). 

When the IgG molecule or its Fab fragment is exposed to a reducing agent in the presence of 6 M guanidine hydrochloride, all disulfide bonds are reduced, native conformation is lost, and the separated chains acquire the configuration of a random coil (17). Despite this complete loss of ordered structure a substantial amount of antibody activity can be recovered if the H and L chains are allowed to renature and recombine under appropriate conditions (17,17a). Since this occurs in the absence of antigen, these important experiments provided the first conclusive evidence that antibody specificity is determined by primary amino acid sequences, which, in turn, specify the final tertiary structure of the folded molecule. 

If the H and L chains are isolated from aAtibody of the same specificity, but from different animals of the same species will they recombine to form molecules with antibody activity ... 

In this section we will describe experiments in which recombinants were prepared from H and L chains derived from antihapten antibody of the same specificity but from different rabbits. Results of two such investigations by Roholt et al. (24,29) are summarized in Table 6.2. It is evident that little antibody activity was restored in recombinants in which the H and L chains came from different rabbits, despite the fact that both chains were isolated from specifically purified anti-p-azoben-zoate antibodies. A large fraction of the initial activity was restored in autologous recombinants. Roholt et al. obtained very similar results when Fd fragments were substituted for intact H chains (29). 

Antibody preparations were reduced and alkylated, dialyzed against I M propionic acid, and passed through Sephadex G-lOO in that solvent to separate H and L chains. Appropriate pairs of chains were mixed at low pH. Recombination occurred when the mixtures were dialyzed against a buffer at neutral pH. 

We will use the phrase autologous chains to denote H and L chains derived from a given population of antibody molecules (not necessarily homogeneous) or from one myeloma protein. The question at issue is whether autologous chains recombine preferentially in a competitive situation in which a polypeptide chain from another antibody or myeloma protein is also present. 

Porter and Weir (18) quantitatively assessed the antibody activity of the H and L chains of horse antidiphtheria toxoid by using I-labeled toxoid and a direct assay. H and L chains retained 20 and 5%, respectively, of the binding capacity of the intact molecule after recombination of the H chains with nonspecific L chains the value was increased to 40%. The measurements were designed to provide information as to binding capacity rather than binding affinity. Very similar results, with respect to activity in isolated chains and in their recombinants, were obtained by Franek et ai, who worked with bovine anti-Dnp antibodies (51). 

Will an H chain, presented with a mixture of its autologous L chains and L chains from a different molecule, preferentially recombine with the autologous chain ... 

Autologous chains (H and L) used for recombination" Competing L chain" Weight ratio of autologous to heterologous L chains in 7 S peak after recombination... 

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