D.R. Stanworth

Biological activities associated with the Facb fragment of rabbit IgG

Abstract Various types of rabbit IgG Fc sub-fragment have been examined for a capacity to fix to heterologous (guinea-pig) skin and to react with rheumatoid anti-IgG-globulins. Facb fragments proved, somewhat surprisingly, to be inactive in both systems; thus resembling pFc′ and tFc′ fragments (derived from the C-terminal end of the Fc region) and in contrast to the active parent Fc fragment. The effect of acid treating whole IgG in a similar way to that employed in preparing Facb fragments was investigated. This treatment per se was found to result in no appreciable loss in biological activity. The demonstration that fragments incorporating the C H 2 or C H 3 structural domains are both inactive suggests the necessity for strict conformational integrity in the expression of at least some biological activities associated with the Fc regions of IgG molecules.

Structural studies of immunoglobulins—I. The role of cysteine in papain hydrolysis

Abstract Two conformational forms of human γG globulin have been distinguished by their differing behavior on incubation with papain in the absence of cysteine. A ‘papain-resistant’ form is separated intact from the products of digestion of the predominant ‘papain-sensitive’ form by gel-filtration on Sephadex G-100. Structural alterations induced in the ‘papain-resistant’ form by pre-treatment with cysteine have been shown to render it susceptible to subsequent proteolysis by the enzyme in the absence of further reducing agent. Myeloma γG globulins with characteristics resembling each of these two forms of normal γG globulin have also been studied. The results thus obtained, by analytical gel-filtration of papain digests, are discussed in relation to methods applied by other investigators for the differentiation of γ G globulin molecules of varying susceptibility to papain proteolysis.

The effect of heterologous antisera and rheumatoid factor on the synthesis of DNA and protein by human peripheral lymphocytes

Abstract Seven antisera prepared by immunizing rabbits with human leucocytes activated human lymphocytes in vitro to varying degrees. The relative activity of the antisera in terms of 14 C-thymidine incorporation after 3 days of culture was the same whatever the source of the cultured lymphocytes. At low concentrations of some antisera considerable stimulation of DNA synthesis occurred even in the presence of complement. At high antiserum concentrations DNA synthesis was adversely affected in the presence, but not in the absence, of complement. Antisera against other human antigens, including immunoglobulins, were not active. Rheumatoid factor did not markedly augment the activity of leucocyte antibody. The level of DNA synthesis of sensitized lymphocytes in culture was markedly influenced by the source of the human serum present in the medium.

Structural studies of immunoglobulins—II the varying susceptibility to papain-digestion of a group of human myeloma γG-globulins

Abstract Sephadex (G-100) filtration and starch gel electrophoresis (at pH 8·8) have been employed in the analysis of eleven human myeloma γG-globulins, showing varying susceptibility to digestion by papain. The results obtained provide further evidence of the existence of two major conformational forms of γG-globulin differing in their net surface charge.

The effect of acid treatment upon the susceptibility of rabbit IgG to proteolytic cleavage with various enzymes.

Abstract The pH of solutions of rabbit IgG was adjusted to 2·7 with HCl and incubated at 37°C for 1 hr after which aliquots were adjusted to the required enzyme pH optimum and digested for 4 hr at 37°C with various proteolytic enzymes. The digestion products obtained were compared with those produced under similar digestion conditions but by omission of the acid treatment step. In all cases it was observed that acid-treated IgG was completely digested irrespective of the enzyme used, whereas non-acid treated IgG exhibited varying susceptibility to cleavage by different enzymes. Trypsin, pepsin and papain digestion of acid-treated IgG each produced a major fragment similar in size and antigenic composition to that of F(ab′) 2 . None of these fragments retained the capacity to react with rheumatoid factor. Fragments similar to the Facb fragment produced by plasmin digestion of acid-treated rabbit IgG were not observed. The significance of these findings is discussed.

The use of baboon IgG-sensitized sheep erythrocytes as an alternative indicator system in the study of the interaction of rheumatoid factor with human IgG

Abstract A haemagglutination-inhibition assay for studying the interaction of rheumatoid factor with monomeric human IgG has been developed. The indicator system employed consists of sheep red blood cells actively sensitized with baboon ( Papio cynocephalus ) IgG. Studies revealed that sub-classes 1, 2 and 4 of human IgG were capable of inhibiting the agglutinatio of baboon IgG-senstized sheep red blood cells by rheumatoid arthritic sera, irrespective of Gm phenotype or the anti-Gm specificity of the rheumatoid arthritic sera employed. The pFc′, F(ab′) 2 and Fab fragments from pooled human IgG failed to inhibit the reaction, in contrast to the Fc fragment. The agglutination titres of a panel of rheumatoid arthritic sera from baboon IgG-sensitized sheep red blood cells were compared with their titres in a conventional Rose-Waaler system (i.e. rabbit IgG-sensitized sheep red blood cells). Such a comparison revealed a close similarity in the titres recorded for each serum examined irrespective of the assay system employed. The significance of these findings is discussed.