Joel W. Goodman

Phage-neutralizing activity in light polypeptide chains of rabbit antibody.

Abstract Neutralization of T1 bacteriophage by fragments and polypeptide chains of rabbit antibody was studied by the use of specific anti-rabbit globulin sera to enhance the efficacy of neutralization. The neutralization given by L polypeptide chains from anti-T1 γ-globulin was markedly enhanced by goat anti-papain fragment I seru which had been absorbed with papain fragment III. The neutralization given by H polypeptide chains was sharly amplified by goat anti-papain fragment III serum which had been absorbed with papain fragment I. The latter antiserum had no demonstrable effect on neutralization by L polypeptide chains, indicating that the activity of L chains was not due to contamination by H chains. The phage neutralizing activity of Fd-piece was also markedly enhanced by fragment III-absorbed anti-fragment I serum. However, when this serum was absorbed with L chains, its enhancing property was entirely abolished. Absorption of the antiserum with Fd-piece eliminated its enhancing effect on neutralization by L chains. Attempts to stimulate production of specific anti-Fd serum in guinea pigs were unsuccesssful.

Antigen specific T cell hybrids—II: T cell hybrids which bind azobenzenearsonate

Abstract T cell hybrids which express specificity for azobenzenearsonate (ABA) have been developed and analysed. Somatic cell hybrids were prepared between BW 5147, a thioguanine resistant AKR mouse T lymphoma and antigen-plate enriched T cells from ABA-mouse gamma globulin immunized A/J mice. Cells which grew in selective medium expressed both parental forms of hypoxanthine guanine phosphoribosyltransferase, isocitrate dehydrogenase and Thy l. T cell hybrids formed rosettes with antigen conjugated sheep and mouse erythrocytes which were inhibited by pre-exposure of the hybrid cells to soluble ABA-human serum albumin but not ovalbumin. T cell hybrids, in contrast to ABA B cell hybrids which were also studied here, did not rosette with antigen coupled human erythrocytes. T cell hybrids which were subcloned on agar maintained their rosetting activity, even to the extent of its inhibition by soluble antigen. These studies set the foundation for future experiments on the molecular analysis of surface membranes and products of antigen-specific T cell hybrids.

Reaction of Rheumatoid Sera with Fragments of Papain-Digested Rabbit Gamma Globulin.∗

Summary Most of the reactivity of rabbit γ-globulin with a pool of sera from rheumatoid arthritics was found to be associated with Fraction III of the papain-digested globulin molecule. Fraction II showed a low degree of reactivity while Fraction I appeared to be completely inactive.

The immune response to glucagon in conjugated form

Abstract Randomly bred rabbits and guinea pigs immunized with bovine glucagon conjugated to proteins using bis-diazotized benzidine made anti-glucagon antibodies with specificity primarily for the car☐y-terminal part of the molecule, whereas animals immunized with free glucagon make amino-terminal-specific antibody (Senyk et al., 1971 a,b). In guina pigs immunized with conjugates of glucagon and bovine serum albumin (G-BSA), glucagon was unable to elicit cellular immune responses (delayed cutaneous reactions, MIF release and stimulation of DNA synthesis by lymphoid cells in culture), in contrast to animals immunized with free glucagon. Animals rendered unresponsive to to BSA by treatment with cyclophosphamide and subsequently immunized with G-BSA still made no response to glucagon, indicating that in this conjugated form glucagon acts only as a haptenic determinant, whereas in free form it is an immunogen. Glucagon conjugated to a poly-γ- d -glutamic acid hapten of mol. wt. 35,000 using a carbodiimide reagent evoked cellular and humoral responses to the glucagon moiety, but no anti-hapten antibody was detected; antiglucagon antibodies from different animals has a variable pattern of specificity with respect to the car☐y-terminal vs. amino-terminal parts of the molecule. The specificity of antibodies made against conjugated glucagon thus depended on the mode of conjugation, a finding which may have a bearing on the development of differential radioimmunoassays for pancreatic glucagon and glucagon-like immunoreactivity in body fluids.

Antigen-specific molecules from murine T lymphocytes and T cell hybridomas☆

Strain A/J mice immunized with azobenzenearsonate-isologous IgG conjugates made strong antigen-specific suppressor T cell responses. The ABA-specific T cells‡ were enriched by affinity purification and an average of 59% of such cells expressed the major idiotype (CRI) associated with anti-ABA specificity in A/J mice. The cellular proteins of affinity enriched, ABA-specific T cells were biosynthetically radiolabelled and ABA-binding molecules from cell lysates were purified by affinity chromatography. SDS-PAGE analysis of T cell-derived ABA-binding molecules revealed a major, invariant component with an apparent mol. wt of about 92,000 daltons (p92). This protein is susceptible to proteolytic degradation as its recovery from cell lysates required the presence of protease inhibitors. Serological analyses of p92 failed to reveal classical immunoglobulin heavy or light chain determinants or Ia markers. Evidence is presented that this class of molecules possesses suppressor regulatory activity, probably in conjunction with a noncovalently associated smaller polypeptide chain. Affinity-enriched ABA-specific suppressor T cells were hybridized with the AKR tumor line BW 5147. Ten hydridomas were obtained which formed rosettes with ABA-SRBC. Rosette formation was inhibitable with soluble ABA-protein conjugates. Three of the lines stained for the CRI in an immunofluorescence assay. Culture supernatants from these lines were tested for modulation of an in vitro primary anti-ABA response and exerted a strong antigen-specific suppressor activity. Thus, hybridoma cell lines have been obtained which express an ABA-specific receptor and secrete antigen-specific regulatory factors. They therefore offer great promise as sources of sufficient material for chemical characterization of antigen specific T cell molecules.

The anatomy of an antigen molecule: functional subregions of L-tyrosine-p-azobenzenearsonate

The structural components of antigen molecules that interact with class II major histocompability complex (MHC) molecules on antigen-presenting cells (APCs) (agretopes) and with antigen receptors of T-lymphocytes (epitopes) in class II restricted T-cell responses have not been precisely defined. This issue was addressed here using murine T-cell clones specific for the simple immunogen L-tyrosine-p-azobenzenearsonate (ABA-tyr) and a series of analogs of the homologous antigen. Two experimental approaches were used. First, APCs were pulsed with analogs and used to stimulate T-cell proliferation. The patterns of stimulation segregated the clones into two specificity groups and indicated that the epitope recognized by the T-cell included the arsonate group and elements in the side chain of tyrosine. Furthermore, the clones manifest different sensitivities to antigen. Second, non-stimulatory analogs were used to block the presentation of ABA-tyr in an effort to define the agretope. Compounds containing the azophenyl group blocked presentation of ABA-tyr in a dose-dependent fashion, whereas p-arsanilic acid and L-tyrosine were ineffective. The blocking was specific inasmuch as the compounds had no effect on the antigen-induced proliferative responses of giant keyhole limpet hemocyanin (KLH) or hen egg white lysozyme (HEL)-reactive T-cell clones. The blocking pattern indicated that the feature required for productive association with the APC centered on the planar structure of the azo-linked aromatic rings, with little or no contribution from either the arsonate moiety or the tyrosyl side chain. We propose that this structure forms an agretope for this family of compounds.

Qualitative and quantitative determination of mixtures of amino acids using 2,4,6-trinitrobenzenesulfonic acid.

Abstract Mixtures of TNP-amino acids may be rapidly resolved by chromatography on thin-layer plates of silica gel. The colored derivatives can be quantitated spectrophotometrically following elution from the plates. The advantages of this method lie in its rapidity and the modest equipment needed.

Antigenic Determinants in Fragments of Gamma Globulin from Rabbit Serum

Peptic digestion of fraction III from papain-digested rabbit serum γ-globulin produces a variety of smaller fragments. Some are too large to pass through a dialysis bag, and these retain the capacity to precipitate with antiserum to rabbit γ-globulin. Others pass through the bag and fail to precipitate with antibody, but they can inhibit the precipitation of antibody with fraction III. This indicates that antigenic determinants of the γ-globulin molecule are carried in these fragments.

The capacity of bifunctional antigens to bridge antibody molecules and to mediate cell cooperation

Abstract The capacity of various bifunctional antigens, some of which induce only cellular immunity whereas others induce both cellular and humoral immunity, to cross-link antibody molecules was assessed by thin-layer gel filtration on Sephadex G-200. DNP-RAT polymerized a mixture of rabbit anti-DNP and anti-RAT antibodies induced anti-DNP responses in guinea pigs, and thus apparently mediates cell cooperation. On the other hand, symmetrical bifunctional RAT antigens with flexible spacers were not effective mediators of cell cooperation but readily polymerized anti-RAT antibody, whereas a bifunctional RAT antigen with a rigid spacer performed both functions. Hence, the capacity of bifunctional immunogens to cross-link antibodies cannot be equated with ability to implement cell cooperation. Possible factors in this discrepancy are discussed.

Immunochemical studies on cross-reactions of antipneumococcl sera—V cross-reactions of horse antipneumococcal type II serum with E. Coli M-II polysaccharide, dextran and hemocyanin-ortho-azophenyl-β-glucuronide

Abstract A capsular polysaccharide of a mucoid strain of E. coli was found to cross-react with horse antiserum to the type specific polysaccharide of pneumococcus type II. Glucuronic acid, which is present as terminal non-reducing residues in the side chains of both the pneumoccoccal and E. coli polysaccharides, is probably responsible for this cross-reactivity. The antiserum also cross-react with dextran; however, the antibody fraction precipitable by the E. coli polysaccharide is distinct from that removed by dextran. Inhibition of precipitation demonstrated that isomaltose is a much more efficient inhibitor than glucoronic acid of the dextran cross-reaction while the reverse is true of the cross-reaction with the E. coli polysaccharide. Keyhole limpet hemocyanin-ortho-azophenyl-β-glucuronide was capable of precipitating almost all of the anti-pneumococcal antibody, demonstrating in these antibodies a common immune specificity for glucuronyl residues and a pivotal role for glucuronic acid in the antigenic determinants of the pneumococcal polysaccharide. Heterogeneity of the antibody in this antiserum was also shown by the finding that the antibody fraction which cross-reacts with E. coli polysaccharide was preferentially absorbed by the hemocyanin conjugate prior to removal of the fraction precipitable by dextran.