Alfred Nisonoff

Separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of disulfide bonds.

Abstract Nonprecipitating active fragments of rabbit antiovalbumin γ-globulin, having approximately the same sedimentation coefficient (3.5 S ) and inhibitory effect on the homologous precipitin reaction as the univalent fragments produced by papain, can be prepared by treatment with pepsin in the presence of a reagent capable of breaking disulfide bonds. The reaction can also be carried out by successive treatments with pepsin and one of several such reagents. Cysteine, 2-mercaptoethylamine, thioglycolate, and cyanide were effective. In the absence of enzyme, but under otherwise identical conditions, these compounds had no apparent effect on the antibody. The combining sites on fragments of an antihapten antibody produced by this method were found, by hapten-binding measurements, to be essentially intact, although the capacity to form specific precipitates was lost. This fact and the close similarity to the univalent fragments formed by the action of papain indicate that the active split products produced by pepsin under these conditions are univalent. Peptic digestion causes a decrease in sedimentation coefficient to about 5 S , but the capacity to form specific precipitates is largely retained, indicating that the 5 S fragment is bivalent. Further treatment with a disulfide-splitting reagent results in the formation of fragments with the properties of univalent antibody and s ≅ 3.5. The results indicate that the proteolytic enzyme splits off an inactive portion of the molecule; and that subsequent to this reaction the bivalent residue can be divided into two univalent fragments by breaking the disulfide bonds which hold them together. Because compounds which react with disulfide groups have always been used with papain, as activators of the enzyme, it appears that a similar mechanism may obtain in the digestion of rabbit antibody by that enzyme. Since Porter has shown that the two active fragments (3.5 S ) released by papain are nearly identical in molecular weight and amino acid composition, the possibility is suggested that the biosynthesis of rabbit antibody may include linkage of these two nearly identical univalent subunits through one or more disulfide bonds.

Implications of the presence of an internal image of the antigen in anti-idiotypic antibodies: Possible application to vaccine production

Abstract There is now strong suggestive evidence that some anti-idiotypic antibodies may possess one or more antigenic determinants (epitopes) that are closely related to epitope(s) on the original antigen (antigen X). Thus, some molecules of anti-anti-X may share an epitope with X. When this occurs a wide range of idiotypic cross-reactions will be observed among anti-X antibodies from various sources and, if X is a hormone, the cross-reactions may include cell surface receptors for the hormone. Such cross-reactions can lead to misinterpretation of the genetic significance of the observed cross-reactive idiotypy. This article reviews the evidence for this phenomenon and its possible relationship to certain autoimmune disease processes involving cell surface receptors. The possibility is discussed of obtaining monoclonal anti-idiotypic antibodies with epitopes similar to those on an infectious agent. Such a monoclonal antibody might elicit protective antibodies when inoculated and thus act as a vaccine.

Properties of the Major Component of a Peptic Digest of Rabbit Antibody

The molecular weight of the active, major component isolated from a peptic digest of rabbit antibody was found to be 106,000. After treatment with a disulfide-splitting reagent, the molecular weight was 56,000, and the products migrated as a single peak in the ultracentrifuge. he univalent fragments thus formed can be partially recombined by passage through IR-120 cation-exchange resin at room temperature or by treatment with a difunctional organic mercurial. Some splitting of the pepsin-treated antibody molecule occurs on carboxymethylcellulose at pH 5.4.

Regulation of Immunity to the Azobenzenearsonate Hapten

Publisher Summary This chapter reviews structural, genetic, and cellular data evaluating the immunological responses to the hapten, azobenzenearsonate, coupled to proteins or cells. The idiotype utilized in the studies reviewed here is that associated with antibodies to the azobenzenearsonate (ABA) hapten in strain A mice. The system is useful because all mice of the A or AL/N strain respond to immunization with KLH-ABA by producing antibodies bearing a major cross-reactive diotype (CRI); in general between 20 and 70% of the anti-ABA antibody population carries the idiotype. In addition to KLH, several protein carriers, when conjugated to ABA and inoculated into strain A mice, induce anti-ABA antibodies with a high content of the CRI. These include ovalbumin, edestin, and p-lactoglobulin. The structure of the protein, at the site to which the ABA is conjugated, nevertheless appears to influence the induction of the idiotype. The administration of rabbit anti-Id antibodies to A/J mice, prior to immunization with KLH-ABA, has little effect on the total anti-ABA response, but can completely suppress the appearance of the CRI. If immunization is started immediately after the administration of anti-Id, say within 2 weeks, the re-emergence of the CRI upon subsequent immunization has never been observed. It might be envisioned that CRI VH genes are present in many mouse strains, but may be utilized to build receptors with different ligand specificities or regulated in some other fashion. Alternatively, BALB/c mice may lack such V genes but nevertheless have T cells with anti-idiotypic receptors.

Hybridization of Half Molecules of Rabbit Gamma Globulin

Specifically purified rabbit antiovalbumin and normal γ-globulin labeled with 1-131 were dissociated into half molecules by reduction and acidification. When a mixture of the two preparations was neutralized, a large proportion of mixed molecules having active combining sites and the same sedimentation coefficient as the original γ-globulins was formed. Since the sulfhydryl groups were inactivated after reduction, the recombined subunits appear to be linked by noncovalent bonds.

Contribution of Allelic Genes Ab4 and Ab5 To Formation of Rabbit 7S γ-Globulins.∗

Summary 1. A method for quantitative analysis of allotypes is presented, based on successive precipitations of specific allotype from I131-labeled 7S γ-globulin with anti-allotype sera. 2. In the homozygotes or heterozygotes, 80–90% of the γ-globulin molecules have Ab4 or Ab5; 10–20% of the γ-globulin molecules have neither Ab4 nor Ab5. 3. An anti-Ab5 serum giving 2 precipitin bands with Ab5 sera was compared with an anti-Ab5 serum giving one precipitin band and was found to precipitate virtually the same quantity of γ-globulin-I131 (82% vs 80%) from the same γ-globulin preparation. This indicates that the 2 precipitin bands represent cross-reacting antigen-antibody systems. 4. In the heterozygote, 64% of the molecules have Ab4, 27% have Ab5. These quantities are independent of the order of precipitation, indicating that Ab4 and Ab5 are not found on the same γ-globuin molecules but only on separate molecules. The limit of overlap is less than 5%. 5. The question as to why allelic genes do not contribute to the same molecule, although both alleles appear to be operating in the synthesis of γ-globulin within the same cell, is discussed. It is suggested that polypeptide chains carrying the allotypic specificity Ab4 form pairs immediately after synthesis; and that the Ab4 pairs do not combine with similarly formed Ab5 pairs.

Structural studies on induced antibodies with defined idiotypic specificities. Viii. Nh2-terminal amino acid sequence analysis of the heavy and light chain variable regions of monoclonal anti- -p-azophenylarsonate antibodies from a/j mice differing with respect to a cross-reactive idiotype.

Abstract Monoclonal antibodies with specificity for the hapten p -azophenyl arsonate (Ar) ∗ were generated by somatic cell hybridization of A/J spleen cells and the non-secreting cell line, Sp2/0-Ag14, derived from a Balb/c myeloma. Of four hybridoma products examined, only one bore the previously described A/J anti-Ar cross-reactive idiotype (CRI). The heavy and light polypeptide chains of this CRI positive molecule along with one of of the CRI negative molecules were subjected to amino-terminal sequence analysis and compared with the induced CRI positive antibodies produced upon immunization of A/J mice. The results confirm the extremely restricted expression of V H and V L frameworks in the A/J anti-Ar population. Unexpectedly, when the hybridoma proteins were compared to the induced population of antibodies, from one to three amino acid substitutions were detected within the first framework segment (1–30) of each chain of both hybridoma products. The complete covalent structure of two monoclonal antibodies known to differ serologically only with respect to idiotype should prove useful in defining the exact structural basis of an idiotypic determinant.

Relative combining affinities of anti-p-azophenylarsonate antibodies bearing a cross-reactive idiotype☆

Abstract All mice of the A/J strain produce anti-p-azophenylarsonate (anti-Ar) antibodies, some of which share a cross-reactive idiotype (CRI). To account for the regular appearance of the CRI, the possibility was considered that such molecules are of high avidity; this could result in selective capture of antigen and stimulation of lymphocytes having molecuels with CRI as receptors. Comparisons were therefore made between antibody preparations with a high or low content of CRI obtained either from different animals or through fractionation by isoelectric focusing. Equilibrium dialysis, using the hapten (p- azobenzenearsonic-acid )-N- acetyl- l -tyrosine , or binding measurement with the radiolabeled immunogen, KLH-Ar, were consistent in indicating that molecules with CRI are somwhat lower in affinity or avidity than the average of the population of molecules lacking CRI. The results suggest that the high of occurence of molecules with CRI must reflect the presence of a relatively large number of precursor lymphocytes with the corresponding receptor.

Amino Acid Composition of Univalent Fragments of Rabbit Antibody

Univalent fragments of γ-globulin preparations from four individual rabbits were fractionated and the amino acid compositions of end fractions were compared. For amino acids with uncharged side chains, differences were consistently less than one residue per fragment of molecular weight 50,000. There were differences in the content of amino acids, other than glutamic acid, with charged side chains; these were in the direction expected on the basis of relative strength of adherence to carboxymethyl cellulose and electrophoretic mobility. Exclusive of any possible differences in amide, the total difference in charged groups was three to four residues per fragment. Sine the net charge of univalent fragments reflects that of the γ-globulin molecule from which the fragments are derived, corresponding differences in composition may also exist among whole γ-globulin molecules.

Immunological suppression of idiotypic specificities.

The suppression of the capacity of an animal to produce antibody of a particular idiotype specificity bears an obvious relationship to the induction of immune tolerance, with the distinction that the tolerant state is restricted to the production of antibody directed to a particular idiotype rather than to an antigen. There is perhaps a closer analogy to the suppression of an allotypic specificity through maternal transfer or direct administration of the corresponding antiallotypic antibody (Dray 1962, Mage 1967). Since most antigens elicit antibodies bearing many different idiotypic specificities, the reagent that must be used to recognize and selectively suppress an individual idiotype is antiidiotypic antibody rather than antigen. The antiidiotypic antibody necessarily exerts its effect through interaction with receptors bearing the idiotype on lymphoid cells. For experimental purposes it is highly advantageous if antibody of the same idiotype is produced in response to a given antigen by different animals of the same species. This permits the use of antiidiotypic antibody, prepared against the antibodies from one animal (or group of animals), to suppress the appearance of the same idiotype in other members of the same species. The occurrence of an intraspecies cross-reactive idiotype was first noted among the antistreptococcal antibodies of partially inbred rabbits by Eichmann & Kindt (1971). Such cross-reactions have proved to be more frequent