Individual Antigenic Determinants Research Articles

Phage antibodies for the immunochemical characterization of Herbaspirillum seropedicae Z78 glycopolymers

Microbial carbohydrate antigens are targets of the immune systems of hosts. In this context, it is of interest to obtain data that will permit judgment of the degree of heterogeneity, chemical makeup, and localization of the antigenic determinants of the Herbaspirillum surface glycopolymers. A sheep single-chain antibody-fragment phage library (Griffin.1, UK) was used to obtain miniantibodies to the exopolysaccharides (EPS-I and EPS-II), capsular polysaccharides (CPS-I and CPS-II) and lipopolysaccharide (LPS) of Herbaspirillum seropedicae Z78. To infer about the presence or absence of common antigenic determinants in the cell-surface polysaccharides of H. seropedicae Z78, we ran a comparative immunoassay using rabbit polyclonal and phage recombinant antibodies to the surface glycopolymers of H. seropedicae Z78. We isolated and purified the exopolysaccharides (EPS-I and EPS-II), capsular polysaccharides (CPS-I and CPS-II), and lipopolysaccharide (LPS) of Herbaspirillum seropedicae Z78. Using rabbit polyclonal antibodies, we found that these cell-surface polysaccharides were of a complex nature. EPS-I, EPS-II, CPS-I, CPS-II, and LPS contained common antigenic determinants. CPS-I, CPS-II, and LPS also contained individual antigenic determinants composed of rhamnose, N-acetyl-d-glucosamine, and N-acetyl-d-galactosamine—sugars responsible for cross-reactions with miniantibodies. The anti-LPS miniantibodies were more specific for the core region of the LPS, in which rhamnose was the most abundant sugar, than they were specific for its O portion. The miniantibodies we isolated can be useful reagents not only in basic biochemical research but also in clinical diagnostic and therapeutic applications.

Electrochemical Differentiation of Epitope-Specific Aptamers

DNA aptamers are promising immunoshielding agents that could protect oncolytic viruses (OVs) from neutralizing antibodies (nAbs) and increase the efficiency of cancer treatment. In the present Article, we introduce a novel technology for electrochemical differentiation of epitope-specific aptamers (eDEA) without selecting aptamers against individual antigenic determinants. For this purpose, we selected DNA aptamers that can bind noncovalently to an intact oncolytic virus, vaccinia virus (VACV), which can selectively replicate in and kill only tumor cells. The aptamers were integrated as a recognition element into a multifunctional electrochemical aptasensor. The developed aptasensor was used for the linear quantification of the virus in the range of 500-3000 virus particles with a detection limit of 330 virions. Also, the aptasensor was employed to compare the binding affinities of aptamers to VACV and to estimate the degree of protection of VACV using the anti-L1R neutralizing antibody in a displacement assay fashion. Three anti-VACV aptamer clones, vac2, vac4, and vac6, showed the best immunoprotection results and can be applied for enhanced delivery of VACV. Another two sequences, vac5 and vac46, exhibited high affinities to VACV without shielding it from nAb and can be further utilized in sandwich bioassays.

Peptide Mapping of a Novel Discontinuous Epitope of the Major Surface Adhesin from Streptococcus mutans

Guy's 13 is a mouse monoclonal antibody that specifically recognizes the major cell-surface adhesion protein SA I/II of Streptococcus mutans, one of the major causative agents of dental caries. Passive immunization with Guy's 13 prevents bacterial colonization in humans. To help elucidate the mechanism of prevention of colonization conferred by this antibody, the SA I/II epitope recognized by Guy's 13 was investigated. It was previously established that the epitope is conformational, being assembled from two non-contiguous regions of SA I/II. In the current study, using recombinant fragments of SA I/II and, ultimately, synthetic peptides, the discontinuous epitope was localized to residues 170-218 and 956-969. This work describes the mapping of a novel discontinuous epitope that requires an interaction between each determinant in order for epitope assembly and recognition by antibody to take place. Guy's 13 binds to the assembled epitope but not to these individual epitope fragments. The assembled epitope results from the interaction between the individual antigenic determinants and can be formed by mixing together determinants present on separate polypeptide chains. The data are consistent with one of the epitope fragments adopting a polyproline II-like helical conformation.

Epitope mapping of neutralizing TSST-1 specific antibodies induced by immunization with toxin or toxoids

Toxic shock syndrome toxin-1 (TSST-1), a superantigen produced by Staphylococcus aureus, is a potent stimulator of the immune system. T-cells are activated by crosslinking of MHC class II molecules on antigen presenting cells with T-cell receptors (TCR). TSST-1 is associated with the majority of the cases of menstrual staphylococcal toxic shock, a severe and life-threatening multisystem disorder. Even though antibody mediated protection has been studied, information on antibody specificity directed to individual antigenic determinants of the protein is incomplete. To obtain immunogens with low toxicity, we generated a double-site mutant ( dmTSST-1), modified at solvent-exposed residues predicted to be important for both MHC class II and TCR binding, and detoxified recombinantly expressed TSST-1 ( rTSST-1) as well as native TSST-1 ( nTSST-1) isolated from Staphylococcus aureus by treatment with formaldehyde. Rabbits were immunized with rTSST-1, nTSST-1, dmTSST-1, and formaldehyde inactivated toxoids. The sera obtained were used to map the antigen-reactive regions of the molecule and to identify specificities of antibodies induced by immunization with the different antigens. To detect linear antigenic epitopes of TSST-1 the reactivity of the sera with 11-meric peptides having an overhang of four residues, covering the entire molecule of TSST-1, have been studied. We found that sera of TSST-1 immunized rabbits predominantly reacted with N-terminal residues 1–15, while sera generated with formaldehyde inactivated toxoid recognized a total of 7 regions located at the N- and C-terminus and internal sites of TSST-1. Despite different specificities all sera were able to inhibit TSST-1 induced proliferation of human mononuclear cells.

Molecular Biology and Immunology for Clinicians 6

The immune response that protects us from pathogens and malignancy is directed at the individual constituent molecules of the invading cells, a response specific for discrete sections of each of these molecules. A unique language is used to describe these individual targets, called epitopes or antigenic determinants. Understanding this language is necessary to better appreciate the character of the challenge and the nature, efficiency, and evolution of the response. As we determine the immune mechanisms of many diseases, e.g., autoimmunity due to molecular mimicry, it is crucial that physicians be able to interpret the immunological literature.Proper interpretation of serological testing, e.g., the use of Western blot to confirm/corroborate less specific tests like ELISA, depends in part on a full understanding of how the specificity of antibodies are determined. Novel therapeutic strategies, e.g., use of intravenous gammaglobulin to alter immune control networks, are being developed; only with an appreciation of the humoral immune response can the clinician make use of these new approaches that seek to manipulate the immune system. The terminology used in defining antigens, the molecules that are the target of the immune response, and the structure of immunoglobulins, and how these structures determine and influence function, are reviewed. This paper serves as a start, a "jumping off" point, for further description of the immune response in later papers in this series.To be successfully immersed in or introduced to immunology, it is important to "speak the language." So, consider this a refresher course, not to recall high school Spanish but medical school immunology.

The generation of MHC class I-associated peptides is only partially inhibited by proteasome inhibitors: involvement of nonproteasomal cytosolic proteases in antigen processing?

The proteasome is believed to participate in the generation of a large percentage of peptide ligands for MHC class I molecules. This conclusion is based largely on the activities of peptidyl aldehydes that block proteasome activity. We tested the ability of a panel of proteasome inhibitors to affect the generation of MHC class I binding peptides in mouse L929 cells. Included in the panel are peptidyl aldehydes and a microbial product, lactacystin, that blocks proteasome activity in a distinct and more specific manner. Contrary to expectations, proteasome inhibitors failed to block the generation of a large portion of high affinity peptides as inferred by measuring cell surface expression of newly synthesized MHC class I molecules. These findings were confirmed by examining the effects of the inhibitors on the presentation of individual antigenic determinants from endogenously synthesized or exogenously delivered influenza virus proteins. Presentation of peptides derived from exogenous basic polymerase 1, endogenous basic polymerase 1, and nonstructural-1 proteins was decreased by inhibitors in a manner consistent with proteasomal involvement. Presentation of peptides derived from endogenous nucleoprotein was not significantly affected by the proteasome inhibitors, while presentation of exogenous hemagglutinin and nucleoprotein was enhanced by the proteasome inhibitors. These data are consistent with the involvement of both proteasomes and nonproteasomal cytosolic proteases in the generation of a significant portion of MHC class I binding peptides.

Genetically permissive recognition of adjacent epitopes from the 19-kDa antigen of Mycobacterium tuberculosis by human and murine T cells.

The specificity of the T cell-immune repertoire at the level of individual antigenic determinants could play a fundamental role in the immunopathogenesis of tuberculous infections. Therefore, we analyzed the immunogenicity, genetic restriction, and epitope core structure of two adjacent, yet distinct, immunodominant T cell determinants from the 19-kDa Ag of Mycobacterium tuberculosis. After immunization with two peptides comprising residues 45 to 64 and 61 to 80, vigorous in vitro proliferative responses to the homologous peptide were elicited in five different strains of C57BL/10 mice (H-2b,k,d,s,f), indicating that both epitopes were recognized in a genetically permissive manner. When immunized with intact 19-kDa protein, lymph node cells from the same mouse strains responded to both peptides, with the exception of H-2b mice, which did not respond to p45-64. Delayed-type hypersensitivity responses in C57BL/10 (H-2b) mice were elicited by p61-80 only, whereas in H-2d mice both peptides were delayed-type hypersensitivity negative, despite eliciting pronounced proliferative responses. Analysis of the proliferative responses of human PBMC in purified protein derivative-positive healthy subjects and tuberculosis patients revealed significant differences in the antigenicity to the two peptides. All purified protein derivative-positive healthy and diseased individuals manifested strong responses to p45-64, indicating HLA permissive recognition. In contrast, pronounced responses to p61-80 were detected only in patients with lymphatic tuberculosis. Epitope core structures, composed of 6 or 7 residues within each peptide, have been mapped with peptides of overlapping sequence. Significantly, for both epitopes, the core sequences recognized by both human and murine T cells were almost identical. We conclude that despite many similarities between murine and human T cell epitope recognition, distinct differences in the responsiveness of the infected host could play a role in pathogenesis. Furthermore, the genetically permissive nature of the identified epitopes is a potentially important attribute for the development of peptide-based diagnostic reagents and vaccines.

Using Monoclonal Antibodies for Phylogenetic Analysis: an Example from the Heliothinae (Lepidoptera: Noctuidae)

Vertebrate antisera have been used for phylogenetic analysis for >85 yr by raising them against molecules from one taxon and reacting them with homologous molecules from other taxa in the groups under consideration. The reactions are evaluated quantitatively and are used to estimate immunological distances between taxa. The strength of these reactions reflects the summation of interactions between the individual antibodies making up the antiserum and the antigenic determinants they recognize on the molecule. The availability of monoclonal antibodies makes possible a new approach for immunological phylogenetic analysis because they recognize individual antigenic determinants that are either present or absent, thus qualifying as character states. We illustrate this approach with monoclonal antibodies to determinants on two larval plasma proteins from Helicoverpa zea (Boddie). The distribution of these two determinants is consistent with current thinking on the close relationship of H. zea and Heliothis armigera (Hubner), and supports the monophyly of those heliothines that we have tested. Immunoblotting may be used with this approach to support the homology of determinants in different taxa by confirming the identity of the molecules on which they reside, after the molecules have been characterized by such assays as native PAGE and isoelectric focusing.

Anti-idiotypic antibodies as immunogens: idiotype-based vaccines.

Numerous studies have documented that antibodies may regulate the immune system and form the basis of vaccines, namely anti-idiotype vaccines. Antibodies carry individual idiotype antigenic determinants against which antibodies can be formed. When the anti-idiotype recognizes the same site that recognizes the primary antigen, a mirror image or combining site antibody may be generated. Other anti-idiotypes which recognize non-combining antigenic determinants have also been used. The evidence is reviewed for the existence of a broad range of anti-idiotypes and details are given of how an anti-idiotype vaccine based on the hepatitis B surface antigen has protected against virus challenge in the most relevant animal model system, namely the chimpanzee. Furthermore, the definition of the CD4 molecule as the conserved binding site for all known human and similar immunodeficiency viruses, (in marked contradiction to their varied neutralizing properties) has led to the raising of anti-idiotypes in mice based on the CD4 receptor which have the capacity to neutralize a broad range of isolates.

I. Induction of proteinuria in the rat by a monoclonal antibody against SGP-115/107

The role of individual antigenic determinants in the pathogenesis of antibody-mediated glomerular injury is, at present, incompletely understood. This study was designed to compare the effect of monoclonal antibodies upon binding to three distinct antigenic determinants present in the glomerular capillary wall. Ten monoclonal antibodies were tested for their nephrotoxic capacity in the rat. Six antibodies directed against basement membrane laminin and three antibodies with specificity for a 129/117 kd antigenic complex present on endothelial and glomerular visceral epithelial cell surfaces did not induce proteinuria or structural injury. A non-complement binding monoclonal antibody that immunoprecipitates a 115/107 kd sialo-glycoprotein (SGP-115/107) from glomerular cell membrane preparations and a 107 kd component from proximal tubules, intestinal cells and liver cells, induces glomerular epithelial cell alterations consisting of focal obliteration of foot process architecture, vacuolar changes in the cytoplasm, microvillous transformation of the cell surface, and focal retraction of podocytes, resulting in detachment of the epithelium from the underlying basement membrane. These structural changes are accompanied by immediate transient proteinuria only in animals given complete Freund's adjuvant at the time of antibody administration. These studies indicate that direct antibody-mediated glomerular injury can be induced in the rat by administration of monoclonal antibodies specific for cell associated antigens.

Idiotypes and Antiidiotypic Antibodies in Health and Disease

The diversity and effectiveness of the humoral immune response depends upon the huge number of different antibodies which a single individual can produce, estimated to be at least 100 million. Although some variation in the function of antibodies depends upon their class (isotype) the greatest part of antibodies' differences among themselves is due to the variable regions of the heavy and light chains, where the antibody combines with its antigen. These particular variations in immunoglobulin structure were detected over a quarter of a century ago by the production of a second generation of antibodies against the antigenic determinants of the polypeptides of the variable region of the first generation of antibodies injected into experimental animals [1-3]. The antigenic structure of the immunoglobulin molecule so defined became known as its 'idiotype', and the antibodies directed against the idiotypes are known as 'antiidiotypic antibodies' or 'antiidiotypes'. The individual antigenic determinants which together make up the idiotype are known as 'idiotopes' (Fig. 1). A single immunoglobulin molecule contains many idiotopes and can therefore give rise to a large number of antiidiotypic antibodies, distinct from each other, but all capable of reacting with the original immunoglobulin. As the antiidiotypes are directed against the Fab region of the first antibody, they are distinct from rheumatoid factors, i.e. antibodies directed against the constant Fc portion. Antiidiotypes may be of two sorts raised experimentally in the same or a foreign species, or arising in the host itself, i.e. an auto-antiidiotypic antibody. In many articles and papers, the prefix 'auto' is not used in the description of these autoantibodies, as it is usually clear from the context that autoantibodies are being described. The recognition of idiotypes and the production in normal and diseased subjects of autoantiidiotypic antibodies has led to concepts which are important in understanding the function of the immune system and, more recently, attempts to use them therapeutically.

Synthetic membrane active polyelectrolytes in design of artificial immunogens and vaccines

AbstractChemical binding or strong complexing of individual antigens (proteins, polysaccharides) or antigenic determinants with non‐natural membrane active polyelectrolytes results in formation of effective immunogens with high protective properties. These artificial antigenic conjugates develop strong immunogenicity also in the absence of T‐cells. The strength of the immune response is not controlled by Ir‐genes. An antigenic moiety of a conjugate is responsible for its “focusing” on B‐lymphocytes of the proper clone. Linear polyion moiety being adsorbed on the cell membrane surface aggregates membrane proteins and induces nonspecific transmembrane ionic permeability. K+‐flux from the cell and Ca+2‐flux into the cell disturb cell homeostasis and thus, pull trigger mechanism of K+, Na+‐ATPase and Ca+2‐ATPase activity. Then the cell synthesizing apparatus starts to operate. The covalent conjugates of Salmonella typhimurium and influenza A‐virus individual antigens with synthetic polyelectrolytes seem to represent a new generation of vaccinating macromolecules.

Clustering of antigenic determinants on H-2 molecules.

The spatial relationship of individual antigenic determinants on H-2Kk and H-2Db molecules was investigated with seven different monoclonal anti-H-2Kk and seven anti-H-2Db antibodies. In these studies the binding of radiolabeled monoclonal anti-H-2 to target cells was competed by addition of various cold anti-H-2 antibodies. The results indicate that on both H-2Kk and H-2Db molecules the antigenic determinants are arranged in two spatially separated clusters. Thus, antibodies to determinants within a cluster show mutual inhibition of binding but do not block the binding of antibodies to the other cluster, and vice versa. Furthermore, in the case of H-2Db antigens it was observed that binding of antibodies to one cluster would considerably enhance the binding of antibodies to the other cluster. A preliminary Scatchard analysis indicated that the enhancing antibody did not alter the affinity of the radiolabeled antibody, but led to an increase of available binding sites on the cell membrane. In addition, binding inhibition studies revealed that the conventional private specificity H-2.2 of H-2Db consists of at least two independent sites on the molecule.

Immunochemical Study of Gangliosides at the Cell Surface of Sea Urchin Embryos

Two main gangliosides (G-1 and G-2) were isolated from eggs and embryos S. intermedius. They contain glucose, N-glucolylneuraminic acids, phytosphyngosine, fatty acids and alpha-hydroxy fatty acids. Molar ratios and sequence of these components are the same for both gangliosides, but G-2 contains sulphate residue which is attached to the terminal neuraminic acid. To obtain specific antisera rabbits were immunized by G-1 or G-2, which were mixed with bovine serum albumin and Freund's adjuvant. Both gangliosides possessed electrophoretic and antigenic heterogeneity. G-1 and G-2 gangliosides have common and individual antigenic determinants. Glucosylceramide of gangliosides is immunologically inactive. Individual antigenic specificity of the gangliosides depends on the presence of N-glycolylneuraminic acid (G-1) and SO3H-group (G-2). Egg gangliosides were demonstrated by immunofluorescence throughout the cell surface. After fertilization of immunofluorescent label was concentrated on one pole of the embryo only. During the development of specific fluorescence was again uniformly distributed at the blastomer surface. The most intense fluorescence was observed in the junction areas of the blastomers.

Effect of monoclonal antibodies on phagocytosis and killing of Toxoplasma gondii by normal macrophages.

Treatment of intact toxoplasma tachyzoites with individual mouse monoclonal antibodies to toxoplasma which are directed against individual membrane-associated antigenic determinants facilitated the phagocytosis of toxoplasma and also prepared the toxoplasma for intracellular destruction by nonelicited mouse peritoneal macrophages. In instances in which the organisms survived intracellularly, their multiplication was significantly reduced. Such monoclonal antibodies should be useful in further elucidating the role of antibody in resistance to toxoplasma and other facultative and obligate intracellular organisms.

Züchtung von salmonella-R-Mutanten in submerskultur 2. einfluß der wachstumsphase auf den gehalt der bakterien an lipopolysaccharid sowie auf die chemische zusammensetzung und die serologischen eigenschaften der lipopolysaccharide

Abstract During the cultivation of microorganisms environmental conditions are known to exert an strong influence on metabolism, morphology and chemical composition of microbial cells. These phenotypic variations are also concerned with the chemical properties and function of the outer membrane. In the case of gram-negative bacteria lipopolysaccharides represent the main antigens of the bacterial cell-surface. At the same time lipopolysaccharides are the endotoxins of these bacteria, their toxicity residing in the lipid part of the molecule, the lipid A. It is therefore interesting whether the conditions of cultivation would also influence the amount of lipopolysaccharide synthesized, or even the biosynthesis of individual antigenic determinants, and finally the chemical composition of the lipid A. Differences in environmental conditions occur, for example, in mass-cultures at the different phases of growth. The present study deals with the cultivation of 5 Salmonella R-mutants of different chemotypes (Ra to Re) in complex medium under constant conditions of temperature, pH and aeration. From the growth curves thus obtained it may be seen that the time required by the various chemotypes to reach the exponential phase increases in the order of Ra to Re ( Download : Download full-size image ). Correspondingly the deep R-mutants require a longer time of cultivation before reaching the stationary phase of growth. Despite the above differences in growth-behaviour the overall yield of bacteria obtained with the 5 mutants was comparable (4.2–4.6 g/l). In the course of cultivation all mutants showed differences in the LPS content of the bacterial mass. A temporary stagnation in LPS biosynthesis, frequently found previously during cultivation of Salmonella S-forms (S. Schlecht, Zbl. Bakt. Hyg., I. Abt. Orig. A 232 [1975] 61–72), was observed here only in the case of an Ra mutant. Highest LPS yields were obtained in the stationary phase (207–334 mg/l culture fluid). Here, the content of extractable LPS was 5.0–6.2% of the bacterial masses. The true LPS content, estimated by measuring the amount of 3-hydroxymyristic acid present in the cells was 8.5–10% indicating that about 40% of the LPS was either not extractable or was lost during the isolation procedure. In the R mutants the molecular distribution of LPS was found to be 2–3 times higher on the bacterial surface than in the S forms, the density increasing from the higher to the lower R-chemotypes (Ra to Re). A small portion of the LPS was found extracellularly. Thus at the end of cultivation the culture medium contained about 5% of the total LPS, as determined in the case of the deeper R-mutants ( Download : Download full-size image ). Chemical analysis on LPS from cultures at different stages of growth, revealed no significant differences in the fatty acid composition of the lipid A or in the sugar composition of the R oligosaccharide ( Download : Download full-size image ). Only in the case of one R-strain at the end of cultivation a low increase in the phosphate content was found. Further, the substitution of lipid A with core oligosaccharide was the same at all stages of growth as indicated by the constant ratio of heptose: β-hydroximyristic acid. Hemagglutination-inhibition studies showed, however, that in the case of a leaky mutant (RcRb2) the Rb2 specificity was expressed more strongly in the exponential phase than at later stages of growth. This suggests, that the biosynthesis of the R-oligosaccharide may, in principle, be influenced by the changing culturing conditions. With all other mutants tested no differences were seen in the serological activity of the LPS at different stages of growth.

Expression of V-region-like determinants on Ig-negative precursors in murine fetal liver and bone marrow

IDIOTYPES, as defined by Oudin1, are antigenic markers associated with antibodies produced by a single individual. The demonstration of ‘individual antigenic determinants’ on human myeloma proteins2 introduced the concept of idiotype as a clonal marker of Igs. However, as the operational definition of an idiotype is based on the specificity of anti-idiotype antisera, the extent of cross-reactivity of a particular idiotype, and therefore the definition of idiotype, depends on the criteria set for the preparation of reagents. In fact, a number of ‘public’ or ‘cross-reactive’ idiotypes are currently described which are produced by all individuals of a strain or species or even by different species3. Yet, idiotypes are exclusively considered as markers of V-gene products defining groups of related Ig molecules. The basic concept of Jerne's network theory4, however, postulates that the universes of idiotypes and of antigens are identical. As a consequence, the finding of idiotype-cross-reactive determinants on non-Ig molecules should be the rule. We have observed5 that a class of polyclonally distributed mitogen receptors on splenic B cells bear determinants cross-reactive with the public idiotype of the J558 myeloma protein of BALB/c origin. We have investigated the possibility that this finding might be related to the mechanism of generation and regulation of the antibody repertoire. Having no indications on the growth regulation of B-cell precursors, we started to investigate the antigenic relationship between Ig idiotypes and mitogen receptors on those cells. We report here the cross-reactivity between several germ-line idiotypes and non-Ig surface structures expressed on cells present in haematopoietic tissues. We hypothesise that growth receptors on precursor B cells have determinants cross-reactive with germ-line V-gene products, and that precursor cells are driven and selected by interactions between these receptors and molecules with anti-idiotype specificity also belonging to an incipient network transmitted in the germline.

Fractionation of rabbit anti-horse cytochrome c—I : Specificity of the isolated fractions

A method has been devised to fractionate rabbit antibodies to horse cytochrome c by affinity chromatography. Immune serum was passed through a series of affinity columns (immunoadsorbents) each prepared with cytochromes c from closely related species. Antibody sub-populations were isolated by selection of the proper sequence of columns, and displayed unitary stoichiometry of reaction (one Fab' molecule bound per cytochrome c molecule) in fluorescence quenching experiments. The localization of the antigenic determinants towards which these tractions were directed was deduced from the pattern of the cross-reactions of such fractions with cytochromes c of known amino acid sequence. Analysis of these fractions reveals that residues 44 (proline). 60 (lysine) and 92 (glutamic acid) influence the structure of individual antigenic determinants of horse cytochrome c.

Role of IJ Region Determinants in Neonatal Tolerance Induction to K and D Region Alloantigens

Abstract Tolerance induction to isolated regional H-2 alloantigenic differences was studied by inoculating neonatal animals with lymphohematopoietic cells from F1 hybrids derived from H-2 congenic pairs, intra-H-2 recombinants. H-2 regions varied considerably in the tolerogenicity of their alloantigenic products, especially when host animals were derived from B10 and B6 genetic backgrounds. Isolated K and D region disparities, especially those resulting from mutation, were highly resistant to tolerance induction, while I region differences alone were highly tolerogenic; ease of tolerance induction across the entire H-2 complex was intermediate between these extremes. Using appropriate recombinants, poorly tolerogenic D and K region determinants, when presented conjointly with IJ region disparity in the tolerance conferring inoculum, proved to be highly specific, not only for the individual antigenic determinants, but apparently also for the composite antigenicity of the tolerance conferring inoculum: tolerance of D alloantigens was restricted to their original IJ region context, i.e. skin grafts from third party donors expressing the tolerated D antigen conjointly with an IJ region determinant unexpressed in the tolerizing inoculum were rejected.

Development of Monoclonal IgA and an Apparent IgG in a Patient with Macroglobulinemia: Sharing of Individually Specific Antigenic Determinants among IgM, IgA, and IgG

Patient CM, who initially was diagnosed as having macroglobulinemia (IgM, kappa) was subsequently found to develop a monoclonal IgA(kappa) protein. Rabbit antisera directed against the patient's IgAm and IgM were rendered specific for individual antigenic (ind) determinants. The anti-IgAm and IgM ind sera reacted with both 131I labeled monoclonal proteins in a double antibody radioimmunoassay (RIA). In addition, both monoclonal immunoglobulins inhibited the reaction between labeled immunoglobulin and both ind antisera, and statistical analysis of the data suggested that the shared ind determinants were identical. The IgG fraction of patient CM's serum also contained a component which competed with both monoclonal IgA (CM) and IgM (CM) in the RIA specific for ind determinants. Analysis of serum samples taken over a 2-year period revealed that, in addition to IgM, both the IgA and IgG components possessing the shared ind determinant(s) were present in low concentrations in the earliest sample, although not detected by conventional techniques. The monoclonal IgA and the IgG component were found to increase in concentration over this time interval with a concomitant decrease in IgM. The regulation of immunoglobulin expression with respect to the proposed models of gene organization in antibody-producing cells was discussed.