Generation Of Specific Antibodies Research Articles

Vibrioantigens of polyvalent vaccines enhance the humoral immune response to Aeromonas salmonicidaantigens in Atlantic salmon ( Salmo salarL.)

Seven experimental oil-adjuvanted vaccines containing Aeromonas salmonicidaand one or more of the following components: Vibrio salmonicida, Vibrio anguillarumserovar O1 and V. anguillarumserovar O2, were compared to a monovalent A. salmonicidavaccine regarding their ability to induce antibody production against A. salmonicidain Atlantic salmon. Enzyme-linked immunosorbent assay (ELISA) tests were used to measure the humoral immune response against various antigen preparations from A. salmonicida; bacterial cell-sonicate, A-layer protein and lipopolysaccharide. The effects of the different Vibriospp. on the generation of specific antibodies were estimated through a factorial statistical design. The results showed that stronger humoral immune responses to A. salmonicidaantigens were induced in fish vaccinated with the various polyvalent vaccines, compared to fish vaccinated with the monovalent furunculosis vaccine. V. salmonicidatended to be the most important component for this enhancement, although V. anguillarumalso contributed. Immunological cross-reactions between A. salmonicidaand V. salmonicidais a possible mechanism.

V λ-light chain genes reconstitute immune responses to defined carbohydrate antigens or haptens by utilizing different V H genes

The contribution of the λ-light chain to the development of peripheral B cell repertoire and generation of specific antibodies to haptens and polysaccharide antigens was studied in genetically manipulated kappa-deficient and λ 2-transgenic mice. The results clearly demonstrate a non-stoichiometric V H gene family expression in the absence of k-light chain and suggest a non-stochastic pairing between V H and V λ genes, expressed in the peripheral B cell repertoire. A shift in V H gene utilization in the case of Vl λ + antibodies was evident in response to β2–6 fructosan and TNP hapten. These observations demonstrate the availability of compensatory mechanisms in the absence of V K genes and are consistent with the hypothesis that V H gene family expression is controlled by genetic factors from inside the V H locus. Furthermore, genetic factors from outside the V H locus, namely restricted available light chain diversity, may lead to a shift in V H gene utilization in the peripheral B cell repertoire.

Gel fiberglass as a new matrix of affinity chromatography columns for isolation of colon cancer-associated antigens

Gel fiberglass (GFG), a new affinity biosensor, was used to isolate human p53 antigen with rabbit anti-rat p53 IgG. The biosensor was prepared as a membrane from glass fibers covered with oxysilanes. A thin layer of protein, trapped in gel glass during its preparation, is deposited on the surface of a lattice of glass fibers. In such conditions, a maximum number of protein molecules may contact external agents percolated through a membrane. The membranes demonstrate high stability and can be stored in dry conditions or several months at room temperature. Columns for affinity chromatography were prepared from the GFG membranes and were used to isolate various proteins, including the tumor-associated antigens (TAA). The capacity of such columns was calculated as the amount mg of protein isolated from 1 ml of TAA-containing serum. In colon cancer patients, up to 5-6 mg TAA were extracted from 1 mi of sera. Two main components of cytoplasmic TAA isolated in our experiments were p64 and p53 proteins. Their concentration was determined by HPLC. The p53 protein has been isolated from the serum of cancer patients in the highest concentration yet reported, up to 3-4 mg/ml. In our previous studies, isolation of p53 protein was based on its affinity reaction with anti-p53 IgG generated against antigens of the same species. Herein, we report for the first time the capability to isolate human p53 antigen using GFG columns with entrapped anti-rat p53 IgG. Blood levels of p53 antigen isolated were very similar in both experiments. This has both theoretical and practical significance, demonstrating that the GFG membranes have great potential for isolating macromolecules utilizing various ligands. The finding facilitates an easy and highly effective method to isolate antigens from different organs, both animal and human, which can be used for important goals including diagnosis, therapy and generation of specific antibodies.

Human Immunoglobulins Produced in hu-PBL-SCID Mice Are Polyclonal Early after Xenotransplantation

Xenotransplantation of human peripheral blood leukocytes (hu-PBL) to severe combined immune-deficient (SCID, hu-PBL-SCID) mice leads to human immunoglobulin (Ig) production in the animals. Here we used a sensitive high-resolution two-dimensional electrophoresis (2D-PAGE) technique to separate, and to analyze, Ig γ and light chains in sera from SCID mice given hu-PBL for less than 35 days. Human Igs in the model appeared polyclonal until 3 weeks after cell transfer. After this period, oligoclonal Igs progressively replaced this polyclonal background. The 2D-PAGE pattern evolution of human Igs was similar when hu-PBL originated from donors with or without previous contact with the Epstein–Barr virus. The broad diversity of human Igs, and therefore the large expressed B cell repertoire, in hu-PBL-SCID mice during a period that allows manipulation (such as immunization) of the human graft has evident implication for the use of this model in the generation of specific antibodies of desired specificity.

Design and development of synthetic peptide antigens

Abstract

Expression of alkaline phosphatase in differentiated rat labyrinthine trophoblast tissue

In this report, we describe the generation of specific antibodies to rat alkaline phosphatase and the temporal and regional characteristics of alkaline phosphatase expression during maturation of the rat chorioallantoic placenta. An antipeptide antiserum was generated to the amino terminal 15 amino acids of rat alkaline phosphatase. The antiserum specifically recognized alkaline phosphatase. Alkaline phosphatase expression was monitored in the junctional and labyrinth zones of the chorioallantoic placenta by Western and Northern blot analyses. Alkaline phosphatase protein and mRNA were present in both the junctional and labyrinth zones on day 13 ofgestation. As gestation advanced, alkaline phosphatase mRNA and protein expression decreased below the limits ofdetection in the junctional zone, while alkaline phosphatase expression increased in the labyrinth zone. Labyrinthine alkaline phosphatase migrated predominantly as a 95-kDa species, whereas rat kidney expressed exclusively the 75-kDa species. Enzymatic deglycosylation of the 75- and 95-kDa alkaline phosphatase species resulted in the generation of a 55-kDa species. In summary, alkaline phosphatase expression is a useful indicator of trophoblast differentiation.

The Biology of Amiloride-Sensitive Sodium Channels

Studies of the molecular nature of epithelial sodium channels are in their infancy and have largely involved experiments in which the interaction between amiloride and this transport process has been examined. Because of the inherent geometric complexity of epithelial tissues, these studies have in large measure been macroscopic in nature, with the molecular details of transport being deduced. In the past five years, however, the molecular biology of these critical ion channels has been studied directly. The development of radioactive high-affinity probes, the application of patch-clamp and reconstitution techniques, the generation of specific antibodies, and the formulation of epithelial cDNA expression libraries have propelled the field of epithelial ion channels into a new era. Now, for the first time, we can rigorously address questions concerning the molecular nature of the amiloride block, the channel's selectivity to alkali metal cations, and the modulation of ion transport through this channel by other ions (such as calcium), hormones (such as vasopressin, aldosterone, and atrial natriuretic factor), or intracellular second messengers (such as cAMP or cGMP). The complexity of the epithelial sodium channel's structure may reflect the constitutive and regulatory role this protein plays in sodium homeostasis. The epithelial sodium channel is continually operating, constantly changing its activity on a second-to-second basis. Hence, its tonic functions are probably modulated by a myriad of factors, most of which are unknown. With the application of molecular techniques, a much clearer understanding of the nature and regulation of epithelial sodium channel processes in health and disease will emerge in the years to come.

Antigen-specific enhancement of the human antibody response by a substituted nucleoside.

Studies from this laboratory have shown that bromination of guanosine endows it with the capacity to induce murine B cells to undergo blast transformation and high level synthesis and secretion of polyclonal immunoglobulin1,2. This and certain other C8-substituted guanine ribonucleosides have been demonstrated to transmit T cell-like signals to B cells, allowing the generation of specific antibodies to a T cell-dependent antigen in the absence of T cells. This type of signal is distinct from signals provided by T cells, and in fact displays a strong synergistic interaction with T cell-derived lymphokines3. Furthermore, the same nucleosides cause marked augmentation of the magnitude of primary antibody responses both in vitro 4 and in vivo 5 in the murine model.KeywordsNone NoneHigh Level SynthesisAdjuvant ActivityIncremental ConcentrationPolyclonal Immunoglobulin1These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Corticotropin Releasing Factor (CRF): Immunocytochemical localization and Radioimmunoassay (RIA)

Recent isolation, structural identification, and synthesis of ovine CRF has made possible the generation of specific antibodies against this hypothalamic peptide. Two fragments of the amino acid sequence corresponding to ovine CRF (CRF 37-41 and CRF 22-41), as well as the full sequence of 41 residues (CRF 1-41), synthesized in our laboratories by solid-phase methods, were coupled to bovine serum albumin (BSA) with glutaraldehyde. New Zealand white rabbits were immunized with the emulsified mixtures of peptide-BSA conjugates and Freund's adjuvant as immunogens. The specificity of the generated antibodies was studied by agar-gel diffusion, absorption tests in the immunohistochemical system, and with the aid of displacement curves in RIA. 125I-Tyr(35)-CRF 36-41 and 125I-Tyr(0)-CRF 1-41 were used as radioligands in the RIA. The minimum detectable dose was 20 pg. The linearity observed in RIA for immunoreactive CRF in extracts of rat hypothalami, together with the immunocytochemical findings in the rat brain, indicate the presence of substance(s) immunologically indistinguishable from CRF. Immunohistochemistry with the peroxidase-antiperoxidase (PAP) technique detected the following CRF-immunoreactive structures in vibratome sections of hypothalami of colchicine-treated rats: CRF-containing cell bodies were observed mainly in smaller neurons of the paraventricular nucleus. CRF-positive nerve fibers and/or terminals were present in the external zone of the median eminence, with some immunoreactive CRF also present in the internal zone. The CRF-positive terminals were localized in the central regions of the median eminence. These morphological data reinforce the view that this polypeptide plays a physiological role in the control of ACTH release.

A monoclonal antibody to human acute lymphoblastic leukaemia antigen.

Previous studies by Greaves and others have demonstrated the existence of an antigen associated with cells from many patients with acute lymphoblastic leukaemia (ALL) and some patients with chronic myelocytic leukamemia (CML) in blast crisis. Antisera to this common ALL antigen (CALLA) have been produced in rabbits and require extensive absorption which limits both the titre and quantity of antisera that can be generated and may result in variable specificity in different laboratories. The method for generation of specific antibody by somatic cell hybridisation introduced by kohler and Milstein has been successfully used to produce monoclonal antibodies against various normal human cell-surface proteins, including beta 2 microglobulin, histocompatibility antigens, thymocyte and peripheral T-cell antigens and Ia-like antigens. The present report describes the generation and characterisation of a monoclonal antibody specific for a common ALL antigen (CALLA) previously identified by conventional heteroantisera.

T Cell Regulation of Specific B Cell Responses

Abstract The role of T cells in the generation of specific antibodies to antigens under Ir gene control was investigated in strain 2 guinea pigs. The specificity of anti-α,Dnp-Lys9 antibody was studied in guinea pigs with intact immune responses and in strain 2 guinea pigs deprived of T cells by adult thymectomy, lethal irradiation, and reconstitution with syngeneic bone marrow cells. Conventional strain 2 guinea pigs developed both T cell responses and highly specific anti-α,Dnp-Lys9 antibody. In contrast, strain 2 “B” guinea pigs failed to develop delayed hypersensitivity and in vitro T cell responses. Nevertheless, these “B” guinea pigs could produce anti-Dnp antibody of restricted heterogeneity and in quantities comparable to those produced by the conventional strain 2 guinea pigs. It was shown that anti-α,Dnp-Lys9 antibody produced in strain 2 “B” guinea pigs lacking specific T cell responses could not distinguish α,Dnp-Lys9 from dinitrophenol. Similar antibodies are produced in strain 13 guinea pigs that are poly-L-lysine, Ir gene negative. Reconstitution of strain 2 “B” guinea pigs with normal T cells enabled them to develop both cell-mediated immune responses and antibody of highly discriminatory power. These findings support the notion that in the absence of specific T cells, comparably specific B cell antibody responses cannot be generated, and perhaps more importantly, that specific T cells are required to facilitate the amplification of unique B cell clones.