Monoclonal Antibodies Against Surface Antigens of Vibrio Anguillarum Serogroup 02

Monoclonal antibodies to Vibrio anguillarum 0-group 2 (MAB 02) were used to compare the cell surface determinants of V. anguillarum 0-groups 1 and 2, and V. ordalii. In the homologous system a significant reactivity was found, while MAB 02 clones showed a very low-graded cross-reactivity to V. anguillarum 01 and V. ordalii. The importance of these aspects in the selection of adequate vaccine strains is discussed, with an emphasis on the improvement of vibriosis vaccines.     

Identification of Specific Antibodies Prepared Against Purified Immobilization Antigens of Paramecium aurelia

SYNOPSIS The identity of antibodies prepared against preparations of isolated immobilization antigen protein of Paramecium aurelia has been studied. Antisera were fractionated by ammonium sulfate precipitation, sucrose gradient centrifugation, and ion exchange chromatography on DEAE cellulose. The different fractions were assayed for the presence of specific antibodies by the immobilization test, and the globulins were identified by immuno-electrophoresis. It is concluded that in the sera studied in this investigation, specific antibodies are excusively of the class Ig G.     

Monoclonal Antibodies Against Vasoactive Intestinal Polypeptide: Studies of Structure and Related Antigens

Abstract: Hybridomas secreting monoclonal anti-vaso-active intestinal polypeptide (VIP) antibodies were constructed from spleen cells sensitized to VIP in vitro . The secreted antibodies were characterized by binding to VIP in indirect radioimmunoassays and enzyme-linked immunosorbent assays. Two monoclonal antibodies, characterized for their binding activities with synthetic fragments of VIP, were found to bind different sites on the VIP molecule. These monoclonal antibodies may recognize tertiary structures of the VIP. A search was conducted for antigens recognized by the monoclonal antibodies in brain: brain proteins separated on polyacrylamide gels were electroblotted onto nitrocellulose filters and were reacted first with the mouse antibody and then with goat anti-mouse imunnoglobulin coupled to horseradish peroxidase as a means of detection. The monoclonal antibodies were found to react with a protein of molecular weight 60,000, which was also recognized by polyclonal antibodies, although the latter reacted with a number of additional proteins. The relationship of the protein of molecular weight 60,000 to VIP is discussed.     

Development of Panel of Monoclonal Antibodies Specific to Urochordate Cell Surface Antigens

Monoclonal antibodies are an important tool in the study of botryllid ascidians’ immunology and developmental biology. Here we describe the development of a panel of 38 monoclonal antibodies that are specific to Botryllus schlosseri (Ascidiacea; subfamily Botryllinae) cell surface antigens. Many of these hybridomas recognize (by enzyme-linked immunosorbent assay and immunohistochemistry) epitopes of Botrylloides subpopulations (SP) II and III from the Mediterranean coast of Israel and show, on blood cell smear assays, reactions with subsets of Botryllus circulating blood cells. Fluorescence-activated cell sorting analyses using antibodies positive for botryllid tissues revealed up to 3.6% positive cells. ELISA screenings were performed with 64 new monoclonal antibodies on 5 different individual botryllid ascidian colonies (B. schlosseri, Botrylloides). The positive antibodies in this panel identified a large number of different antigenic determinants, some of which distinguish Botryllus versus Botrylloides colonies, and other, different colonies within these two species, or different cell types within tissues, embryos, and buds of individual colonies. Only 21 monoclonal antibodies tested positive with all colonies. Cross-reactivity with at least one Botrylloides colony was recorded in 49 hybridomas that identified Botryllus cells. This wide panel of monoclonal antibodies is the first such detailed set of monoclonals available for studies on botryllid ascidians.     

Antinuclear Antibodies and Nuclear Antigens

The lupus erythematosus (LE) cell factor is one of a variety of heterogeneous antibodies directed against many nuclear antigens. These antinuclear antibodies are found in a wide variety of clinical disorders. As detected by immunofluorescence techniques, they are so frequently found in association with systemic lupus erythematosus that their absence essentially excludes that diagnosis.It is suggested that in certain situations other than systemic lupus, antinuclear antibodies may be the result of some inflammatory and destructive processes. Antinuclear antibodies may be detected in certain lower animals frequently in association with disease. In lower animals, immunization with nuclear antigens appropriately complexed^* to protein carriers consistently results in the induction of antinuclear antibodies. In both man and animals, antinuclear antibodies are often detected without associated disease. Furthermore, antinuclear antibodies are not injurious to intact tissue culture cells. Nonetheless, antigen-antibody complexes consisting of nuclear antigens and antinuclear antibodies may contribute to the propagation of certain diseases, particularly lupus nephritis.The use of antinuclear antibodies as immunochemical tools holds great promise for the better understanding of such nuclear antigens as are found in viruses and in the nuclei of certain malignant cells.     

Induction of Strain-Transcending Antibodies Against Group A PfEMP1 Surface Antigens from Virulent Malaria Parasites

Sequence diversity in pathogen antigens is an obstacle to the development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. Here we identified the PfEMP1 variants transcribed by P. falciparum strains selected for a virulence-associated adhesion phenotype (IgM-positive rosetting). The parasites transcribed a subset of Group A PfEMP1 variants characterised by an unusual PfEMP1 architecture and a distinct N-terminal domain (either DBLα1.5 or DBLα1.8 type). Antibodies raised in rabbits against the N-terminal domains showed functional activity (surface reactivity with live infected erythrocytes (IEs), rosette inhibition and induction of phagocytosis of IEs) down to low concentrations (<10 µg/ml of total IgG) against homologous parasites. Furthermore, the antibodies showed broad cross-reactivity against heterologous parasite strains with the same rosetting phenotype, including clinical isolates from four sub-Saharan African countries that showed surface reactivity with either DBLα1.5 antibodies (variant HB3var6) or DBLα1.8 antibodies (variant TM284var1). These data show that parasites with a virulence-associated adhesion phenotype share IE surface epitopes that can be targeted by strain-transcending antibodies to PfEMP1. The existence of shared surface epitopes amongst functionally similar disease-associated P. falciparum parasite isolates suggests that development of therapeutic interventions to prevent severe malaria is a realistic goal.     

Monoclonal Antibodies to Toxoplasma Cell Membrane Surface Antigens Protect Mice from Toxoplasmosis1,2

Groups of mice were given an intraperitoneal injection of one of six monoclonal antibodies to Toxoplasma gondii, a mixture of equal amounts of five monoclonal antibodies to T. gondii, or the murine myeloma protein MOPC 21, and challenged with either a highly virulent or moderately virulent parasite strain. Two monoclonal antibodies (FMC 19 and FMC 22) conferred total protection against the moderately virulent challenge, with all mice surviving, whereas 90% of control mice died. FMC 19 and FMC 22 also conferred significant protection against the highly virulent challenge as indicated by a prolonged mean time to death (MTD) of immunized compared with control groups of mice. One monoclonal antibody (FMC 23) and the mixture of five antibodies gave significant protection against the moderately virulent challenge only. Passive immunization with dilutions of FMC 22 antibody indicated that the lowest serum titer needed to confer significant protection to mice against a moderately virulent Toxoplasma challenge was 1/640. Mice challenged with highly virulent tachyzoites that had been preincubated with FMC 22 had a significantly longer MTD than mice challenged with highly virulent tachyzoites that had been preincubated with MOPC 21 or phosphate buffered saline, pH 7.2 (PBS). Immunoprecipitation and autoradiography of radiolabeled tachyzoites confirmed that FMC 19 was directed against a 35,000 molecular weight (mol. wt.) antigen and FMC 22 was directed against a 14,000 mol. wt. fraction. The potential for use of single antigens as protective immunogens in preventing toxoplasmosis is raised.     

Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules

To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.     

Monoclonal Antibodies Against Glutaraldehyde-Conjugated Dopamine

Four mice were immunized with dopamine (DA)-glutaraldehyde (G)--protein conjugates over a period of 8-10 weeks. Polyclonal antisera, obtained at various intervals, were tested using an enzyme-linked immunosorbent assay (ELISA). All had anti-conjugated DA antibodies. As soon as good antibody affinity was detected between 10(-10) and 10(-6) M, the mouse yielding the highest apparent affinity was killed, and the spleen was dissected out. Hybridomas were obtained from spleen cells fused with SP2/O/Ag myeloma cells. Supernatant culture media of hybridomas were tested for the presence of anti-conjugated DA antibodies with the ELISA method. Selected hybridomas giving good antibody affinity and specificity were then cloned by the limiting dilution technique. The resulting supernatant culture media were again tested by ELISA. Clones that gave a high antibody affinity (10(-10)-10(-8)M) for G-conjugated DA were used for histochemical localization of DA in rat brain. G-fixed rat brains were sectioned from the telencephalon to the mesencephalon, reduced with sodium borohydride, and prepared for peroxidase-antiperoxidase immunocytochemistry using supernatant (diluted 1:100) or ascites fluid (diluted 1:50,000). Dense networks of very fine fibers were observed in the striatum, septum, and cortex. Numerous immunoreactive cell bodies were found in the ventral tegmental area, the substantia nigra, the hypothalamus, and the dorsal raphe. The ELISA tests and adsorption controls suggested that the monoclonal antibody allowed highly specific detection of DA in tissues.     

Human Carcinoma Antigens cross reacting with Anti-embryonic Antibodies

EMBRYONAL antigens found in malignant neoplasms but not in homologous normal adult tissues have been much studied. Recently, universal embryonal tissue components were detected in different types of carcinomas of mice¹. Our work indicates that immunologically definable embryonic cell components are universally present in human carcinomas.     

Generation of Monoclonal Antibodies against Highly Conserved Antigens

Background

Therapeutic antibody development is one of the fastest growing areas of the pharmaceutical industry. Generating high-quality monoclonal antibodies against a given therapeutic target is very crucial for the success of the drug development. However, due to immune tolerance, some proteins that are highly conserved between mice and humans are not very immunogenic in mice, making it difficult to generate antibodies using a conventional approach.

Methodology/Principal Findings

In this report, the impaired immune tolerance of NZB/W mice was exploited to generate monoclonal antibodies against highly conserved or self-antigens. Using two highly conserved human antigens (MIF and HMGB1) and one mouse self-antigen (TNF-alpha) as examples, we demonstrate here that multiple clones of high affinity, highly specific antibodies with desired biological activities can be generated, using the NZB/W mouse as the immunization host and a T cell-specific tag fused to a recombinant antigen to stimulate the immune system.

Conclusions/Significance

We developed an efficient and universal method for generating surrogate or therapeutic antibodies against “difficult antigens” to facilitate the development of therapeutic antibodies.     

Expanded Polyfunctional T Cell Response to Mycobacterial Antigens in TB Disease and Contraction Post-Treatment

Background

T cells producing multiple factors have been shown to be required for protection from disease progression in HIV but we have recently shown this not to be the case in TB. Subjects with active disease had a greater proportion of polyfunctional cells responding to ESAT-6/CFP-10 stimulation than their infected but non-diseased household contacts (HHC). We therefore wanted to assess this profile in subjects who had successfully completed standard TB chemotherapy.

Methods

We performed a cross-sectional study using PBMC from TB cases (pre- and post-treatment) and HHC. Samples were stimulated overnight with TB antigens (ESAT-6/CFP-10 and PPD) and their CD4+ and CD8+ T cells were assessed for production of CD107a, IFN-γ, IL-2 and TNF-α and the complexity of the responses was determined using SPICE and PESTLE software.

Results and Conclusions

We found that an increase in complexity (i.e., production of more than 1 factor simultaneously) of the T cell profile was associated with TB disease and that this was significantly reduced following TB treatment. This implies that T cells are able to respond adequately to TB antigens with active disease (at least initially) but the ability of this response to protect the host from disease progression is hampered, presumably due to immune evasion strategies by the bacteria. These findings have implications for the development of new diagnostics and vaccine strategies.     

自身免疫性疾病治疗性疫苗研究进展

自身免疫性疾病的发生与体内自身反应性T／B细胞的异常活化有关。病理性自身免疫应答是多发性硬化、重症肌无力等自身免疫性疾病的主要致病原因。针对已感染致病原的无症状携带者或发病的患者的自身免疫性疾病治疗性疫苗能特异性地调节异常的免疫应答,具有重要的理论价值和应用前景。     

Humoral Responses to Diverse Autoimmune Disease-Associated Antigens in Multiple Sclerosis

To compare frequencies of autoreactive antibody responses to endogenous disease-associated antigens in healthy controls (HC), relapsing and progressive MS and to assess their associations with clinical and MRI measures of MS disease progression.

Methods

The study analyzed 969 serum samples from 315 HC, 411 relapsing remitting MS (RR-MS), 128 secondary progressive MS (SP-MS), 33 primary progressive MS (PP-MS) and 82 patients with other neurological diseases for autoantibodies against two putative MS antigens CSF114(Glc) and KIR4.1a and KIR4.1b and against 24 key endogenous antigens linked to diseases such as vasculitis, systemic sclerosis, rheumatoid arthritis, Sjogren’s syndrome, systemic lupus erythematosus, polymyositis, scleroderma, polymyositis, dermatomyositis, mixed connective tissue disease and primary biliary cirrhosis. Associations with disability and MRI measures of lesional injury and neurodegeneration were assessed.

Results

The frequencies of anti-KIR4.1a and anti-KIR4.1b peptide IgG positivity were 9.8% and 11.4% in HC compared to 4.9% and 7.5% in RR-MS, 8.6% for both peptides in SP-MS and 6.1% for both peptides in PP-MS (p = 0.13 for KIR4.1a and p = 0.34 for KIR4.1b), respectively. Antibodies against CSF114(Glc), KIR4.1a and KIR4.1b peptides were not associated with MS compared to HC, or with MS disease progression. HLA DRB1*15:01 positivity and anti-Epstein Barr virus antibodies, which are MS risk factors, were not associated with these putative MS antibodies.

Conclusions

Antibody responses to KIR4.1a and KIR4.1b peptides are not increased in MS compared to HC nor associated with MS disease progression. The frequencies of the diverse autoreactive antibodies investigated are similar in MS and HC.