Neutralization Of Biological Activity Research Articles

Beyond B-Cell Epitopes: Curating Positive Data on Antipeptide Paratope Binding to Support Peptide-Based Vaccine Design.

B-cell epitope prediction is a computational approach originally developed to support the design of peptide-based vaccines for inducing protective antibody-mediated immunity, as exemplified by neutralization of biological activity (e.g., pathogen infectivity). Said approach is benchmarked against experimentally obtained data on paratope-epitope binding; but such data are curated primarily on the basis of immune-complex structure, obscuring the role of antigen conformational disorder in the underlying immune recognition process. This work aimed to critically analyze the curation of epitope-paratope binding data that are relevant to B-cell epitope prediction for peptide-based vaccine design. Database records on neutralizing monoclonal antipeptide antibody immune-complex structure were retrieved from the Immune Epitope Database (IEDB) and analyzed in relation to other data from both IEDB and external sources including the Protein Data Bank (PDB) and published literature, with special attention to data on conformational disorder among paratope-bound and unbound peptidic antigens. Data analysis revealed key examples of antipeptide antibodies that recognize conformationally disordered B-cell epitopes and thereby neutralize the biological activity of cognate targets (e.g., proteins and pathogens), with inconsistency noted in the mapping of some epitopes due to reliance on immune-complex structural details, which vary even among experiments utilizing the same paratope-epitope combination (e.g., with the epitope forming part of a peptide or a protein). The results suggest an alternative approach to curating paratope-epitope binding data based on neutralization of biological activity by polyclonal antipeptide antibodies, with reference to immunogenic peptide sequences and their conformational disorder in unbound antigen structures.

Monoclonal antibodies against vascular endothelial growth factor 165 (VEGF165): neutralization of biological activity and recognition of the epitope.

Five antibodies against vascular endothelial growth factor 165 (VEGF165) were obtained. These antibodies, Ab-2, Ab-20, Ab-153, Ab-309, Ab-342, were able to recognize not only native VEGF165, but also reducing VEGF165. Three of the antibodies, Ab-153, Ab-309, Ab-342, were identified as VEGF165 neutralizing antibody on the basis of its ability to inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) induced by VEGF165 and the binding of [125I]-VEGF165 to receptors on HUVEC. The fragments of E1 (VEGF120-135) and E2 (VEGF46-60) recognized by neutralizing antibodies may be related the receptor binding domain of VEGF.

Neutralization of biological activity and inhibition of receptor binding by antibodies against human thrombopoietin.

Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D8 to Q28, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S1 to R117 and A60 to K122 of hTPO, indicating that the epitope(s) of TN1 is localized in residues A60 to R117 of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S47 to D62, L108 to A126, N172 to A190, S262 to T284, and P306 to G332 of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D8 to Q28 and A60 to R117 of hTPO, may contain the domains associated with its receptor, C-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.

Monoclonal Antibodies against Heparin-Binding Growth Facto 1: Neutralization of Biological Activity and Recognition of Specific Amino Acid Sequence

A panel of three monoclonal antibodies against heparin-binding growth factor-1 (HBGF-1) was obtained. These antibodies, Ab-47α, Ab-15, Ab-29, were able to recognize HBGF-1 but not HBGF-2. One of the antibodies, Ab-47α, was identified as a HBGF-1 neutralizing antibody on the basis of its ability to inhibit the binding of [125I]HBGF-1 to receptors on HepG-2 cells and the proliferation of fetal bovine heart endothelial cells induced by HBGF-1. Ab-15 reacted with truncated HBGF-1(Mr= 16,000), intact HBGF-1( Mr= 18,000) and mutant HBGF-1U which lacks a putative nuclear translocation sequence (amino acid residues 21 to 27 of HBGF-1). Ab-29 reacted with only truncated HBGF-1 and was thought to recognize the putative nuclear translocation sequence of HBGF-1. The three monoclonal antibodies did not inhibit the binding of [125I]HBGF-1 to heparin. These data indicate that each monoclonal antibody recognizes a distinct epitope of HBGF-1 and identifies them as useful reagents for evaluating functional domains and biological roles of HBGF-1.

High titre anticytokine antibodies obtained by intralymphnode immunization with low amounts of antigen

Intralymphnode immunization was performed on rabbits to obtain anticytokine antibodies using low or very low amounts of the following purified cytokines: CSF-1 (or M-CSF: 10, 2 or 0.2 microgram/injection), GM-CSF (10 micrograms/injection), IL-2 (10 micrograms/injection) and HILDA/LIF (10 micrograms for the first injection and 5 micrograms/injection for boosts). This technique is easily performed by dissection of the popliteal lymphnode. Specific high titre antibodies were obtained after the first or second boost for antigen doses between 10 (for all cytokines tested) and 0.2 microgram (for CSF-1) per injection. In most cases, these antibodies could be used for immunoprecipitation, competition assays, dot immunoblotting, neutralization of biological activity and receptor binding inhibition. Some applications show that these tools are useful for cytokine research projects. For newly identified cytokines available in limited amounts, this method of obtaining specific polyclonal antibodies is an interesting alternative to the expensive, time-consuming and technically more demanding monoclonal antibody method.

Fibroblast stimulation in schistosomiasis. XI. Purification to apparent homogeneity of fibroblast-stimulating factor-1, an acidic heparin-binding growth factor produced by schistosomal egg granulomas.

Liver fibrosis is the most serious complication of infection with Schistosoma mansoni and Schistosoma japonicum and is responsible for severe morbidity and mortality in hundreds of thousands of patients in many Third World nations. The pathogenesis of this condition remains to be elucidated. We proposed that certain cytokines produced by cells that comprise the chronic granulomas that surround the helminth eggs within the liver initiate hepatic fibrogenesis. We now report our successful purification to apparent homogeneity of the egg granuloma-derived fibroblast mitogen. The high affinity of this factor for heparin (elutes from heparin-Sepharose with 1.5 M NaCl) facilitates its purification by a two-step procedure, and identifies the cytokine as a heparin-binding growth factor (HBGF). Furthermore, because it has an isoelectric point approximately equal to 6.2, it has one of the characteristics of a class 1 (acidic) HBGF. We immunized rabbits with the purified factor and observed that the resulting antibodies (IgG) detected the factor but not acidic fibroblast growth factor (the prototypic class 1 HBGF) either by dot-blot ELISA or neutralization of biologic activity. The granuloma product and fibroblast growth factor also differ in target-cell specificity and amino acid composition. On the basis of these distinctions, we have designated the granuloma-derived mitogen fibroblast-stimulating factor-1. With the availability of purified fibroblast-stimulating factor-1 and the future analysis of its amino acid sequence, its structural relationship to other mesenchymal growth factors can be determined.

Production of monoclonal antibodies that detect Hodgkin's high molecular weight transforming growth factor-beta

High molecular weight transforming growth factor-beta (TGF beta) is a physiologically active TGF secreted by nodular sclerosing Reed- Sternberg cells. Five monoclonal murine antibodies were prepared that distinguished Hodgkin's TGF beta from platelet-derived TGF beta using an enzyme-linked immunosorbent assay, neutralization of biologic activity, and Western blotting. These monoclonal antibodies directed at unique antigenic determinants (epitopes) of Hodgkin's TGF beta will allow further characterization of the role of Hodgkin's TGF beta in Hodgkin's disease and related entities.

Production of Monoclonal Antibodies That Detect Hodgkin's High Molecular Weight Transforming Growth Factor-β

High molecular weight transforming growth factor-beta (TGF beta) is a physiologically active TGF secreted by nodular sclerosing Reed-Sternberg cells. Five monoclonal murine antibodies were prepared that distinguished Hodgkin's TGF beta from platelet-derived TGF beta using an enzyme-linked immunosorbent assay, neutralization of biologic activity, and Western blotting. These monoclonal antibodies directed at unique antigenic determinants (epitopes) of Hodgkin's TGF beta will allow further characterization of the role of Hodgkin's TGF beta in Hodgkin's disease and related entities.

Towards large-scale purification of natural CSF-1 from human placenta tissue extracts.

The monocyte-macrophage colony-stimulating factor (colony-stimulating factor 1) is characterized and partially purified from industrially processed human tissues for the first time. A five-step purification procedure using placenta tissue extracts furnished a 13,620-fold enrichment of biological activity. This procedure includes a "pilot" scale anion-exchange chromatography at pH 4.5, gel permeation, and lectin affinity separation followed by HPLC steps (hydrophobic interaction and C18 reverse-phase chromatographies). The purified bioactive material, which stimulates only monocyte-macrophage progenitors and mature cells, showed an Mr of 58,000-62,000 (gel filtration) and an isoelectric point of 3.8-4.0. The hydrophobicity of the molecule was low, and the biological activity was eluted at 50% acetonitrile on a C18 reverse-phase HPLC column. It was totally inactivated by 2-beta-mercaptoethanol reduction and heat treatment. Immunoprecipitation and neutralization of biological activity with specific anti-CSF-1 antibodies (not shown) demonstrated that this material was CSF-1. Step 5 of this protocol yielded two silver-stained bands on 12.5% SDS-PAGE: a major 55-kDa band (96%) and a minor 33-kDa band (4%). CSF-1 was detected exclusively in a band of 52-62 kDa by both Western immunoblotting and bioassays. Immunoaffinity techniques using antibodies directed against selective epitopes on the placental CSF-1 are now considered to purify this material to homogeneity. This approach to the mass production of natural CSF-1 from human tissue has advantages with respect to both the difficulty of post-translational processing of bioactive material in procaryotes and the cost of eucaryotic cell cultures.

Epitope mapping of IL-1β: Identification of a discontinuous epitope of human IL-1 associated with neutralization of biological activity

Epitope mapping of IL-1β: Identification of a discontinuous epitope of human IL-1 associated with neutralization of biological activity

Porcine heart valves produce a protein that induces cell-mediated connective tissue degradation: I. Biological properties.

Cultured porcine heart valves secrete a factor that induces cells to degrade their extracellular matrix. This activity was routinely monitored by measuring the release of glycosaminoglycans from proteoglycan in cultured bovine nasal cartilage. It was observed that factor-mediated matrix degradation was dose dependent and required live target cells, while factor production by valves was inhibited by cycloheximide and declined with reduced cell viability. The effect of various steroidal and nonsteroidal anti-inflammatory drugs on the production and activity of the factor is discussed with reference to similar cytokines. It is concluded that there is a close similarity between the cardiac catabolic factor described here and catabolin, a protein isolated from porcine synovia and leukocytes, as shown by the neutralization of biological activity with antisera raised to porcine catabolin (interleukin-1).

Identification and Neutralization of Biological Activities Associated with Venom from the Brown Recluse Spider, Loxosceles reclusa *

Venom from the brown recluse spider, Loxosceles reclusa, reacted with human erythrocytes to form venom-sensitized erythrocytes. These cells were agglutinated specifically by high dilutions of adsorbed rabbit antivenin or were lysed by normal blood group compatible human sera. The specific rabbit antivenin prevented venom from attaching to erythrocytes, from interacting with serum complement, and from producing dermonecrotic lesions in rabbits. Results of experiments involving heat inactivation and adsorption to erythrocytes provide circumstantial evidence to suggest that the three biological activities of venom could be associated with a single component or few components with similar properties. The component interacting with serum complement is immunologically distinct from a factor in cobra venom which possesses similar biological activities.

Neutralization of primate chorionic gonadotropin activities by antisera against the subunits of human chorionic gonadotropin in radioimmunoassay and bioassay.

Antisera to the subunits (a and p) of human chorionic gonadotropin (hCG) were used in radioimmunoassay (RIA) and in neutralization of biological activity measured by rat uterine weight bioassay (RUW) to test for the presence of common antigenic determinants between the subunits and chorionic gonadotropins from the human, gorilla (gCG), chimpanzee (chCG), and rhesus monkey (mCG). Urinary kaolin—acetone extracts of gCG and chCG, as well as native hCG, inhibited antibody binding of both 125I labeled hCG—a and hCG—P in their respective immunoassay systems, but mCG was ineffective in RIA for either of the hCG subunits. Although RUW responses to hCG, gCG and chCG were neutralized by varying quantities of each antiserum, neither antiserum to hCG—a nor hCG—P inhibited the biological activity of mCG. Evidence was obtained by both RIA and RUW neutralization studies for similarity of antigenic determinants in preparations of gCG, chCG and hCG and the subunits of hCG. (Endocrinology 92: 705, 1973)

Similarity of antigenic determinants in pit- uitary and chorionic gonadotropins from primates.

Radioimmunoassay (RIA) and neutralization of biological activity in bioassays were used to test antisera for identification of common antigenic determinants among pituitary and chorionic gonadotropins in extracts of pituitary and urine, and in sera from man and other higher primates. Extracts of human and baboon pituitaries, pregnancy sera from rhesus monkeys, baboons and a chimpanzee, and extracts of chimpanzee urine displaced 131I-labeled human luteinizing hormone from combination with antiserum against human chorionic gonadotropin (HCG) made in a rabbit. In addition, an antiserum against HCG neutralized the biological activity of these materials. Despite the fact that not all of these materials were equally effective in the radioimmunoassay or biological test systems utilized, evidence was obtained for similarity of antigenic determinants among these preparations. (Endocrinology 88: 702, 1971)

Human Chorionic Thyrotropin: Further Characterization and Study of Its Secretion During Pregnancy1

The partial purification and immunological characterization of the human chorionic thyrotropin (HCT) was described. The behavior of the chorionic factor during the steps of purification was similar to that of pituitary thyrotropin (TSH) although its chromatographic and electrophoretic properties suggested that HCT was less acidic than the human pituitary hormone. The specific activity of the most purified fraction was 3500 times that of the starting extract. Double diffusion experiments showed the extent of immunological cross-reactivity of HCT with human pituitary thyrotropin to be low, in confirmation of earlier results, but a close immunological relationship between the chorionic factor, bovine and porcine TSH was found when both were reacted against highly specific antibovine or antiporcine TSH serum. This rather surprising difference in the degree of cross-reactivity between HCT with human compared to that with porcine and bovine TSH was confirmed by experiments on the neutralization of bio logical activity and by radioimmunological studies: HCT interfered very poorly in a 131I-human TSH-antihuman TSH antibody system, while it strongly inhibited the binding of labeled bovine TSH to antibovine or to antiporcine TSH antibody; HCT also inhibited the binding of labeled human TSH to antiporcine TSH antibody. The circulating thyroid-stimulating activity in pregnant women was found significantly higher than in serum of nonpregnant women, and particularly at the beginning of gestation. Radioimmunoassay of pregnancy serum extracts with high thyroid-stimulating activity gave results fitting in with the behavior of HCT in the 131I-human TSH-antihuman TSH as well as in the 13lI-human TSH-antiporcine TSH systems. It was thus suggested that HCT is actually secreted during pregnancy and responsible for the high thyroid stimulating activity of pregnancy serum.

Antibody to human follicle-stimulating hormone: cross-reactivity with three other hormones.

Antibodies directed against human follicle-stimulating hormone (FSH) were demonstrated in rabbit serum by neutralization of biological activity. Antibodies that bound FSH-(131)I were produced in rabbits and guinea pigs by repeated injections of FSH. By (131)I immunochemical methods, we found that at least 90% of the FSH-(131)I-binding antibody failed to distinguish the four human glycoprotein hormones: FSH, luteinizing hormone, chorionic gonadotropin, and thyrotropin, purified as well as endogenous hormone in plasma. Neither growth hormone, adrenocorticotropin, nor a variety of glycoproteins or animal plasmas were able to react with these antibodies.