Variable regions of antibodies to synthetic polypeptides--I. Characterization of an idiotope expressed on antibodies and T-cell factors

Spleen cells from a Lewis rat immunized with affinity-purified B10 anti-(T,G)-A-L antibody were fused with the non-secreting murine hybridoma SP2/0. Cell lines secreting monoclonal antibodies specific for mu- and kappa-chains, as well as an idiotope on anti-(T,G)-A-L antibodies, were isolated and characterized. The anti-mu and -kappa antibodies, are true anti-isotypes, reacting with sera from all strains of mice tested. The anti-idiotope antibodies recognize a determinant on antibodies binding a GT-containing epitope. The proportion of anti-GAT antibody bearing the idiotope varies markedly in different murine strains. A 1000-fold higher level of antibody from Igha mice than from Ighb and Ighe mice is required to give an equivalent inhibition of the idiotope-anti-idiotope reaction. Analysis of monoclonal antibodies expressing the idiotope indicates that the affinity of binding between idiotope and anti-idiotope can vary by as much as two orders of magnitude. Immunoadsorbants prepared with anti-idiotope antibody bind suppressor factor secreted by a GAT-specific T-cell hybridoma.     

Comparison of radioimmunoassay and ELISA methods for detection of antibodies to chromatin components

A solid phase radioimmunoassay has been compared with an enzyme-linked immunosorbent assay (ELISA) for efficacy in measuring anti-chromatin antibodies. The low backgrounds achieved with the radioimmunoassay method produced a high signal-to-noise ratio and enabled detection of the human test antiserum at a dilution of 1:102,400. By contrast, the ELISA could detect the same antiserum only at a dilution of 1:3200 and above. The radioimmunoassay was consistently more sensitive than the ELISA for detection of anti-chromatin antibodies in a number of human and mouse sera and ascites fluid containing a monoclonal antibody. Factors affecting sensitivity in both assays are discussed.     

High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100

Advances in molecular evolution strategies have made it possible to identify antibodies with exquisite specificities and also to fine-tune their biophysical properties for practically any specified application. Depending on the desired function, antibody/antigen interactions can be long-lived or short-lived and, therefore, particular attention is needed when seeking to identify antibodies with specific reaction-rate and affinity properties. Surface plasmon resonance (SPR) biosensors routinely generate sensitive and reliable kinetic data from antibody/antigen interactions for both therapeutic and diagnostic applications. However, many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To ameliorate this problem, we developed a rapid and reliable assay for characterising recombinant scFv antibody fragments, directly from crude bacterial lysates. Ninety-six scFv antibodies derived from chickens immunised with C-reactive protein (CRP) were selected by phage display and evaluated using the Biacore A100 protein interaction array system. Antibodies were captured from crude bacterial extracts on the sensor chip surface and ranked based on the percentage of the complex left (% left) after dissociation in buffer. Kinetic rate constants (k(a) and k(d)) and affinity (K(D)) data were obtained for six clones that bound monomeric CRP across a broad affinity range (2.54 x 10(-8) to 3.53 x 10(-10) M). Using this assay format the A100 biosensor yielded high quality kinetic data, permitting the screening of nearly 400 antibody clones per day.     

Use of a self-generating percoll gradient and single cell cytotoxicity assay to identify tumor-lytic properties of inflammatory neutrophils

Within a murine model of regional immunotherapy, the cytolytic potential of peritoneal neutrophils could not be confirmed or quantified using routine techniques of cell separation and chromium release assays. We, therefore, developed procedures for the enrichment of neutrophils and estimation of the frequency of killer cells. Peritoneal exudate cells from mice injected with Corynebacterium parvum were fractionated on a self-generating Percoll gradient to enrich for neutrophils and deplete macrophages. A significant enrichment of neutrophils (greater than 90%) was obtained in a band corresponding to a density of 1.088 with a recovery of 35-50% of input. Neutrophil-enriched cell populations were then mixed with tumor cells to examine neutrophil-target interactions at the single cell level. Conjugates of neutrophils and tumor targets were obtained and the majority were lytic. With the aid of trypan blue staining and safranin counterstaining, it was possible to distinguish effector cells from targets and neutrophils from other host cells. The frequency of conjugates was dependent upon the effector to target cell ratio and was not affected by changes in temperature (range 4-30 degrees C). The post-binding lytic events were initiated rapidly after conjugation and tumor lysis was completed within 30 min. The lytic events occurred optimally between 25 degrees and 37 degrees C. The present studies support the role of neutrophils in tumor lysis following administration of an immunoadjuvant. The techniques described are important to further study the role of neutrophils in disease states as well as the underlying mechanisms of neutrophil-mediated tumor cytotoxicity.     

Allergens in Hymenoptera venom XIII: Isolation and purification of protein components from three species of vespid venoms

Pure venoms were collected from individual insects of the species Dolichovespula maculata, white-faced hornet, Vespula squamosa, southern yellow jacket, and Polistes exclamans, paper wasp (one species). The venoms were first fractionated by high-resolution gel filtration on a 1.6 m column of Sephadex G-75 superfine, and the components were then purified by high-performance, ion-exchange chromatography on a Mono-S cation exchange column followed by a further gel filtration step. The isolated components were evaluated for purity by sodium dodecyl sulfate polyacrylamide gel electrophoresis by use of two different types of silver stains, by assays for enzyme activities, and by immunodiffusion with the use of rabbit antisera. The protein components were isolated in highly purified states by these techniques. Only three significant proteins were found in V. squamous venom: phospholipase (PL) A and B, hyaluronidase (HYAL), and antigen 5 (Ag 5). D. maculata venom contained HYAL, Ag 5, two isozymes of PL A and B, a high-molecular-weight protein, and several trace proteins. No significant amounts of proteases were found in D. maculata venom. P. exclamans venom contained HYAL, PL A and B, Ag 5, a high-molecular-weight protein, and several minor proteins. In all three venoms the PL A and B activities were found to be in the same molecule and did not separate. Trace components with apparent PL A activity were observed in the venoms. The venoms were screened for a variety of esterases, proteases, peptidases, glucosidases, and phosphatases, and none were detected in more than trace amounts. Vespid venoms do not appear to contain significant amounts of acid phosphatases as bee venoms do.     

Interaction of immunoglobulin with actin

Actin can form specific, direct associations with immunoglobulin resulting in soluble complexes or cross-linked matrices. This interaction can be detected by four in vitro assays using purified components: (1) actin enhances the cytophilic activity of guinea pig IgG2; (2) in solutions of low ionic strength, actin and IgG2 co-precipitate: (3) soluble complexes exist in 0.1 M KCl as revealed by the displacement of actin from its expected sedimentation pattern in a gradient of sucrose when in the presence of IgG 1, IgG2, or IgM; (4) immunoglobulin (IgG1, IgG2, BGG)‡: increases the viscosity of F-actin solutions, presumably by crosslinking F-actin filaments. These data suggest that direct interaction of a cytoskeletal protein with a cell surface receptor is possible.     

Production and characterization of monoclonal antibodies to guinea pig lymphoid differentiation antigens

We have prepared 2 mouse monoclonal antibodies which react with differentiation antigens on guinea pig lymphoid cells. Monoclone 5AB2 recognizes an antigen expressed on both T and B lymphocytes and absent on macrophages. It has proven useful in the preparation of populations of antigen presenting cells which are free of T and B lymphocytes. The second monoclonal, 8BE6, is specific for peripheral T cells and 10% of thymocytes. It reacts with a 68,000 dalton molecule which is also expressed on the guinea pig B cell leukemia, EN-L2C. 8BE6 has proven to be lytic for peripheral T cells in the presence of rabbit complement and has been used to deplete T cells from heterogenous cell populations.     

Trypanosoma brucei: Effect of inhibition of N-linked glycosylation on the nearest neighbor analysis of the major variable surface coat glycoprotein

As an assay for the surface deposition of newly synthesized major variable surface coat glycoprotein (VSCG) we have treated intact Trypanosoma brucei cells with the cleavable cross-linking reagent dithiobis-(succinimidyl propionate). Under appropriate conditions, surface VSCG is converted to oligomers of n not less than 8. The oligomeric protein, apparent molecular weight greater than 4 × 10⁵, does not migrate more than 1 to 2 mm into a 3–15% linear polyacrylamide gradient gel containing 0.1% sodium dodecyl sulfate, hence the appearance of newly synthesized radiolabeled protein in the top 2 mm of the gel indicates the translocation of VSCG from the site of synthesis to the surface and the gross establishment of normal interactions among the molecules. In addition, purified VSCG treated with the cross-linking reagent yielded a dimeric product on gel electrophoresis. To examine the role of N-linked carbohydrate in the translocation of the protein and in intermolecular interactions we have allowed trypanosomes to incorporate L-[¹⁴C]serine into protein in the presence of the antibiotic tunicamycin. Our results show that N-linked carbohydrate is not essential to the transfer of VSCG to the cell surface nor does its absence interfere with gross intermolecular interactions in the short term. On the other hand N-linked carbohydrate does appear to play an essential role in dimer formation.     

Neuronal gastrin-releasing peptide in the mammalian gut and pancreas

Immunoreactive gastrin releasing peptide (GRP) was demonstrated in neuronal elements in the porcine pancreas and in the gut of several mammals. Immunoreactive endocrine cells could not be detected. The results of radioimmunochemical analysis agreed well with those of immunocytochemistry. The occurrence of gastrin-releasing peptide-containing nerve cell bodies in the myenteric ganglia all along the gut indicates that gastrin-releasing peptide fibers are intramural in origin. The distribution of gastrin-releasing peptide fibers in all layers of the gut wall suggests multiple functions of gastrin-releasing peptide, including a role in the regulation of intramural neuronal activities, smooth muscle tone and in secretory and absorptive processes.