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Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening.

ABSTRACT: We describe here a method, based on iterative colony filter screening, for the rapid isolation of binding specificities from a large synthetic repertoire of human antibody fragments in single-chain Fv configuration. Escherichia coli cells, expressing the library of antibody fragments, are grown on a porous master filter, in contact with a second filter coated with the antigen, onto which antibodies secreted by the bacteria are able to diffuse. Detection of antigen binding on the second filter allows the recovery of a number of E.coli cells, including those expressing the binding specificity of interest, which can be submitted to a second round of screening for the isolation of specific monoclonal antibodies. We tested the methodology using as antigen the ED-B domain of fibronectin, a marker of angiogenesis. From an antibody library of 7 x 10(8) clones, we recovered a number of specifically-binding antibodies of different aminoacid sequence. The antibody clone showing the strongest enzyme-linked immunosorbent assay signal (ME4C) was further characterised. Its epitope on the ED-B domain was mapped using the SPOT synthesis method, which uses a set of decapeptides spanning the antigen sequence synthesised and anchored on cellulose. ME4C binds to the ED-B domain with a dissociation constant K:(d) = 1 x 10(-7) M and specifically stains tumour blood vessels, as shown by immunohistochemical analysis on tumour sections of human and murine origin.

SUBMITTER: Giovannoni L

PROVIDER: S-EPMC29740 | biostudies-literature | 2001 Mar

REPOSITORIES: biostudies-literature

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Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening.

Giovannoni L L Viti F F Zardi L L Neri D D

Nucleic acids research 20010301 5

We describe here a method, based on iterative colony filter screening, for the rapid isolation of binding specificities from a large synthetic repertoire of human antibody fragments in single-chain Fv configuration. Escherichia coli cells, expressing the library of antibody fragments, are grown on a porous master filter, in contact with a second filter coated with the antigen, onto which antibodies secreted by the bacteria are able to diffuse. Detection of antigen binding on the second filter al ...[more]

PMID: 11222778

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Project description:Obesity is a risk factor for severe disease and mortality for both influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. While previous studies show that individuals with obesity generate antibody responses following influenza vaccination, infection rates within the obese group were twice as high as those in the healthy-weight group. The repertoire of antibodies raised against influenza viruses following previous vaccinations and/or natural exposures is referred to here as baseline immune history (BIH). To investigate the hypothesis that obesity impacts immune memory to infections and vaccines, we profiled the BIH of obese and healthy-weight adults vaccinated with the 2010-2011 seasonal influenza vaccine in response to conformational and linear antigens. Despite the extensive heterogeneity of the BIH profiles in both groups, there were striking differences between obese and healthy subjects, especially with regard to A/H1N1 strains and the 2009 pandemic virus (Cal09). Individuals with obesity had lower IgG and IgA magnitude and breadth for a panel of A/H1N1 whole viruses and hemagglutinin proteins from 1933 to 2009 but increased IgG magnitude and breadth for linear peptides from the Cal09 H1 and N1 proteins. Age was also associated with A/H1N1 BIH, with young individuals with obesity being more likely to have reduced A/H1N1 BIH. We found that individuals with low IgG BIH had significantly lower neutralizing antibody titers than individuals with high IgG BIH. Taken together, our findings suggest that increased susceptibility of obese participants to influenza infection may be mediated in part by obesity-associated differences in the memory B-cell repertoire, which cannot be ameliorated by current seasonal vaccination regimens. Overall, these data have vital implications for the next generation of influenza virus and SARS-CoV-2 vaccines. IMPORTANCE Obesity is associated with increased morbidity and mortality from influenza and SARS-CoV-2 infection. While vaccination is the most effective strategy for preventing influenza virus infection, our previous studies showed that influenza vaccines fail to provide optimal protection in obese individuals despite reaching canonical correlates of protection. Here, we show that obesity may impair immune history in humans and cannot be overcome by seasonal vaccination, especially in younger individuals with decreased lifetime exposure to infections and seasonal vaccines. Low baseline immune history is associated with decreased protective antibody responses. Obesity potentially handicaps overall responses to vaccination, biasing it toward responses to linear epitopes, which may reduce protective capacity. Taken together, our data suggest that young obese individuals are at an increased risk of reduced protection by vaccination, likely due to altered immune history biased toward nonprotective antibody responses. Given the worldwide obesity epidemic coupled with seasonal respiratory virus infections and the inevitable next pandemic, it is imperative that we understand and improve vaccine efficacy in this high-risk population. The design, development, and usage of vaccines for and in obese individuals may need critical evaluation, and immune history should be considered an alternate correlate of protection in future vaccine clinical trials.

Project description:BackgroundThe objective of this study is to assess the frequency of autoantibodies against 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR) in a single center myositis cohort and to analyze associations with statin exposure, clinical features, and outcome of disease course.MethodsA total of 312 patients with idiopathic inflammatory myopathies (IIMs) followed at the rheumatology clinic, Karolinska University Hospital, were identified in the Euromyositis registry between 1988 and 2014 and were classified according to the 2017 European Alliance of Associations for Rheumatology/American College of Rheumatology (EULAR/ACR) criteria. Available serum samples were analyzed for anti-HMGCR autoantibodies by ELISA. Positive sera were confirmed by immunoprecipitation. Clinical data were extracted from Euromyositis registry and medical records. Muscle samples were examined by two pathologists blinded to the subjects' autoantibody status.ResultsOf 312 patients, 13 (4.3%) were positive for anti-HMGCR. Two of the 13 (15%) anti-HMGCR-positive patients had histories of statin use versus 12 (4.2%) in the anti-HMGCR-negative group. In the anti-HMGCR-positive group, five (38%) had a clinical phenotype compatible with dermatomyositis. Muscle biopsies of patients with HMGCR autoantibodies showed findings consistent with immune-mediated necrotizing myopathy in all cases except for one. Five (38%) patients required treatment with intravenous immunoglobulin compared to seven (2.3%) without this antibody. At the last visit, seven patients had chronic, active disease course, and five of 13 patients were in remission, including three without treatment.ConclusionsPatients with IIM related to anti-HMGCR autoantibodies may present with a wide range of symptoms, more than previously anticipated. When a broad approach to screening for these antibodies is applied, only a minority of patients was found to have previous statin exposure. The results of this study justify the addition of anti-HMGCR autoantibodies to routine diagnostic procedures in patients with myositis.

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Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening.

Publications

Isolation of anti-angiogenesis antibodies from a large combinatorial repertoire by colony filter screening.

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