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      Antibodies on demand: a fast method for the production of human scFvs with minimal amounts of antigen

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          Abstract

          Background

          Antibodies constitute a powerful tool to study protein function, protein localization and protein-protein interactions, as well as for diagnostic and therapeutic purposes. High-throughput antibody development requires faster methodologies with lower antigen consumption.

          Results

          Here, we describe a novel methodology to select human monoclonal recombinant antibodies by combining in vitro protein expression, phage display antibody libraries and antibody microarrays. The application of this combination of methodologies permitted us to generate human single-chain variable fragments (scFvs) against two proteins: green fluorescent protein (GFP) and thioredoxin (Trx) in a short time, using as low as 5 μg of purified protein. These scFvs showed specific reactivity against their respective targets and worked well by ELISA and western blot. The scFvs were able to recognise as low as 31 ng of protein of their respective targets by western blot.

          Conclusion

          This work describes a novel and miniaturized methodology to obtain human monoclonal recombinant antibodies against any target in a shorter time than other methodologies using only 5 μg of protein. The protocol could be easily adapted to a high-throughput procedure for antibody production.

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          Disease proteomics.

          Sam Hanash (2003)
          The sequencing of the human genome and that of numerous pathogens has opened the door for proteomics by providing a sequence-based framework for mining proteomes. As a result, there is intense interest in applying proteomics to foster a better understanding of disease processes, develop new biomarkers for diagnosis and early detection of disease, and accelerate drug development. This interest creates numerous opportunities as well as challenges to meet the needs for high sensitivity and high throughput required for disease-related investigations.
          Article 0 comments Cited 140 times – based on 0 reviews     Review now
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            From genomics to proteomics.

            Mike Tyers, Matthias Mann (2003)
            Proteomics is the study of the function of all expressed proteins. Tremendous progress has been made in the past few years in generating large-scale data sets for protein-protein interactions, organelle composition, protein activity patterns and protein profiles in cancer patients. But further technological improvements, organization of international proteomics projects and open access to results are needed for proteomics to fulfil its potential.
            Article 0 comments Cited 132 times – based on 0 reviews     Review now
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              High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules.

              J. Marks, Howard Weiner, Elizabeth McCall … (2001)
              Antitumor monoclonal antibodies must bind to tumor antigens with high affinity to achieve durable tumor retention. This has spurred efforts to generate high affinity antibodies for use in cancer therapy. However, it has been hypothesized that very high affinity interactions between antibodies and tumor antigens may impair efficient tumor penetration of the monoclonal antibodies and thus diminish effective in vivo targeting (K. Fujimori et al., J. Nucl. Med., 31: 1191-1198, 1990). Here we show that intrinsic affinity properties regulate the quantitative delivery of antitumor single-chain Fv (scFv) molecules to solid tumors and the penetration of scFv from the vasculature into tumor masses. In biodistribution studies examining a series of radioiodinated scFv mutants with affinities ranging from 10(-7)-10(-11) M, quantitative tumor retention did not significantly increase with enhancements in affinity beyond 10(-9) M. Similar distribution patterns were observed when the scFv were evaluated in the absence of renal clearance in anephric mice, indicating that the rapid renal clearance of the scFv was not responsible for these observations. IHC and IF evaluations of tumor sections after the i.v. administration of scFv affinity mutants revealed that the lowest affinity molecule exhibited diffuse tumor staining whereas the highest affinity scFv was primarily retained in the perivascular regions of the tumor. These results indicate that antibody-based molecules with extremely high affinity have impaired tumor penetration properties that must be considered in the design of antibody-based cancer therapies.
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                Author and article information

                Journal
                Journal ID (nlm-ta): BMC Biotechnol
                Title: BMC Biotechnology
                Publisher: BioMed Central
                ISSN (Electronic): 1472-6750
                Publication date Collection: 2011
                Publication date (Electronic): 2 June 2011
                Volume: 11
                Page: 61
                Affiliations
                [1 ]Functional Proteomics Laboratory. Centro de Investigaciones Biológicas (CIB-CSIC). Ramiro de Maeztu 9, Madrid 28040, Spain
                Article
                Publisher ID: 1472-6750-11-61
                DOI: 10.1186/1472-6750-11-61
                PMC ID: 3125328
                PubMed ID: 21635725
                SO-VID: 3deba080-815a-41e3-988c-c7bfb1e701f5
                Copyright statement: Copyright ©2011 Babel et al; licensee BioMed Central Ltd.
                License:

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 10 December 2010
                Date accepted : 2 June 2011
                Product
                Self URI: http://www.biomedcentral.com/1472-6750/11/61
                Categories
                Subject: Research Article

                ScienceOpen disciplines: Biotechnology
                Keywords: in vitro protein expression,scfv antibodies,antibody microarrays,phage display
                Data availability:
                ScienceOpen disciplines: Biotechnology
                Keywords: in vitro protein expression, scfv antibodies, antibody microarrays, phage display

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