An improved phage display vector for antibody repertoire cloning by construction of combinatorial libraries

Università degli Studi di Trieste, Trst, Friuli Venezia Giulia, Italy
Research in Virology 03/1997; 148(2):161-4. DOI: 10.1016/S0923-2516(97)89903-7
Source: PubMed


Phagemid pComb3 is a widely used vector for molecular cloning of the antibody repertoire and for production of phage display libraries. However, in practical use, the utilization of this vector has some drawbacks. In this work we describe the construction of pComb3/TIG, an improved, easily manipulated vector for the cloning and display of antibody fragment libraries on the surface of filamentous phage. The two small "stuffer" fragments at the cloning sites were replaced with long DNA fragments, for easier differentiation of the correctly cut forms of the vector. Moreover, in pComb3/TIG the fragment at the heavy-chain-fragment cloning site contains an acid phosphatase-encoding gene. This feature allows the easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid instead of the heavy-chain fragment coding cDNA in a simple plate histochemical assay.

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    • "The extreme versatility of the technique itself, subsequently improved on through the introduction of multiple peptide libraries and affinity selection procedures (“biopanning”) [2], was of immediate interest to the scientific community because of the potential variety of its uses. In particular, the possibility to construct more complex protein libraries, such as antibody fragments libraries [3], opened new perspectives for the specific targeting of human microbial pathogens by monoclonal antibodies (mAbs) obtained through affinity selection strategies. Since the last century, the therapeutic potential of mAbs against human pathogens has been regularly reported. "
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    ABSTRACT: In the last two decades, several phage display-selected monoclonal antibodies (mAbs) have been described in the literature and a few of them have managed to reach the clinics. Among these, the anti-respiratory syncytial virus (RSV) Palivizumab, a phage-display optimized mAb, is the only marketed mAb directed against microbial pathogens. Palivizumab is a clear example of the importance of choosing the most appropriate strategy when selecting or optimizing an anti-infectious mAb. From this perspective, the extreme versatility of phage-display technology makes it a useful tool when setting up different strategies for the selection of mAbs directed against human pathogens, especially when their possible clinical use is considered. In this paper, we review the principal phage display strategies used to select anti-infectious mAbs, with particular attention focused on those used against hypervariable pathogens, such as HCV and influenza viruses.
    International Journal of Molecular Sciences 12/2012; 13(7):8273-92. DOI:10.3390/ijms13078273 · 2.86 Impact Factor
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    • "This procedure was performed well before the emergence of the novel S-OIV strain. Fifteen B-cell lines producing antibodies reacting in immunofluorescence with influenza A-infected cells were obtained by Epstein-Barr Virus (EBV) transformation (Cole et al., 1984), and subsequently cDNA coding for Fab fragments were PCR amplified and cloned in appropriate bacterial expression vector (Burioni et al., 1997, 1998a) for avoiding instability of antibody Fig. 5 "
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    ABSTRACT: The new H1N1 swine-origin influenza virus (S-OIV) strain is a global health problem. The elucidation of the virus-host relationship is crucial for the control of the new infection. Two human monoclonal antibody Fab fragments (HMab) neutralizing the novel H1N1 influenza strain at very low concentrations were cloned before the emergence of S-OIV from a patient who had a broad-range H1N1 serum neutralizing activity. The two HMabs neutralized all tested H1N1 strains, including S-OIV and a swine strain with IC(50) ranging from 2 to 7 microg/ml. Data demonstrate that infection with previously circulating H1N1 strains can elicit antibodies neutralizing S-OIV. Finally, the human genes coding for the neutralizing HMabs could be used for generating full human monoclonal IgGs that can be safely administered being potentially useful in the prophylaxis and the treatment of this human infection.
    Virology 03/2010; 399(1):144-52. DOI:10.1016/j.virol.2009.12.014 · 3.32 Impact Factor
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    ABSTRACT: Demonstration of antibodies inhibiting key viral functions is the basis for the design of an effective vaccine. Dissection of the human antibody response by repertoire cloning may be a powerful means to address this issue. In this study, a panel of human monoclonal recombinant Fab fragments specific for hepatitis C virus (HCV) E2 envelope protein was generated. The selection procedure was designed to select for cross-genotype reactive antibodies. Sequences coding five different human recombinant Fabs specific for the HCV/E2 protein were cloned and characterized. The ability of the cloned antibody fragments to inhibit adhesion of recombinant envelope E2 protein to target cells was assayed. While affinity of the different antibody fragments appeared similar, activity in inhibiting E2 binding to target cells varied considerably from one Fab fragment to another. Two Fabs were not able to inhibit E2 binding at high concentration (40 μg/mL), while three other Fab clones were active in neutralizing 50% of the E2 binding at concentrations ranging from 3 to 0.35 μg/mL.
    Hepatology 09/1998; 28(3):810 - 814. DOI:10.1002/hep.510280331 · 11.06 Impact Factor
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