Abstract

We present a bispecific antibody that recognizes an antigen and a hapten and can be applied to various biological assays, including immunoblotting and immunoprecipitation. In immunoblot analysis of serum, an anti-C5 × anti-cotinine bispecific tandem single-chain variable fragment (scFv)-Fc fusion protein and cotinine-conjugated horseradish peroxidase (HRP) generated a clean signal without the high background that was observed in a parallel experiment using HRP-conjugated goat anti-rabbit immunoglobulin G (Fc-specific) antibody. In immunoprecipitation analysis of serum, use of the bispecific tandem scFv-Fc fusion protein and cotinine-crosslinked magnetic beads significantly reduced the amount of protein contaminants compared with a parallel experiment done with protein A agarose beads. In subsequent immunoblot analysis, use of cotinine–HRP as the secondary probe instead of HRP-conjugated goat anti-rabbit IgG (Fc-specific) antibody successfully eliminated the band corresponding to the bispecific tandem scFv-Fc fusion protein.

Highlights

  • Antibodies are used in a wide variety of biological experiments such as immunoblot analysis, immunoprecipitation, flow cytometry and affinity purification

  • In the wells incubated with cotinine–horseradish peroxidase (HRP), the anti-C5 Â anti-cotinine bispecific tandem scFv-Fc fusion protein showed a dose-dependent reactivity in the range of 4–4000 mg ml À1

  • The challenge in immunoblot analysis of biological samples lies in the heterogeneity of proteins in these samples, in terms of diversity and in wide dynamic range

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Summary

Introduction

Antibodies are used in a wide variety of biological experiments such as immunoblot analysis, immunoprecipitation, flow cytometry and affinity purification. In immunoblot analysis and flow cytometry, a secondary antibody labeled with enzyme or fluorescent dye is frequently used to detect or quantify the primary antibody. Reactivity of secondary antibody and protein A with immunoglobulins (Igs) from a range of diverse species frequently raises the issues of background in immunoblot and flow cytometry analysis and of protein contaminants in immunoprecipitation and affinity purification, especially when the sample contains Ig. To reduce nonspecific binding of secondary antibody or protein A, direct chemical crosslinking of primary antibody to enzyme, fluorescent dyes and solid supports has been used. For affinity purification of proteins and cells, either biotinlabeled anti-protein antibody or anti-cell surface molecule antibody paired with avidin-crosslinked solid support can be employed.[1,2] even in this system, there remains the issues of background and contamination because of the presence of proteins reactive to biotin and avidin in biological samples.[3]

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