Construction of an HRP-streptavidin bound antigen and its application in an ELISA for porcine circovirus 2 antibodies

Abstract

A fusion protein SBP-Cap∆41, consisting of Cap∆41 (without 41 amino acids at the N-terminus) protein of porcine circovirus 2 (PCV2) and a streptavidin binding peptide (SBP), was constructed. This fusion protein binds to HRP-labeled streptavidin (HRP-SA) through high affinity between SBP and SA, forming an HRP-streptavidin bound antigen (Hsb-Ag) with both immunoreactivity and enzymatic activity, which can be used in a double-antigen sandwich ELISA for detection of PCV2 antibodies. Comparison of the characteristics of the HSb-Cap∆41 and chemical conjugates of the recombinant Cap∆41 protein showed that the HSb-Cap∆41 based double-antigen sandwich ELISA (HBDS-ELISA) had higher specificity and sensitivity. Use of the HBDS-ELISA detected PCV2-IgG in 9 injected pigs as early as 10 days p.i., 3 days earlier than both a double-antigen sandwich ELISA (DS-ELISA) based on a chemically conjugated antigen, and a commercial indirect ELISA kit.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0473-3) contains supplementary material, which is available to authorized users.

A nanobody‐horseradish peroxidase fusion protein‐based competitive ELISA for rapid detection of antibodies against porcine circovirus type 2

“…Conventional antibodies, polyclonal and monoclonal antibodies, are often used as reagents in ELISA reactions. In recent decades, different types of ELISAs, including indirect ELISA [ 14 – 18 ], competitive ELISA [ 19 – 21 ], blocking ELISA [ 22 ], and double-antigen sandwich ELISA [ 23 , 24 ], have been widely applied to detect PCV2 antibodies in large-scale blood or feces samples. However, these assays are based on PCV2-specific monoclonal or polyclonal antibodies that require more support cost and exhibit low expression yields and high levels of instability.…”

Strategies for Making Multimeric and Polymeric Bifunctional Protein Conjugates and Their Applications as Bioanalytical Tools

“…The enzymes must be conjugated with binding modules such as biotin, antigens, and binding proteins, e.g., antibodies, immunoglobulinbinding proteins (IBPs), and streptavidin (SA). 12 Enzymelinked molecular probes are generally produced by chemical conjugation reactions; such reactions often deactivate the enzymes or binding modules by random modifications, resulting in low yields and a loss of functionality. Because the signal outputs of detection probes are derived from enzymes, the conjugation of multiple proteins is a promising strategy for the creation of highly sensitive detection probes.…”

A Nanobody-horseradish Peroxidase Fusion Protein-based Competitive ELISA for Rapid Detection of Antibodies Against Porcine Circovirus Type 2

“…Conventional antibodies, polyclonal and monoclonal antibodies, are often used as reagents in ELISA reactions. In recent decades, different types of ELISAs, including indirect ELISA [14][15][16][17][18], competitive ELISA [19][20][21], blocking ELISA [22], and double-antigen sandwich ELISA [23,24], have been widely applied to detect PCV2 antibodies in large-scale blood or feces samples. However, these assays are based on PCV2-speci c monoclonal or polyclonal antibodies that require more support cost and exhibit low expression yields and high levels of instability.…”