Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method

Thromb Res. 2003;111(4-5):285-91. doi: 10.1016/j.thromres.2003.09.007.

Abstract

We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • Antibodies / blood*
  • Antibodies / genetics
  • Antibodies / immunology
  • Blood Chemical Analysis / methods*
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Mice
  • Plasminogen Activator Inhibitor 1 / blood*
  • Plasminogen Activator Inhibitor 1 / immunology
  • Rabbits
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Vaccines, DNA / genetics
  • Vaccines, DNA / immunology

Substances

  • Antibodies
  • Plasminogen Activator Inhibitor 1
  • Recombinant Proteins
  • Vaccines, DNA