title: Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method.
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.
authors.affiliation: Department of Medical Technology, Nagoya University School of Health Sciences, 1-1-20 Daiko-Minami, Higashi, Nagoya 461-8673, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Yamada
authors.forename: Akira
authors.initials: A
authors.lastname: Takagi
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Takeshita
authors.forename: Koji
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Masafumi
authors.initials: M
authors.lastname: Ito
authors.forename: Tadashi
authors.initials: T
authors.lastname: Matsushita
authors.forename: Takashi
authors.initials: T
authors.lastname: Murate
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Saito
authors.forename: Tetsuhito
authors.initials: T
authors.lastname: Kojima
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood* / Q000235:genetics / Q000276:immunology; D001774:Blood Chemical Analysis / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D051379:Mice; D017395:Plasminogen Activator Inhibitor 1 / Q000097:blood* / Q000276:immunology; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology
chemical_list: D000906:Antibodies; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D019444:Vaccines, DNA