An ELISA for the detection of TIMP-1 based on recombinant single chain Fv fusion proteins.
BACKGROUND
Altered serum levels of TIMP-1 are an indicator of various pathological states. To quantitate TIMP-1 in biological samples, we have previously isolated TIMP-1 specific single-chain Fv fragments (scFvs) using phage-display. In the work presented here, we have used these scFvs to establish a TIMP-1 ELISA based exclusively on recombinant scFv fusion proteins.


METHODS
Two distinct TIMP-1 specific scFvs were used as the antigen binding components after being genetically fused to the N-termini of two different fusion protein constructs. One fusion protein, comprising a CL domain, serves as a coating reagent, while the second fusion protein with a modified form of bacterial alkaline phosphatase is used as a detection reagent. A double antibody sandwich-ELISA was then established and optimized.


RESULTS
An ELISA for the quantitation of tissue inhibitor of metalloproteinase (TIMP)-1, based entirely on recombinant antibody fragments, was developed as an alternative to assays using polyclonal antisera or monoclonal antibodies. Its performance was shown to compare well with a conventional ELISA. Finally, TIMP-1 concentrations in the sera of sixty healthy individuals were determined.


CONCLUSIONS
The assay described here provides a standardized, reliable and readily available means of quantitation of TIMP-1 in a large number of blood samples.
Wozniak
Gordana
G
Institute of Applied Microbiology, BOKU-University of Natural Resources and Applied Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
Obermayr
Eva
E
Jeras
Matjaz
M
Knezevic
Mio
M
Rüker
Florian
F
D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis* / Q000097:blood / Q000276:immunology
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1