Summary
An enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against
human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity
of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and
found to be 100, 74, 94, 92 and 8l% for free tPA and tPA in complex with PAI-1, PAI-2,
α2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma
resulted in rapid and total loss of tPA activity without detectable loss of ELISA
response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about l00%.
Three different treatments of citrated plasma samples (acidification/reneutralization,
addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted
in increased tPA levels. The fact that the increase was equally large in all three
cases along with good analytical recovery of tPA added to plasffi, supported the notion
that all tPA antigen present in plasma samples is measured by the ELISA. Analysis
by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing
tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular
weight degradation products of tPA. Determinations of tPA antigen were made at seven
French clinical laboratories on coded and randomized plasma samples with known tPA
antigen content. For undiluted samples there was no significant difference between
the tPA levels found and those known to be present. The between-assay coefficient
of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination
of total tPA antigen in human plasma samples.
Keywords
Tissue plasminogen activator - tPA - Immunoreactivity - Immunological determination
- Elisa - Multicenter validation
