Enzyme-linked immunosorbent assay for the specific detection of angiostatin-like plasminogen moieties in biological samples.
An enzyme-linked immunosorbent assay (ELISA) was developed for the specific detection of human angiostatin-like plasminogen moieties (comprising kringles 1-4) in biological samples. The assay involves prior removal of all other plasminogen moieties by immunoadsorption of diluted samples (to about 10 ng/ml plasminogen) with a mixture of insolubilized MA-42B12 (directed against kringle 5) and MA-31E9 (directed against the proteinase domain). The recovery of angiostatin during this procedure is > or = 95%. Subsequently, angiostatin-like fragments are detected in an ELISA, based on two monoclonal antibodies reacting with nonoverlapping epitopes in the kringle 1-3 domain: MA-36E6 for capture and MA-34D3 for tagging. The assay has a lower detection limit of about 0.1 ng/ml and is performed with intra- and interassay coefficient of variation of 2.4% and 15%. In tumor fluids obtained from cancer patients (n = 10), angiostatin levels ranged between 0.24 and 6.7 microg/ml (1.62+/-0.60 microg/ml; mean+/-S.E.M.) The identity of angiostatin was confirmed by immunoblotting using specific monoclonal antibodies. A weak correlation (r = .66) was observed with the total plasminogen concentration in these samples. This ELISA thus appears suitable for the specific quantitation of angiostatin-like plasminogen moieties in biological samples, and may be useful to study its (patho)physiological relevance.
Lijnen
H R
HR
Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O&N, Herestraat 49, B-3000, Louvain, Belgium. roger.lijnen@med.kuleuven.ac.be
Bloemmen
F
F
Vereecken
A
A
Collen
D
D
D043168:Angiostatins; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D000918:Antibody Specificity; D001826:Body Fluids / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards; D000939:Epitopes; D006801:Humans; D009369:Neoplasms / Q000737:chemistry; D010446:Peptide Fragments / Q000378:metabolism*; D010958:Plasminogen / Q000032:analysis* / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010446:Peptide Fragments; D043168:Angiostatins; D010958:Plasminogen