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 kring les 1-4) in biological samples. The assay involves prior removal of all oth er plasminogen moieties by immunoadsorption of diluted samples (to about 10 ng/ml plasminogen) with a mixture of insolubilized MA-42B12 (directed agai nst kringle 5) and MA-31E9 (directed against the proteinase domain). The re covery of angiostatin during this procedure is greater than or equal to 95% . Subsequently, angiostatin-like fragments are detected in an ELISA, based on two monoclonal antibodies reacting with nonoverlapping epitopes in the k ringle 1-3 domain: MA-36E6 for capture and MA-34D3 for tagging. The assay h as 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 ob tained from cancer patients (n=10), angiostatin levels ranged between 0.24 and 6.7 mug/ml (1.62 +/-0.60 mug/ml; mean +/-S.E.M.) The identity of angios tatin was confirmed by immunoblotting using specific monoclonal antibodies. A weak correlation (r=.66) was observed with the total plasminogen concent ration 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. (C) 2001 E lsevier Science Ltd. All rights reserved.