Patent ID: 6022699
Filing Date: 2000-02-08
Classification: G01N

Abstract:
An endotoxin-induced inflammation assay for evaluating a drug comprising the steps of:a) administering the drug to ocular tissue of an experimental animal;b) inducing inflammation in the animal by administration of an endotoxin;c) isolating the ocular tissue approximately 16-30 hours after administration of the endotoxin;d) homogenizing the ocular tissue in a buffered composition comprising a non-interfering thiol reactive compound, wherein the buffered composition has a pH of about 5-8;e) centrifuging the composition of step (d) to recover neutrophils (PMN) and myeloperoxidase (MPO) in a pellet;f) solubilizing the pellet of step (e) in a buffered composition containing hexadecyltrimethylammonium bromide (HTA-Br) in order to release MPO from PMN azurophil granules, wherein the buffered composition has a pH of about 5-7;g) centrifuging the composition of step (f) to recover MPO in the supernatant;h) preparing a buffered composition comprising the supernatant of step (g), o-dianisidine (o-DA), H.sub.2 O.sub.2 and HTA-Br, wherein the buffered composition has a pH of about 5-7, preferably about pH 6; andi) determining MPO activity by spectrophotometrically monitoring at about 460 nm change in absorbance of the composition of step (h) due to o-DA oxidation.