Abstract: By making a bilirubin oxidase derived from the genus Pleurotus to act on a living body fluid sample in the presence of at least one of a cationic surfactant such as alkyltrimethylammonium salt, etc. and a nonionic surfactant such as polyoxyethylenealkyl ether, etc., only bilirubin non-.delta. fractions of the living body fluid sample can be oxidized, while keeping a bilirubin .delta. fraction remaining as unreacted. Thus, the bilirubin non-.delta. fractions and .delta. fraction of the living body fluid sample can be exactly quantitatively determined by action of the bilirubin oxidase on the sample.

Abstract: Total bilirubin or direct bilirubin is measured by allowing nitrous acid as an oxidizing agent to act on a sample of living body fluid, and measuring optical changes of the sample. For measuring the direct bilirubin, nitrous acid is allowed to act on the sample in the presence of thiourea as a reaction inhibitor for indirect bilirubin. For measuring the total bilirubin, nitrous acid is allowed to act on the sample in the presence of cetyltrimethylammonium bromide as a reaction accelerator. Or, direct bilirubin is measured by allowing an oxidizing agent to act on a sample of living body fluid in the presence of a specific non-ionic surfactant. These methods have a good correlation to the conventional enzymatic method and a good reagent stability and provide safe methods for measuring bilirubin with less danger to environmental pollution.

Abstract: There is provided a method for determination of direct bilirubin which comprises the steps of contacting a bilirubin oxidase with a sample suspected of containing bilirubin; and, measuring direct bilirubin in the sample by optical changes of the sample, characterized in that bilirubin oxidase is allowed to act in the presence of a indirect bilirubin reaction inhibitor selected from a thiocyanate ion, a hydrazide, reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate and a potassium ion of 100 mM to 800 mM. By completely avoiding interference of indirect bilirubin, direct bilirubin is selectively and precisely determined using a reagent kit in the form of solution. The method and reagent of the invention are safe and free of environmental pollution due to unnecessary waste liquid treatment.