Methods for determination of hydrogen peroxide by chemiluminescence analysis are well-known in the prior art. For example, Luminol-peroxidase methods have been described by DeChatelet, L. R. et al., J. Immunol., 129, 1589-1593 (1982); Descamps-Latscha, B. et al., Ann. Immunol., 133, 349-364; and De Sole, P. et al., Adv. Exp. Med. Biol., 141, 591-601 (1982). A Lucigenin-peroxidase method has been described by Minkenberg, I. and Ferber, E., J. Immunol. Methods, 71, 61-67 (1984). However, these prior art methods have the disadvantage that their sensitivities are insufficient for determination of hydrogen peroxide unless they are performed under basic conditions. Moreover, when the above known chemiluminescence analysis methods are used for biochemical tests in the field of clinical diagnosis, they are severely affected by components in samples from a living body such as serum, urine, and the like, which result not only in reduction of light emission activity but also in low reliability of data due to variability. Although attempts have been made to overcome this problem by reducing sample amount to reduce living body component amount, this results in the disadvantage of lowering sensitivity.
In order to obtain a novel highly sensitive method for determination of hydrogen peroxide by chemiluminescence analysis without any interference from living body components, the present inventors have carried out an intensive study. As a result, it was found that the reaction of an oxalic acid di-ester with a fluorescent substance and hydrogen peroxide makes it possible to determine the amount of light emmision quantitatively without any adverse effect from body components. However, this reaction had to be performed in an organic solvent and is therefore unsuitable for use in clinical diagnostic tests because such tests are usually performed in an aqueous system. Then, the present inventors carried out a further intensive study and found that it is possible to determine hydrogen peroxide in an aqueous reaction system quantitatively with high sensitivity without any serum catalase effect by reacting hydrogen peroxide in an aqueous reaction system with an oxidizable non-fluorescent substance in the presence of an oxidizing catalyst to give a fluorescent substance, and then reacting the resulting fluorescent substance with an oxalic acid di-ester and freshly added hydrogen peroxide in an organic solvent under conditions for inhibition of the oxidizing catalyst to determine the amount of light emission. In addition, it has been also found that this reaction sequence can be utilized not only for determination of hydrogen peroxide per se contained in samples directly but also for determination of various components other than hydrogen peroxide in samples indirectly through quantitative formation of hydrogen peroxide from various reactions of the components and suitable reagents. For example, this reaction sequence can be utilized in enzyme immunoassay and other clinical diagnostic tests for determination of various components in samples from a living body. Further, it has been also found that the method of the present invention can be applied to evaluation of bactericidal activity in phagocytosis of leukocytes such as macrophages and neutrophilis because they release hydrogen peroxide and/or superoxide anion (O.sub.2.sup.-) when they exhibit bactericidal activity.