This invention relates to a process for selective inhibition of properties of endotoxin (hereinafter abbreviated as "ET"), such as a property of reacting with horseshoe crab hemocyte lysate (hereinafter abbreviated as "AL solution") to cause activation reaction of an enzyme (e.g. protease) contained in said solution (hereinafter abbreviated as "enzyme activation reaction") or gelation reaction; and a process for measuring at least one substance other than ET among substances having ET activity (hereinafter abbreviated as "activating substances for AL solution") (the substance other than ET is hereinafter abbreviated as "ET analogous substance") which is contained in a sample treated by the above inhibiting process.
ET's are lipopolysaccharides (LPS) present in cell walls of Gram-negative bacteria and are known as potent pyrogens. Therefore, the detection of ET in parenteral drugs and the like is considered important, and test methods for endotoxin are described in the U.S. Pharmacopoeia and the Japanese Pharmacopoeia. ET is considered a main cause of shock in Gram-negative bacterial infections. In clinical diagnoses, the measurement of endotoxin in plasma is employed, for example, for diagnoses of Gram-negative bacterial infections, judgement of curative effect on and prognosis of Gram-negative bacterial infections, and early diagnosis of endotoxin shock. AL solution has a property of being activated by ET to cause activation reaction of an enzyme (e.g. protease) or gelation reaction. Simple, low-cost ET detecting methods utilizing this property, for example, so-called Limulus test including a method of measuring the degree of activation of the enzyme (e.g. protease) by a colorimetric method, or utilizing the gelation reaction (in the present invention, the "Limulus test" includes these methods mentioned above) are widely employed in the fields of medical science, pharmacy and microbiology.
Reagents used for the Limulus test, however, have been disadvantageous in that since they react also with an ET analogous substance(s) such as (1.fwdarw.3)-.beta.-D-glucan and/or a derivative thereof (hereinafter abbreviated as ".beta.G") [Kakinuma et al., Biochem. Biophys. Res. Commun., vol. 101, 434-439 (1981), and Morita et al., FEBS Lett., vol. 129, 318-321 (1981)], the presence of .beta.G together with ET in a sample for measurement causes positive errors in measured values.
Although, .beta.G's are interfering substances which cause positive errors in ET measurement, the detection of .beta.G by use of AL solution is considered utilizable for diagnoses of infectious diseases caused by Eumycetes, because they are components of cell wall of Eumycetes such as yeast and mold. Hence the development of a method for measurement of .beta.G is investigated. However, since commercially available reagents usable for the Limulus test, of course, react with ET, there has been a problem in that when .beta.G is measured using the reagents, ET in a sample affects measured values.
For solving the above problems, there have been reported, for example, a process for removing a factor which is present in AL solution and reacts with ET to cause activation reaction of an enzyme (e.g. protease) or gelation reaction (hereinafter abbreviated as "ET-sensitive factor"), by treating AL solution by various chromatographies (Japanese Patent Unexamined Publication (JP-A) No. 59-27828, JP-A H2-138193 and JP-A H4-76459), a process for inhibiting ET-sensitive factors by adding a peptide having affinity for ET to AL solution (JP-A H2-207098), and a process for inhibiting ET-sensitive factors by adding antibodies against ET-sensitive factor to AL solution (JP-A H4-52558). All of these processes, however, are disadvantageous in that since they comprise treating AL solution itself, there is a strong fear that during the treatment, AL solution may be contaminated with ET or .beta.G, which are widely present in a usual environment. Moreover, these processes require sterile facilities and complicated sterile operations for avoiding the contamination, and the peptide having affinity for ET and the antibodies against ET-sensitive factor are difficult to obtain and are expensive. As is clear from these facts and the like, the processes involve many economical and technical problems.
For inactivating ET by treatment of not AL solution but a sample for measurement, a process for heat-treating the sample has been reported (JP-A H2-141666). This process is free from the defects of the processes described above, but is disadvantageous in that a long treatment time is required for sufficient inactivation of ET. Therefore, it cannot be said to be a preferable process.