Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1992-05-28
1995-01-03
Wityshyn, Michael G.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 721, 435 734, 435 74, 435 29, C12Q 134, C12Q 100, C12Q 102
Patent
active
053786106
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a method for assaying an endotoxin in plasma or serum. More particularly, it relates to pretreatment for assaying an endotoxin with high accuracy.
BACKGROUND OF THE INVENTION
There has been known a method for assaying an endotoxin by using limulus amoebocyte lysate components (hereinafter referred to as the limulus test), which has been frequently employed in the examination on the contamination of drugs and water and in clinical tests. The limulus test is suited for the detection of a trace amount of endotoxin in a biological specimen because of its high detection sensitivity.
A biological specimen contains an endotoxin adsorbed by, for example, proteins. It is therefore required prior to the limulus test to perform a pretreatment of the specimen so that the endotoxin could be efficiently liberated from the proteins. It is known to pretreat a biological specimen (for example, plasma, serum) containing granulocytes by treating a sample with an acid, having a pKa of 3 or below at 25.degree. C., at a pH value of 3 or below, removing the denatured precipitate by centrifugation, collecting the supernatant and neutralizing it with an alkali to serve as a sample solution (JP-B-63-55671; the term "JP-B" as used herein means an "examined Japanese patent publication"). However this method is disadvantageous in that the separation procedure is complicated and requires a long period of time for completing the whole procedure and that the specimen would be possibly contaminated.
DISCLOSURE OF THE INVENTION
It is an object of the present invention to provide a method of efficiently, conveniently and rapidly assaying an endotoxin in plasma or serum with extremely high accuracy.
The present invention relates to a method of assaying an endotoxin in plasma or serum using limulus amoebocyte lysate components, wherein a mixed aqueous solution comprising: polyoxyethylene sorbitans, n-alkylglucopyranosides and dodecyl sulfates; to the plasma or serum prior to the assay.
Among the surfactants usable in the present invention, examples of the polyoxyethylene ethers include polyoxyethylene-p-tert-octyl (or iso-octyl) phenyl ether (degree of polymerization: 8-40), polyoxyethylene-4-tert-octyl (or iso-octyl) cyclohexyl ether (degree of polymerization: 8-40), polyoxyethylene-p-nonyl phenyl ether (degree of polymerization: 9-15), polyoxyethylene heptamethyl hexyl ether (degree of polymerization: 10-20) and polyoxyethylene dodecyl ether (degree of polymerization: 10-29). Examples of the n-alkylglucopyranosides include n-(heptyl, octyl, nonyl, decyl or dodecyl)(.alpha.- or .beta.-)D-glucopyranosides. Examples of the polyoxyethylene sorbitans include polyoxyethylene sorbitan (degree of polymerization: about 20) monolaurate, monopalmitate, monostearate, monooleate and trioleate. Examples of the dodecyl sulfates include sodium dodecyl sulfate, lithium dodecyl sulfate and calcium dodecyl sulfate.
Examples of the compound having an imidazolyl or amino group include histamine dihydrochloride, L-histidine dihydrochloride, poly-L-histidine hydrochloride (molecular weight: 15,000-50,000), poly-L-lysine hydrochloride (molecular weight: 2,000-70,000), poly-L-arginine hydrochloride (molecular weight: 5,000-150,000), polyethyleneimine (molecular weight: 1,000-70,000), adenine hydrochloride and cytosine hydrochloride.
The content of the surfactant a) in the mixed aqueous solution may vary depending on the type but preferably ranges from 0.04 to 0.40% (w/v). The content of the compound b) in the mixed aqueous solution having an imidazolyl or amino group preferably ranges from 0.03 to 0.3% (w/v), though it may vary depending on the type. The contents of the alkaline earth metal salt and the alkaline metal hydroxide in the mixed aqueous solution range from 0.005 to 0.05 mol/l and from 0.05 to 0.5 mol/l respectively.
Further, a clear sample solution without any turbidity can be obtained by adding to the mixed aqueous solution N,N-bis(2-hydroxyethyl)glycine so as to give a concentration of from 0.005
REFERENCES:
patent: 4096091 (1978-06-01), Hopkins, II
patent: 4276050 (1981-06-01), Firca
patent: 4322217 (1982-03-01), Dikeman
patent: 4406832 (1983-09-01), Mills
patent: 4495294 (1985-01-01), Nakahara
patent: 4808314 (1989-02-01), Karplus
Chemical Abstracts, vol. 92, No. 21, May 21, 1980, abstract No. 17696 Du Bose, D. A. et al.
Chemical Abstracts, vol. 111, No. 11, Sep. 11, 1989, abstract No. 91769 Yajima, Y. et al.
Tamura Hiroshi
Tanaka Shigenori
Gitomer Ralph
Seikagaku Kogyo Kabushiki Kaisha (Seikagaku Corporation)
Wityshyn Michael G.
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