Chemistry of carbon compounds – Miscellaneous organic carbon compounds – C-metal
Patent
1982-09-09
1986-03-18
Phillips, Delbert R.
Chemistry of carbon compounds
Miscellaneous organic carbon compounds
C-metal
C07C10352
Patent
active
045767459
DESCRIPTION:
BRIEF SUMMARY
The subject of the present invention is a method for the fluorimetric determination of endotoxins (bacterial lipopolysaccharides or lipopolyosides) using a peptide carrying a fluorophorous substance. The invention also relates to new peptides carrying a fluorophorous substance usable in the said method and their method of preparation.
It is known that endotoxins can be determined by measuring the rise in temperature caused in rabbits (cf. for example French Pharmacopoeia, 9th edition, II-235 to II-238), by measuring the time of formation of a gel from a Limule amebocytes lysate (cf. for example Cooper, Levin, Wagner, J. Lab. Cli. Med., July 1971, 138-148) and by colorimetry with the aid of p-nitroaniline (cf. Iwanaga et al., Haemostasis 7, 183-188, 1978; Harada et al., Progress in Clinical and Biological Research, vol. 29, 1979, 209-220). These methods are generally not quantitative and in addition their sensitivity is not sufficient to enable very small quantities of endotoxins to be detected (quantities in the neighborhood of a picogram).
It has also been suggested (cf. U.S. Pat. No. 4,188,264) that endotoxins could be determined by a fluorimetric method using a peptide substrate of the formula: group is protected by a protective group, and R' is the residue of beta-naphthylamine or a fluorescent hydroxylated compound selected from alpha and beta naphthol, indoxyl, N-methyl indoxyl, 4-methyl umbelliferone and resorufin. The peptide substrates of formula (A) for which R' represents the residue of a fluorescent hydroxylated compound have the disadvantage of hydrolyzing spontaneously in aqueous solution.
A method for determining endotoxins by fluorimetry in liquids has now been found in accordance with the present invention which enables endotoxins to be determined quantitatively with a very high sensitivity. This method does not have the disadvantage pointed out above for the fluorimetric method using peptide substrates of formula (A) for which R' is the residue of a fluorescent hydroxylated compound. In addition, it has a markedly greater sensitivity than the fluorimetric method using peptide substrates of formula (A) for which R' is the residue of beta-baphthylamine.
The method according to the invention consists in bringing the liquid to be analyzed into contact, at a temperature between 20.degree. C. to 40.degree. C., with a limule amebocytes lysate and an aqeuous solution of a peptide of the formula: ##STR3## in which [Pept--designates a hydrophobic peptide chain, n is equal to 1, 2, 3, 4 or 5, B is a radical: ##STR4## designating hydrogen atoms or methyl groups; --NH--Ar] designates the remainder of a fluorescent heteroaromatic amine Ar--NH.sub.2 containing one or several heteroatoms selected from the oxygen, sulfur and nitrogen atoms, whose fluorescence spectrum is markedly displaced by acylation of the amine, and X.sup..crclbar. designates an anion, in particular the Cl.sup..crclbar. anion, in adjusting if necessary the pH of the medium thereby obtained to a value between 5 and 10, the determining by fluorimetry the heteroaromatic amine Ar--NH.sub.2 formed.
In the method according to the invention, the contact is established preferably at a temperature in the range of 25.degree. C. to 37.degree. C. and the pH of the medium is preferably adjusted to a value in the range of 6-7.5.
By a fluorescent heteroaromatic amine Ar--NH.sub.2 whose fluorescence spectrum is markedly displaced by acylation of the amine is intended, within the scope of the present invention, any primary heteroaromatic amine whose fluorescence intensity at the emission wavelength of said free amine is at least 100 times greater than the fluorescence intensity, at this same wavelength, of the corresponding compounds of formula (I). For this condition to be met, it is generally necessary for the wavelength corresponding to the maximum emission of the free amine to be shifted by at least 50 nm towards the longest wavelength with respect to the wavelength corresponding to the maximum emission of the corresponding compounds of formula (I
REFERENCES:
The Embo Journal, vol. 1 (1982) 303-306.
FEBS Letters 157, No. 2 (1983) 265-270.
Centre National de la Recherche Scientifique
Phillips Delbert R.
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