Substrate for assaying β-glucan and/or endotoxin and assay method
View Patent ↗An object of the present invention is to provide a peptide derivative for determining β-glucan or endotoxin which allows high sensitivity measurement, and a method for determining β-glucan and/or endotoxin using the same. The present invention relates to (1) a peptide derivative represented by the following general formula [1]: X-A 1 -Gly-Arg-A 2 -E-D [1], (2) a reagent for determining β-glucan and/or endotoxin comprising the above-described peptide derivative, (3) a method for determining β-glucan and/or endotoxin, characterized in that a sample containing β-glucan and/or endotoxin, an amebocyte lysate of a horseshoe crab and the above-described peptide derivative are reacted each other, then the resulting released compound represented by the following general formula [2]: H-A 2 -E-D [2] is separated from unreacted substance and quantified, and the determination is made based on this value, and (4) a reagent kit for determining β-glucan and/or endotoxin, comprising an amebocyte lysate of a horseshoe crab and the above-described peptide derivative as constituents thereof.
1. A peptide derivative represented by formula (1):
X-A 1 -Gly-Arg-Gly-E-D (1)
wherein
X represents an N-terminal protecting group of amino acid,
A 1 represents a leucine residue, isoleucine residue, serine residue, or threonine residue,
Gly represents a glycine residue,
Arg represents an arginine residue,
D represents a fluorescent dye comprising a functional group, and
E represents a binding arm or a spacer which is derived from a compound represented by formula (3):
R 1 -A-R 2 (3)
wherein R 1 represents a group having reactivity with a carboxyl group, A represents a straight-chain alkylene group having 1 to 6 carbon atoms, and R 2 represents a group having reactivity with the functional group present in the fluorescent dye represented by D.
2. The peptide derivative according to claim 1 , wherein A 1 is a threonine residue.
3. The peptide derivative according to claim 1 , wherein the N-terminal protecting group represented by X is selected from acetyl group, benzoyl group, benzyloxycarbonyl group, tosyl group, glutaryl group and t-butoxycarbonyl group.
4. The peptide derivative according to claim 1 , wherein the dye in the fluorescent dye represented by D has an excitation wavelength of 500 nm to 800 nm.
5. The peptide derivative according to claim 1 , wherein the fluorescent dye represented by D is cyanine dye.
6. The peptide derivative according to claim 1 , wherein the peptide derivative represented by formula (1) is a compound represented by the formula:
Boc-Thr-Gly-Arg-Gly-NH—(CH 2 ) 4 —NH-D
wherein Boc represents t-butoxycarbonyl group and Thr represents a threonine residue.
7. A reagent for determining the presence or absence of, or for quantifying, β-glucan and/or endotoxin in a sample, comprising the peptide derivative according to claim 1 .
8. A method for detecting or quantifying β-glucan and/or endotoxin comprising
reacting a sample containing β-glucan and/or endotoxin, an amebocyte lysate of a horseshoe crab, and the peptide derivative according to claim 1 under conditions suitable to cause the release of a compound represented by formula (2):
H-Gly-E-D (2)
separating the compound represented by formula (2) from unreacted substance, and
quantifying the amount of the compound represented by formula (2),
thereby detecting or quantifying β-glucan and/or endotoxin based on the amount of the compound represented by formula (2).
9. The method according to claim 8 , wherein the separation and quantification use capillary electrophoresis or liquid chromatography.
10. A reagent kit for detecting or quantifying β-glucan and/or endotoxin, comprising an amebocyte lysate of a horseshoe crab and the peptide derivative according to claim 1 .