Immunoassay method and assay reagent used in said method

Patent metadata

Field	Value
Publication number	US-2018246090-A1
Application number	US-201615553054-A
Country	US
Kind code	A1
Filing date	Feb 25, 2016
Priority date	Feb 25, 2015
Publication date	Aug 30, 2018
Grant date	—

Abstract

An object of the present invention is to provide a more accurate and sensitive measurement method lowering the possibility of occurrence of nonspecific reaction in immunological measurement of L-FABP. More specifically, an object is to provide a measurement method that is accurate and sensitive even when the concentration of an analyte is low (e.g., an L-FABP concentration is around the normal value). In an immunological measurement method of L-FABP using an anti-L-FABP antibody, nonspecific reaction can easily be suppressed by a polypeptide consisting of amino acids No. 419 to No. 607 of the amino acid sequence of DnaK, a heat shock protein (HSP), derived from E. coli as set forth in SEQ ID NO: 1 or a polypeptide having at least 90% sequence identity with the polypeptide even in highly sensitive measurement.

First claim

1 . A method of detecting with an anti-L-FABP antibody L-FABP (liver-type fatty acid binding protein) in a sample, comprising the step of: bringing
a sample suspected of containing L-FABP, particles having an immobilized anti-L-FABP antibody, a compound having a partial structure of NH 2 —C═N— in a molecule, and a polypeptide consisting of amino acids No. 419 to No. 607 of the amino acid sequence of DnaK, a heat shock protein (HSP), derived from E. coli as set forth in SEQ ID NO: 1 or a polypeptide having at least 90% sequence identity with the polypeptide, into contact with each other.

2 . The method according to claim 1 , wherein the compound having a partial structure of NH 2 —C═N— in a molecule is one or two or more selected from a compound represented by Formula (1) or a salt or ester thereof and a compound represented by Formula (2) or a salt thereof:
the compound of Formula (1)

[in Formula (1), R 1 is a hydrogen atom, a hydroxyl group, or an alkyl group having the carbon number of 1, 2, or 3 that may be branched, and R 2 to R 6 each independently represent a hydrogen atom, a halogen atom, an alkyl group having the carbon number of 1, 2, or 3 that may be branched, a hydroxyl group, a carboxy group, an amino group, or —SR 7 (R 7 represents a hydrogen atom, a hydroxyl group, or an alkyl group having the carbon number of 1, 2, or 3 that may be branched and, when a plurality of R 7 s are present, R 7 s may be the same groups as or different groups from each other)] or a salt or ester thereof; and
the compound of Formula (2)

[in Formula (2), R 11 to R 14 each independently represent a hydrogen atom, a halogen atom, an alkyl group having the carbon number of 1, 2, or 3 that may be branched, an amino group, a phenyl group that may be substituted with a halogen atom, or —SR 16 (R 16 represents a hydrogen atom, a hydroxyl group, or an alkyl group having the carbon number of 1, 2, or 3 that may be branched and, when a plurality of R 16 s are present, R 16 s may be the same groups as or different groups from each other), wherein R 11 and R 12 present in the same molecule may form a carbonyl group together and R 13 and R 14 present in the same molecule may form a carbonyl group together,
R 15 is a hydrogen atom, a halogen atom, or an alkyl group having the carbon number of 1, 2, or 3 that may be branched,
X 11 is a nitrogen atom or a sulfur atom,
X 12 and X 13 are each independently a carbon atom or a nitrogen atom, and
l1, l2, m1, m2, and n are each independently 0 or 1,
the double broken line between X 11 and X 13 and the double broken line between X 12 and X 13 are each independently a single bond or a double bond, wherein
the values of l1, l2, m1, m2, and n as well as the bonds of the double broken line between X 11 and X 13 and the double broken line between X 12 and X 13 indicate values and bonds determined in conformity with the valences of X 11 to X 13 ], or a salt thereof.

3 . The method according to claim 2 , wherein the compound represented by Formula (2) or a salt thereof is one or two or more selected from the following compound or a salt thereof:
the compound of Formula (2)

[In Formula (2), R 11 to R 14 each independently represent a hydrogen atom, a halogen atom, an alkyl group having the carbon number of 1, 2, or 3 that may be branched, an amino group, a phenyl group that may be substituted with a halogen atom, or —SR 16 (R 16 represents a hydrogen atom, a hydroxyl group, or an alkyl group having the carbon number of 1, 2, or 3 that may be branched and, when a plurality of R 16 s are present, R 16 s may be the same groups as or different groups from each other), wherein R 11 and R 12 present in the same molecule may form a carbonyl group together and R 13 and R 14 present in the same molecule may form a carbonyl group together,
R 15 is a hydrogen atom, a halogen atom, or an alkyl group having the carbon number of 1, 2, or 3 that may be branched], wherein
in a combination of X 11 to X 13 , l1+l2, m1+m2, n (l1, l2, m1, m2, and n each independently represent 0 or 1), and double broken lines,
(a) X 11 is a sulfur atom, X 12 and X 13 are carbon atoms, l1+l2 is 2, m1+m2 is 2, n is 0, and the double broken lines between X 11 and X 13 and between X 12 and X 13 are single bonds,
(b) X 11 is a sulfur atom, X 12 and X 13 are carbon atoms, l1+l2 is 1, m1+m2 is 1, n is 0, the double broken line between X 11 and X 13 is a single bond, and the double broken line between X 12 and X 13 is a double bond,
(c) X 11 is a nitrogen atom, X 12 and X 13 are carbon atoms, l1+l2 is 2, m1+m2 is 2, n is 1, and the double broken lines between X 11 and X 13 and between X 12 and X 13 are single bonds,
(d) X 11 is a nitrogen atom, X 12 and X 13 are carbon atoms, l1+l2 is 1, m1+m2 is 1, n is 1, the double broken line between X 11 and X 13 is a single bond, and the double broken line between X 12 and X 13 is a double bond,
(e) X 11 and X 12 are nitrogen atoms, X 13 is a carbon atom, l1+l2 is 1, m1+m2 is 1, n is 0, the double broken line between X 11 and X 13 is a double bond, and the double broken line between X 12 and X 13 is a single bond, or
(f) X 11 , X 12 , and X 13 are nitrogen atoms, l1+l2 is 0, m1+m2 is 0, n is 1, the double broken line between X11 and X13 is a single bond, and the double broken line between X 12 and X 13 is a double bond, or a salt thereof.

4 . The method according to claim 1 , wherein the compound having a partial structure of NH 2 —C═N— in a molecule is either benzamidine or 2-amino-2-thiazoline.

5 . The method according to claim 1 , wherein
at the step of bringing a sample suspected of containing L-FABP, an anti-L-FABP antibody, a compound having a partial structure of NH 2 —C═N— in a molecule, and a polypeptide consisting of amino acids No. 419 to No. 607 of the amino acid sequence of DnaK, a heat shock protein (HSP), derived from E. coli as set forth in SEQ ID NO: 1 or a polypeptide having at least 90% sequence identity with the polypeptide into contact with each other, the step of bringing the sample suspected of containing L-FABP, the compound, and the polypeptide into contact with each other is followed by the step of bringing the sample into contact with the anti-L-FABP antibody.

6 . The method according to claim 5 , wherein the concentration of the polypeptide at the step of bringing the sample suspected of containing L-FABP, the compound, and the polypeptide into contact with each other is 0.04 mmol/L to 1.36 mmol/L.

7 . The method according to claim 1 , wherein the anti-L-FABP antibody is two or more monoclonal antibodies having recognition sites different from each other.

8 . The method according to claim 1 , wherein the particles are latex particles.

9 . The method according to claim 7 , wherein the two or more anti-L-FABP monoclonal antibodies having recognition sites different from each other are respectively immobilized on the latex particles, and wherein L-FABP is detected by a latex turbidimetric immunoassay.

10 . The method according to claim 7 , wherein one monoclonal antibody of the two or more monoclonal antibodies having recognition sites different from each other is labeled with a labeling substance while the other monoclonal antibody or antibodies are immobilized on a solid phase or solid phases, and wherein L-FABP is detected by immunochromatography.

Assignees

• Sekisui Medical Co Ltd

Inventors

• Kobayashi Koji
• Matsumoto Takuji
• Yamamoto Mitsuaki

Classifications

• G01N33/54313

  the carrier being characterised by its particulate form · CPC title

• G01N33/53

  Immunoassay; Biospecific binding assay; Materials therefor · CPC title

• G01N2333/47

  Assays involving proteins of known structure or function as defined in the subgroups · CPC title

• G01N33/54393 Primary

  Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding · CPC title

• G01N33/6893

  related to diseases not provided for elsewhere · CPC title