REAGENT KIT USED FOR DETECTING LIPOPROTEIN-ASSOCIATED PHOSPHOLIPASE A2, AND PREPARATION METHOD AND APPLICATION FOR REAGENT KIT

• US 20170363627A1
• Filed: 02/10/2015
• Published: 12/21/2017
• Est. Priority Date: 02/10/2015
• Status: Active Grant

• Current Assignee: Shenzhen New Industries Biomedical Engineering Co., Ltd.
• Original Assignee: Shenzhen New Industries Biomedical Engineering Co., Ltd.
• Inventors: RAO, Wei, YUAN, Jinyun, LI, Qin, HUANG, Ke, LUO, Kai, LIN, Dongxia, LI, Tinghua
• Granted Patent: US 10,451,624 B2
• Time in Patent Office: Days
• CPC Class Codes: C12Q 1/44 involving esterase
• CPC Class Codes: C12Y 301/01004 Phospholipase A2 (3.1.1.4)
• CPC Class Codes: G01N 2333/918 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes

Abstract

A reagent kit used for detecting lipoprotein-associated phospholipase A2, and a preparation method for the reagent kit. The reagent kit comprises one or a plurality of first anti-lipoprotein-associated phospholipase A2 antibodies used for binding lipoprotein-associated phospholipase A2 to be measured, and one or a plurality of second anti-lipoprotein-associated phospholipase A2 antibodies marked with a trace marker and binding with the lipoprotein-associated phospholipase A2 to be measured at another site, different from the binding site of the lipoprotein-associated phospholipase A2 to be measured and the first anti-lipoprotein-associated phospholipase A2 antibodies. The reagent kit also comprises a displacing agent, so as to further increase the detection accuracy of the reagent kit. A method using the reagent kit for the detection of lipoprotein-associated phospholipase A2 may take serum as a detection sample, has high repeatability and high accuracy, and measures the concentration of lipoprotein-associated phospholipase A2 in the sample in a highly sensitive manner.

Claims

1. A kit for detecting lipoprotein-associated phospholipase A2, comprising one or more first anti-lipoprotein-associated phospholipase A2 antibodies coated on a magnetic sphere for binding with lipoprotein-associated phospholipase A2 to be detected, and one or more second anti-lipoprotein-associated phospholipase A2 antibodies labeled with a trace marker for binding with the lipoprotein-associated phospholipase A2 to be detected at other binding sites different from the binding sites of the first anti-lipoprotein-associated phospholipase A2 antibodies with the lipoprotein-associated phospholipase A2 to be detected;

wherein the trace marker is at least one selected from the group consisting of luminol and derivatives thereof, isoluminol and derivatives thereof, and acridinium esters; and

wherein the kit further comprises a displacer including water as a solvent and the following components;

at least one selected from the group consisting of 3-[3-(cholamidopropyl) dimethylammonio] propanesulfonate, 8-anilino-1-naphthalenesulfonic acid, sodium deoxycholate, barbiturate, and acetic acid;

newborn bovine serum;

goat serum and/or horse serum;

dithiothreitol;

tris (hydroxymethyl) aminomethane;

hydrated 2-morpholinoethanesulfonic acid;

casein; and

disodium ethylenediaminetetraacetate.

2. The kit according to claim 1, wherein the first anti-lipoprotein-associated phospholipase A2 antibodies and the second anti-lipoprotein-associated phospholipase A2 antibodies are independently anti-lipoprotein-associated phospholipase A2 monoclonal antibodies and/or anti-lipoprotein-associated phospholipase A2 polyclonal antibodies.

4. The kit according to claim 2, wherein the trace marker is N-(4-aminobutyl)-N-ethylisoluminol.

5. The kit according to claim 2, wherein the magnetic sphere is a complex of Fe₂O₃ or Fe₃O₄ magnetic particles and an organic polymeric material and has a particle size of 0.1 to 5 microns;

and, the magnetic sphere are optionally modified by surface modification to carry one or more active functional groups.

6. The kit according to claim 2, wherein the first anti-lipoprotein-associated phospholipase A2 antibodies are directly or indirectly coated on the magnetic sphere, and the indirect coating forms include indirect coating via a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system;

and

the trace marker directly or indirectly labels the second anti-lipoprotein-associated phospholipase A2 antibodies and the indirect labeling forms include indirect labeling via a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system.

11. The kit according to claim 2, wherein the concentrations of the first anti-lipoprotein-associated phospholipase A2 antibodies and the second anti-lipoprotein-associated phospholipase A2 antibodies in the kit are each 10 to 200 μ

g/ml, the concentration of the trace marker is 0.1 to 1 mg/ml, and the concentration of the magnetic sphere is 0.1 to 5 mg/ml.

3. (canceled)

7-8. -8. (canceled)

9. The kit according to claim 81, wherein the kit comprises the following components in an amount relative to the total amount of the solvent:

at least one selected from the group consisting of 0.1 to 5 wt % of 3-[3-(cholamidopropyl) dimethylammonio] propanesulfonate, 0.1 to 2 wt % of 8-anilino-1-naphthalenesulfonic acid, 0.1 to 2 wt % of sodium deoxycholate, 0.1 to 2 wt % of barbiturate, and 0.1 to 2 vol % of acetic acid;

1 to 50 vol % of newborn bovine serum;

0.1 to 10 vol % of goat serum and/or 0.1 to 10 vol % of horse serum;

0.1 to 10 wt % of dithiothreitol;

0.1 to 10 wt % of tris (hydroxymethyl) aminomethane;

0.1 to 10 wt % of monohydrate 2-morpholinoethanesulfonic acid;

0.01 to 1 wt % of casein; and

0.01 to 1 wt % of disodium ethylenediaminetetraacetate.

10. The kit of claim 9, wherein the kit further comprises a low-point calibrator and a high-point calibrator of lipoprotein-associated phospholipase A2 and optionally a buffer.

12. A method for preparing a kit for detecting lipoprotein-associated phospholipase A2, comprising:

directly or indirectly coating a first anti-lipoprotein-associated phospholipase A2 antibody on magnetic sphere, and directly or indirectly labeling a second anti-lipoprotein-associated phospholipase A2 antibody with a trace marker, to obtain the kit including one or more first anti-lipoprotein-associated phospholipase A2 antibodies coated on magnetic sphere for binding with lipoprotein-associated phospholipase A2 to be detected, and one or more second anti-lipoprotein-associated phospholipase A2 antibodies labeled with a trace marker for binding with lipoprotein-associated phospholipase A2 to be detected at other binding sites different from the binding sites of the first anti-Lipoprotein-associated phospholipase A2 antibodies with lipoprotein-associated phospholipase A2 to be detected;

wherein the trace marker is at least one selected from the group consisting of luminol and its derivatives, isoluminol and its derivatives, and acridinium esters; and

wherein the method further comprises a step of preparing a displacer as a component of the kit, including;

mixing water as a solvent and the following components;

at least one selected from the group consisting of 3-[3-(cholamidopropyl) dimethylammonio] propanesulfonate, 8-anilino-1-naphthalenesulfonic acid, sodium deoxycholate, barbiturate, and acetic acid;

newborn bovine serum;

goat serum and/or horse serum;

dithiothreitol;

tris (hydroxymethyl) aminomethane;

hydrated 2-morpholinoethanesulfonic acid;

casein; and

disodium ethylenediaminetetraacetate.

13. The method according to claim 12, wherein

the indirect coating includes coating the first anti-lipoprotein-associated phospholipase A2 antibody indirectly via a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system;

and

the indirect labeling includes labeling the second anti-lipoprotein-associated phospholipase A2 antibody via a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system.

15. A process according to claim 12, wherein the displacer comprises the following components in an amount relative to the total amount of the solvent:

at least one selected from the group consisting of 0.1 to 5 wt % of 3-[3-(cholamidopropyl) dimethylammonio] propanesulfonate, 0.1 to 2 wt % of 8-anilino-1-naphthalenesulfonic acid, 0.1 to 2 wt % of sodium deoxycholate, 0.1 to 2 wt % of barbiturate, and 0.1 to 2 vol % of acetic acid;

1 to 50 vol % of newborn bovine serum;

0.1 to 10 vol % of goat serum and/or 0.1 to 10 vol % of horse serum;

0.1 to 10 wt % of dithiothreitol;

0.1 to 10 wt % of tris (hydroxymethyl) aminomethane;

0.1 to 10 wt % of monohydrate 2-morpholinoethanesulfonic acid;

0.01 to 1 wt % of casein; and

0.01 to 1 wt % of disodium ethylenediaminetetraacetate.

18. The method according to claim 12, wherein the trace marker is N-(4-aminobutyl)-N-ethylisoluminol.

19. The method according to claim 12, wherein the magnetic sphere is a complex of Fe₂O₃ or Fe₃O₄ magnetic particles and an organic polymeric material and has a particle size of 0.1 to 5 microns.

20. The method according to claim 12, wherein, in the kit prepared, the concentrations of the first anti-lipoprotein-associated phospholipase A2 antibodies and the second anti-lipoprotein-associated phospholipase A2 antibodies are each 10 to 200 μ

g/ml, the concentration of the trace marker is 0.1 to 1 mg/ml, and the concentration of the magnetic sphere is 0.1 to 5 mg/ml.

14. (canceled)

16-17. -17. (canceled)

21. A method for detecting lipoprotein-associated phospholipase A2, wherein the method comprises using a kit for detecting lipoprotein-associated phospholipase A2 to detect a concentration of lipoprotein-associated phospholipase A2 in a subject sample by chemiluminescence immunoassay, wherein the kit includes one or more first anti-lipoprotein-associated phospholipase A2 antibodies coated on magnetic sphere for binding with lipoprotein-associated phospholipase A2 to be detected, and one or more second anti-lipoprotein-associated phospholipase A2 antibodies labeled with a trace marker for binding with lipoprotein-associated phospholipase A2 to be detected at other binding sites different from the binding sites of the first anti-Lipoprotein-associated phospholipase A2 antibodies with lipoprotein-associated phospholipase A2 to be detected;

wherein the kit further comprises a displacer including water as a solvent and the following components;

at least one selected from the group consisting of 3-[3-(cholamidopropyl) dimethylammonio] propanesulfonate, 8-anilino-1-naphthalenesulfonic acid, sodium deoxycholate, barbiturate, and acetic acid;

newborn bovine serum;

goat serum and/or horse serum;

dithiothreitol;

tris (hydroxymethyl) aminomethane;

hydrated 2-morpholinoethanesulfonic acid;

casein; and

disodium ethylenediaminetetraacetate.

22. The method according to claim 21, wherein the trace marker is N-(4-aminobutyl)-N-ethylisoluminol.

23. The method according to claim 21, wherein the magnetic sphere is a complex of Fe₂O₃ or Fe₃O₄ magnetic particles and an organic polymeric material and has a particle size of 0.1 to 5 microns.

24. The method according to claim 21, wherein the first anti-lipoprotein-associated phospholipase A2 antibody is directly or indirectly coated on the magnetic sphere, and the indirect coating forms include coating the first anti-lipoprotein-associated phospholipase A2 antibody indirectly via a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system;

and

the trace marker directly or indirectly labels the second anti-lipoprotein-associated phospholipase A2 antibody, and the indirect labeling forms include labeling the second anti-lipoprotein-associated phospholipase A2 antibody via a fluorescein isothiocyanate and anti-fluorescein isothiocyanate antibody system or a streptavidin and biotin system.

26. The method according to claim 21, wherein the kit comprises the following components in an amount relative to the total amount of the solvent:

at least one selected from the group consisting of 0.1 to 5 wt % of 3-[3-(cholamidopropyl) dimethylammonio] propanesulfonate, 0.1 to 2 wt % of 8-anilino-1-naphthalenesulfonic acid, 0.1 to 2 wt % of sodium deoxycholate, 0.1 to 2 wt % of barbiturate, and 0.1 to 2 vol % of acetic acid;

1 to 50 vol % of newborn bovine serum;

0.1 to 10 vol % of goat serum and/or 0.1 to 10 vol % of horse serum;

0.1 to 10 wt % of dithiothreitol;

0.1 to 10 wt % of tris (hydroxymethyl) aminomethane;

0.1 to 10 wt % of monohydrate 2-morpholinoethanesulfonic acid;

0.01 to 1 wt % of casein; and

0.01 to 1 wt % of disodium ethylenediaminetetraacetate.

27. The method according to claim 21, wherein, in the kit, the concentration of the first anti-lipoprotein-associated phospholipase A2 antibodies and the second anti-lipoprotein-associated phospholipase A2 antibodies are each 10 to 200 μ

g/ml, the concentration of the trace marker is 0.1 to 1 mg/ml, and the concentration of the magnetic sphere is 0.1 to 5 mg/ml.

25. (canceled)