1. A latex immunoturbidimetric second reagent comprising 0.05-5% (w/v) choline substance.

2. The latex immunoturbidimetric second reagent of claim 1,

the choline substance is a choline derivative obtained by replacing a hydrogen bond on carbon number 2 or a hydrogen bond on a hydroxyl group of choline by a chemical group, and preferably comprises one or more of phosphorylcholine, L-alpha-glycerophosphorylcholine, L-alpha-phosphatidylcholine, methacryloyloxyethyl trimethylammonium chloride, succinyl L-carnitine lithium salt, (S) - (-) - (3-chloro-2-hydroxypropyl) trimethylammonium chloride, valeryl L-carnitine, polyethylene glycol-phosphorylcholine, poly (phosphorylcholine ethylene glycol acrylate), biolipidure103, biolipidure203, biolipidure206, biolipidure405, biolipidure406 and biolipidure 1002;

and/or the latex immunoturbidimetric second reagent further comprises latex microspheres connected with antibodies, wherein the antibodies are monoclonal antibodies or polyclonal antibodies, and preferably comprise one or more of serum amyloid A antibodies, urinary transferrin antibodies, growth stimulator-expressing protein 2 antibodies, procalcitonin antibodies, heparin-binding protein antibodies, retinol-binding protein antibodies, C-reactive protein antibodies, alpha 1-microglobulin antibodies, beta 2-microglobulin antibodies, neutrophil gelatinase-associated apolipoprotein antibodies, pepsinogen I antibodies, pepsinogen II antibodies, apolipoprotein E antibodies and salivary liquefaction sugar chain antigen antibodies.

3. The latex immunoturbidimetric second reagent of claim 2, wherein the latex microspheres comprise carboxyl microspheres, the carboxyl microspheres having a particle size of 50-450 nm; and/or the presence of a gas in the gas,

the choline substances comprise one or more of biolipidure103, biolipidure405, valeryl L-carnitine, methacryloyloxyethyl trimethyl ammonium chloride and succinyl L-carnitine lithium salt; and/or the presence of a gas in the gas,

the antibodies include apolipoprotein E antibodies, neutrophil gelatinase-associated apolipoprotein antibodies, and urinary transferrin antibodies.

4. The latex immunoturbidimetric second reagent according to claim 3, further comprising a buffer having a pH of 4 to 10, preferably a buffer N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer, a buffer N, N-bis (2-hydroxyethyl) glycine or a buffer N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid).

5. The latex immunoturbidimetric second reagent of claim 4, wherein the buffer is N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer, the latex microspheres are carboxyl microspheres, the antibody is apolipoprotein E antibody, and the choline is biolipidure 103; wherein the mass-volume ratio of the biolipidure103 is preferably 0.5-2%, more preferably 1%; the concentration of the N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer solution is preferably 20-70mmol/L, and more preferably 50 mmol/L; the pH is preferably 7-8, and more preferably 7.2; the particle size of the carboxyl microsphere is preferably 80-100nm, or,

the buffer solution is N, N-di (2-hydroxyethyl) glycine buffer solution, the antibody is neutrophil gelatinase-associated apolipoprotein antibody, the latex microspheres are carboxyl microspheres, and the choline substances are valeryl L-carnitine and methacryloyloxyethyl trimethyl ammonium chloride; wherein, the mass volume ratio of the valeryl L-carnitine is preferably 0.05-1%, and more preferably 0.75%; the mass volume ratio of the methacryloyloxyethyl trimethyl ammonium chloride is preferably 0.1-1%, and more preferably 0.6%; the concentration of the N, N-di (2-hydroxyethyl) glycine buffer solution is preferably 50-80mmol/L, more preferably 70mmol/L, and the pH is preferably 7-9, more preferably 8.5; the particle size of the carboxyl microsphere is preferably 180-250nm, or,

the buffer solution is N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer solution, the latex microspheres are carboxyl microspheres, the antibody is urine transferrin antibody, and the choline substances are succinyl L-carnitine lithium salt and biolipidure 405; wherein the mass volume ratio of the succinyl L-carnitine lithium salt is 0.7-2%, and more preferably 1.5%; the mass-to-volume ratio of the biolipidure405 is preferably 0.05 to 1%, more preferably 0.7%; the concentration of the N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer solution is preferably 70-120mmol/L, more preferably 90mmol/L, and the pH is preferably 7-8, more preferably 7.5; the particle size of the carboxyl microsphere is preferably 60-400 nm.

6. A combination comprising the latex immunoturbidimetric second reagent of any of claims 1-5.

7. The combination of claim 6, wherein said combination is in the form of a solution, said combination preferably further comprising a first agent; or the like, or, alternatively,

the combination is in the form of a kit, said combination preferably further comprising independently a first agent;

the first reagent preferably comprises a phosphate buffer, a tris buffer or a glycine buffer.

8. A method of preparing a second reagent of a latex immunoturbidimetric reagent, comprising the steps of:

mixing the latex microspheres connected with the antibody with a solution of a choline substance, wherein the concentration of the choline substance in the second reagent is 0.05-5% (w/v); wherein the content of the first and second substances,

preferably, the choline base substance is a choline derivative obtained by replacing a hydrogen bond on carbon number 2 or a hydrogen bond on a hydroxyl group of choline with a chemical group; preferably comprises one or more of phosphorylcholine, L-alpha-glycerophosphorylcholine, L-alpha-phosphatidylcholine, methacryloyloxyethyl trimethylammonium chloride, succinyl L-carnitine lithium, (S) - (-) - (3-chloro-2-hydroxypropyl) trimethylammonium chloride, valeryl L-carnitine, polyethylene glycol-phosphorylcholine, poly (phosphorylcholine) ethylene glycol acrylate, biolipidure103, biolipidure203, biolipidure206, biolipidure405, biolipidure406 and biolipidure 1002; more preferably comprises one or more of biolipidure103, biolipidure405, valeryl L-carnitine, methacryloyloxyethyl trimethyl ammonium chloride and succinyl L-carnitine lithium salt;

and/or, preferably, the antibody is a monoclonal antibody or a polyclonal antibody; preferably, the antibody comprises serum amyloid A antibody, urinary transferrin antibody, growth stimulating factor expression protein 2 antibody, procalcitonin antibody, heparin-binding protein antibody, retinol-binding protein antibody, C-reactive protein antibody, alpha 1-microglobulin antibody, beta 2-microglobulin antibody, neutrophil gelatinase-associated apolipoprotein antibody, pepsinogen I antibody, pepsinogen II antibody, apolipoprotein E antibody and salivary fluid carbohydrate chain antigen antibody; more preferably, an apolipoprotein E antibody, a neutrophil gelatinase-associated apolipoprotein antibody and a urinary transferrin antibody are included.

9. The method of claim 8, wherein the antibody-linked latex microspheres are prepared by:

a. mixing buffer 1, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and the latex microspheres, and centrifuging;

the buffer solution 1 is preferably 2-morpholine ethanesulfonic acid buffer solution, 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution, 3-morpholine propanesulfonic acid buffer solution or bis (2-hydroxyethyl) imino Tris (hydroxymethyl) methane buffer solution;

the latex microspheres are preferably carboxyl microspheres, the particle size of the carboxyl microspheres is preferably 50-450nm, and the mixing is preferably performed in a volume ratio of 5: (0.01-0.03): (0.01-0.04): 1, mixing the raw materials, namely mixing the raw materials,

b. adding a buffer solution 1 into the precipitate obtained in the step a for resuspension, adding the antibody, and centrifuging;

the volume ratio of buffer to antibody is preferably 5: (0.08 to 0.12),

and/or the presence of a gas in the gas,

the solution of choline substances is prepared by the following steps:

adding the choline substances into a buffer solution, wherein the buffer solution is an N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer solution, an N, N-bis (2-hydroxyethyl) glycine buffer solution or an N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer solution.

10. The method of claim 9, wherein the buffer 1 is 2-morpholinoethanesulfonic acid buffer, the buffer is N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer, the latex microspheres are carboxyl microspheres, the antibody is apolipoprotein E antibody, the choline is biolipidure 103; wherein the mass-volume ratio of the biolipidure103 is preferably 0.5-2%, more preferably 1%; the concentration of the N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer solution is preferably 20-70mmol/L, and more preferably 50 mmol/L; the pH is preferably 7-8, and more preferably 7.2; the particle size of the carboxyl microsphere is preferably 80-100nm, or,

the buffer solution 1 is a 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution, the buffer solution is an N, N-di (2-hydroxyethyl) glycine buffer solution, the antibody is a neutrophil gelatinase-associated apolipoprotein antibody, the latex microspheres are carboxyl microspheres, and the choline substances are valeryl L-carnitine and methacryloyloxyethyl trimethyl ammonium chloride; wherein, the mass volume ratio of the valeryl L-carnitine is preferably 0.05-1%, and more preferably 0.75%; the mass volume ratio of the methacryloyloxyethyl trimethyl ammonium chloride is preferably 0.1-1%, and more preferably 0.6%; the concentration of the N, N-di (2-hydroxyethyl) glycine buffer solution is preferably 50-80mmol/L, more preferably 70mmol/L, and the pH is preferably 7-9, more preferably 8.5; the particle size of the carboxyl microsphere is preferably 180-250nm, or,

the buffer solution 1 is 3-morpholine propanesulfonic acid buffer solution and bis (2-hydroxyethyl) imino Tris (hydroxymethyl) methane buffer solution, the buffer solution is N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer solution, the latex microspheres are carboxyl microspheres, the antibody is a urinary transferrin antibody, and the choline substances are succinyl L-carnitine lithium salt and biolipidure 405; wherein, the mass volume ratio of the succinyl L-carnitine lithium salt is preferably 0.7-2%, and more preferably 1.5%; the mass-to-volume ratio of the biolipidure405 is preferably 0.05 to 1%, more preferably 0.7%; the concentration of the N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer solution is preferably 70-120mmol/L, more preferably 90mmol/L, and the pH is preferably 7-8, more preferably 7.5; the particle size of the carboxyl microsphere is preferably 60-400 nm.

Background

In the field of in vitro diagnosis, latex immunoturbidimetry includes latex immunoturbidimetry transmission and latex immunoturbidimetry scattering, both of which are methods of labeling antibodies on the surface of latex microspheres by means of chemical coupling or physical adsorption to form a latex reagent of the antibody-microsphere conjugate, i.e. a second reagent. In a buffer solution system of a first reagent, an antibody-microsphere conjugate in a second reagent reacts with an antigen to be detected to form an antigen-antibody-microsphere complex, the complex causes the reagent to generate turbidity, and the change of the turbidity is reflected by the change of an instrument signal value. And (3) drawing a standard curve by using a standard substance with known concentration and concentration, measuring a sample with unknown concentration to obtain a signal value, and obtaining the corresponding concentration of the sample to be measured on the standard curve corresponding to the signal value.

For the second reagent comprising latex microspheres and antibodies, the latex reagent is typically a thermodynamically unstable system, and the surface area of the particles in the reagent is large, and therefore the corresponding surface energy is large, and in order to reduce the high surface energy, the particles in the reagent will reduce the surface energy by aggregation, and as time passes, the particles in the reagent that gradually agglutinate eventually settle.

On the other hand, the latex microspheres used for connecting antibodies in the latex immunoturbidimetric reagent have negative charges, the antibodies have positive charges, when excessive positively charged antibodies are added, the positively charged antibodies are connected with the negatively charged latex microspheres, and the chemically connected antibodies and the physically adsorbed antibodies exist on the surfaces of the microspheres, so that the surface space of the antibody-microsphere conjugate still has a plurality of positive charges. Since the gravitational effect of the antibody-microsphere conjugate is greater than the repulsive effect of positive charges between the microspheres, the antibody-microsphere conjugate in the reagent will slowly settle down over time.

Due to surface energy, electric charge and the like, most latex immunoturbidimetric reagents in a liquid state still have the problem of agglutination and sedimentation at room temperature or 2-8 ℃. When the latex reagent undergoes coagulation sedimentation, the basic performance of the reagent is obviously damaged, such as the reagent blank is increased, the reagent repeatability is reduced, the linear range is reduced, and the like.

Patent document 1 (jp 61-222531 a) describes an antifreeze agent to be added when an unsensitized latex is freeze-dried, and includes polysaccharides having a large molecular weight, and surfactants such as PVP and Tween 20. This method enables stable storage of the latex reagent. However, this method is only suitable for freeze-dried reagents, and freeze-dried reagents are cumbersome to operate and inconvenient for users.

Patent document 2 (application No. 201480011899.5) describes that the use of trimethylglycine makes it possible to solve the problem of coagulation and sedimentation of latex particles after freezing and thawing. However, this method is also applicable only to a latex immunoturbidimetric reagent which is not sensitized and which is a phenomenon in which aggregation and precipitation occur due to a temperature change.

Therefore, it is necessary to provide a latex immunoturbidimetric second reagent which has a wide applicability and high anti-settling stability.

Disclosure of Invention

The invention aims to solve the technical problem of overcoming the defects of lack of latex immunoturbidimetric reagents with wide applicability and high anti-settling stability in the prior art, and provides a latex immunoturbidimetric second reagent, a composition containing the same and a preparation method thereof.

In order to achieve the above purpose, one of the technical solutions provided by the present invention is: a second reagent for immunoturbidimetric latex assay, which comprises 0.05-5% (w/v) of choline substances.

The choline substance is a choline derivative obtained by replacing a hydrogen bond on the No. 2 carbon or a hydrogen bond on a hydroxyl group of choline by a chemical group; preferably comprises one or more of phosphorylcholine, L-alpha-glycerophosphorylcholine, L-alpha-phosphatidylcholine, methacryloyloxyethyl trimethylammonium chloride, succinyl L-carnitine lithium, (S) - (-) - (3-chloro-2-hydroxypropyl) trimethylammonium chloride, valeryl L-carnitine, polyethylene glycol-phosphorylcholine, poly (phosphorylcholine) ethylene glycol acrylate, biolipidure103, biolipidure203, biolipidure206, biolipidure405, biolipidure406 and biolipidure 1002; more preferably comprises one or more of biolipidure103, biolipidure405, valeryl L-carnitine, methacryloyloxyethyl trimethyl ammonium chloride and succinyl L-carnitine lithium salt.

The latex immunoturbidimetric second reagent of the present invention preferably further comprises latex microspheres linked with antibodies, wherein the antibodies are monoclonal antibodies or polyclonal antibodies. The antibody preferably comprises a serum amyloid A antibody, a urinary transferrin antibody, a growth stimulating factor expressing protein 2 antibody, a procalcitonin antibody, a heparin binding protein antibody, a retinol binding protein antibody, a C-reactive protein antibody, an alpha 1-microglobulin antibody, a beta 2-microglobulin antibody, a neutrophil gelatinase-associated apolipoprotein antibody, a pepsinogen I antibody, a pepsinogen II antibody, an apolipoprotein E antibody, a salivary fluid carbohydrate chain antigen antibody; more preferably, an apolipoprotein E antibody, a neutrophil gelatinase-associated apolipoprotein antibody and a urinary transferrin antibody are included.

The latex microspheres of the present invention preferably comprise carboxyl microspheres, preferably having a particle size of 50-450 nm.

The latex immunoturbidimetric second reagent according to the invention preferably further comprises a buffer having a pH of 4 to 10, preferably an N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer, an N, N-bis (2-hydroxyethyl) glycine buffer or an N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer.

In a preferred embodiment of the present invention, the buffer is N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer, the latex microspheres are carboxyl microspheres, the antibody is apolipoprotein E antibody, and the choline substance is biolipidure 103; wherein the mass-volume ratio of the biolipidure103 is preferably 0.5-2%, more preferably 1%; the concentration of the N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer solution is preferably 20-70mmol/L, and more preferably 50 mmol/L; the pH is preferably 7-8, and more preferably 7.2; the particle size of the carboxyl microsphere is preferably 80-100 nm.

In a preferred embodiment of the invention, the buffer is bicine buffer, the antibody is neutrophil gelatinase-associated apolipoprotein antibody, the latex microsphere is carboxyl microsphere, and the choline substances are valeryl L-carnitine and methacryloyloxyethyl trimethyl ammonium chloride; wherein, the mass volume ratio of the valeryl L-carnitine is preferably 0.05-1%, and more preferably 0.75%; the mass volume ratio of the methacryloyloxyethyl trimethyl ammonium chloride is preferably 0.1-1%, and more preferably 0.6%; the concentration of the N, N-di (2-hydroxyethyl) glycine buffer solution is preferably 50-80mmol/L, more preferably 70mmol/L, and the pH is preferably 7-9, more preferably 8.5; the particle size of the carboxyl microsphere is preferably 180-250 nm.

In a preferred embodiment of the invention, the buffer is N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer, the latex microspheres are carboxyl microspheres, the antibody is a urinary transferrin antibody, and the choline substances are succinyl L-carnitine lithium salt and biolipidure 405; wherein, the mass volume ratio of the succinyl L-carnitine lithium salt is preferably 0.7-2%, and more preferably 1.5%; the mass-to-volume ratio of the biolipidure405 is preferably 0.05 to 1%, more preferably 0.7%; the concentration of the N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer solution is preferably 70-120mmol/L, more preferably 90mmol/L, and the pH is preferably 7-8, more preferably 7.5; the particle size of the carboxyl microsphere is preferably 60-400 nm.

The second technical scheme provided by the invention is as follows: a combination comprising a latex immunoturbidimetric second reagent as described in one of the preceding claims.

The combination according to the invention is for example in the form of a solution or a kit. Specifically, the method comprises the following steps:

when the combination is in the form of a solution, the invention is intended to indicate that the combination is a solution system; the combination preferably further comprises a first agent.

When the combination is in the form of a kit, the kit preferably further comprises a separate first reagent;

the first reagent in the present invention preferably comprises a phosphate buffer, a tris buffer or a glycine buffer.

In a preferred embodiment of the present invention, the buffer is N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer, the latex microspheres are carboxyl microspheres, the antibody is apolipoprotein E antibody, the choline substance is biolipidure103, and the first reagent is phosphate buffer; wherein the mass-volume ratio of the biolipidure103 is preferably 0.5-2%, more preferably 1%; the concentration of the N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer solution is preferably 20-70mmol/L, and more preferably 50 mmol/L; the pH is preferably 7-8, and more preferably 7.2; the particle size of the carboxyl microsphere is preferably 80-100 nm.

In a preferred embodiment of the present invention, the buffer is bicine buffer, the antibody is neutrophil gelatinase-associated apolipoprotein antibody, the latex microsphere is carboxyl microsphere, the choline substances are valeryl L-carnitine and methacryloyloxyethyl trimethyl ammonium chloride, and the first reagent is tris buffer; wherein, the mass volume ratio of the valeryl L-carnitine is preferably 0.05-1%, and more preferably 0.75%; the mass volume ratio of the methacryloyloxyethyl trimethyl ammonium chloride is preferably 0.1-1%, and more preferably 0.6%; the concentration of the N, N-di (2-hydroxyethyl) glycine buffer solution is preferably 50-80mmol/L, more preferably 70mmol/L, and the pH is preferably 7-9, more preferably 8.5; the particle size of the carboxyl microsphere is preferably 180-250 nm.

In a preferred embodiment of the present invention, the buffer is N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer, the latex microspheres are carboxyl microspheres, the antibody is urine transferrin antibody, the choline substances are succinyl L-carnitine lithium salt and biolipidure405, and the first reagent is glycine buffer; wherein, the mass volume ratio of the succinyl L-carnitine lithium salt is preferably 0.7-2%, and more preferably 1.5%; the mass-to-volume ratio of the biolipidure405 is preferably 0.05 to 1%, more preferably 0.7%; the concentration of the N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer solution is preferably 70-120mmol/L, more preferably 90mmol/L, and the pH is preferably 7-8, more preferably 7.5; the particle size of the carboxyl microsphere is preferably 60-400 nm.

The third technical scheme provided by the invention is as follows: a second reagent method for preparing a latex immunoturbidimetric reagent, comprising the steps of:

mixing the latex microspheres connected with the antibody with a choline substance solution, wherein the concentration of the choline substance in the second reagent is 0.05-5% (w/v);

wherein the content of the first and second substances,

the choline base is preferably a choline derivative obtained by replacing the hydrogen bond of choline with a chemical group; preferably comprises one or more of phosphorylcholine, L-alpha-glycerophosphorylcholine, L-alpha-phosphatidylcholine, methacryloyloxyethyl trimethylammonium chloride, succinyl L-carnitine lithium, (S) - (-) - (3-chloro-2-hydroxypropyl) trimethylammonium chloride, valeryl L-carnitine, polyethylene glycol-phosphorylcholine, poly (phosphorylcholine) ethylene glycol acrylate, biolipidure103, biolipidure203, biolipidure206, biolipidure405, biolipidure406 and biolipidure 1002; more preferably comprises one or more of biolipidure103, biolipidure405, valeryl L-carnitine, methacryloyloxyethyl trimethyl ammonium chloride and succinyl L-carnitine lithium salt;

the antibody is preferably a monoclonal antibody or a polyclonal antibody; preferably comprises serum amyloid A antibody, urinary transferrin antibody, growth stimulating factor expressing protein 2 antibody, procalcitonin antibody, heparin binding protein antibody, retinol binding protein antibody, C-reactive protein antibody, alpha 1-microglobulin antibody, beta 2-microglobulin antibody, neutrophil gelatinase associated apolipoprotein antibody, pepsinogen I antibody, pepsinogen II antibody, apolipoprotein E antibody, salivary fluid carbohydrate chain antigen antibody; more preferably, an apolipoprotein E antibody, a neutrophil gelatinase-associated apolipoprotein antibody or a urinary transferrin antibody is included.

The antibody-linked latex microspheres of the present invention can be prepared by:

a. mixing the buffer solution 1, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the N-hydroxysuccinimide and the latex microspheres, centrifuging,

wherein the buffer 1 is preferably 2-morpholinoethanesulfonic acid buffer, 3- (N-morpholino) -2-hydroxypropanesulfonic acid buffer, 3-morpholinopropanesulfonic acid buffer or bis (2-hydroxyethyl) iminoTris (hydroxymethyl) methane buffer,

wherein the latex microspheres are preferably carboxyl microspheres, the particle size of the carboxyl microspheres is preferably 50-450nm, and the mixing is preferably performed in a volume ratio of 5: (0.01-0.03): (0.01-0.04): 1, mixing;

b. and (b) adding buffer 1 into the precipitate obtained in the step a for resuspension, adding the antibody, and centrifuging.

Wherein the volume ratio of buffer to antibody is preferably 5: (0.08-0.12).

The solution of choline substances in the present invention can be prepared by the following steps:

adding the choline substances into a buffer solution, wherein the buffer solution is an N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer solution, an N, N-bis (2-hydroxyethyl) glycine buffer solution or an N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer solution.

In one embodiment of the present invention, the buffer 1 is 2-morpholinoethanesulfonic acid buffer, the buffer is N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer, the latex microspheres are carboxyl microspheres, the antibody is apolipoprotein E antibody, and the choline substance is biolipidure 103; wherein the mass-volume ratio of the biolipidure103 is preferably 0.5-2%, more preferably 1%; the concentration of the N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer solution is preferably 20-70mmol/L, and more preferably 50 mmol/L; the pH is preferably 7-8, and more preferably 7.2; the particle size of the carboxyl microsphere is preferably 80-100 nm.

In one embodiment of the present invention, the buffer 1 is 3- (N-morpholino) -2-hydroxypropanesulfonic acid buffer, the buffer is N, N-bis (2-hydroxyethyl) glycine buffer, the antibody is neutrophil gelatinase-associated apolipoprotein antibody, the latex microspheres are carboxyl microspheres, and the choline substances are valeryl l-carnitine and methacryloyloxyethyl trimethylammonium chloride; wherein, the mass volume ratio of the valeryl L-carnitine is preferably 0.05-1%, and more preferably 0.75%; the mass volume ratio of the methacryloyloxyethyl trimethyl ammonium chloride is preferably 0.1-1%, and more preferably 0.6%; the concentration of the N, N-di (2-hydroxyethyl) glycine buffer solution is preferably 50-80mmol/L, more preferably 70mmol/L, and the pH is preferably 7-9, more preferably 8.5; the particle size of the carboxyl microsphere is preferably 180-250 nm.

In one embodiment of the invention, the buffer 1 is 3-morpholinopropanesulfonic acid buffer and bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane buffer, the buffers are N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer, the latex microspheres are carboxyl microspheres, the antibodies are urine transferrin antibodies, and the choline substances are succinyl L-carnitine lithium salt and biolipidure 405; wherein, the mass volume ratio of the succinyl L-carnitine lithium salt is preferably 0.7-2%, and more preferably 1.5%; the mass-to-volume ratio of the biolipidure405 is preferably 0.05 to 1%, more preferably 0.7%; the concentration of the N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer solution is preferably 70-120mmol/L, more preferably 90mmol/L, and the pH is preferably 7-8, more preferably 7.5; the particle size of the carboxyl microsphere is preferably 60-400 nm.

As used herein, the term "mass to volume ratio" or "w/v" refers to the ratio of the mass (g) of a particular class of choline-like substance to the volume (ml) of latex immunoturbidimetric second agent.

The numerical values after the buffer in the present invention (e.g., "1" in buffer 1) have no practical meaning, but are merely to distinguish the same terms.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The latex immunoturbidimetric second reagent provided by the invention has the following advantages: under the condition that other properties (such as linearity of a calibrator, reagent blank and repeatability) of the immunoturbidimetric reagent are not influenced, the second reagent has excellent anti-settling stability and can be placed for at least 18 months at normal temperature; and can be placed in any environment and is suitable for most latex microspheres obtained by a chemical coupling mode.

Drawings

FIG. 1 is a graph of data from a calibration curve of an example of apolipoprotein E (APOE) and a control agent over various time periods.

FIG. 2 is a graph of data from a calibration curve of an example of neutrophil gelatinase-associated apolipoprotein (NGAL) with control reagents at various time periods.

FIG. 3 is a graph of calibration curve data for an example of urinary transferrin (uTRF) and control agents over various time periods.

Detailed Description

The present invention will be described in further detail with reference to specific examples, which are intended to illustrate only some, but not all, of the embodiments of the present invention. Other embodiments, which can be derived by one of ordinary skill in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Example 1: reagent for preparing detection apolipoprotein E (APOE)

The detection reagent includes a first reagent and a second reagent. Example 1 and control 1 used the same set of first reagents.

The method comprises the following steps:

(1) preparation of apolipoprotein E antibody-microsphere conjugates: mixing 2-morpholine ethanesulfonic acid buffer solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 80-100nm carboxyl microspheres to activate the carboxyl microspheres, mixing at room temperature for 30min at a volume ratio of 5:0.01:0.02:1, and centrifuging to remove supernatant. Adding 2-morpholine ethanesulfonic acid buffer solution into the precipitate for resuspension, adding sheep anti-human apolipoprotein E polyclonal antibody (purchased from Fipeng biological corporation), mixing the buffer solution and the antibody at a volume ratio of 5:0.1 for 2 hours at room temperature, adding BSA for sealing, sealing for 1 hour, and centrifuging to remove supernatant, thereby obtaining the apolipoprotein E antibody-microsphere conjugate. This step used the same process and materials to prepare two sets of apolipoprotein E antibody-microsphere conjugates, one for example 1 and one for control 1.

(2) Solutions of the group of example 1 were prepared: in 20-70mmol/L of N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonate buffer solution with pH 7-8, 0.5-2% biolipidure103 is added. Specifically, the concentration of N, N-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid buffer solution was 50mmol/L, pH was 7.2, and the concentration of biolipidure103 was 1%.

(3) Preparation of the solution of control 1: preparing N, N-di (2-hydroxyethyl) -2-aminoethanesulfonate buffer solution with the concentration of 50mmol/L and the pH value of 7.2, and adding no choline substances.

(4) Preparing a second reagent: and (3) adding the solution prepared in the step (2) into the solution prepared in the step (1) for the group 1 antibody-microsphere conjugate prepared in the step (1) and carrying out resuspension to obtain the second reagent of the group 1 of the apolipoprotein E latex reagent. And (3) adding the solution of the control group 1 prepared in the step (3) into the antibody-microsphere conjugate of the other group 1 and carrying out resuspension to obtain a second reagent of the apolipoprotein E control group 1.

(5) Preparation ofDetecting apolipoprotein E (APOE)A first reagent: phosphate buffer was prepared at a concentration of 100mmol/L and pH 7.0.

Example 2: reagent for preparing neutrophil gelatinase associated apolipoprotein (NGAL) for detection

The detection reagent includes a first reagent and a second reagent. Example group 2 and control group 2 used the same set of first reagents.

The method comprises the following steps:

(1) preparing a neutrophil gelatinase-associated apolipoprotein antibody-microsphere conjugate: the carboxyl microspheres are activated by mixing 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 180-ion 250nm carboxyl microspheres in a volume ratio of 5:0.03:0.01:1, and after mixing for 30min at room temperature, the supernatant is centrifuged off. Adding 3- (N-morpholinyl) -2-hydroxypropanesulfonic acid buffer solution into the precipitate for resuspension, adding a mouse anti-human neutrophil gelatinase-associated apolipoprotein monoclonal antibody (purchased commercially from Shenzhen ya century science and technology Co., Ltd.), wherein the volume ratio of the buffer solution to the antibody is 5:0.12, mixing at room temperature for 2 hours, adding BSA for sealing for 1 hour, and centrifuging to remove the supernatant, thereby obtaining the neutrophil gelatinase-associated apolipoprotein antibody-microsphere conjugate. This step used the same process and raw materials to prepare two sets of neutrophil gelatinase-associated apolipoprotein antibody-microsphere conjugates, one for example 2 and one for control 2.

(2) Solutions of the group of example 2 were prepared: adding 0.05-1% of valeryl L-carnitine and 0.1-1% of methacryloyloxyethyl trimethyl ammonium chloride into 50-80mmol/L of N, N-di (2-hydroxyethyl) glycine buffer solution with pH of 7-9. Specifically, the concentration of N, N-bis (2-hydroxyethyl) glycine buffer solution is 70mmol/L, the pH is 8.5, the concentration of valeryl L-carnitine is 0.75%, and the concentration of methacryloyloxyethyl trimethyl ammonium chloride is 0.6%.

(3) Preparation of the solution of control 2: n, N-di (2-hydroxyethyl) glycine buffer solution with the concentration of 70mmol/L and the pH value of 8.5 is prepared, and choline substances are not added.

(4) Preparing a second reagent: and (3) adding the solution prepared in the step (2) into the antibody-microsphere conjugate in the group 1 to obtain the latex reagent in the group 2 of the neutrophil gelatinase-related apolipoprotein latex reagent in the group 1, and performing resuspension on the solution to obtain the neutrophil gelatinase-related apolipoprotein latex reagent in the group 2. And (3) adding the solution of the control group 2 prepared in the step (3) into the other 1 group of antibody-microsphere conjugates and carrying out resuspension to obtain the neutrophil gelatinase-associated apolipoprotein control group 2.

(5) Preparation ofDetection of neutrophil gelatinase-associated apolipoprotein (NGAL)A first reagent: tris buffer solution with a concentration of 50mmol/L and pH of 8.0 was prepared.

Example 3: reagent for preparing urine transferrin (uTRF)

The detection reagent includes a first reagent and a second reagent. Example group 3 and control group 3 used the same set of first reagents.

The method comprises the following steps:

(1) preparation of a urinary transferrin antibody-microsphere conjugate, this project requires labeling with two latex microspheres and two antibodies:

(1-1) mixing 3-morpholine propanesulfonic acid buffer solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 60-90nm carboxyl microspheres to activate the carboxyl microspheres, mixing the mixture at the volume ratio of 5:0.02:0.04:1 for 30min at room temperature, and centrifuging to remove the supernatant. Adding 3-morpholine propanesulfonic acid buffer solution into the precipitate for resuspension, adding rabbit anti-human urinary transferrin polyclonal antibody (purchased from Dako company) at a volume ratio of the buffer solution to the antibody of 5:0.08, mixing at room temperature for 2 hours, adding BSA for sealing for 1 hour, centrifuging and removing supernatant to obtain the urinary transferrin antibody-small particle size microsphere conjugate. The same process and raw materials were used in this step to prepare two groups of urinary transferrin antibody-small particle size microsphere conjugates, one group for example 3 and one group for control 3.

(1-2) mixing bis (2-hydroxyethyl) iminoTris (hydroxymethyl) methane buffer solution, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and carboxyl microspheres with the size of 300-400nm to activate the carboxyl microspheres, mixing the mixture at the volume ratio of 5:0.01:0.01:1 for 30min at room temperature, and centrifuging to remove the supernatant. Adding bis (2-hydroxyethyl) imino Tris (hydroxymethyl) methane buffer solution into the precipitate for resuspension, adding goat anti-human urinary transferrin polyclonal antibody (purchased from Dako corporation) with the volume ratio of 5:0.15, mixing at room temperature for 2 hours, adding BSA for blocking for 1 hour, and centrifuging to remove the supernatant, thus obtaining the urinary transferrin antibody-large particle size conjugate. The same process and raw materials are used in the step, and two groups of urine transferrin antibody-large particle size microsphere conjugates are prepared, one group is used in the example 3 group, and the other group is used in the control group 3.

(2) Solutions of the group of example 3 were prepared: in 70-120mmol/L of pH 7-8N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer, 0.7-2% of succinyl L-carnitine lithium salt and 0.05-1% of biolipidure405 are added. Specifically, the N- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer solution concentration was 90mmol/L, the pH was 7.5, the succinyl L-carnitine lithium salt concentration was 1.5%, and the biolipidure405 concentration was 0.7%.

(3) Preparation of the solution of control 2: n- (2-hydroxyethyl) piperacha-N/- (2-ethanesulfonic acid) buffer solution with a concentration of 70mmol/L and a pH of 7.5 was prepared without adding any choline substances.

(4) Preparing a second reagent: and (2) adding the solution prepared in the step (2) into 1 group of the antibody-small particle size microsphere conjugates and the antibody-large particle size microsphere conjugates in the urine transferrin antibody-microsphere conjugates prepared in the step (1), and performing resuspension to obtain the latex reagents of the group 3 in the urine transferrin latex reagent, wherein the volume ratio of the large particle size conjugates to the large particle size conjugates is 2.5:1: 50. And (3) adding the control group 3 solution prepared in the step (3) into the other 1 groups of antibody-small particle size microsphere conjugates and antibody-large particle size microsphere conjugates and carrying out resuspension to obtain the urinary transferrin control group 3, wherein the volume ratio of the large particle size conjugates to the small particle size conjugates to the solution is 2.5:1: 50.

(5) Preparation ofMethod for producing urinary transferrin (uTRF)A first reagent: a glycine buffer solution was prepared at a concentration of 80mmol/L and pH 8.3.

Effects of the embodiment

The latex reagents of the various examples were subjected to the following performance tests two days after completion of the formulation, 15 days after completion of the formulation, 1 month after completion of the formulation, 2 months after completion of the formulation, 4 months after completion of the formulation, 6 months after completion of the formulation, and 12 months after the formulation:

1. and (3) appearance inspection: under the condition of sufficient light, whether the latex reagent has the phenomenon of coagulation sedimentation is checked visually.

2. Calibration graph: the calf serum is used to dilute at least 5 concentrations of the corresponding antigen of different antibodies, and the highest concentration of the antigen is required to be the concentration close to the upper limit of the linear interval of the corresponding product.

3. Reagent blank absorbance: the assay was repeated 2 times with purified water as the sample and the reagent blank absorbance was recorded.

4. Repeatability: for two samples with antigen concentration (APOE antigen concentration of 40mg/L and 100mg/L, NGAL antigen concentration of 45 μ g/L and 300 μ g/L, and uTRF antigen concentration of 2mg/L and 7mg/L), the test was repeated 10 times, and the average of 10 measurements was calculatedAnd Standard Deviation (SD), calculated varianceThe coefficient of variation CV:

if the reagent has the phenomenon of agglutination and sedimentation, the reagent can be re-suspended and then subjected to calibration, reagent blank absorbance and repeatability tests.

Table 1 shows the results of appearance inspection of the examples and the control group at different time periods. As is clear from Table 1, the second reagent of the example to which choline was added did not cause aggregation and sedimentation over a period of 12 months. The second reagent of the control group to which no choline was added showed coagulation and sedimentation after one or two months. Even if resuspension is carried out again after the precipitation is found, agglutination and sedimentation are found at the later observation time.

TABLE 1

Table 2 shows the calibration curve data of the examples and the control group at different time periods. As can be seen from the data in Table 2, the calibration curve data of the example did not change significantly in the period of 12 months, while the control group started to show a continuous decrease in absorbance corresponding to the antigen with high concentration after 1 or 2 months from the completion of the preparation, and the time is prolonged. That is, the latex reagent that agglutinates the sediment, even after resuspension, still has some antibody-microsphere conjugates that can not bind to the antigen, resulting in a reduction in the linear range of the reagent.

TABLE 2

Table 3 shows reagent blanks for the examples and the control group at different time periods. As can be seen from the data in table 3, the absorbance of the reagent blank in the examples did not change significantly over the period of 12 months, but the reagent blank in the control group started to increase continuously after 1 month or 2 months. The data are shown in FIGS. 1-3.

TABLE 3

Table 4 shows the repeatability data of the examples and the control group at different time periods.

TABLE 4

As can be seen from the data in Table 4, the CV values of the examples were not significantly changed by more than 10% over a period of 12 months. However, the CV value of the control group after 1 month or 2 months tends to continuously increase, that is, as the agglutination and sedimentation phenomenon of the control group becomes more serious with the increase of time, the repeatability of the control group reagent is continuously deteriorated.