CN110596405A

CN110596405A - Kit for detecting content of heart-type fatty acid binding protein by latex enhanced immunoturbidimetry

Info

Publication number: CN110596405A
Application number: CN201910892074.3A
Authority: CN
Inventors: 王丽娇
Original assignee: Shanghai Jiemen Biotechnology Co Ltd
Current assignee: Shanghai Jiemen Biotechnology Co Ltd
Priority date: 2019-09-20
Filing date: 2019-09-20
Publication date: 2019-12-20

Abstract

The invention relates to a kit for detecting the content of heart-type fatty acid binding protein by a latex enhanced immunoturbidimetry method. In particular the kit comprises reagent R1 and reagent R2; wherein the reagent R1 contains buffer solution, electrolyte, turbidity-increasing agent and preservative; the reagent R2 contains horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles, a stabilizer, a buffer solution, an electrolyte, a protective agent and a preservative. The kit has high sensitivity, good stability and high accuracy, and can be used for various biochemical analysis instruments.

Description

Kit for detecting content of heart-type fatty acid binding protein by latex enhanced immunoturbidimetry

Technical Field

The invention relates to the technical field of biology, in particular to a kit for detecting the content of cardiotype fatty acid binding protein by using a latex enhanced immunoturbidimetry method, and more particularly relates to a kit for detecting the content of cardiotype fatty acid binding protein in human serum by using a latex enhanced immunoturbidimetry method, and a preparation method and application thereof.

Background

Heart-type fatty acid binding protein (h-FABP) is an acidic novel small cell protein with isoelectric Point (PI) of 5.1 which is rich in heart. Consists of 132 amino acids and has a molecular weight of 15 kDa. It is highly heart-specific, and is mainly present in cardiomyocytes, but is also expressed at low concentrations in tissues other than the heart, such as in trace amounts in skeletal muscle. The h-FABP can be combined with the long-chain fatty acid in the myocardium, transports the long-chain fatty acid into mitochondria, and finally is oxidized and decomposed into adenosine triphosphate ATP to provide energy for myocardial activity. Therefore, h-FABP is also an important carrier protein of energy source fatty acid. Since normal human plasma and urine contain no or only a very small amount of h-FABP, when myocardial cells are damaged, h-FABP is rapidly released into blood and urine, and the content has a good correlation with infarct size. The specificity and the sensitivity of the protein are better than those of myoglobin. And because the concentration change of the h-FABP can be quickly appeared in blood after the myocardial damage, the concentration change of the h-FABP can be used as a better index for the early diagnosis of acute myocardial infarction AMI; meanwhile, the method is widely applied to prediction of the recurrence of AMI, prediction of the area of AMI and assessment of the prognosis of heart failure.

The conventional methods for detecting heart-type fatty acid binding protein (h-FABP) are as follows: fluorescence Immunoassay (FIA), Radioimmunoassay (RIA), time-resolved fluorescence immunoassay (TRFIA), enzyme-linked immunoassay (ELISA), immunosensor technology, immunocolloidal gold technology, latex-enhanced immunoturbidimetry, etc., wherein latex-enhanced immunoturbidimetry is a relatively stable and accurate assay method which has emerged in recent years. It is largely classified into two types. One is a scattering turbidimetric assay; the other is a turbidimetric assay. The basic principle of the two methods is very similar, both methods are that the antibody is crosslinked on the surface of the polymer latex microsphere, and when the microsphere crosslinked with the antibody is combined with the antigen, the antibody can be rapidly aggregated together in a short time, so that the light-scattering property or the light-transmitting property of the reaction solution is changed. Moreover, the change of the light-scattering property or the light-transmitting property (i.e., the absorbance) of the reaction solution has strong correlation with the concentration of the antigen to be detected, and the concentration of the antigen to be detected can be reflected in a certain range. The method is simple to operate, can quickly and accurately detect the content of serum protein within a few minutes, truly reflects the progress of the disease and evaluates the treatment effect, has important clinical significance for early diagnosis and treatment of diseases, is not easily interfered by manual operation and external factors, has good detection stability and repeatability, can be used for detection by using a common biochemical analyzer, is easy to realize automation, and can be popularized and applied in basic medical institutions at all levels. However, the existing latex-enhanced immunoturbidimetry also has great technical difficulties, such as low detection sensitivity; the reagent has poor storage stability and self-coagulation phenomenon; low detection accuracy and the like.

Disclosure of Invention

The invention aims to provide a kit for measuring the content of the cardiovascular fatty acid binding protein in human serum by adopting a latex enhanced immunoturbidimetry method, which has high sensitivity, good stability and high precision.

In a first aspect, the present invention provides a kit for determining the content of cardiac fatty acid binding protein by a latex-enhanced immunoturbidimetry method, the kit comprising a reagent R1 and a reagent R2; wherein the content of the first and second substances,

the reagent R1 contains buffer solution, electrolyte, turbidity-increasing agent and preservative;

the reagent R2 contains horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles, a stabilizer, a buffer solution, an electrolyte, a protective agent and a preservative.

In another preferred embodiment, the kit further comprises a cardiac fatty acid binding protein calibrator.

In another preferred embodiment, the cardiac fatty acid binding protein calibrator comprises a cardiac fatty acid binding protein antigen, a buffer, a protective agent, a stabilizer, and a preservative.

In another preferred embodiment, the calibration material for heart-type fatty acid binding protein comprises HEPES buffer solution, sucrose, bovine serum albumin and sodium azide.

In another preferred embodiment, the calibration product of heart-type fatty acid binding protein comprises the following components:

in another preferred embodiment, the latex particles are carboxylated polystyrene latex particles, hydroxylated polystyrene latex particles, sulfonated polystyrene latex particles, diazotized polystyrene latex particles, or azido polystyrene latex particles.

In another preferred embodiment, the latex particles are carboxylated polystyrene latex particles.

In another preferred embodiment, the latex particles are latex particles with the diameter of 100 +/-10 nm; preferably, it is a latex particle with a diameter of 100 nm.

In another preferred example, the reagent R1 contains the following components:

in another preferred example, the reagent R1 contains the following components:

in another preferred embodiment, the process for preparing the reagent R1 comprises the steps of: the components are formulated according to their respective concentrations or contents.

In another preferred example, the reagent R2 contains the following components:

in another preferred example, the reagent R2 contains the following components:

in another preferred embodiment, the process for preparing the reagent R2 comprises the steps of:

firstly, dissolving a buffer solution, an electrolyte, a protective agent and a preservative in water according to the content of each component, thereby obtaining a horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particle protective agent;

then, the heart-type fatty acid binding protein monoclonal antibody latex particles marked by horseradish peroxidase are mixed with the protective agent according to the content of the latex particles, and the reagent R2 is obtained.

then, a part (for example, 4ml) of the protecting agent is mixed with horseradish peroxidase-labeled monoclonal antibody latex particles (for example, 3mg) of the cardiac fatty acid binding protein, and then the mixture is mixed with another part (for example, 36ml) of the protecting agent according to the content of the protecting agent, so that the reagent R2 is obtained.

In another preferred embodiment, the buffer is phosphate buffer or Tris-HCl buffer.

In another preferred embodiment, the pH of the buffer is 6 to 8.

In another preferred embodiment, the pH of the buffer is 6 or 8.

In another preferred embodiment, the electrolyte is selected from the group consisting of: sodium chloride, potassium chloride, or a combination thereof.

In another preferred embodiment, the stabilizer is selected from the group consisting of: bovine serum albumin, casein, skim milk powder, or combinations thereof.

In another preferred embodiment, the clouding agent is selected from the group consisting of: polyethylene glycol-8000, polyethylene glycol-6000, polyethylene glycol-4000 or combinations thereof.

In another preferred embodiment, the preservative is selected from the group consisting of: sodium azide, Proclin300, or a combination thereof.

In another preferred embodiment, the protective agent is selected from the group consisting of: sucrose, mannitol, trehalose, or a combination thereof.

In another preferred example, in the reagent R1, the buffer is a phosphate buffer.

In another preferred example, in the reagent R1, the electrolyte is sodium chloride.

In another preferred example, in the reagent R1, the turbidity-increasing agent is polyethylene glycol-6000.

In another preferred embodiment, in the reagent R1, the preservative is sodium azide.

In another preferred example, in the reagent R2, the stabilizer is bovine serum albumin.

In another preferred example, in the reagent R2, the buffer is a phosphate buffer.

In another preferred example, in the reagent R2, the electrolyte is sodium chloride.

In another preferred example, in the reagent R2, the protective agent is sucrose.

In another preferred embodiment, in the reagent R2, the preservative is sodium azide.

In another preferred embodiment, the horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles are prepared by a method comprising the following steps of:

(1) labeling the heart-type fatty acid binding protein monoclonal antibody by using horseradish peroxidase so as to obtain the heart-type fatty acid binding protein monoclonal antibody labeled by the horseradish peroxidase;

(2) carrying out cross-linking reaction on the horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody and latex particles in a cross-linking buffer solution in the presence of a chemical cross-linking agent; after the crosslinking reaction is finished, adding ethanolamine into the reaction mixture for reaction, and collecting precipitate after the reaction is finished; then adding a sealing agent into the precipitate for dispersion and reaction; and after the reaction is finished, collecting the precipitate, thereby obtaining the horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles.

In another preferred embodiment, the chemical cross-linking agent is selected from the group consisting of: hydrazide, isocyanate, carbodiimide, N-hydroxysuccinimide, N-hydroxythiosuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, or combinations thereof.

In another preferred embodiment, the chemical crosslinking agent is a combination of N-hydroxythiosuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

In another preferred embodiment, the pH of the crosslinking buffer is between 6 and 8.

In another preferred embodiment, the crosslinking buffer has a pH of 6.

In another preferred embodiment, the crosslinking buffer is selected from the group consisting of: MES buffer, HEPES buffer, phosphate buffer, and the like.

In another preferred embodiment, the crosslinking buffer is MES buffer with pH 6.0 and 50 mmol/L.

In another preferred embodiment, the blocking agent is selected from the group consisting of: bovine serum albumin, casein, skim milk powder, gelatin, bovine serum, or a combination thereof.

In another preferred example, the step (2) includes the steps of:

(2-1) reacting the latex particles with a chemical cross-linking agent aqueous solution in a cross-linking buffer solution (for example, 10-30 minutes), collecting a precipitate after the reaction is finished, continuously adding the cross-linking buffer solution to disperse the precipitate, and then adding a horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody to perform a cross-linking reaction (for example, 1-10 hours or 1-3 hours);

(2-2) after the crosslinking reaction is finished, adding ethanolamine into the reaction mixture for reaction (for example, 10 to 30 minutes), and collecting precipitates after the reaction is finished;

(2-3) adding a blocking agent to the precipitate collected in the step (2-2) to perform dispersion and reaction (for example, 1 to 10 hours or 1 to 3 hours); and after the reaction is finished, collecting the precipitate, thereby obtaining the horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles.

In another preferred example, in step (2-1), the latex particles and the aqueous solution of the chemical crosslinking agent are reacted in a crosslinking buffer by: firstly, selecting a latex particle aqueous solution with the particle size of 100nm, centrifuging and collecting a precipitate; then adding a crosslinking buffer solution to disperse and precipitate; then adding a chemical cross-linking agent aqueous solution to react.

In another preferred embodiment, in the latex particle aqueous solution, the solid content of latex particles is 0.5% -5%; preferably 0.5% -2%; more preferably 1%.

In another preferred embodiment, the horseradish peroxidase-labeled monoclonal antibody to the heart-type fatty acid binding protein is coated in an amount of 3mg/ml of the aqueous latex particle solution.

In another preferred embodiment, the dispersion is carried out by using ultrasound.

In another preferred embodiment, the crosslinking buffer has a pH of 6.

In another preferred embodiment, in step (2) or (2-2), 1 to 5ul (preferably 2.5ul) of ethanolamine is added per ml of the reaction mixture after the completion of the crosslinking reaction.

In another preferred embodiment, the blocking agent is selected from the group consisting of: bovine serum albumin, casein, skim milk powder, gelatin and bovine serum.

In another preferred example, the step (1) includes the steps of:

(1-1) mixing horseradish peroxidase (HRP) with a sodium periodate solution in water and standing in the dark (for example, standing for 20-40 minutes); adding ethylene glycol aqueous solution, and standing at room temperature (for example, standing for 20-40 minutes); then adding the heart-type fatty acid binding protein monoclonal antibody and mixing; then, the reaction is carried out in a carbonate buffer solution with the pH value of 9.5 and protected from light (for example, 2 to 24 hours or 4 to 12 hours);

(1-2) after the reaction is finished, adding a sodium borohydride solution into the reaction solution, and standing in a dark place (for example, standing for 1-4 hours or 2-3 hours); and slowly adding an equal volume of saturated ammonium sulfate solution, standing (for example, at 4 ℃ for 20-40 minutes), centrifuging, and collecting the precipitate to obtain the horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody.

In another preferred example, the pellet obtained in step (1-2) is further dissolved in PBS and dialyzed overnight in PBS, followed by centrifugation, and the supernatant is collected and used in step (2).

In a second aspect, the present invention provides a use of the kit of the first aspect for detecting the amount of cardiac fatty acid binding protein.

In a third aspect, the present invention provides a method of making a kit according to the first aspect; the preparation method comprises the following steps:

preparing each component in the reagent R1 according to the respective concentration or content; thus obtaining reagent R1; and

firstly, dissolving a buffer solution, an electrolyte, a protective agent and a preservative in a reagent R2 in water according to the content of each component, thereby obtaining a horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particle protective agent; then, horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles were mixed with the above-mentioned protective agent in accordance with the content thereof, thereby obtaining a reagent R2.

In another preferred embodiment, the horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles are prepared by the method as above.

The main advantages of the invention include:

1. in the kit, the heart-type fatty acid binding protein monoclonal antibody is labeled by enzyme, so that the antigen-antibody reaction is promoted, the reaction is complete, and the sensitivity of the kit is effectively improved.

2. In the kit, ethanolamine is introduced when the heart-type fatty acid binding protein monoclonal antibody is coated, so that ethanolamine reacts with redundant carboxyl groups after protein is added for reaction, thereby improving the storage stability of the reagent and solving the self-coagulation phenomenon.

3. In the kit, the precision of the kit is improved while the detection sensitivity is improved.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows a line graph of the kit of example 1.

FIG. 2 shows a line graph of the kit of comparative example 1.

FIG. 3 shows a line graph of the kit of comparative example 2.

Fig. 4 shows a line graph of the comparative example 3 kit.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1

The kit is a liquid double reagent comprising a reagent R1 and a reagent R2, wherein the ratio of the reagent R1 to the reagent R2 is 1: 1. Wherein the content of the first and second substances,

the formulation of reagent R1 is as follows:

the reagent R2 contains the following components:

preparation of a reagent:

1. preparation of the R1: dissolving the components in purified water according to the content, uniformly mixing, and fixing the volume.

2. The preparation of the horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody comprises the following steps:

(1) 5mg of horseradish peroxidase (HRP) was dissolved in 0.5ml of distilled water, and freshly prepared 0.06mol/L sodium periodate (NaIO) was added₄) 0.5ml of the solution is mixed evenly and placed in a refrigerator to be protected from light for 30 min.

(2) Taking out, adding 0.5ml of 0.16mol/L ethylene glycol aqueous solution, and standing at room temperature for 30 min.

(3) Adding 1mL of 5mg/mL h-FABP monoclonal antibody, mixing uniformly, filling into a dialysis bag, and dialyzing in 50mmol/L carbonate buffer solution with pH value of 9.5 for 6h or overnight in a dark place under slow stirring to allow iodine to bind to the antibody.

(4) The liquid was aspirated and 5mg/mL sodium borohydride (NaBH) was added₄) 0.2ml of the solution was added,and placing the glass in a refrigerator to prevent light for 2 h.

(5) Taking out, slowly adding equal volume of saturated ammonium sulfate solution while stirring, and placing in a refrigerator at 4 deg.C for 30 min.

(6) Centrifuging at 20000r for 10min to obtain precipitate as enzyme-labeled antibody (i.e. horseradish peroxidase-labeled h-FABP monoclonal antibody), dissolving with small amount of PBS, and dialyzing in PBS overnight while changing fresh PBS for 2-3 times.

(7) And (4) centrifuging to remove insoluble substances, wherein the supernatant is the PBS solution of the enzyme-labeled antibody. Adding 30% or 1% bovine serum albumin as protective agent, subpackaging and freezing for use.

3. The preparation of 3g/L of horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles comprises the following steps:

selecting 1ml of an aqueous solution of carboxylated polystyrene latex particles with the particle size of 100nm and the solid content of 1%, centrifuging at 12000r for 10min, discarding the supernatant, collecting the precipitate, adding 1ml of MES buffer solution with the pH value of 6.050 mmol/L, performing ultrasonic dispersion precipitation, adding a 300 mu L N-hydroxythiosuccinimide (sulfo-NHS) aqueous solution (50mg/ml), mixing uniformly, adding 150 mu L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) aqueous solution (50mg/ml), shaking uniformly quickly, and placing in a rotary mixer to react at room temperature for 15 min; centrifuging at 6000r for 10min, removing the supernatant, adding 2ml of MES buffer solution with the pH value of 6.050 mmol/L for washing twice, then adding 2ml of MES buffer solution with the pH value of 6.050 mmol/L for ultrasonic dispersion and precipitation, adding 3mg/ml (antibody coating amount) of PBS solution of the horseradish peroxidase-labeled h-FABP monoclonal antibody prepared in the step 2, rapidly mixing uniformly, and placing a rotary mixing machine for reacting at room temperature for 2 h; adding 2.5ul ethanolamine per ml of reaction mixture, mixing, continuing to rotate for 10min, centrifuging at 6000r for 10min, removing unbound antibody and ethanolamine from supernatant, adding 2ml bovine serum albumin confining liquid, performing ultrasonic dispersion and precipitation, and continuing to rotate for 2h at room temperature in a rotary mixer; centrifuge at 12000r for 10min, discard the supernatant. The obtained precipitate is 3g/L horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles.

4. Preparation of the reagent R2:

dissolving each component of the reagent R2 with pH of 6.020 mmo/L phosphate buffer solution, 4mmol/L bovine serum albumin, 21.2g/L sodium chloride, 20g/L sucrose and 1% sodium azide in purified water, mixing uniformly, and fixing the volume to 1L to be used as a protective agent for horseradish peroxidase-labeled monoclonal antibody latex particles of the heart-type fatty acid binding protein for later use. Mixing 4ml of the protective agent with the horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles prepared in the step (3), and performing ultrasonic dispersion to obtain horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particle solution; and adding the 4ml of horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particle solution into 36ml of the protective agent, and fully and uniformly mixing to obtain the reagent R2.

5. Preparation of calibrator

Firstly, preparing a heart-type fatty acid binding protein calibrator diluent, wherein the formula is as follows:

dissolving the components of the calibrator diluent with pH of 7.250 mmo/LHEPES buffer solution, 4mmol/L bovine serum albumin, 20g/L sucrose and 1% sodium azide in purified water, mixing uniformly, and fixing the volume to 1L for later use.

Diluting the cardiovascular fatty acid binding protein antigen by using the calibration diluent, wherein the specification of the calibration product is as follows: 6 points for 0.5ml liquid, the calibration concentrations are as follows:

example 2

the formulation of reagent R1 is as follows:

the reagent R2 contains the following components:

preparation of a reagent:

(4) The liquid was aspirated and 5mg/mL sodium borohydride (NaBH) was added₄) 0.2ml of the solution is put in a refrigerator to be protected from light for 2 hours.

selecting 1ml of an aqueous solution of carboxylated polystyrene latex particles with the particle size of 100nm and the solid content of 1%, centrifuging at 12000r for 10min, discarding the supernatant, collecting the precipitate, adding 1ml of MES buffer solution with the pH value of 6.050 mmol/L, performing ultrasonic dispersion precipitation, adding a 300 mu L N-hydroxythiosuccinimide (sulfo-NHS) aqueous solution (50mg/ml), mixing uniformly, adding 150 mu L of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) aqueous solution (50mg/ml), shaking uniformly quickly, and placing in a rotary mixer to react at room temperature for 15 min; centrifuging at 6000r for 10min, removing the supernatant, adding 2ml of MES buffer solution with the pH value of 6.050 mmol/L for washing twice, then adding 2ml of MES buffer solution with the pH value of 6.050 mmol/L for ultrasonic dispersion and precipitation, adding 3mg/ml (antibody coating amount) of PBS solution of the horseradish peroxidase-labeled h-FABP monoclonal antibody prepared in the step 2, rapidly mixing uniformly, and placing a rotary mixing machine for reacting at room temperature for 2 h; adding 2.5ul ethanolamine per ml of reaction mixture, mixing, continuing to rotate for 10min, centrifuging at 6000r for 10min, removing unbound antibody and ethanolamine from supernatant, adding 2ml bovine serum albumin confining liquid, performing ultrasonic dispersion and precipitation, and continuing to rotate for 2h at room temperature in a rotary mixer; centrifuge at 12000r for 10min, discard the supernatant. The obtained precipitate is 3g/L of horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles.

4. Preparation of the reagent R2:

dissolving each component of the reagent R2 with pH of 6.0100 mmo/L phosphate buffer solution, 4mmol/L bovine serum albumin, 21.2g/L sodium chloride, 20g/L sucrose and 1% sodium azide in purified water, mixing uniformly, and fixing the volume to 1L to be used as a horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particle protective agent for later use. Taking 4ml of the protective agent, mixing the protective agent with the horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles prepared in the step (3), and performing ultrasonic dispersion to obtain horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particle solution; and adding the 4ml of horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particle solution into 36ml of the protective agent, and fully and uniformly mixing to obtain the reagent R2.

5. Preparation of a calibrator: the procedure was as in step 5 of example 1.

Example 3

the formulation of reagent R1 is as follows:

the reagent R2 contains the following components:

preparation of a reagent:

4. Preparation of the reagent R2:

dissolving each component of the reagent R2 with pH of 8.020 mmo/L tris-HCl buffer solution, 4mmol/L bovine serum albumin, 21.2g/L sodium chloride, 20g/L sucrose and 1% sodium azide in purified water, uniformly mixing, and fixing the volume to 1L to be used as a protective agent of horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles for later use. Mixing 4ml of the protective agent with the horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles prepared in the step (3), and performing ultrasonic dispersion to obtain horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particle solution; and adding the 4ml of horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particle solution into 36ml of the protective agent, and fully and uniformly mixing to obtain the reagent R2.

5. Preparation of a calibrator: the procedure was as in step 5 of example 1.

Example 4

The procedure of example 1, step 3, was changed from "2.5 ul ethanolamine/ml mixing" to "2 ul ethanolamine/ml mixing" in the reaction mixture, and the rest was the same as in example 1.

Example 5

The procedure of example 1, step 3, was changed from "2.5 ul ethanolamine/ml mixing" to "3 ul ethanolamine/ml mixing" in the example 1, and the rest was the same as in example 1.

Comparative example 1

The method is characterized in that the heart-type fatty acid binding protein monoclonal antibody latex particles marked by horseradish peroxidase in the example 1 are changed into heart-type fatty acid binding protein monoclonal antibody latex particles, namely, the horseradish peroxidase marking is omitted, the heart-type fatty acid binding protein monoclonal antibody is directly coated on the 100nm latex particles, ethanolamine is not added after the antibody coating is finished, and the rest steps are the same as the example 1.

Comparative example 2

The heart-type fatty acid binding protein monoclonal antibody latex particles marked by the horseradish peroxidase in the example 1 are changed into heart-type fatty acid binding protein monoclonal antibody latex particles, namely the horseradish peroxidase marking is omitted, the heart-type fatty acid binding protein monoclonal antibody is directly coated on the 100nm latex particles, and the rest steps are the same as the example 1.

Comparative example 3

The step of adding ethanolamine after the coating is finished in the embodiment 1 is omitted, namely ethanolamine is not added after the coating is finished, the ultrasonic treatment is directly carried out by centrifugation, and the rest steps are the same as the embodiment 1.

EXAMPLE 6 Performance testing of the kits

The detection is carried out by using Hitachi 7180 biochemical instrument. The test basic parameters are shown in table 1. The test conditions were as follows: two-point endpoint method or fixed time method. First, the reagent R1 and the sample are incubated at 37 ℃ for 300 seconds to read A1; then, a reagent R2 was added, and the reaction was carried out at 37 ℃ for 300 seconds to read a2,. DELTA.A. 2-A1, and then the concentration of the analyte was determined by comparing the standard value with the measured value of. DELTA.A.

TABLE 1 basic parameters

Sample size	4μl
		Reagent amount (R1/R2)	100ul/100ul
Reaction temperature	37℃
		Reaction time (T1/T2)	300 seconds/300 seconds
Wavelength of light	700nm
		Unit of	ng/mL
Reaction direction	Increate (upward)
		Standard curve simulation equation	6-point scaling using non-linear computation modes, e.g. log/Spline

1. Sensitivity of the kit

Samples of known concentration (5.0 ± 1.0) ng/mL were tested and the absorbance difference Δ a recorded, converted to an absorbance difference Δ a' at a concentration of 5.0ng/mL, Δ a ═ 5.0ng/mL (Δ a/known concentration).

The results are given in table 2 below:

TABLE 2 sensitivity of reagents

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION	Example 1	Example 2	Example 3	Comparative example 1	Comparative example 2	Comparative example 3
							ΔA’	0.038	0.033	0.030	0.112	0.108	0.035

As can be seen from table 2, the sensitivity of the kits of example 1, example 2, example 3 and comparative example 3 was good, but the sensitivity of comparative example 1 and comparative example 2 was poor. The reagent coated by the enzyme-labeled antibody can effectively improve the sensitivity of the kit.

2. Reagent kit linearity

The high concentration sample near the linear range and the low concentration sample near the lower limit of the linear range were mixed to 6 dilution gradients (Xi) and detected with the above-mentioned kit. Each dilution gradient is measured 3 times, the mean value (Yi) of the detection result is obtained, the dilution gradient (Xi) is used as an independent variable, the mean value (Yi) of the detection result is used as a dependent variable, a linear regression equation is obtained, and the linear regression correlation coefficient (r) is calculated, wherein the results are shown in table 3 and fig. 1, table 4 and fig. 2, table 5 and fig. 3, table 6 and fig. 4.

Table 3 linearity of the kit of example 1

TABLE 4 linearity of the kit of comparative example 1

TABLE 5 linearity of the kit of comparative example 2

TABLE 6 linearity of the kit of comparative example 3

As can be seen from the results, the kit prepared by the method of the invention has better linear effect.

3. Study of the stability of the opened bottle

And (3) opening the bottle of the kit, placing the kit in an instrument reagent disk, calibrating, reserving a sample, observing for 7 days, and measuring and controlling the quality every day. The results are shown in the following table.

Table 7 example 1 kit open bottle stability test

Table 8 example 2 kit open bottle stability test

TABLE 9 EXAMPLE 3 kit open bottle stability test

TABLE 10 open bottle stability test of comparative example 1 kit

TABLE 11 open bottle stability test of comparative example 2 kit

TABLE 12 open bottle stability test of comparative example 3 kit

From the results, it can be seen that the stability of examples 1, 2 and 3 is the best; the comparative example 2 has relatively poor stability, and the comparative examples 1 and 3 have the worst stability, which shows that the preparation method of the invention can effectively improve the stability of the kit.

4. 37 ℃ thermal accelerated stability test

2ml of the reagent in the kits of example 1, example 4 and example 5 was collected by a centrifuge tube on day 1, day 2, day 3, day 6 and day 7, respectively, and 2ml of the reagent in the kit of comparative example 3 was collected by a centrifuge tube on day 1, day 3, day 5, day 6 and day 7, respectively, and placed in an oven at 37 ℃ with respective seals, and each reagent was tested on day 8. Blank is the reagent background. The test results are shown in table 13, table 14, table 15 and table 16.

TABLE 13 example 1 kit 37 ℃ accelerated stability test

Number of days	blank	S1	S2	S3	S4	S5	S6
								Acceleration at 37 deg.C	7	10145	-10	224	440	933	3990	6626
6	10211	-9	219	456	941	4053	6730
									3	10245	4	228	458	969	4155	6838
2	10236	6	231	461	963	4189	6737
									1	10133	-14	230	445	962	4126	6759
0	10163	3	228	453	963	4089	6739
								Loss of power	7	-0.18％	-433.33％	-1.75％	-2.87％	-3.12％	-2.42％	-1.68％
6	0.47％	-400.00％	-3.95％	0.66％	-2.28％	-0.88％	-0.13％
									3	0.81％	33.33％	0.00％	1.10％	0.62％	1.61％	1.47％
2	0.72％	100.00％	1.32％	1.77％	0.00％	2.45％	-0.03％
									1	-0.30％	-566.67％	0.88％	-1.77％	-0.10％	0.90％	0.30％
7 to 1	0.12％	-28.57％	-2.61％	-1.12％	-3.01％	-3.30％	-1.97％

TABLE 14 comparative example 3 kit 37 deg.C thermal accelerated stability test

Number of days	blank	S1	S2	S3	S4	S5	S6
								Acceleration at 37 deg.C	7	9832	-7	169	397	864	3216	5598
6	9924	9	174	402	883	3563	5839
									5	10589	6	181	423	942	3823	6283
3	10996	-5	189	432	964	3974	6398
									1	11212	-19	205	435	999	4068	6475
0	11224	-16	224	444	959	4030	6545
								Loss of power	7	-12.40％	-56.25％	-24.55％	-10.59％	-9.91％	-20.20％	-14.47％
6	-11.58％	-156.25％	-22.32％	-9.46％	-7.92％	-11.59％	-10.79％
									5	-5.66％	-137.50％	-19.20％	-4.73％	-1.77％	-5.14％	-4.00％
3	-2.03％	-68.75％	-15.63％	-2.70％	0.52％	-1.39％	-2.25％
									1	-0.11％	18.75％	-8.48％	-2.03％	4.17％	0.94％	-1.07％
7 to 1	-12.31％	-63.16％	-17.56％	-8.74％	-13.51％	-20.94％	-13.54％

TABLE 15 example 4 kit 37 ℃ accelerated stability test

TABLE 16 example 5 kit 37 ℃ accelerated stability test

Number of days	blank	S1	S2	S3	S4	S5	S6
								Acceleration at 37 deg.C	7	10832	-7	207	409	910	3893	6208
6	10983	5	211	415	923	3866	6324
									3	10911	6	227	423	942	3924	6525
2	11096	-5	213	432	964	3974	6398
									1	11212	-19	205	435	999	4068	6475
0	11212	-16	224	444	959	4030	6545
								Loss of power	7	-3.39％	-56.25％	-7.59％	-7.88％	-5.11％	-3.40％	-5.15％
6	-2.04％	-131.25％	-5.80％	-6.53％	-3.75％	-4.07％	-3.38％
									3	-2.68％	-137.50％	1.34％	-4.73％	-1.77％	-2.63％	-0.31％
2	-1.03％	-68.75％	-4.91％	-2.70％	0.52％	-1.39％	-2.25％
									1	0.00％	18.75％	-8.48％	-2.03％	4.17％	0.94％	-1.07％
7 to 1	-3.39％	-63.16％	0.98％	-5.98％	-8.91％	-4.30％	-4.12％

As can be seen from the results, the stability of the kit prepared by the invention is obviously improved.

5. General storage conditions of the kit

The kit is stored at 4 ℃, the detection is carried out every 3 months, the appearance of the reagent is observed, and the blank absorbance, the sensitivity and the accuracy of the reagent are tested (wherein, the quality control level 1 is 10.60ng/ml, and the quality control level 2 is 40.13 ng/ml). Wherein, the accuracy is the deviation between the three calculated detection values and the target value of the test quality control, and the test result is as follows:

table 17 example 1 test results

TABLE 18 test results of comparative example 1

TABLE 19 test results of comparative example 2

TABLE 20 test results of comparative example 3

The results show that in the kit prepared by the invention, the reagent has uniform appearance without foreign matters, the blank absorbance of the test reagent is low, the sensitivity is higher and the accuracy is higher.

6. Precision degree

The two quality control levels were repeatedly measured 10 times each to calculate the Coefficient of Variation (CV), and the test results were as follows:

table 21 example 1 precision testing

Number of tests	Quality control 1(10.25ng/ml)	Quality control 2(39.81ng/ml)
			1	10.25	39.55
2	10.24	39.72
			3	10.25	39.67
4	10.21	39.70
			5	10.22	39.56
6	10.23	39.58
			7	10.22	39.36
8	10.22	39.54
			9	10.25	39.21
10	10.26	40.02
			AV	10.24	39.59
SD	0.0156	0.1652
			CV	0.15％	0.42％

Table 22 example 2 precision testing

Table 23 example 3 precision testing

Number of tests	Quality control 1(10.25ng/ml)	Quality control 2(39.81ng/ml)
			1	10.50	39.94
2	10.75	39.81
			3	10.23	39.51
4	10.23	39.91
			5	10.44	39.64
6	10.70	39.27
			7	10.40	39.84
8	10.34	39.51
			9	10.46	39.37
10	10.45	39.56
			AV	10.45	39.64
SD	0.1826	0.2437
			CV	1.75％	0.61％

TABLE 24 precision test of comparative example 1

Number of tests	Quality control 1(10.25ng/ml)	Quality control 2(39.81ng/ml)
			1	11.29	39.95
2	10.48	39.64
			3	11.21	41.97
4	9.12	42.13
			5	10.88	41.52
6	10.69	42.33
			7	11.37	40.21
8	10.25	43.94
			9	10.75	43.41
10	10.33	43.94
			AV	10.64	41.90
SD	0.6921	1.5063
			CV	6.51％	3.59％

TABLE 25 precision measurement of comparative example 2

TABLE 26 precision test of comparative example 3

Number of tests	Quality control 1(10.25ng/ml)	Quality control 2(39.81ng/ml)
			1	10.30	40.28
2	10.17	40.37
			3	10.26	39.73
4	10.10	39.18
			5	10.25	39.06
6	10.78	38.99
			7	11.09	39.91
8	10.15	39.51
			9	10.18	39.82
10	9.96	40.12
			AV	10.32	39.70
SD	0.3382	0.5053
			CV	3.28％	1.27％

As can be seen from the results, the kit prepared by the invention has higher precision.

The kit disclosed by the invention coats the enzyme-labeled antibody on the latex microsphere, the catalytic action of enzyme is utilized to promote the complete reaction of the antigen and the antibody so as to improve the detection sensitivity, ethanolamine is introduced in the coating process to react with the excessive carboxyl groups added after the antibody is added for reaction, so that the problems of poor storage stability and reagent self-coagulation existing in the existing reagent are solved, finally, the appearance and detection performance of the reagent meet the requirements after the reagent is stored in a 37-DEG oven for 7 days and then taken out, the reagent can be stably stored for more than 12 months in a 4-DEG environment, the self-coagulation phenomenon is not generated any more, and the CV value of the reagent can reach less than or equal to 5 percent and even can reach less than or equal to 3.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A kit for detecting the content of heart-type fatty acid binding protein by using a latex enhanced immunoturbidimetry method is characterized by comprising a reagent R1 and a reagent R2; wherein the content of the first and second substances,

2. The kit of claim 1, wherein the latex particles are carboxylated polystyrene latex particles, hydroxylated polystyrene latex particles, sulfonated polystyrene latex particles, diazotized polystyrene latex particles, or azido polystyrene latex particles.

3. The kit according to claim 1, wherein the reagent R1 comprises the following components:

4. the kit according to claim 1, wherein the reagent R2 comprises the following components:

0.1-0.05g/L of horseradish peroxidase-labeled heart-type fatty acid binding protein monoclonal antibody latex particles

5. The kit of claim 1, wherein the buffer is a phosphate buffer or a Tris-HCl buffer.

6. The kit of claim 1, wherein the electrolyte is selected from the group consisting of: sodium chloride, potassium chloride, or a combination thereof.

7. The kit of claim 1, wherein the stabilizer is selected from the group consisting of: bovine serum albumin, casein, skim milk powder, or combinations thereof.

8. The kit of claim 1, wherein the turbidifying agent is selected from the group consisting of: polyethylene glycol-8000, polyethylene glycol-6000, polyethylene glycol-4000 or combinations thereof.

9. The kit of claim 1, wherein the preservative is selected from the group consisting of: sodium azide, Proclin300, or a combination thereof.

10. The kit of claim 1, wherein the horseradish peroxidase-labeled monoclonal antibody latex particles of heart-type fatty acid binding protein are prepared by a method comprising the steps of: