CN111505303A

CN111505303A - Kit for detecting heart-type fatty acid binding protein by chemiluminescence method and use method thereof

Info

Publication number: CN111505303A
Application number: CN201910099809.7A
Authority: CN
Inventors: 焉丽波; 王立杰; 艾冰花; 常青侠; 赵京超; 陈蒙蒙
Original assignee: Avioq Biology Technology Co ltd
Current assignee: Avioq Biology Technology Co ltd
Priority date: 2019-01-31
Filing date: 2019-01-31
Publication date: 2020-08-07

Abstract

The invention is suitable for the field of biotechnology, and particularly relates to a detection kit for detecting heart-type fatty acid binding protein by a chemiluminescence method. The detection kit for detecting the heart-type fatty acid binding protein by the chemiluminescence method provided by the embodiment of the invention has high accuracy and good sensitivity; the uniformity is good, and the stability is high; the specificity is good; the rapid quantitative detection of H-FABP in clinical serum samples can be realized, so that the screening method can be used for simply, conveniently and rapidly excluding and screening the disease of AMI patients in the early stage, and is simple to operate and free from pollution.

Description

Kit for detecting heart-type fatty acid binding protein by chemiluminescence method and use method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a kit for detecting heart-type fatty acid binding protein by a chemiluminescence method and a using method thereof.

Background

Acute Myocardial Infarction (AMI) is a common critical clinical condition. AMI usually has sudden and rapid onset, and early discovery and treatment have great significance for reducing infarct size and protecting cardiac function, so that AMI is a key point for saving the life of a patient and improving the prognosis of the patient. ESC and ACC combinations in 1999 suggested detection of abnormal changes in cardiac markers as a prerequisite for diagnosis of acute onset of AMI. The biochemical detection markers commonly used in the clinic at present are cTnI and CK-MB, however, the release time of cTnI and CK-MB into blood is relatively late, so that the detection markers are not ideal as early diagnosis of AMI. In recent years, myocardial fatty acid binding protein (H-FABP) appears in blood early and has high specificity, and has become one of the biochemical indicators of early diagnosis of AM I which are concerned at home and abroad.

H-FABP is an important intracellular fatty acid binding protein, exists in a large amount in myocardial tissues, is a soluble protein, has a small molecular weight of only 15KDa, can be quickly released into blood when myocardial cells are damaged, and can detect high-level serum H-FABP in the early stage (0-3H) of AMI. When myocardial cells are ischemic and anoxic, the utilization of fatty acid is mobilized to supply energy, so that the H-FABP in the myocardial cells is rapidly increased, and the H-FABP can be quickly released into blood due to small molecular weight. The H-FABP level in blood plasma can be increased due to the heart failure after the acute myocardial infarction, and the increased degree is in direct proportion to the degree of the heart failure, so the H-FABP is not only an early indicator of myocardial damage, but also can be used as an indicator for evaluating the cardiac function.

The H-FABP level detection has higher sensitivity and specificity for early diagnosis of AMI, and has certain clinical value in the aspects of determining severity, prognosis evaluation, curative effect judgment and the like. Because H-FABP is a protein without enzyme activity, the qualitative detection and the quantitative detection of H-FABP both need to be carried out by means of an immunological method. From the earliest radioimmunoassay to the latest latex particle enhanced immunoturbidimetry and the application of colloidal gold immunoassay to H-FABP determination, the H-FABP clinical determination is subject to a development process with faster and faster determination speed and higher determination sensitivity.

The first report on the quantitative detection of FABP was the radioimmunoassay adopted in 1974 by Ockner et al in the study of I-FABP, in which Ockner et al first obtained purified I-FABP by gel filtration column chromatography and isoelectric focusing electrophoresis, and then immunized rabbits with purified I-FABP to obtain I-FABP antiserum and radiolabeled with 14C, the 14C-labeled antiserum reacted with the specimen to be tested, and the concentration of the corresponding I-FABP was determined by measuring the specific activity of radioactivity in the immunoprecipitates. The method reported by Ockner et al has the disadvantages of complicated preparation process and measurement process, high price, radioactive pollution and lack of practical value in clinical detection, but lays a foundation for the improvement of the FABP measurement method.

After Ockner, many researchers report a plurality of FABP measuring methods based on the E L ISA principle on the basis of preparing various FABP monoclonal antibodies, the methods have no radioactive pollution, are simple, convenient and quick to operate, the measuring time is between 2-16 h, the stability and the repeatability of the result are good, and the application value is high.

In 1998, Rober et al have reported an immunoturbidimetry method for measuring FABP in plasma, which is based on the principle that monoclonal antibodies against various FABPs are adsorbed to latex particles by physical adsorption to prepare a specific latex detection reagent, and when the specific FABP in a specimen to be measured reacts with the corresponding monoclonal antibody, the latex particles agglutinate, and then the concentration of the corresponding FABP can be detected according to the turbidity change after latex agglutination, and the immunoturbidimetry method can be fully automated by combining a widely clinically used turbidimeter such as COBASMIRA, etc., wherein the detection of one specimen only requires 3min, compared with the E L ISA method, the immunoturbidimetry method has high automation, lower measurement cost, and simpler and faster operation, and may replace the E L ISA method to become a conventional method for clinically detecting FABP.

Although a great number of experiments and clinical researches have shown in recent years that H-FABP has higher sensitivity, specificity and accuracy in the aspects of ACS early diagnosis, long-term prognosis prediction and the like, it still cannot be widely popularized in clinic at present, and the reasons for analyzing the H-FABP are as follows: 1. there is no unified standard for H-FABP laboratory detection values at home and abroad, and more detailed research and series detection are still needed for clinically measuring the critical value of H-FABP increase; 2. because the H-FABP detection value is influenced by factors such as age, renal function and the like, the application of the H-FABP detection value is still limited to a certain extent; 3. commercial detection means are used for early detection of H-FABP of ACS patients in clinic at present, but the detection cost is relatively high; 4. although there are many subtypes of FABPs and there is tissue specificity among the subtypes, it should be theoretically possible to accurately identify different subtypes, but there are larger and the same molecular groups among the subtypes of human FABPs, and the current detection means has insufficient resolution capability for each tissue subtype.

In view of the above, there is a need for a method for detecting H-FABP, which is applicable to clinical use, has high accuracy, simple operation, high specificity and sensitivity, and is free from contamination.

Disclosure of Invention

The embodiment of the invention provides a detection kit for quantitatively detecting heart-type fatty acid binding protein by a chemiluminescence method, and the kit is used for carrying out H-FABP clinical detection, and has the advantages of high accuracy, simple operation, high specificity and sensitivity and no pollution.

The detection kit for detecting heart-type fatty acid binding protein by using a chemiluminescence method comprises a luminescent plate coated with an anti-H-FABP monoclonal antibody, an anti-H-FABP monoclonal antibody solution for detection marked by horseradish peroxidase, a standard, a sample diluent, a standard diluent, a concentrated washing solution, a luminescent solution A, a luminescent solution B and a detection antibody diluent.

The embodiment of the invention also provides a use method of the detection kit for detecting the heart-type fatty acid binding protein by the chemiluminescence method, which comprises the following steps:

(1) the following solutions were prepared:

diluting the detection H-FABP monoclonal antibody marked by the horseradish peroxidase by using a detection antibody diluent, wherein the ratio of the detection H-FABP monoclonal antibody marked by the horseradish peroxidase to the antibody diluent is 1: 5000;

dissolving a standard substance by using a standard substance diluent to obtain a plurality of standard substance solutions with gradient changes in concentration;

and diluting the concentrated washing solution with distilled water or deionized water according to the volume ratio of 1:20 to obtain the washing solution.

(2) Drawing a standard curve

Respectively adding 50 mu L of calibrator solution into corresponding holes of the ELISA plate coated with the anti-H-FABP monoclonal antibody, and lightly shaking and uniformly mixing;

sealing the plate with a sealing plate film, incubating at 37 ℃ for 30 minutes, spin-drying the liquid in the hole, fully washing for 5 times with a washing solution, and drying;

adding 100 mu L of horseradish peroxidase-labeled anti-H-FABP monoclonal antibody into each hole, and lightly shaking and uniformly mixing;

sealing the plate with a sealing plate film, then incubating for 60 minutes at 37 ℃, fully washing for 5 times with a washing solution, and drying;

adding 25 mu L of luminous liquid A, B into each hole, and lightly shaking and uniformly mixing;

adjusting the zero point by using a blank hole, and measuring the optical density value of each hole by using a luminometer;

performing data processing by using a log-log linear fitting mode, and drawing by using the log of the optical density value of each calibrator as a vertical coordinate and the log of the concentration of each calibrator as a horizontal coordinate to obtain a standard curve;

(3) detection of a sample to be tested

Respectively adding 50 mu L samples to be tested into corresponding holes of the ELISA plate coated with the anti-H-FABP monoclonal antibody, and lightly shaking and uniformly mixing;

adding 100 mu L of horseradish peroxidase-labeled anti-H-FABP monoclonal antibody, and lightly shaking and uniformly mixing;

adding 25 mu L of luminous liquid A, B liquid respectively, and lightly shaking and uniformly mixing;

and substituting the optical density value of the sample solution to be detected into the standard curve to obtain the content of H-FABP in the sample solution to be detected.

The detection kit for detecting the heart-type fatty acid binding protein by the chemiluminescence method provided by the embodiment of the invention has the advantages that the minimum detection limit is as low as 1pg/m L, the accuracy is high, the sensitivity is good, the uniformity and the stability of the kit are good, the intra-batch variation coefficient is less than 10%, the inter-batch variation coefficient is less than 5%, the specificity is good, the cross reaction of the kit and serum total bilirubin TBI L and triglyceride TG which are easy to interfere with the test in serum is very low, the interference on the measurement of H-FABP is avoided, the rapid quantitative detection of the H-FABP in a clinical serum sample can be realized, the kit can be used for early, simple and rapid exclusion screening of the AMI patient, the operation is simple, and no pollution is caused.

Drawings

FIG. 1 is a schematic diagram of a calibration curve provided by an embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the embodiment of the invention, the luminescent plate coated with the anti-H-FABP monoclonal antibody is prepared by dissolving the anti-H-FABP monoclonal antibody in a buffer solution until the concentration of the anti-H-FABP monoclonal antibody is 10mg/m L to obtain a mixed solution, adding the mixed solution into the holes of a micro-porous plate, covering the mixed solution with 0.1m L per hole, placing the micro-porous plate at 2-8 ℃ for 18-24 hours, washing the micro-porous plate with 0.5% Tween20-PBS for 5 times, adding a sealing solution into each hole, wherein the sealing solution is 0.12m L per hole, placing the micro-porous plate at 2-8 ℃ for 18-24 hours, washing the micro-porous plate with 0.5% Tween20-PBS for 5 times, placing the micro-porous plate at 25-35 ℃ for drying at a humidity of less than 40%, drying for 20-24 hours, storing the micro-porous plate in an aluminum foil bag at 2-8 ℃, wherein the solvent of the buffer solution is 100 mmol/L, the pH is 9.6, the carbonate buffer solution, the solvent of the sealing solution is 20-24% of a NaCl, the preservative solution, the mass fraction of the preservative is 0.3, and the mass fraction of the sodium bicarbonate is 300% of the sodium bicarbonate in the mass fraction of the sodium bicarbonate solution of the sodium bicarbonate, the sodium bicarbonate of the sodium bicarbonate.

In the embodiment of the invention, an anti-H-FABP monoclonal antibody solution for detecting horseradish peroxidase labeling is prepared by purifying and quantifying an anti-H-FABP monoclonal antibody, dialyzing the purified and quantified anti-H-FABP monoclonal antibody in a carbonate buffer solution with the pH of 9.0-9.5 at 0.01 mol/L to obtain an anti-H-FABP monoclonal antibody solution with the final concentration of 5mg/m L, dissolving horseradish peroxidase in water to obtain an horseradish peroxidase aqueous solution with the final concentration of 5mg/m L, adding NaIO4 to the horseradish peroxidase aqueous solution with the concentration of 37.5mg/m L, stirring the horseradish peroxidase aqueous solution at room temperature in a dark place for 20min to obtain an oxidized enzyme solution, dialyzing the oxidized enzyme solution in an acetate buffer solution with the pH of 1 mmol/L and the pH of 4.4 overnight at the temperature of 4 ℃, replacing the solution for 3 times, taking out the dialysis bag solution, adding 0.2 mol/L Na2CO3 buffer solution to the oxidized enzyme solution, adjusting the pH of 9.5, adding the bovine serum albumin to the pH of 9.4, quickly dialyzing the dialyzed enzyme solution at the overnight at the temperature of 4 in an acetate buffer solution at the temperature of 1 mmol/L, adding albumin solution at the pH of 1-10000, stirring time, adding a fresh albumin solution, stirring time, and mixing the bovine serum albumin solution at the temperature of L to obtain a fresh albumin solution, and quickly mixing rate of 364, and stirring the albumin solution at the concentration of 1-364 to obtain a mixture, and stirring time, and stirring solution at the concentration of the mixture of 1-10000H, and the concentration of the supernatant of.

In the present example, the polyclonal antibody against H-FABP is preferably produced by HyTest corporation of Finland.

In the examples of the present invention, the standard is an H-FABP antigen, preferably produced by HyTest corporation of Finland.

In the present example, the sample diluent was a carbonate buffer solution of 20 mmol/L, pH 6.0, and the solutes and their concentrations in the sample diluent were 0.2% gelatin, 0.2% casein, 150 mmol/L NaCl, 0.01% Tween-20, 5% sucrose, 0.5/ten thousand brompotash phenol purple, and 0.1% preservative Proclin-300.

In the embodiment of the invention, the solvent of the composition of the concentrated washing solution is water, and the solute and the concentration thereof in the concentrated washing solution are as follows, 116 g/L of Na2HPO 4.12H 2O, 11.84 g/L of NaH2PO 4.2H 2O, 180 g/L of NaCl, 5m L/L of Tween20 and 1m L/L of preservative Proclin-300.

In the embodiment of the invention, the solvent of the luminescent liquid A is Tri-HCl buffer solution with the pH value of 8.0 and the concentration of 0.2 mol/L, the solute and the concentration thereof in the luminescent liquid A are luminol 3mg/m L, iodophenol 3mg/m L and sodium tetraphenylborate 0.5mg/m L, the solvent of the luminescent liquid B is Tri-HCl buffer solution with the pH value of 8.0 and the concentration thereof in the luminescent liquid B is carbamide peroxide 0.5mg/m L.

In the embodiment of the invention, the solvent of the standard dilution is a carbonate buffer solution with 20 mmol/L and pH 7.4, and the solute and the concentration thereof in the standard dilution are casein with mass fraction of 1%, trehalose with mass fraction of 8%, mannitol with mass fraction of 3%, EDTA with mass fraction of 1 mmol/L, glycine with mass fraction of 0.5%, NaCl with mass fraction of 150 mmol/L and a preservative Proclin-300 with mass fraction of 0.1%.

In the embodiment of the invention, the solvent of the test antibody diluent is carbonate buffer solution with the pH value of 7.4 and the mass fraction of bovine serum albumin of 20 mmol/L, the mass fraction of casein of 0.1%, NaCl of 150 mmol/L, the mass fraction of Tween20 of 0.01%, the mass fraction of amino-oxazoline of 5/1 ten thousand, the mass fraction of dye of 1/20 ten thousand and the mass fraction of preservative Proclin-300 of 0.1%.

In embodiments of the present invention, the dye is preferably food red.

As follows, the embodiment of the present invention provides a method for drawing a standard curve, in which an Optical Density (OD) value of a sample solution to be tested is substituted into the standard curve, so as to obtain the content of the central fatty acid binding protein in the sample solution to be tested.

(1) The following solutions were prepared:

dissolving the standard substance with the standard substance diluent to obtain standard substance solutions with concentrations of 0pg/m L, 5pg/m L, 15pg/m L, 40pg/m L, 130pg/m L and 300pg/m L respectively;

(2) Drawing a standard curve

performing data processing by using a log-log linear fitting mode, and drawing by using the log of the optical density value of each calibrator as a vertical coordinate and the log of the concentration of each calibrator as a horizontal coordinate to obtain a standard curve; as shown in fig. 1, the equation for the standard curve is that y is 0.9355x +4.3686 and the correlation coefficient is 0.9950.

The embodiment of the invention also provides a method for detecting the content of H-FABP in the sample solution to be detected.

In the embodiment of the invention, the sample to be tested is a fresh serum or fresh plasma sample, and is separated within 24 hours after the venous blood collection. Serum plasma samples should not be stored at 4 ℃ for more than 1 week. If the measurement can not be carried out within 1 week after blood sampling, the serum sample is sealed and placed below-20 ℃ to avoid repeated freeze thawing. Severely hemolyzed, lipemic samples were not available for detection.

The kits provided in the examples of the present invention were subjected to performance tests by the following methods. And respectively selecting three batches of kits, namely a batch one, a batch two and a batch three, and carrying out performance test.

Firstly, the method comprises the following steps: minimum limit of detection

Adding 50 mu L standard substance into each well of a luminescent plate coated with an anti-H-FABP monoclonal antibody, gently shaking and uniformly mixing, incubating at 37 ℃ for 60 minutes, washing the plate for 5 times, adding 100 mu L enzyme conjugate into each well, incubating at 37 ℃ for 30 minutes, fully washing for 5 times by using a washing solution, draining, adding 25 mu L luminescent solution A and 25 mu L luminescent solution B into each well, and measuring the luminescent value of each well by using a luminometer.

Wherein, each kit to be tested, the blank standard substance solution is provided with 20 compound holes, other standard substances are provided with double holes, the light absorption value of each hole is shown in table 1, and the light emitting value of each hole is shown in table 1.

Calculating the Mean value (Mean) and Standard Deviation (SD) of the corresponding luminescence values of the 50 mu L S0 solution of the 20 holes, and calculating the concentration value of the luminescence value of Mean +2 × SD as the lowest detection limit by a fitting equation.

The lowest detection limit of the three batches of kits is below 1pg/m L.

Table 1:

second, repeatability and batch-to-batch differences

The repeatability is the Coefficient of Variation (CV) obtained by measuring a sample by using the same batch of kit, each standard sample needs to be subjected to 10-hole precision measurement randomly in one plate, the average concentration (Mean) and the Standard Deviation (SD) of the measurement result are calculated, and the intra-batch Coefficient of Variation (CV) is 100 percent of SD/Mean × 100.

The inter-batch difference is the repeatability among different batches of kits, three batches of kits are randomly drawn, the standard substance is measured for 3 times by using the kits, the average concentration (Mean) and the Standard Deviation (SD) of the measurement result are calculated, the inter-batch variation Coefficient (CV) is SD/Mean × 100% and the standard substance is H-FABP.

The specific detection process is as follows:

the luminescence plate coated with the anti-H-FABP monoclonal antibody is taken, 50 mu L specific solution is added into each hole, the mixture is gently shaken and uniformly mixed, the mixture is incubated for 30 minutes at 37 ℃ and washed for 5 times, horseradish peroxidase labeled anti-H-FABP monoclonal antibody solution 100 mu L is added into each hole, the mixture is incubated for 60 minutes at 37 ℃, the mixture is fully washed for 5 times by washing liquid and dried, 25 mu L luminescence solution A and 25 mu L luminescence solution B are added into each hole, and the luminescence value of each hole is measured by using a luminometer.

The specific solution is a specific solution A or a specific solution B.

The specific solution A is an S0 solution (the concentration of a standard is 0pg/m L), an S1 solution (the concentration of the standard is 10pg/m L), an S2 solution (the concentration of the standard is 100pg/m L), an S3 solution (the concentration of the standard is 200pg/m L), an S4 solution (the concentration of the standard is 500pg/m L) and an S5 solution (the concentration of the standard is 1000pg/m L).

The specific solution B is a standard solution with different concentration from the specific solution A.

The preparation method of the standard solution comprises the steps of dissolving a quality control substance in 20mM PBS buffer solution with the pH value of 7.4 to obtain 100pg/m L low-concentration quality control substance solution (QC1) and 500pg/m L high-concentration quality control substance solution (QC2), and each specific solution B is provided with 10 compound wells.

The luminescence values of the wells with the particular solution A are shown in Table 2.

Table 2:

the luminescence values of the wells with the particular solution B are shown in Table 3.

Table 3:

and performing data processing by using a log-log linear fitting mode, calculating the H-FABP concentration in the specific solution B according to a standard curve, wherein the results are shown in tables 4 and 5, the three batches of the kit are used for measuring high concentration and low concentration quality control, and the variation coefficient in batches is less than 10%, so that the kit is good in uniformity and has repeatability.

Table 4:

table 5:

	QC1	QC2
			first batch	295515	1199070
Second batch	275270	1174860
			Third batch	291455	1251640
Mean value of	287413.3333	1208286.7
			SD	10710.57	60706.64
CV	3.7％	5.0％

Three batches of kits are used for measuring two quality control products with high concentration and low concentration, and the batch-to-batch variation coefficient is less than 5 percent, which shows that the kits in different batches have small variation and the measurement result has repeatability.

In summary, the main performance indexes of the kit provided by the invention have the following standards:

the lowest detection limit is not higher than 1pg/m L;

repeatability: the intra-batch variation coefficient is not higher than 10%;

inter-batch difference: the inter-batch coefficient of variation is not higher than 5%.

Third, specificity

Adding 50 mu L specific solution into each well of a luminescent plate coated with an anti-H-FABP monoclonal antibody, gently shaking and uniformly mixing, incubating at 37 ℃ for 30 minutes, washing the plate for 5 times, adding 100 mu L enzyme conjugate into each well, incubating at 37 ℃ for 60 minutes, fully washing for 5 times by using a washing solution, draining, adding 25 mu L luminescent solution A and 25 mu L luminescent solution B into each well, and measuring the luminescent value of each well by using a luminometer.

The specific solutions were S0 solution (standard concentration of 0pg/m L), S1 solution (standard concentration of 10pg/m L), S2 solution (standard concentration of 100pg/m L), S3 solution (standard concentration of 200pg/m L), S4 solution (standard concentration of 500pg/m L), S5 solution (standard concentration of 1000pg/m L), sample solution with serum total bilirubin TBI L of 40. mu. mol/L, and sample solution with triglyceride TG of 4 mmol/L.

L og-L og fitting is carried out according to the photometric value of each calibration point and the corresponding concentration, and the measured value of H-FABP of each cross-reaction substance is calculated according to a standard curve, wherein the concentration is the intersection value of the reagent and TBI L and the intersection value of the reagent and TG.

The absorbance and back-extrapolated concentration for each well are shown in Table 6.

Table 6:

the substances which easily interfere with the measurement of H-FABP in human blood are serum total bilirubin TBI L and

triglyceride TG. Experiments were performed with high concentrations of these substances and referring to tables 6 and 7, the kit showed low cross-reactivity with these substances and did not interfere with the measurement of H-FABP.

Table 7:

reactive substance	Concentration of	Cross reaction value
			Serum total bilirubin (TBI L)	40μmol/mL	<5pg/mL
Triglycerides (TG)	4mmol/L	<5pg/mL

In conclusion, the kit has high sensitivity, the minimum detection limit of the kit is as low as 1pg/m L, the kit has good uniformity and high stability, the intra-batch variation coefficient is less than 10%, the inter-batch variation coefficient is less than 5%, the specificity is good, the kit has very low cross reaction with serum total bilirubin TBI L and triglyceride TG which are easy to interfere with the test in serum, the measurement of H-FABP cannot be interfered, the rapid quantitative detection of H-FABP in clinical serum samples can be realized, the kit can be used for simply, conveniently and rapidly excluding and screening the early onset of disease of AMI patients, the accuracy is high, the operation is simple, and no pollution is caused.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A detection kit for detecting heart-type fatty acid binding protein by a chemiluminescence method is characterized by comprising a luminescent plate coated with an anti-H-FABP monoclonal antibody, an anti-H-FABP monoclonal antibody solution for detection marked by horseradish peroxidase, a standard substance, a sample diluent, a standard substance diluent, a concentrated washing solution, a luminescent solution A, a luminescent solution B and a detection antibody diluent.

2. The detection kit for the quantitative detection of heart-type fatty acid binding protein by chemiluminescence as defined in claim 1, wherein the luminescent plate coated with an anti-H-FABP monoclonal antibody is produced by the following method:

dissolving the H-FABP-resistant monoclonal antibody in a buffer solution until the concentration of the H-FABP-resistant monoclonal antibody is 2mg/m L to obtain a mixed solution;

adding the mixed solution into the holes of a microporous plate, wherein each hole is 0.1m L, covering a plate membrane, standing at the temperature of 2-8 ℃ for 18-24 hours, and washing for 5 times by using a washing solution;

adding sealing liquid into each well, wherein each well has a thickness of 0.12m L, standing at 2-8 deg.C for 18-24 hr, and washing with washing liquid for 5 times;

drying at 25-35 deg.C with humidity less than 40% for 20-24 hr, packaging with aluminum foil bag, and storing at 2-8 deg.C;

the buffer is carbonate buffer with the pH of 9.6 and the concentration of 100 mmol/L;

the solvent of the sealing liquid is carbonate buffer solution with the pH value of 7.4 of 20 mmol/L, and the solute and the concentration thereof in the sealing liquid are as follows, wherein the mass fraction of the bovine serum albumin is 3%, the mass fraction of the casein is 0.5%, the mass fraction of the sucrose is 8%, the mass fraction of the NaCl is 150 mmol/L%, and the mass fraction of the preservative Proclin-300 is 0.1%.

3. The detection kit for the quantitative detection of heart-type fatty acid binding protein by the chemiluminescence method according to claim 1, wherein the detection of horseradish peroxidase marker is carried out by using an anti-H-FABP monoclonal antibody solution prepared by the following method:

purifying and quantifying the anti-H-FABP monoclonal antibody, and dialyzing in carbonate buffer solution with the pH of 9.0-9.5 at 0.01 mol/L to obtain anti-H-FABP monoclonal antibody solution with the final concentration of about 5mg/m L;

dissolving horseradish peroxidase in water to obtain horseradish peroxidase water solution with final concentration of 5mg/m L;

adding NaIO into the horseradish peroxidase aqueous solution₄，NaIO₄The concentration of (2) is 37.5mg/m L, and the mixture is stirred for 20min at room temperature in the dark to obtain an oxidized enzyme solution;

dialyzing the oxidized enzyme solution in acetate buffer solution with the pH value of 4.4 of 1 mmol/L at 4 ℃ overnight, and changing the solution for 3 times;

the solution in the dialysis bag was taken out and 0.2 mol/L of Na was added₂CO₃Adjusting the pH of the buffer solution to 9.0-9.5, quickly mixing the buffer solution with the anti-H-FABP monoclonal antibody solution according to the volume ratio of 1:1, and slightly stirring the mixture for 2 hours at room temperature in a dark place;

adding new 6mg/m L NaBH₄Mixing 100ul of the solution, reacting at 4 deg.C for 2h, dialyzing in 0.15 mol/L carbonate buffer solution with pH of 7.4 at 4 deg.C overnight, and changing the solution for 3 times;

adding bovine serum albumin to make the final concentration of bovine serum albumin be 2-3mg/m L, mixing at rotation speed of 10000rpm for 3min, collecting supernatant, packaging, and freezing.

4. The detection kit for the quantitative detection of cardiac fatty acid binding protein by the chemiluminescence method according to claim 1, wherein the standard substance is H-FABP antigen.

5. The detection kit for the quantitative detection of heart-type fatty acid binding protein by the chemiluminescence method according to claim 1, wherein the solvent of the sample diluent is 20 mmol/L, and the pH is 6.0 carbonate buffer solution, and the solute and the concentration thereof in the sample diluent are 0.2% of gelatin, 0.2% of casein, 150 mmol/L of NaCl, 0.01% of Tween-20, 5% of sucrose, 0.5/ten thousand of brompotash phenol violet, and 0.1% of Proclin-300 as a preservative.

6. The detection kit for the quantitative detection of heart-type fatty acid binding protein by chemiluminescence method according to claim 1, wherein the solvent of the composition of the concentrated washing solution is water, and the solute and the concentration thereof in the concentrated washing solution are as follows 29 g/L of Na₂HPO₄·12H₂O, NaH 2.96 g/L₂PO₄·2H₂O, NaCl 234 g/L, and Tween20 of 20m L/L.

7. The detection kit for the quantitative detection of the heart-type fatty acid binding protein by the chemiluminescence method according to claim 1, wherein the solvent of the luminescence liquid A is Tri-HCl buffer solution with pH 8.0 and 0.2 mol/L, the solutes and the concentrations thereof in the luminescence liquid A are luminol 3mg/m L, paraiodophenol 3mg/m L and sodium tetraphenylborate 0.5mg/m L, the solvent of the luminescence liquid B is Tri-HCl buffer solution with pH 8.0 and 0.2 mol/L, and the solutes and the concentrations thereof in the luminescence liquid B are carbamide peroxide 0.5mg/m L.

8. The detection kit for the quantitative detection of the cardioid fatty acid binding protein by the chemiluminescence method according to claim 1, wherein the solvent of the standard dilution is a carbonate buffer solution with the pH value of 7.4 and the solute and the concentration thereof in the standard dilution are casein with the mass fraction of 1%, trehalose with the mass fraction of 8%, mannitol with the mass fraction of 3%, EDTA with the mass fraction of 1 mmol/L, glycine with the mass fraction of 0.5%, NaCl with the mass fraction of 150 mmol/L and Proclin-300 with the mass fraction of 0.1%.

9. The detection kit for the quantitative detection of heart-type fatty acid binding protein by the chemiluminescence method according to claim 1, wherein the solvent of the detection antibody diluent is a carbonate buffer solution with pH of 7.4 and 20 mmol/L, and the solute and the concentration thereof in the detection antibody diluent are bovine serum albumin with mass fraction of 2%, casein with mass fraction of 0.1%, NaCl with mass fraction of 150 mmol/L, Tween20 with mass fraction of 0.01%, aminopyrine with mass fraction of 5/1 ten thousand, dye with mass fraction of 1/20 ten thousand, and Proclin-300 preservative with mass fraction of 0.1%.

10. A method of using the chemiluminescent assay kit for detecting cardiotype fatty acid binding protein according to any one of claims 1-9 comprising the steps of:

(1) the following solutions were prepared:

(2) Drawing a standard curve

(3) detection of a sample to be tested