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