CN115015556A - Liver type fatty acid binding protein (L-FABP) detection kit and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of kits, and particularly relates to a liver fatty acid binding protein (L-FABP) detection kit and a preparation method thereof. Polyethylene glycol (PEG) is introduced into the liver type fatty acid binding protein (L-FABP) detection kit to perform magnetic bead surface second modification, the polyethylene glycol spacer arm is used for functionalizing the solid phase surface, so that the combination of non-specific protein can be obviously reduced, and then bis (sulfosuccinimidyl) suberate (BS3) is matched as a cross-linking agent, so that the steric hindrance effect can be effectively avoided, the sensitivity of the reagent is improved, and the solubility is increased due to the fact that the bis (sulfosuccinimidyl) suberate has large molecular property, so that the storage stability of the kit is enhanced.

Description

Liver type fatty acid binding protein (L-FABP) detection kit and preparation method thereof

Technical Field

The invention belongs to the technical field of kits, and particularly relates to a liver fatty acid binding protein (L-FABP) detection kit and a preparation method thereof.

Background

L-FABP (hepatic fatty acid binding protein), one of the members of the Fatty Acid Binding Protein (FABP) family, is a group of low molecular weight, highly conserved cytoplasmic proteins that can bind long chain fatty acids. In studying the regulation of fatty acid absorption in the small intestine of rats by Ockner et al, California university in the 20 th century 70 years, fatty acid-binding proteins (FABP) were found in the intestinal mucosa. FABP is a member of lipoprotein-binding protein superfamily, widely exists in various histiocytes of mammal such as small intestine, heart, brain, fat, skeletal muscle, liver, etc., and accounts for 3% -8% of the total amount of soluble protein in the cells. FABPs are primarily involved in the uptake, transport, metabolic regulation of intracellular long chain fatty acids and in the protection of cells from the toxic effects of free fatty acids. 9 different types of fatty acid binding proteins have been isolated so far and named, depending on the source, heart-type (H-FABP), adipocyte-type (A-FABP), liver-type (L-FABP), intestine-type (I-FABP), brain cell-type (B-FABP), ileum-type (I1-FABP), epithelial cell-type (E-FABP), myelin-type (M-FABP), testis-pill-type (T-FABP), mainly expressed in liver, small intestine and kidney. In the kidney, L-FABP is mainly expressed in the proximal convoluted tubule, and when kidney diseases are damaged by a large amount of proteinuria, ischemia, toxicity and the like, it is presumed that L-FABP participates in reabsorption of free fatty acids in the urinary night, and promotes β -oxidation energy supply, thereby alleviating oxygen stress injury and protecting the kidney. The L-FABP molecule has small mass of only 14.4KD, and can quickly overflow due to the permeability change of cell membranes when liver cells and renal tubule cells are damaged, so that the L-FABP can be used as a sensitive and specific tissue damage marker and can well reflect the damage of the renal tubules.

In the protein overload kidney damage animal test of Kamijo et al, the up-regulation of the expression of the proximal tubular L-FABP can obviously reduce the inflammatory reaction of the tubulointerstitial tissue and slightly inhibit the progression of the tubulointerstitial tissue damage. Later, the research of the rat model of Unilateral Ureteral Obstruction (UUO) proves that the L-FABP can reduce the oxidative stress of the UUO rat and reduce the renal interstitial injury, so that the L-FABP can reduce the oxidative stress injury by regulating the metabolism of FFAs and exerting the antioxidant effect during the acute renal injury, thereby playing the role of protecting the kidney. In chronic kidney disease, secretion of L-FABP increases in the proximal tubule and urine. Kamijo et al found that along with the deterioration of kidney function, the secretion of L-FABP in urine is increased, and the L-FABP in urine is not influenced by L-FABP in serum, so that the L-FABP in urine can possibly become a clinical marker for monitoring CKD, and other studies show that the urine L-FABP level diagnosis acute kidney injury sensitivity is 74.5%, and the specificity is 77.6%; predicted mortality, sensitivity 93.2%; specificity 78.8%; the occurrence of AKI can be well predicted by urine L-FABP, and the higher the urine L-FABP content is, the worse the prognosis of the patient is.

With the gradual introduction of other researches, the urinary L-FABP shows that the urinary L-FABP has better prediction effects in the aspects of detecting focal glomerular necrosis, nephropathy caused by coronary contrast agent, acute renal injury after cardiac bypass, diabetic nephropathy and renal injury caused by renal transplantation ischemia-reperfusion. Therefore, urinary L-FABP can serve as a better biomarker for diagnosing AKI and predicting AKI.

As a biomarker for diagnosing AKI, a chemiluminescence method in the prior art adopts a carboxyl one-step method for coupling, and a conventional chemical reagent is added into an alkaline phosphatase complex, so that the long-term stability of the reagent cannot be ensured, and the following method is introduced; using PEG-based reagents for magnetic bead surface modification, polyethylene glycol (PEG) compounds can provide linkers of known molecular dimensions for the generation of biocompatible planar surfaces or particles. In particular, PEG reagents containing a carboxylate group at one end and a thiol or lipoamide group at the other end can effectively act as a hydrophilic linkage between the adsorption surface and the ligand. MT (PEG) to be reacted with thiol reagent in surface modification ₈ A combination of PEG capped with methyl ether and PEG with carboxyl groups exposed periodically can be used to form a hydrophilic "lawn". The exposed carboxyl groups can be coupled to affinity ligands using a coupling reaction of carbodiimide with EDC and Sulfo-NHS. Carboxylic acids are reactive towards carbodiimide (EDC) and conventional methods are such that magnetic beads of carboxyl groups (-COOH) are coupled using EDC or EDC and NHS mixed in a certain ratio. Typically, this results in a high level of antigen binding on the carrier protein. However, if an antigenic determinant within the antigenic peptide sequence contains primary amines (lysine residues) or carboxylates (aspartic acid and glutamic acid residues), the epitope may be bound by EDC-mediated conjugationBlocking often results in random aggregation of the polypeptide. Meanwhile, EDC is a zero-length cross-linking agent which directly couples carboxylic acid (-COOH) with primary amine (-NH2), and the cross-linking mode causes steric hindrance effect because atoms or groups close to a reaction center in a molecule occupy a certain spatial position, so that partial binding sites are blocked, and the reactivity of the molecule is influenced, which is most obvious when a ligand is a small molecule. For the reasons, the traditional detection reagent has low sensitivity, poor repeatability and unsatisfactory stability.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provide a liver fatty acid binding protein (L-FABP) detection kit which has the advantages of high sensitivity, wide linear range, good repeatability, high precision, good stability, small batch difference and low cost.

The purpose of the invention can be realized by the following technical scheme: a liver type fatty acid binding protein (L-FABP) detection kit comprises an R1 reagent, an R2 reagent and an R3 reagent;

the R1 reagent comprises a magnetic particle-conjugated L-FABP antibody;

the R2 reagent comprises an alkaline phosphatase-labeled L-FABP antibody;

the R3 reagent comprises the following components: 3-4g/L MES, 45-55g/L bovine serum albumin, 5-15g/L trehalose, 25-35g/L sucrose, 15-25g/L mannitol, 5-15g/L polyethylene glycol 8000, 0.5-1.5g/L T-X405, 0.5-1.5g/L surfactant S7, 3-5g/L Proclin300 and 3-5g/L gentamycin.

Preferably, the magnetic particles are one of tosyl magnetic beads, amino magnetic beads, carboxyl magnetic beads and streptavidin magnetic beads having a particle size of 1.0 to 3.0 μm.

Preferably, the L-FABP antibody is a monoclonal antibody.

In the above liver fatty acid binding protein (L-FABP) detection kit, the preparation method of the magnetic particle-coupled L-FABP antibody comprises the following steps:

s1, activating the magnetic particles by a buffer solution, and then uniformly mixing the magnetic particles with the L-FABP antibody;

s2, sequentially adding bis (sulfosuccinimidyl) suberate and polyethylene glycol 20000 for reaction;

s3, adding a blocking agent to suspend the reaction, and finally diluting and storing the reaction product by using a buffer solution.

In the above liver fatty acid binding protein (L-FABP) detection kit, the mass ratio of the magnetic beads in step S1 to the L-FABP antibody is 10: (0.01-0.3)

In the detection kit for liver fatty acid binding protein (L-FABP), the concentration of bis (sulfosuccinimidyl) suberate in step S2 is 5-15mg/ml, and the addition amount is 0.5-2 times of the total volume of the magnetic particles and the antibody.

According to the invention, a coupling arm is added after PEG 20000 is introduced to couple magnetic beads, bis (sulfosuccinimidyl) suberate (BS3) is used as a cross-linking agent, BS3 has an amine-reactive N-hydroxysulfosuccinimidyl (NHS) ester at each end of a spacer arm with 8 carbon atoms, NHS ester can react with primary amine at the pH of 7-9 to form a stable amido bond and release a leaving group of N-hydroxysulfosuccinimidyl, an L-FABP antibody has a plurality of primary amines at the side chain of a lysine (K) residue and the N end of each polypeptide, and the primary amines can be used as targets of NHS ester cross-linking reagents, so that the water solubility is enhanced, and the steric hindrance effect is effectively avoided during antibody coupling.

Preferably, the blocking agent of step S3 is 0.05M HEPES buffer containing one or more of BSA, fish skin gelatin, and sodium caseinate.

Preferably, the concentration of the L-FABP antibody coupled with the magnetic particles after dilution and preservation in the buffer solution of the step S3 is 0.2-2.0 mg/ml.

In the above liver fatty acid binding protein (L-FABP) detection kit, the buffer solution in step S3 has a pH of 7-8, and comprises the following components: 3-3.5g/LMES, 8-12g/L fish skin gelatin, 45-55g/L bovine serum albumin, 5-10g/L zinc chloride, 0.5-1.5g/L LT-X405, 1.5-2.5g/L Proclin300 and 18-22g/L gentamicin.

In the above liver fatty acid binding protein (L-FABP) detection kit, the preparation method of the L-FABP antibody labeled by alkaline phosphatase comprises the following steps:

s1, adding the L-FABP antibody into a buffer solution without amino groups and sulfhydryl groups, then adding tris (2-carbonylethyl) phosphate for reaction, and adding glycine for reaction to obtain an activated antibody solution;

s2, adding alkaline phosphatase into a buffer solution without amino groups and sulfhydryl groups, then adding a DMF solution containing SMCC, and adding glycine to react to obtain an alkaline phosphatase solution;

and S3, uniformly mixing the activated antibody solution with the alkaline phosphatase solution, adding a magnesium chloride solution for reaction, and finally diluting and storing by using an enzyme diluent.

In the above liver fatty acid binding protein (L-FABP) detection kit, the mass ratio of the L-FABP antibody to the alkaline phosphatase is (3-5): 1.

preferably, in step S1, the addition amount of tris (2-carbonylethyl) phosphonium hydrochloride is 0.5-2.5 per thousand microliter per milliliter of the L-FABP antibody volume.

Preferably, the glycine concentration in step S1 is 1M, and the addition amount is 0.5-2.5 ‰ microliter per milliliter based on the volume of the L-FABP antibody.

Preferably, the concentration of the DMF solution containing SMCC is 5-8 mg/ml, and the addition amount of the DMF solution is 2.5-7.5 per mill of the volume of the alkaline phosphatase.

In the above liver fatty acid binding protein (L-FABP) detection kit, the pH of the enzyme diluent of step S3 is 7-8, and the kit comprises the following components: 5-6g/L MES, 45-55g/L bovine serum albumin, 1.5-2.5g/L sodium chloride, 8-12mol/L magnesium chloride, 0.8-1.2mol/L zinc chloride, 1.5-2.5g/L Proclin300, 18-22g/L gentamicin, and 0.3-0.8g/L tert-butyl hydroquinone ethanol.

The detection kit for the liver fatty acid binding protein (L-FABP) also comprises a calibrator and a quality control product,

the calibrator comprises protein solutions with L-FABP antigen concentrations of 0, 5, 20, 50, 200 and 500ng/mL respectively;

the quality control product comprises protein solutions with L-FABP antigen concentrations of 5 ng/mL and 20ng/mL respectively.

Preferably, the protein solution comprises the following components: 3-3.5g/LMES, 45-55g/L fish skin gelatin, 5-15g/L bovine serum albumin, 25-35g/L sucrose, 15-5g/L mannitol, 5-15g/L polyethylene glycol 8000, 3-8g/L methyl fiber, 0.5-1.5g/L T-X405, 0.5-1.5g/L surfactant S7, 3-5g/L Proclin300 and 3-5g/L gentamicin.

In the above-mentioned liver fatty acid binding protein (L-FABP) detection kit, bovine serum albumin is subjected to enzyme removal treatment.

The invention also provides a use method of the liver fatty acid binding protein (L-FABP) detection kit, which comprises the following steps: reacting the sample to be detected with the R1 reagent, the R2 reagent and the R3 reagent for 5-15min, then placing the sample in a magnetic field for separation, adding chemiluminescent substrate liquid after washing, and detecting the intensity of chemiluminescent photons.

Preferably, the volume ratio of the sample to be tested to the R1 reagent, the R2 reagent and the R3 reagent is 1: (1.5-2.5): (4.5-5.5): (4.5-5.5).

Compared with the prior art, the kit has the following advantages:

1. polyethylene glycol (PEG) is introduced into the liver type fatty acid binding protein (L-FABP) detection kit to perform magnetic bead surface second modification, the polyethylene glycol spacer arm is used for functionalizing the solid phase surface, so that the combination of non-specific protein can be obviously reduced, and then bis (sulfosuccinimidyl) suberate (BS3) is matched as a cross-linking agent, so that the steric hindrance effect can be effectively avoided, the sensitivity of the reagent is improved, and the solubility is increased due to the fact that the bis (sulfosuccinimidyl) suberate has large molecular property, so that the storage stability of the kit is enhanced.

2. The lowest detection limit of the detection kit for the liver fatty acid binding protein (L-FABP) is 0.2 ng/ml; the linear range is 0.2-500ng/ml, the linear correlation coefficient is R0.99, and the kit has the advantages of high sensitivity, wide linear range, good repeatability, high precision, good stability and short detection time with small batch difference.

Drawings

FIG. 1 is a test luminescence standard curve of the calibrator of example 1.

FIG. 2 is a graph showing the correlation between the detection results of the kit prepared in example 1 and the detection results of the foreign RD (RD SYSTEMS Co.) kit (subjected to CE certification).

Detailed Description

The following are specific embodiments of the present invention and the technical solutions of the present invention will be further described with reference to the drawings, but the present invention is not limited to these embodiments.

The following examples used raw material ingredients and sources:

carboxyl magnetic particles were purchased from JSR corporation;

the L-FABP antibody is a self-developed antibody of Ningbo Hai Biotechnology Limited.

Example 1:

preparation of R1 reagent:

s1, adding 10mg of carboxyl magnetic particles into an EP tube;

s2, placing an EP tube on a magnetic frame, washing the EP tube for 1-5 times by using a coupling buffer solution, and fixing the volume to 1000 mu L; the coupling buffer is 0.05M MES buffer with pH 8.0;

s3, adding 100 mu L of L-FABP antibody with the concentration of 2.0mg/mL, then sequentially adding 800 mu L of bis (sulfosuccinimidyl) suberate (BS3) with the concentration of 10mg/mL and 30 mu L of polyethylene glycol 20000 with the concentration of 1%, wherein the adding amount of the bis (sulfosuccinimidyl) suberate (BS3) is 0.8 times of the total volume of the magnetic particles and the antibody, and then suspending at 28 ℃ for 10 h.

S4, adding 0.2ml of sealant, suspending for 20h at 30 ℃, replacing with a magnetic bead preservation solution, wherein the final concentration of magnetic particles is 10mg/ml, and finally diluting with a buffer solution to 1.0mg/ml for preservation; blocking agent is 10% BSA + 0.01% fishskin gelatin 0.05M HEPES buffer solution; a 0.05M HEPES buffer solution with the pH value of the magnetic bead preservation solution being 7.5; the buffer has a pH of 7.5 and comprises the following components: 3.25g/LMES, 10g/L fish skin gelatin, 50g/L bovine serum albumin, 8g/L zinc chloride, 1g/L T-X405, 2g/L Proclin300 and 20g/L gentamicin.

Preparation of R2 reagent:

s1, replacing 1mg of L-FABP antibody with a buffer solution without amino and sulfhydryl groups by dialysis, and concentrating to 3mg/ml by a concentration tube;

s2, adding TCEP (tris (2-carbonylethyl) phosphate), reacting at room temperature for 20min, then adding glycine solution with the pH value of 7.3 and the concentration of 1M, reacting at room temperature for 8min, replacing buffer solution by using a PD-10 desalting column, and concentrating to 3mg/ml to obtain activated antibody solution for later use; TCEP (tris (2-carbonylethyl) phosphate) and glycine are added in an amount of 1 ‰ microliter per milliliter of antibody;

s3, replacing 0.25mg of alkaline phosphatase with a buffer solution without amino and sulfhydryl groups by dialysis, and concentrating to 3mg/ml by a concentration tube;

s4, adding 6mg/mL DMF solution containing SMCC to react for 15min at room temperature, then adding 1M glycine with pH7.3 to react for 15min at room temperature, replacing buffer solution by using a PD-10 desalting column, and concentrating to 3mg/mL to obtain alkaline phosphatase solution for later use; the DMF solution containing SMCC and glycine are added in an amount which is 5 per mill of the volume of the alkaline phosphatase.

S5, mixing the activated antibody solution and the alkaline phosphatase solution, adding 0.3M magnesium chloride solution, adding 3 per mill ml of volume of the total volume of the reaction, reacting for 12 hours at 5 ℃, and finally purifying and storing at 2-8 ℃;

s6, when used, diluted to 0.8 mg/mL.

Preparation of R3 reagent:

0.325g of MES, 5g of bovine serum albumin, 1g of trehalose, 3g of sucrose, 2g of mannitol, 1g of polyethylene glycol 8000, 0.1g T-X405,0.1g of surfactant S7, 0.4g of Proclin300 and 0.4g of gentamicin are put into a glass container, 100ml of purified water is added, and the mixture is stirred uniformly to obtain the R3 reagent.

Preparing a calibration material and a quality control material:

0.325g of MES, 5g of fish skin gelatin, 1g of bovine serum albumin, 3g of sucrose, 2g of mannitol, 1g of polyethylene glycol 8000, 0.5g of methylcellulose, 0.1g T-X405,0.1g of surfactant S7, 0.4g of Proclin300 and 0.4g of gentamicin are put into a glass container, 100ml of purified water is added, and the mixture is stirred uniformly to obtain a buffer solution.

The L-FABP antigen was prepared into calibrators with concentrations of 0, 5, 20, 50, 200, and 500ng/mL using a buffer solution.

And preparing the L-FABP antigen into quality control products with the concentrations of 5 ng/mL and 20ng/mL respectively by using a buffer solution.

Example 2:

the only difference from example 1 was that the PEG + BS3 conjugation in the R1 reagent was replaced with PEG + SMCC conjugation.

Example 3:

the only difference from example 1 is that the PEG + BS3 coupling in the R1 reagent was replaced by PEG + EDC coupling.

Example 4:

the only difference from example 1 is that the PEG + BS3 coupling in the R1 reagent was replaced by PEG + smdpp coupling.

Application examples 1 to 4:

10ul of sample to be tested is respectively taken to react with 20ul of R1 reagent, 50ul of R2 reagent and 50ul of R3 reagent in the kit prepared in the embodiment 1 to 4 for 10min, then magnetic separation and cleaning are carried out, finally alkaline phosphatase substrate luminescent liquid is added, and the luminescent value in a reaction tube is measured.

Table 1: EXAMPLES 1-4 kit calibrator test luminescence results

FIG. 1 is a standard curve of luminescence for the calibrator test in example 1, and in combination with the data in Table 1, the liver fatty acid binding protein kit for detecting calibrator prepared in example 1 has better reaction gradient, lower signal-to-noise ratio and curve fitting degree R ² When the drug is 0.99983, the improvement effect is obvious.

And (3) testing the performance of the lowest detection limit and the detection limit:

and parallelly measuring the luminescence value (RLU) of the 20-time zero-value calibrator, calculating the average value (M) and the Standard Deviation (SD) of the RLU, obtaining the RLU value corresponding to M +2SD, performing two-point regression fitting according to the concentration-luminescence value (RLU) result between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation, substituting the luminescence value of the M +2SD into the equation, and calculating the corresponding concentration value, wherein the result is not higher than the minimum detection limit value (0.2 ng/mL). The results are shown in Table 2.

Table 2: examples 1-4 kit minimum detection limit and detection limit performance tests

Number of tests	Example 1	Example 2	Example 3	Example 4
					1	7040	22492	37256	20810
2	7059	25138	30578	22527
					3	7024	19625	35850	17807
4	7618	24697	40419	25530
					5	7047	25579	37256	19309
6	7032	26241	34796	23814
					7	7146	21389	28469	25101
8	7035	19625	37607	23599
					9	7037	21830	36904	21239
10	7067	20728	42176	22956
					11	7145	21389	33038	18236
12	7052	20728	35498	24458
					13	7050	22933	35498	25530
14	7048	20728	33390	19738
					15	7030	21389	35850	20167
16	7039	24256	34444	25530
					17	7018	26241	34093	22741
18	7047	24477	29875	22956
					19	7035	23595	41122	22312
20	7066	22272	39013	21025
					Minimum limit of detection	0.007	0.530	0.053	0.358

The results in table 2 show that the 20 zero value calibrator test using PEG + EDC coupled with PEG + BS3 meets the 0.2ng/ml requirement, but the PEG + BS3 has a minimum detection limit of 0.007, with obvious advantages.

And (3) linear verification:

taking a high-concentration sample close to the upper limit of the linear range and a low-concentration sample close to the lower limit of the linear range, and mixing the high-concentration sample and the low-concentration sample into at least 5 diluted concentrations according to a certain proportion. Each dilution concentration was tested 3 times and the mean of the test results calculated. Using the dilution concentration as independent variable, using the mean value of the detection result as dependent variable, calculating linear regression equation, calculating correlation coefficient r, and obtaining the result such as

Shown in table 3.

Table 3: examples 1-4 kit Linear verification results

The results in Table 3 show that the PEG + BS3 coupling has a correlation coefficient r >0.99 in the concentration range of 0.2-500 ng/ml. The method is coupled through PEG + BS3, the detection range of the L-FABP kit is 0.2-500ng/ml, and the data have obvious advantages compared with PEG + BS 3.

And (3) coupling repeatability verification:

the repeatability was tested using samples of different concentrations, each sample was tested for 10 consecutive analytical measurements CV, and the results are shown in table 4.

Table 4: example 1 kit reproducibility test results

Precision in the day:

the precision between days was tested by using samples of different concentrations, the samples were frozen, and the test was performed once in the morning, in the middle of the day, and in the evening, for 7 days, each sample obtained 21 data, and the CV of the test results was analyzed, and the results are shown in table 5.

Table 5: example 1 results of inter-day precision measurement of kit

Thermal stability:

the kit was placed in an accelerated destruction environment at 37 ℃ and taken out on days 3, 7 and 14, respectively, the luminescence values of the calibrators were measured, and the deviations were calculated, the results of which are shown in table 6.

Table 6: example 1 kit thermal stability test results

Long-term stability:

the kit is stored at 2-8 ℃, and the test calibrator is taken out at 3 months, 7 months, 12 months, 15 months and 24 months respectively to calculate the deviation of the luminescence value, and the result is shown in table 7.

Table 7: example 1 Long term stability test results of the kit

ng/ml	Initial value	3 months old	Amplitude reduction	7 months old	Amplitude reduction	12 months old	Amplitude reduction	15 months old	Amplitude reduction	24 months	Amplitude reduction
													0	7524	7363	-2.14％	7152	-4.94％	7082	-5.87％	7222	-4.01％	7222	-4.01％
5	245695	246229	0.22％	246229	0.22％	239057	-2.70％	248619	1.19％	234276	-4.65％
												20	878928	923308	5.05％	896928	2.05％	896928	2.05％	870548	-0.95％	905721	3.05％
50	2141008	2196195	2.58％	2261428	5.62％	2239684	4.61％	2174450	1.56％	2174450	1.56％
												200	7256794	7376001	1.64％	7092309	-2.27％	7376001	1.64％	7305078	0.67％	7234155	-0.31％
500	16116238	16233463	0.73％	16233463	0.73％	15915160	-1.25％	16074312	-0.26％	16551766	2.70％

Inter-batch difference test:

4 batches of the carboxymagnetic bead-liver fatty acid binding protein monoclonal antibody and the alkaline phosphatase-liver fatty acid binding protein monoclonal antibody were continuously coupled to produce 4 batches of kits, 5 samples were tested for luminescence value to evaluate the reagent batch CV, and the results are shown in Table 8.

Table 8: EXAMPLE 1 kit run-to-run test results

FIG. 2 is a graph showing the correlation between the detection results of the kit prepared in example 1 and the detection results of the foreign RD (RD SYSTEMS Co.) kit (CE certified). As can be seen from the figure, the kit prepared in example 1 and the RD comparison kit are better in comparison correlation _。

In conclusion, the sensitivity of the liver fatty acid binding protein kit can reach 0.2ng/ml, the linear range can reach 0.2-500ng/ml, and the batch repeatability can be controlled within 2%; the precision in the day can be controlled within 3 percent; the thermal accelerated destruction is carried out for 14 days at 37 ℃ and the product is stored for 15 months at 2-8 ℃, and the reduction amplitude can be controlled within 10 percent; the inter-batch difference can be controlled within 3%, and the test result is more accurate.

The technical scope of the invention claimed by the embodiments herein is not exhaustive and new solutions formed by equivalent replacement of single or multiple technical features in the embodiments are also within the scope of the invention, and all parameters involved in the solutions of the invention do not have mutually exclusive combinations if not specifically stated.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims (10)

1. A liver type fatty acid binding protein (L-FABP) detection kit is characterized by comprising an R1 reagent, an R2 reagent and an R3 reagent;

the R1 reagent comprises a magnetic particle-conjugated L-FABP antibody;

the R2 reagent includes an alkaline phosphatase-labeled L-FABP antibody.

The R3 reagent comprises the following components: 3-4g/L MES, 45-55g/L bovine serum albumin, 5-15g/L trehalose, 25-35g/L sucrose, 15-25g/L mannitol, 5-15g/L polyethylene glycol 8000, 0.5-1.5g/L T-X405, 0.5-1.5g/L surfactant S7, 3-5g/L Proclin300 and 3-5g/L gentamycin.

2. The liver-type fatty acid binding protein (L-FABP) detection kit according to claim 1, wherein the preparation method of the magnetic particle-coupled L-FABP antibody comprises the following steps:

s1, activating the magnetic particles by buffer solution, and then uniformly mixing the magnetic particles with the L-FABP antibody;

s2, sequentially adding bis (sulfosuccinimidyl) suberate and polyethylene glycol 20000 for reaction;

s3, adding a blocking agent for suspension reaction, and finally diluting and storing by using a buffer solution.

3. The liver-type fatty acid binding protein (L-FABP) detection kit according to claim 2, wherein the mass ratio of the magnetic beads to the L-FABP antibody in step S1 is 10: (0.01-0.3).

4. The detection kit for liver-type fatty acid binding protein (L-FABP) according to claim 2, wherein the concentration of bis (sulfosuccinimidyl) suberate of step S2 is 5-15mg/ml, and the amount added is 0.5-2 times of the total volume of the magnetic particles and the antibody.

5. The liver-type fatty acid binding protein (L-FABP) detection kit according to claim 2, wherein the pH of the buffer solution of step S3 is 7-8, and the kit comprises the following components: 3-3.5g/LMES, 8-12g/L fish skin gelatin, 45-55g/L bovine serum albumin, 5-10g/L zinc chloride, 0.5-1.5g/L T-X405, 1.5-2.5g/L Proclin300 and 18-22g/L gentamicin.

6. The liver fatty acid binding protein (L-FABP) detection kit according to claim 1, wherein the preparation method of the alkaline phosphatase-labeled L-FABP antibody comprises the following steps:

s1, adding the L-FABP antibody into an amino-free and sulfhydryl-free buffer solution, then adding tris (2-carbonylethyl) phosphate for reaction, and adding glycine for reaction to obtain an activated antibody solution;

s2, adding alkaline phosphatase into a buffer solution without amino and sulfhydryl groups, then adding a DMF solution containing SMCC, and adding glycine to react to obtain an alkaline phosphatase solution;

s3, uniformly mixing the activated antibody solution with the alkaline phosphatase solution, adding a magnesium chloride solution for reaction, and finally diluting and storing by using an enzyme diluent.

7. The liver-type fatty acid binding protein (L-FABP) detection kit according to claim 6, wherein the mass ratio of the L-FABP antibody to the alkaline phosphatase is (3-5): 1.

8. the liver-type fatty acid binding protein (L-FABP) detection kit according to claim 6, wherein the pH of the enzyme diluent of step S3 is 7-8, and the kit comprises the following components: 5-6g/L MES, 45-55g/L bovine serum albumin, 1.5-2.5g/L sodium chloride, 8-12mol/L magnesium chloride, 0.8-1.2mol/L zinc chloride, 1.5-2.5g/L Proclin300, 18-22g/L gentamicin, and 0.3-0.8g/L tert-butyl hydroquinone ethanol.

9. The detection kit for liver-type fatty acid binding protein (L-FABP) according to claim 1, further comprising a calibrator and a quality control,

the calibrator comprises protein solutions with L-FABP antigen concentrations of 0, 5, 20, 50, 200 and 500ng/mL respectively;

the quality control product comprises protein solutions with L-FABP antigen concentrations of 5 ng/mL and 20ng/mL respectively.

10. The liver-type fatty acid binding protein (L-FABP) detection kit according to claim 9, wherein the protein solution comprises the following components: 3-3.5g/LMES, 45-55g/L fish skin gelatin, 5-15g/L bovine serum albumin, 25-35g/L sucrose, 15-5g/L mannitol, 5-15g/L polyethylene glycol 8000, 3-8g/L methyl fiber, 0.5-1.5g/L T-X405, 0.5-1.5g/L surfactant S7, 3-5g/L Proclin300 and 3-5g/L gentamicin.

Images