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

Abstract

The present invention belongs to the technical field of kits, and in particular, relates to a liver-type fatty acid binding protein (L-FABP) detection kit and a preparation method thereof. The liver-type fatty acid binding protein (L-FABP) detection kit of the present invention introduces polyethylene glycol (PEG) to modify the surface of magnetic beads, and functionalizing the solid phase surface with a polyethylene glycol spacer arm can significantly reduce the binding of nonspecific proteins. Then, bis(sulfosuccinimidyl) suberate (BS3) is used as a cross-linking agent to effectively avoid steric effects and improve reagent sensitivity. Because bis(sulfosuccinimidyl) suberate has macromolecular properties, it will increase solubility and enhance the stability of the kit storage.

Description

Technical Field

[0001] The invention belongs to the technical field of kits, and in particular relates to a liver-type fatty acid binding protein (L-FABP) detection kit and a preparation method thereof. Background Art

[0002] L-FABP (liver fatty acid binding protein) is a member of the fatty acid binding protein (FABP) family. It is a group of low molecular weight, highly conserved cytoplasmic proteins that can bind long-chain fatty acids. In the 1970s, Ockner et al. from the University of California, USA, discovered fatty acid binding protein (FABP) in the intestinal mucosa when studying the regulation of small intestinal fatty acid absorption in rats. FABP is a member of the lipoprotein binding protein superfamily and is widely present in various tissue cells such as the small intestine, heart, brain, fat, skeletal muscle, and liver of mammals, accounting for 3%-8% of the total soluble protein in cells. FABP is mainly involved in the uptake, transport, and metabolic regulation of intracellular long-chain fatty acids and protects cells from the toxic effects of free fatty acids. So far, 9 different types of fatty acid binding proteins have been isolated and named according to their sources: heart type (H-FABP), adipocyte type (A-FABP), liver type (L-FABP), intestinal type (I-FABP), brain cell type (B-FABP), ileum type (I1-FABP), epithelial cell type (E-FABP), myelin type (M-FABP), and testis type (T-FABP). They are mainly expressed in the liver, small intestine, and kidney. In the kidney, L-FABP is mainly expressed in the proximal tubule. When kidney disease causes a large amount of proteinuria, ischemia, and toxicity, it is speculated that L-FABP participates in the reabsorption of free fatty acids in urine at night, promotes β-oxidation energy supply, and thus reduces oxygen stress damage, thereby protecting the kidney. The molecular weight of L-FABP is relatively small, only 14.4KD. When liver cells and renal tubular cells are damaged, changes in cell membrane permeability can cause it to overflow quickly. Therefore, L-FABP can be used as a sensitive and specific tissue damage marker, which can well reflect renal tubular damage.

[0003] In an animal experiment on renal damage caused by protein overload, Kamijo et al. found that the upregulation of L-FABP expression in the proximal tubules could significantly reduce the inflammatory response in the tubulointerstitial and slightly inhibit the progression of tubulointerstitial damage. Later, they used a rat model of unilateral ureteral obstruction (UUO) and found that L-FABP could reduce oxidative stress and alleviate renal interstitial damage in UUO rats. Therefore, in the process of acute renal injury, L-FABP may reduce oxidative stress damage by regulating the metabolism of FFAs and exerting antioxidant effects, thereby protecting the kidneys. In chronic kidney disease, the secretion of L-FABP in the proximal tubules and urine increases. Kamijo et al. found that with the deterioration of renal function, the secretion of L-FABP in urine increased and was not affected by L-FABP in serum. L-FABP in urine may become a clinical marker for monitoring CKD. Other studies have shown that the sensitivity of urinary L-FABP level in diagnosing acute kidney injury is 74.5%, and the specificity is 77.6%. For predicting mortality, the sensitivity is 93.2% and the specificity is 78.8%. Urinary L-FABP can well predict the occurrence of AKI. The higher the urine L-FABP content, the worse the patient's prognosis.

[0004] As other studies have gradually deepened, urinary L-FABP has shown good predictive effects in detecting focal glomerular necrosis, coronary contrast agent-induced nephropathy, acute kidney injury after cardiac bypass surgery, diabetic nephropathy, and renal injury caused by ischemia-reperfusion in renal transplantation. Therefore, urinary L-FABP can be used as a good biomarker for diagnosing and predicting AKI.

[0005] As a biomarker for diagnosing AKI, the chemiluminescence method in the prior art uses a carboxyl one-step method for coupling, and conventional chemical reagents added to the alkaline phosphatase complex cannot guarantee the long-term stability of the reagent, so the following method is introduced; using a PEG-based reagent for magnetic bead surface modification, polyethylene glycol (PEG) compounds can provide a linker with a known molecular size for generating a biocompatible planar surface or particle. In particular, the PEG reagent contains a carboxylic acid ester group at one end and a thiol or lipoamide group at the other end, which can effectively serve as a hydrophilic connection bond between the adsorption surface and the ligand. In the surface modification, the combination of MT (PEG) 8 with the thiol reagent can form a hydrophilic "lawn" composed of methyl ether-capped PEG and periodically exposed carboxyl-containing PEG. Using the coupling reaction of carbodiimide with EDC and Sulfo-NHS, the exposed carboxyl group can be coupled to the affinity ligand. Carboxylic acid is reactive to carbodiimide (EDC), and the conventional method is that the carboxyl (-COOH) magnetic bead coupling uses EDC or EDC and NHS in a certain proportion. Typically, this approach results in a high level of antigen binding on the carrier protein. However, if the antigenic determinant within the antigenic peptide sequence contains primary amines (lysine residues) or carboxylates (aspartic acid and glutamic acid residues), the epitope may be blocked by EDC-mediated binding, often resulting in random polymerization of the polypeptide. At the same time, EDC is a zero-length cross-linking agent that directly couples carboxylic acids (-COOH) with primary amines (-NH2). This cross-linking method causes a steric effect because the atoms or groups close to the reaction center in the molecule occupy a certain spatial position, resulting in the blocking of some binding sites and affecting the molecular reactivity, which is most obvious when the ligand is a small molecule. For the above reasons, traditional detection reagents have low sensitivity, poor repeatability, and unsatisfactory stability. Summary of the invention

[0006] The purpose of the present invention is to provide a liver-type fatty acid binding protein (L-FABP) detection kit with high sensitivity, wide linear range, good repeatability, high precision, good stability, small batch difference and low cost in view of the above problems existing in the prior art.

[0007] The object of the present invention can be achieved by the following technical scheme: a liver-type fatty acid binding protein (L-FABP) detection kit, the kit comprising R1 reagent, R2 reagent, and R3 reagent;

[0008] The R1 reagent includes L-FABP antibody coupled to magnetic particles;

[0009] The R2 reagent includes an alkaline phosphatase-labeled L-FABP antibody;

[0010] The R3 reagent includes the following ingredients: 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 gentamicin.

[0011] Preferably, the magnetic particles are one of tosyl magnetic beads, amino magnetic beads, carboxyl magnetic beads and streptavidin magnetic beads with a particle size of 1.0 to 3.0 μm.

[0012] Preferably, the L-FABP antibody is a monoclonal antibody.

[0013] In the above-mentioned liver fatty acid binding protein (L-FABP) detection kit, the method for preparing the L-FABP antibody coupled with magnetic particles comprises the following steps:

[0014] S1, magnetic particles activated by buffer solution and evenly mixed with L-FABP antibody;

[0015] S2, adding bis(sulfosuccinimidyl) suberate and polyethylene glycol 20000 in sequence for reaction;

[0016] S3. Then add the blocking agent to suspend the reaction, and finally dilute with buffer for storage.

[0017] In the above-mentioned liver-type fatty acid binding protein (L-FABP) detection kit, the mass ratio of the magnetic beads to the L-FABP antibody in step S1 is 10:(0.01-0.3)

[0018] In the above-mentioned liver-type fatty acid binding protein (L-FABP) detection kit, the concentration of bis(sulfosuccinimidyl) suberate in step S2 is 5-15 mg / ml, and the added amount is 0.5 to 2 times the total volume of the magnetic particles and the antibody.

[0019] The invention adds coupling arms after introducing PEG 20000 coupling magnetic beads, and uses bis(sulfosuccinimidyl) suberate (BS3) as a cross-linking agent. BS3 has an amine-reactive N-hydroxysulfosuccinimide (NHS) ester at each end of the 8-carbon atom spacer arm. The NHS ester reacts with primary amines under pH 7-9 to form a stable amide bond and releases the N-hydroxysulfosuccinimide leaving group. The L-FABP antibody has a plurality of primary amines on the side chain of the lysine (K) residue and the N-terminus of each polypeptide. The primary amines can be used as targets of the NHS ester cross-linking agent, thereby enhancing water solubility and effectively avoiding steric effects when the antibody is coupled.

[0020] Preferably, the blocking agent in step S3 is a 0.05M HEPES buffer containing one or more of BSA, fish skin gelatin, and sodium caseinate.

[0021] Preferably, the concentration of L-FABP antibody coupled to the magnetic particles after dilution and storage with the buffer in step S3 is 0.2-2.0 mg / ml.

[0022] In the above-mentioned liver-type fatty acid binding protein (L-FABP) detection kit, the pH of the buffer in step S3 is 7-8, and it includes the following ingredients: 3-3.5g / L MES, 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.

[0023] In the above-mentioned liver-type fatty acid binding protein (L-FABP) detection kit, the preparation method of the L-FABP antibody labeled with alkaline phosphatase comprises the following steps:

[0024] S1. Add L-FABP antibody to an amino-free and thiol-free buffer, then add tris(2-carbonylethyl)phosphine hydrochloride to react, and then add glycine to react to obtain an activated antibody solution;

[0025] S2, adding alkaline phosphatase to an amino-free and thiol-free buffer, then adding a DMF solution containing SMCC, and then adding glycine to react to obtain an alkaline phosphatase solution;

[0026] S3. Evenly mix the activated antibody solution and alkaline phosphatase solution, then add magnesium chloride solution to react, and finally dilute with enzyme diluent for storage.

[0027] In the above-mentioned liver-type fatty acid binding protein (L-FABP) detection kit, the mass ratio of L-FABP antibody to alkaline phosphatase is (3-5):1.

[0028] Preferably, in step S1, the amount of tri(2-carbonylethyl)phosphine hydrochloride added is 0.5 to 2.5‰ microliters per milliliter of the L-FABP antibody volume.

[0029] Preferably, the glycine concentration in step S1 is 1 M, and the amount added is 0.5 to 2.5‰ microliters per milliliter of the L-FABP antibody volume.

[0030] Preferably, the concentration of the DMF solution containing SMCC is 5-8 mg / ml, and the added amount is 2.5‰-7.5‰ of the volume of alkaline phosphatase.

[0031] In the above-mentioned liver-type fatty acid binding protein (L-FABP) detection kit, the pH of the enzyme dilution solution in step S3 is 7-8, and includes 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-butylhydroquinone ethanol.

[0032] The above-mentioned liver-type fatty acid binding protein (L-FABP) detection kit also contains calibration products and quality control products.

[0033] The calibrators include protein solutions with L-FABP antigen concentrations of 0, 5, 20, 50, 200, and 500 ng / mL, respectively;

[0034] The quality control products include protein solutions with L-FABP antigen concentrations of 5 and 20 ng / mL, respectively.

[0035] Preferably, the protein solution comprises the following ingredients: 3-3.5 g / L MES, 45-55 g / L fish skin gelatin, 5-15 g / L bovine serum albumin, 25-35 g / L sucrose, 15-5 g / L mannitol, 5-15 g / L polyethylene glycol 8000, 3-8 g / L methyl cellulose, 0.5-1.5 g / L T-X405, 0.5-1.5 g / L surfactant S7, 3-5 g / L Proclin300, and 3-5 g / L gentamicin.

[0036] In the above-mentioned liver-type fatty acid binding protein (L-FABP) detection kit, bovine serum albumin is subjected to deenzyme treatment.

[0037] The present invention also provides a method for using the above-mentioned liver-type fatty acid binding protein (L-FABP) detection kit, which comprises the following steps: reacting the sample to be tested with R1 reagent, R2 reagent and R3 reagent for 5-15 minutes, then placing it in a magnetic field for separation, adding chemiluminescent substrate solution after washing, and detecting the chemiluminescent photon intensity.

[0038] 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).

[0039] Compared with the prior art, the kit of the present invention has the following advantages:

[0040] 1. The liver-type fatty acid binding protein (L-FABP) detection kit of the present invention introduces polyethylene glycol (PEG) to modify the magnetic bead surface, and functionalizes the solid surface with a polyethylene glycol spacer arm to significantly reduce the binding of nonspecific proteins. In addition, bis(sulfosuccinimidyl) suberate (BS3) is used as a cross-linking agent to effectively avoid the steric effect and improve the sensitivity of the reagent. In addition, because bis(sulfosuccinimidyl) suberate has a large molecular weight, it will increase the solubility and enhance the storage stability of the kit.

[0041] 2. The minimum detection limit of the liver-type fatty acid binding protein (L-FABP) detection kit of the present invention for detecting liver-type fatty acid binding protein is 0.2 ng / ml; the linear range is 0.2-500 ng / ml, and the linear correlation coefficient is R0.99. It can be seen that the kit of the present invention has high sensitivity, wide linear range, good repeatability, high precision, good stability, small batch difference and short detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The luminescence standard curve was tested for the calibrator of Example 1.

[0043] Figure 2 The test results of the kit prepared in Example 1 are correlated with those of a foreign RD (RD SYSTEMS) kit (CE certified). DETAILED DESCRIPTION

[0044] The following are specific embodiments of the present invention, and the technical solutions of the present invention are further described in conjunction with the accompanying drawings, but the present invention is not limited to these embodiments.

[0045] The raw materials used in the following examples are as follows:

[0046] Carboxyl magnetic particles were purchased from JSR Corporation;

[0047] L-FABP antibody is a self-developed antibody of Ningbo Haiyi Biotechnology Co., Ltd.

[0048] Embodiment 1:

[0049] Preparation of R1 reagent:

[0050] S1, add 10 mg of carboxyl magnetic particles into the EP tube;

[0051] S2, EP tubes were placed on a magnetic rack and washed 1 to 5 times with coupling buffer, and the volume was fixed to 1000 μL; the coupling buffer was 0.05 M MES buffer with a pH of 8.0;

[0052] S3, add 100 μL of L-FABP antibody with a concentration of 2.0 mg / mL, and then add 800 μL of 10 mg / ml bis(sulfosuccinimidyl) suberate (BS3) and 30 μL of 1% polyethylene glycol 20000 in sequence. The amount of bis(sulfosuccinimidyl) suberate (BS3) added is 0.8 times the total volume of the magnetic particles and the antibody, and then suspend at 28°C for 10 hours.

[0053] S4, add 0.2ml of blocking agent and suspend at 30°C for 20h, replace with magnetic bead preservation solution, the final concentration of magnetic particles is 10mg / ml, and finally dilute with buffer solution to a concentration of 1.0mg / ml for preservation; the blocking agent is 0.05M HEPES buffer with 10% BSA+0.01% fish skin gelatin; the magnetic bead preservation solution has a pH of 7.5 in 0.05M HEPES buffer; the buffer solution has a pH of 7.5 and includes the following ingredients: 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.

[0054] Preparation of R2 reagent:

[0055] S1. Take 1 mg of L-FABP antibody and replace it with amino-free and thiol-free buffer by dialysis, and concentrate it to 3 mg / ml using a concentrator tube;

[0056] S2, add TCEP (tris(2-carbonylethyl) phosphine hydrochloride), react at room temperature for 20 minutes, then add 1M glycine solution at pH 7.3 and react at room temperature for 8 minutes, use PD-10 desalting column to replace buffer, concentrate to 3 mg / ml to obtain activated antibody solution for standby use; TCEP (tris(2-carbonylethyl) phosphine hydrochloride) and glycine are added in an amount of 1‰ microliter per milliliter of antibody;

[0057] S3, take 0.25 mg of alkaline phosphatase and replace it with amino-free and thiol-free buffer by dialysis, and concentrate it to 3 mg / ml using a concentrator tube;

[0058] S4. Add 6 mg / mL DMF solution containing SMCC and react at room temperature for 15 min, then add 1 M glycine at pH 7.3 and react at room temperature for 15 min, use a PD-10 desalting column to replace the buffer, and concentrate to 3 mg / ml to obtain an alkaline phosphatase solution for use; the amount of DMF solution containing SMCC and glycine added is 5‰ of the volume of alkaline phosphatase.

[0059] S5, the activated antibody solution and alkaline phosphatase solution were mixed, and 0.3M magnesium chloride solution was added, the volume of which was 3‰ml of the total reaction volume, and the mixture was reacted at 5°C for 12h, and finally purified and stored at 2-8°C;

[0060] S6. Dilute before use to a concentration of 0.8 mg / mL.

[0061] Preparation of R3 reagent:

[0062] Put 0.325g MES, 5g bovine serum albumin, 1g trehalose, 3g sucrose, 2g mannitol, 1g polyethylene glycol 8000, 0.1g T-X405, 0.1g surfactant S7, 0.4g Proclin300, and 0.4g gentamicin into a glass container, add 100ml purified water and stir evenly to obtain R3 reagent.

[0063] Preparation of calibrators and quality control products:

[0064] Put 0.325g MES, 5g fish skin gelatin, 1g bovine serum albumin, 3g sucrose, 2g mannitol, 1g polyethylene glycol 8000, 0.5g methylcellulose, 0.1g T-X405, 0.1g surfactant S7, 0.4g Proclin300, and 0.4g gentamicin into a glass container, add 100ml purified water and stir well to obtain a buffer solution.

[0065] L-FABP antigen was prepared into calibrators with concentrations of 0, 5, 20, 50, 200, and 500 ng / mL using a buffer solution.

[0066] L-FABP antigen was prepared into quality control products with concentrations of 5 and 20 ng / mL using buffer.

[0067] Embodiment 2:

[0068] The only difference from Example 1 is that the coupling of PEG+BS3 in the R1 reagent is replaced by the coupling of PEG+SMCC.

[0069] Embodiment 3:

[0070] The only difference from Example 1 is that the coupling of PEG+BS3 in the R1 reagent is replaced by PEG+EDC.

[0071] Embodiment 4:

[0072] The only difference from Example 1 is that the coupling of PEG+BS3 in the R1 reagent is replaced by the coupling of PEG+SMDCP.

[0073] Application Examples 1-4:

[0074] Take 10ul of the sample to be tested and react with 20ul R1 reagent, 50ul R2 reagent and 50ul R3 reagent in the kit prepared in Example 1-4 for 10 minutes, then perform magnetic separation and cleaning, and finally add alkaline phosphatase substrate luminescent liquid to measure the luminescence value in the reaction tube.

[0075] Table 1: Luminescence value results of the kit calibrators tested in Examples 1-4

[0076]

[0077] Figure 1 The luminescence standard curve of the calibrator test in Example 1 is shown in Table 1. The liver-type fatty acid binding protein kit prepared in Example 1 has a better reaction gradient, a lower signal-to-noise ratio, and a curve fitting degree R 2 =0.99983, the improvement effect is obvious.

[0078] Minimum detection limit and detection limit performance test:

[0079] The luminescence value (RLU) of the zero-value calibrator was measured in parallel 20 times, and its mean value (M) and standard deviation (SD) were calculated to obtain the RLU value corresponding to M+2SD. A linear equation was obtained by performing a two-point regression fitting based on the concentration-luminescence value (RLU) results between the zero-concentration calibrator and the adjacent calibrator. The M+2SD luminescence value was substituted into the above equation to obtain the corresponding concentration value, and the result was not higher than the minimum detection limit (0.2 ng / mL). The results are shown in Table 2.

[0080] Table 2: Minimum detection limit and detection limit performance test of the kit of Examples 1-4

[0081] 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 detection limit 0.007 0.530 0.053 0.358

[0082] The results in Table 2 show that the 20 zero-value calibrator tests using PEG+EDC and PEG+BS3 for coupling met the 0.2 ng / ml requirement, but the minimum detection limit of PEG+BS3 was 0.007, which had a clear advantage.

[0083] Linearity Verification:

[0084] Take 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 mix them into at least 5 dilution concentrations according to a certain ratio. Test each dilution concentration 3 times and calculate the mean of the test results. Take the dilution concentration as the independent variable and the mean of the test results as the dependent variable, find the linear regression equation, and calculate the correlation coefficient r. The results are as follows:

[0085]

[0086] As shown in Table 3.

[0087] Table 3: Linearity verification results of kits of Examples 1-4

[0088] The results in Table 3 show that when PEG+BS3 is coupled, the correlation coefficient r>0.99 in the concentration range of 0.2-500 ng / ml. It can be seen that the present invention couples with PEG+BS3, and the detection range of L-FABP kit is 0.2-500 ng / ml. The above data show that PEG+BS3 has obvious advantages.

[0089] Coupling repeatability verification:

[0090] The repeatability was tested using samples of different concentrations. Each sample was tested 10 times in succession for the CV of the analytical value. The results are shown in Table 4.

[0091] Table 4: Repeatability verification results of the kit of Example 1

[0092]

[0093]

[0094] Day precision:

[0095] Different concentrations of samples were used to test the inter-day precision. The samples were packaged and frozen. One sample was taken for testing once in the morning, noon, and evening every day. The test was continued for 7 days. A total of 21 data points were obtained for each sample. The CV of the test results was analyzed. The results are shown in Table 5.

[0096] Table 5: Test results of the day-to-day precision of the test kit in Example 1

[0097]

[0098] Thermal stability:

[0099] The kit was placed in an accelerated destruction environment at 37°C and taken out on the 3rd, 7th and 14th days respectively. The luminescence value of the calibration sample was measured and the deviation was calculated. The results are shown in Table 6.

[0100] Table 6: Thermal stability test results of the test kit of Example 1

[0101]

[0102]

[0103] Long-term stability:

[0104] The test kit was stored at 2-8°C, and the luminescence values ​​of the test calibrators were taken out at the 3rd month, 7th month, 12th month, 15th month, and 24th month to calculate the deviation. The results are shown in Table 7.

[0105] Table 7: Long-term stability test results of the kit of Example 1

[0106] ng / ml Initial value 3 months Decline 7 months Decline 12 months Decline 15 months Decline 24 months Decline 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％

[0107] Batch-to-batch test:

[0108] Four batches of carboxyl magnetic beads-liver fatty acid binding protein monoclonal antibody and alkaline phosphatase-liver fatty acid binding protein monoclonal antibody were coupled continuously to produce four batches of test kits. The luminescence values ​​of five samples were tested to evaluate the batch CV of the reagents. The results are shown in Table 8.

[0109] Table 8: Test results of batch difference of the kit in Example 1

[0110]

[0111] Figure 2 The correlation between the test results of the kit prepared in Example 1 and the foreign RD (RD SYSTEMS) kit (CE certified) is shown in the figure. As can be seen from the figure, the test results of the kit prepared in Example 1 and the RD comparison kit are well correlated.

[0112] In summary, the liver-type fatty acid binding protein test kit of the present invention has a sensitivity of 0.2 ng / ml, a linear range of 0.2-500 ng / ml, and an intra-batch repeatability that can be controlled within 2%; the inter-day precision can be controlled within 3%; the decrease can be controlled within 10% after 14 days of thermal accelerated destruction at 37°C and 15 months of storage at 2-8°C; the inter-batch difference can be controlled within 3%, and the test results are more accurate.

[0113] The parts of the technical scope of the present invention that are not exhausted by the midpoint value of the embodiment and the new technical solutions formed by the equivalent replacement of single or multiple technical features in the technical solutions of the embodiments are also within the scope of protection claimed by the present invention, and unless otherwise specified, there is no unique and irreplaceable combination between all the parameters involved in the solutions of the present invention.

[0114] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0115] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention.

Claims

1. A liver-type fatty acid binding protein L-FABP detection kit, characterized in that: The kit includes R1 reagent, R2 reagent and R3 reagent; The R1 reagent includes L-FABP antibody coupled to magnetic particles; The R2 reagent includes an alkaline phosphatase-labeled L-FABP antibody; The R3 reagent includes the following ingredients: 3-4 g / L MES, 45-55 g / L bovine serum albumin, 5-15 g / L trehalose, 25-35 g / L sucrose, 15-25 g / L mannitol, 5-15 g / L polyethylene glycol 8000, 0.5-1.5 g / L T-X405, 0.5-1.5 g / L surfactant S7, 3-5 g / L Proclin300 and 3-5 g / L gentamicin; The method for preparing magnetic particle-coupled L-FABP antibody comprises the following steps: S1, magnetic particles activated by buffer solution and evenly mixed with L-FABP antibody; S2, adding bis(sulfosuccinimidyl) suberate and polyethylene glycol 20000 in sequence for reaction; S3, then add the blocking agent to suspend the reaction, and finally dilute with buffer for storage; Step S1: The mass ratio of magnetic beads to L-FABP antibody is 10:(0.01-0.3); In step S2, the concentration of bis(sulfosuccinimidyl) suberate is 5-15 mg / ml, and the amount added is 0.5 to 2 times the total volume of the magnetic particles and the antibody.

2. A liver-type fatty acid binding protein L-FABP detection kit according to claim 1, characterized in that: The pH of the buffer in step S3 is 7-8, and it includes the following components: 3-3.5 g / L MES, 8-12 g / L fish skin gelatin, 45-55 g / L bovine serum albumin, 5-10 g / L zinc chloride, 0.5-1.5 g / L T-X405, 1.5-2.5 g / L Proclin300 and 18-22 g / L gentamicin.

3. A liver-type fatty acid binding protein L-FABP detection kit according to claim 1, characterized in that: The method for preparing alkaline phosphatase-labeled L-FABP antibody comprises the following steps: S1. Add L-FABP antibody to an amino-free and thiol-free buffer, then add tris(2-carbonylethyl)phosphine hydrochloride to react, and then add glycine to react to obtain an activated antibody solution; S2, adding alkaline phosphatase to an amino-free and thiol-free buffer, then adding a DMF solution containing SMCC, and then adding glycine to react to obtain an alkaline phosphatase solution; S3. Evenly mix the activated antibody solution and alkaline phosphatase solution, then add magnesium chloride solution to react, and finally dilute with enzyme diluent for storage.

4. A liver-type fatty acid binding protein L-FABP detection kit according to claim 3, characterized in that: The mass ratio of L-FABP antibody to alkaline phosphatase is (3-5):

1.

5. A liver-type fatty acid binding protein L-FABP detection kit according to claim 3, characterized in that: The pH of the enzyme dilution solution in step S3 is 7-8, and it includes the following components: 5-6 g / L MES, 45-55 g / L bovine serum albumin, 1.5-2.5 g / L sodium chloride, 8-12 mol / L magnesium chloride, 0.8-1.2 mol / L zinc chloride, 1.5-2.5 g / L Proclin 300, 18-22 g / L gentamicin and 0.3-0.8 g / L tert-butylhydroquinone ethanol.

6. A liver-type fatty acid binding protein L-FABP detection kit according to claim 1, characterized in that: Also contains calibrators and quality controls. The calibrators include protein solutions with L-FABP antigen concentrations of 0, 5, 20, 50, 200 and 500 ng / mL; The quality control products include protein solutions with L-FABP antigen concentrations of 5 and 20 ng / mL, respectively.

7. A liver-type fatty acid binding protein L-FABP detection kit according to claim 6, characterized in that: The protein solution includes the following ingredients: 3-3.5 g / L MES, 45-55 g / L fish skin gelatin, 5-15 g / L bovine serum albumin, 25-35 g / L sucrose, 15-5 g / L mannitol, 5-15 g / L polyethylene glycol 8000, 3-8 g / L methyl cellulose, 0.5-1.5 g / L T-X405, 0.5-1.5 g / L surfactant S7, 3-5 g / L Proclin300 and 3-5 g / L gentamicin.

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