Buffer solution, beta collagen degradation product chemiluminescence detection kit and preparation method of beta collagen degradation product chemiluminescence detection kit

Abstract

The invention relates to a buffer solution. The buffer solution contains one of a non-mammalian protein, an enzymolysis product of the non-mammalian protein or a protein-free sealing agent. The invention also relates to a chemiluminiscence detection kit for the beta collagen degradation product and a preparation method of the chemiluminiscence detection kit. Through a large number of research experiments, the inventor finds that when the mammalian proteins such as BSA are applied to the beta collagen degradation product chemiluminiscence detection kit, the sensitivity and gradient are poor. One of non-mammalian proteins, enzymatic hydrolysate of the non-mammalian proteins or a protein-free sealing agent is adopted to replace BSA in the kit, non-specific reactions introduced in test reactions can be greatly reduced, the background of the item is effectively reduced, the gradient and sensitivity of the test are improved, and the stability of the test is enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnostic detection, and in particular to a chemiluminescent detection kit for beta collagen degradation products. Background Art

[0002] Type I collagen is an important component of bone matrix, and its degradation products are the most commonly used bone resorption markers. During normal bone metabolism, mature type I collagen is degraded, and the small fragments formed enter the bone bloodstream and are excreted through the kidneys. When physiological or pathological bone resorption is enhanced (such as in the elderly or osteoporosis), the degradation of type I collagen also increases, and the content of collagen fragments in the blood also increases accordingly. Particularly relevant is the β-isomerized C (carboxyl)-terminal cross-linked telopeptide (β-CTx) produced by osteoclast hydrolysis of type I collagen, i.e., the degradation product of β collagen. The β isomer form is related to protein aging, and its content increases with the aging of type I collagen molecules in bone; in patients with increased bone resorption, the serum level of type I collagen iso C-telopeptide increases, and then returns to normal levels during anti-resorptive treatment. At present, the commonly used methods for detecting β collagen degradation products in clinics and laboratories include electrochemiluminescence, fluorescence chromatography, enzyme-linked immunosorbent assay, and non-magnetic particle chemiluminescence immunoassay.

[0003] The commonly used chemiluminescent immunoassay for testing β-collagen degradation products has problems with gradient and poor sensitivity. Summary of the invention

[0004] The present invention overcomes the shortcomings of the prior art and provides a buffer solution capable of improving the gradient and sensitivity of the test, a chemiluminescent detection kit for beta collagen degradation products and a preparation method thereof.

[0005] The specific technical solutions of the present invention are as follows:

[0006] A buffer comprises one of a non-mammalian protein, an enzymatic hydrolysate of a non-mammalian protein or a protein-free blocking agent.

[0007] Furthermore, the buffer is selected from one of MES buffer, PB buffer, TRIS buffer and HEPES buffer.

[0008] Furthermore, the non-mammalian protein is selected from enzymatically hydrolyzed deep-water fish skin gelatin or hydrolyzed chicken albumin.

[0009] The present invention also relates to a chemiluminescent detection kit for beta collagen degradation products, comprising reagent L1, reagent L2 and reagent L3, wherein reagent L1 is a streptavidin-coated carboxyl magnetic nanoparticle solution containing a buffer, reagent L2 is an acridinium ester-labeled beta collagen degradation product monoclonal antibody 1 solution containing a buffer, and reagent L3 is a biotin-labeled beta collagen degradation product monoclonal antibody 2 solution containing a buffer.

[0010] Furthermore, the concentration of streptavidin-coated carboxyl magnetic nanoparticles in reagent L1 is 0.8-2.5 mg / ml, the concentration of acridinium ester-labeled β-collagen degradation product monoclonal antibody 1 in reagent L2 is 0.5-1 μg / ml, and the concentration of biotin-labeled β-collagen degradation product monoclonal antibody 2 in reagent L3 is 5-10 μg / ml.

[0011] The present invention also relates to a method for preparing a chemiluminescent detection kit for β-collagen degradation products, comprising the following steps:

[0012] Preparation of reagent L1: preparing carboxyl magnetic nanoparticles into a carboxyl magnetic nanoparticle suspension;

[0013] activating the carboxyl magnetic nanoparticles in the carboxyl magnetic nanoparticle suspension to obtain activated carboxyl magnetic nanoparticles;

[0014] adding streptavidin to the carboxyl magnetic nanoparticle suspension to react with the activated carboxyl magnetic nanoparticles, removing the supernatant to obtain streptavidin-coated carboxyl magnetic nanoparticles, and adjusting the working concentration of the streptavidin-coated carboxyl magnetic nanoparticles with a buffer;

[0015] Preparation of reagent L2: using acridinium ester to label the β-collagen degradation product specific antibody 1 to obtain acridinium ester labeled β-collagen degradation product monoclonal antibody 1; adjusting the working concentration of acridinium ester labeled β-collagen degradation product monoclonal antibody 1 with buffer.

[0016] Preparation of reagent L3: using biotin to label the β-collagen degradation product specific antibody 2 to obtain the biotin-labeled β-collagen degradation product monoclonal antibody 2; adjusting the working concentration of the biotin-labeled β-collagen degradation product monoclonal antibody 2 with a buffer solution.

[0017] Furthermore, when preparing reagent L1, EDC and NHS are used to activate the carboxyl magnetic nanoparticles in the carboxyl magnetic nanoparticle suspension, and the mass ratio of the carboxyl magnetic nanoparticles to EDC and NHS is 1:0.5-1:0.5-1.

[0018] Furthermore, when preparing reagent L1, streptavidin is added to the carboxyl magnetic nanoparticle suspension to react with the activated carboxyl magnetic nanoparticles, and the mass ratio of the added streptavidin to the activated carboxyl magnetic nanoparticles is 1:(10-100); the working concentration of the streptavidin-coated carboxyl magnetic nanoparticles is adjusted to 0.8-2.5 mg / mL with a buffer solution.

[0019] Furthermore, when preparing reagent L2, the molar ratio of β-collagen degradation product specific antibody 1 to acridinium ester is 1:(1-20); the working concentration of acridinium ester labeled β-collagen degradation product monoclonal antibody 1 is adjusted to 0.5-1 μg / mL with buffer.

[0020] Furthermore, when preparing reagent L3, the molar ratio of β-collagen degradation product specific antibody 2 to biotin is 1:(1-20); the working concentration of biotin-labeled β-collagen degradation product monoclonal antibody 2 is adjusted to 5-10 μg / mL with a buffer solution.

[0021] The beneficial effects of the present invention are as follows:

[0022] The inventors have found through a large number of research experiments that mammalian proteins such as BSA have poor sensitivity and gradient when used in a chemiluminescent detection kit for β-collagen degradation products. Using non-mammalian proteins, enzymatic hydrolysates of non-mammalian proteins, or a protein-free blocking agent to replace BSA in the kit can greatly reduce the nonspecific reaction introduced in the test reaction, effectively reduce the background of the project, improve the gradient and sensitivity of the test, and enhance its stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a graph showing the accelerated stability changes of the embodiments and comparative examples of the present invention. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The parts not mentioned in the present invention are all prior art.

[0025] The present invention relates to a buffer, wherein the buffer contains one of a non-mammalian protein, an enzymatic hydrolysate of the non-mammalian protein or a protein-free blocking agent. The non-mammalian protein of the present invention can be any non-mammalian protein and can also be a synthetic protein.

[0026] Furthermore, the buffer can be any conventional buffer, preferably, the buffer is selected from MES buffer, PB buffer, TRIS buffer, HEPES buffer. The buffer contains 1-5% non-mammalian protein or its enzymatic hydrolysate, or no protein blocking agent.

[0027] Furthermore, the non-mammalian protein may be any conventional non-mammalian protein. Preferably, the non-mammalian protein is selected from enzymatically hydrolyzed deep-water fish skin gelatin or hydrolyzed chicken albumin.

[0028] The present invention also relates to a chemiluminescent detection kit for beta collagen degradation products, comprising reagent L1, reagent L2 and reagent L3, wherein reagent L1 is a streptavidin-coated carboxyl magnetic nanoparticle solution containing a buffer, reagent L2 is an acridinium ester-labeled beta collagen degradation product monoclonal antibody 1 solution containing a buffer, and reagent L3 is a biotin-labeled beta collagen degradation product monoclonal antibody 2 solution containing a buffer.

[0029] Furthermore, the concentration of streptavidin-coated carboxyl magnetic nanoparticles in reagent L1 is 0.8-2.5 mg / ml, the concentration of acridinium ester-labeled β-collagen degradation product monoclonal antibody 1 in reagent L2 is 0.5-1 μg / ml, and the concentration of biotin-labeled β-collagen degradation product monoclonal antibody 2 in reagent L3 is 5-10 μg / ml.

[0030] The present invention also relates to a method for preparing the chemiluminescent detection kit for β-collagen degradation products, comprising the following steps:

[0031] Preparation of reagent L1: preparing carboxyl magnetic nanoparticles into a carboxyl magnetic nanoparticle suspension;

[0032] activating the carboxyl magnetic nanoparticles in the carboxyl magnetic nanoparticle suspension to obtain activated carboxyl magnetic nanoparticles;

[0033] adding streptavidin to the carboxyl magnetic nanoparticle suspension to react with the activated carboxyl magnetic nanoparticles, removing the supernatant to obtain streptavidin-coated carboxyl magnetic nanoparticles, and adjusting the working concentration of the streptavidin-coated carboxyl magnetic nanoparticles with a buffer;

[0034] Preparation of reagent L2: using acridinium ester to label the β-collagen degradation product specific antibody 1 to obtain acridinium ester labeled β-collagen degradation product monoclonal antibody 1; adjusting the working concentration of acridinium ester labeled β-collagen degradation product monoclonal antibody 1 with buffer.

[0035] Preparation of reagent L3: using biotin to label the β-collagen degradation product specific antibody 2 to obtain the biotin-labeled β-collagen degradation product monoclonal antibody 2; adjusting the working concentration of the biotin-labeled β-collagen degradation product monoclonal antibody 2 with a buffer solution.

[0036] Furthermore, when preparing reagent L1, EDC and NHS are used to activate the carboxyl magnetic nanoparticles in the carboxyl magnetic nanoparticle suspension, and the mass ratio of the carboxyl magnetic nanoparticles to EDC and NHS is 1:(0.5-1):(0.5-1).

[0037] Furthermore, when preparing reagent L1, streptavidin is added to the carboxyl magnetic nanoparticle suspension to react with the activated carboxyl magnetic nanoparticles, and the mass ratio of the added streptavidin to the activated carboxyl magnetic nanoparticles is 1:(10-100); the working concentration of the streptavidin-coated carboxyl magnetic nanoparticles is adjusted to 0.8-2.5 mg / mL with a buffer solution.

[0038] Furthermore, when preparing reagent L2, the molar ratio of β-collagen degradation product specific antibody 1 to acridinium ester is 1:(1-20); the working concentration of acridinium ester labeled β-collagen degradation product monoclonal antibody 1 is adjusted to 0.5-1 μg / mL with buffer.

[0039] Furthermore, when preparing reagent L3, the molar ratio of β-collagen degradation product specific antibody 2 to biotin is 1:(1-20); the working concentration of biotin-labeled β-collagen degradation product monoclonal antibody 2 is adjusted to 5-10 μg / mL with a buffer solution.

[0040] Experimental reagents

[0041] β-Collagen degradation product monoclonal antibody 1, β-Collagen degradation product monoclonal antibody 2, and carboxyl magnetic nanoparticles were all provided by GeneDx Biotechnology Co., Ltd., acridinium ester and biotin were purchased from Aladdin, enzymatic deep-water fish skin gelatin and chicken hydrolyzed albumin were purchased from Shanghai Xibao Biotechnology, and protein-free blocking agent FP MAP was purchased from Feipeng Biotechnology with the catalog number CC00101B20. Other reagents and consumables were purchased from Sigma-Aldrich unless otherwise specified.

[0042] The sealing agent used in the embodiment can be any conventional sealing agent in the art.

[0043] Example 1

[0044] A chemiluminescent detection kit for β-collagen degradation products, comprising reagent L1, reagent L2, and reagent L3. The preparation method of the kit comprises the following steps:

[0045] Preparation of reagent L1

[0046] (1) Take 1 mL of carboxyl magnetic nanoparticles with a particle size of 1 μm and place them in a centrifuge tube and place them on a micromagnetic separation rack for magnetic separation. After about 1 minute, aspirate the clarified liquid, add 1 mL of 50 mM pH = 5.5 MES buffer, and disperse them again by blowing to prepare a carboxyl magnetic nanoparticle suspension.

[0047] (2) EDC and NHS were added to the carboxyl magnetic nanoparticle suspension at a mass ratio of carboxyl magnetic nanoparticles: EDC: NHS = 1:0.5:0.5, and the reaction was carried out for 30 minutes. After the reaction was completed, the suspension was washed three times to obtain activated carboxyl magnetic nanoparticles, which were dispersed in 50 mM pH = 5.5 MES buffer to obtain dispersed magnetic microspheres.

[0048] (3) Slowly drop streptavidin into the dispersed magnetic microspheres at a mass ratio of streptavidin: activated carboxyl magnetic nanoparticles = 1:10; fully stir and react at room temperature for 3 hours, add a blocking agent to the system to block the excess sites on the magnetic microspheres, fully stir and block for 1.5 hours, and then wash three times with PBS-T; dilute with MES buffer containing 1% by volume of enzymatically hydrolyzed deep-water fish skin gelatin to a working concentration of 2.5 mg / mL.

[0049] Preparation of reagent L2

[0050] Acridinium ester was dissolved in DMSO to a concentration of 1 mg / ml, and the β-collagen degradation product-specific antibody 1 was diluted with 50 mM phosphate buffer with a pH of 7.6 to a concentration of 1 mg / mL according to a molar ratio of β-collagen degradation product-specific antibody 1: acridinium ester=1:10, and then the acridinium ester solution was added to react at 37 degrees Celsius for 1 hour, and unbound acridinium ester was removed by dialysis to obtain acridinium ester-labeled β-collagen degradation product monoclonal antibody 1. Acridinium ester-labeled β-collagen degradation product monoclonal antibody 1 was diluted with MES buffer containing enzymatic deep-water fish skin gelatin with a volume concentration of 1% to a working concentration of 0.5 μg / mL.

[0051] Preparation of reagent L3

[0052] Biotin was dissolved in DMF to a concentration of 10 mg / ml, and the β-collagen degradation product-specific antibody 2 was diluted to a concentration of 1 mg / mL with 50 mM carbonate buffer at pH 9.0 according to a molar ratio of β-collagen degradation product-specific antibody 2:biotin=1:10, and then the biotin solution was added, and the reaction was carried out at 37 degrees Celsius for 0.5 hours, and unbound biotin was removed by dialysis to obtain biotin-labeled β-collagen degradation product monoclonal antibody 2. The biotin-labeled β-collagen degradation product monoclonal antibody 2 was diluted to a working concentration of 5 μg / mL with MES buffer containing enzymatically hydrolyzed deep-water fish skin gelatin at a volume concentration of 1%.

[0053] Example 2

[0054] A chemiluminescent detection kit for β-collagen degradation products, comprising reagent L1, reagent L2, and reagent L3. The preparation method of the kit comprises the following steps:

[0055] Preparation of reagent L1

[0056] (1) Take 1 mL of carboxyl magnetic nanoparticles with a particle size of 1 μm and place them in a centrifuge tube and place them on a micromagnetic separation rack for magnetic separation. After about 1 minute, aspirate the clarified liquid, add 1 mL of 50 mM pH = 5.5 MES buffer, and disperse them again by blowing to prepare a carboxyl magnetic nanoparticle suspension.

[0057] (2) EDC and NHS were added to the carboxyl magnetic nanoparticle suspension at a mass ratio of carboxyl magnetic nanoparticles: EDC: NHS = 1:0.8:1, and the reaction was carried out for 30 minutes. After the reaction was completed, the suspension was washed three times to obtain activated carboxyl magnetic nanoparticles, which were dispersed in 50 mM pH = 5.5 MES buffer to obtain dispersed magnetic microspheres.

[0058] (3) Slowly add streptavidin to the dispersed magnetic microspheres at a mass ratio of streptavidin: activated carboxyl magnetic nanoparticles = 1:30; fully stir and react at room temperature for 3 hours, add a blocking agent to the system to block the excess sites on the magnetic microspheres, fully stir and block for 1.5 hours, and then wash three times with PBS-T; dilute with MES buffer containing 3% volume concentration of protein-free blocking agent FP MAP to a working concentration of 2 mg / mL.

[0059] Preparation of reagent L2

[0060] Acridinium ester was dissolved in DMSO to a concentration of 1 mg / ml, and the β-collagen degradation product-specific antibody 1 was diluted with 50 mM phosphate buffer with a pH of 7.6 to a concentration of 1 mg / mL according to a molar ratio of β-collagen degradation product-specific antibody 1: acridinium ester=1:15, and then the acridinium ester solution was added to react at 37 degrees Celsius for 1 hour, and unbound acridinium ester was removed by dialysis to obtain acridinium ester-labeled β-collagen degradation product monoclonal antibody 1. Acridinium ester-labeled β-collagen degradation product monoclonal antibody 1 was diluted with TRIS buffer containing enzymatic hydrolyzed deep-water fish skin gelatin at a volume concentration of 5% to a working concentration of 1 μg / mL.

[0061] Preparation of reagent L3

[0062] Biotin was dissolved in DMF to a concentration of 10 mg / ml, and the β-collagen degradation product-specific antibody 2 was diluted to a concentration of 1 mg / mL with a 50 mM carbonate buffer solution of pH=9.0 according to a molar ratio of β-collagen degradation product-specific antibody 2:biotin=1:20, and then the biotin solution was added, and the reaction was carried out at 37 degrees Celsius for 0.5 hours, and unbound biotin was removed by dialysis to obtain biotin-labeled β-collagen degradation product monoclonal antibody 2. The biotin-labeled β-collagen degradation product monoclonal antibody 2 was diluted to a working concentration of 8 μg / mL with a MES buffer solution containing enzymatically hydrolyzed deep-water fish skin gelatin at a volume concentration of 2%.

[0063] Example 3

[0064] A chemiluminescent detection kit for β-collagen degradation products, comprising reagent L1, reagent L2, and reagent L3. The preparation method of the kit comprises the following steps:

[0065] Preparation of reagent L1

[0066] (1) Take 1 mL of carboxyl magnetic nanoparticles with a particle size of 1 μm and place them in a centrifuge tube and place them on a micromagnetic separation rack for magnetic separation. After about 1 minute, aspirate the clarified liquid, add 1 mL of 50 mM pH = 5.5 MES buffer, and disperse them again by blowing to prepare a carboxyl magnetic nanoparticle suspension.

[0067] (2) EDC and NHS were added to the carboxyl magnetic nanoparticle suspension at a mass ratio of carboxyl magnetic nanoparticles: EDC: NHS = 1:1:0.7, and the reaction was carried out for 30 minutes. After the reaction was completed, the suspension was washed three times to obtain activated carboxyl magnetic nanoparticles, which were dispersed in 50 mM PH = 5.5 MES buffer to obtain dispersed magnetic microspheres.

[0068] (3) Slowly drop streptavidin into the dispersed magnetic microspheres at a mass ratio of streptavidin: activated carboxyl magnetic nanoparticles = 1:50; fully stir and react at room temperature for 3 hours, add a blocking agent to the system to block the excess sites on the magnetic microspheres, fully stir and block for 1.5 hours, and then wash three times with PBS-T; dilute with PB buffer containing 5% by volume of enzymatically hydrolyzed deep-water fish skin gelatin to a working concentration of 1 mg / mL.

[0069] Preparation of reagent L2

[0070] Acridinium ester was dissolved in DMSO to a concentration of 1 mg / ml, and the β-collagen degradation product-specific antibody 1 was diluted with 50 mM phosphate buffer with a pH of 7.6 to a concentration of 1 mg / mL according to a molar ratio of β-collagen degradation product-specific antibody 1: acridinium ester=1:20, and then the acridinium ester solution was added to react at 37 degrees Celsius for 1 hour, and unbound acridinium ester was removed by dialysis to obtain acridinium ester-labeled β-collagen degradation product monoclonal antibody 1. Acridinium ester-labeled β-collagen degradation product monoclonal antibody 1 was diluted with MES buffer containing enzymatic deep-water fish skin gelatin with a volume concentration of 2% to a working concentration of 0.8 μg / mL.

[0071] Preparation of reagent L3

[0072] Biotin was dissolved in DMF to a concentration of 10 mg / ml, and the beta collagen degradation product specific antibody 2 was diluted to a concentration of 1 mg / mL with 50 mM carbonate buffer at pH 9.0 according to a molar ratio of beta collagen degradation product specific antibody 2: biotin = 1:18, and then the biotin solution was added, and the reaction was carried out at 37 degrees Celsius for 0.5 hours, and unbound biotin was removed by dialysis to obtain biotin-labeled beta collagen degradation product monoclonal antibody 2. The biotin-labeled beta collagen degradation product monoclonal antibody 2 was diluted to a working concentration of 10 μg / mL with a HEPES buffer containing enzymatically hydrolyzed deep-water fish skin gelatin at a volume concentration of 5%.

[0073] Example 4

[0074] A chemiluminescent detection kit for β-collagen degradation products, comprising reagent L1, reagent L2, and reagent L3. The preparation method of the kit comprises the following steps:

[0075] Preparation of reagent L1

[0076] (1) Take 1 mL of carboxyl magnetic nanoparticles with a particle size of 1 μm and place them in a centrifuge tube and place them on a micromagnetic separation rack for magnetic separation. After about 1 minute, aspirate the clarified liquid, add 1 mL of 50 mM pH = 5.5 MES buffer, and disperse them again by blowing to prepare a carboxyl magnetic nanoparticle suspension.

[0077] (2) EDC and NHS were added to the carboxyl magnetic nanoparticle suspension in a mass ratio of carboxyl magnetic nanoparticles: EDC: NHS = 1:0.8:0.6, and the reaction was carried out for 30 minutes. After the reaction, the suspension was washed three times to obtain activated carboxyl magnetic nanoparticles, which were dispersed in 50 mM PH = 5.5 MES buffer to obtain dispersed magnetic microspheres.

[0078] (3) Slowly add streptavidin to the dispersed magnetic microspheres at a mass ratio of streptavidin: activated carboxyl magnetic nanoparticles = 1:100; fully stir and react at room temperature for 3 hours, add a blocking agent to the system to block the excess sites on the magnetic microspheres, stir and block for 1.5 hours, then wash three times with PBS-T; dilute to a working concentration of 0.8 mg / mL with MES buffer containing 4% chicken albumin hydrolyzate by volume.

[0079] Preparation of reagent L2

[0080] Acridinium ester was dissolved in DMSO to a concentration of 1 mg / ml, and the β-collagen degradation product-specific antibody 1 was diluted with 50 mM phosphate buffer at pH 7.6 to a concentration of 1 mg / mL according to a molar ratio of β-collagen degradation product-specific antibody 1: acridinium ester=1:18, and then the acridinium ester solution was added to react at 37 degrees Celsius for 1 hour, and unbound acridinium ester was removed by dialysis to obtain acridinium ester-labeled β-collagen degradation product monoclonal antibody 1. Acridinium ester-labeled β-collagen degradation product monoclonal antibody 1 was diluted with TRIS buffer containing enzymatic deep-water fish skin gelatin at a volume concentration of 4% to a working concentration of 0.7 μg / mL.

[0081] Preparation of reagent L3

[0082] Biotin was dissolved in DMF to a concentration of 10 mg / ml, and the β-collagen degradation product-specific antibody 2 was diluted to a concentration of 1 mg / mL with 50 mM carbonate buffer at pH 9.0 according to a molar ratio of β-collagen degradation product-specific antibody 2:biotin=1:1, and then the biotin solution was added, reacted at 37 degrees Celsius for 0.5 hours, and unbound biotin was removed by dialysis to obtain biotin-labeled β-collagen degradation product monoclonal antibody 2. The biotin-labeled β-collagen degradation product monoclonal antibody 2 was diluted to a working concentration of 7 μg / mL with HEPES buffer containing 3% chicken albumin hydrolyzate by volume.

[0083] Example 5

[0084] A chemiluminescent detection kit for β-collagen degradation products, comprising reagent L1, reagent L2, and reagent L3. The preparation method of the kit comprises the following steps:

[0085] Preparation of reagent L1

[0086] (1) Take 1 mL of carboxyl magnetic nanoparticles with a particle size of 1 μm and place them in a centrifuge tube and place them on a micromagnetic separation rack for magnetic separation. After about 1 minute, aspirate the clarified liquid, add 1 mL of 50 mM pH = 5.5 MES buffer, and disperse them again by blowing to prepare a carboxyl magnetic nanoparticle suspension.

[0087] (2) EDC and NHS were added to the carboxyl magnetic nanoparticle suspension in a mass ratio of carboxyl magnetic nanoparticles: EDC: NHS = 1:0.6:0.9, and the reaction was carried out for 30 minutes. After the reaction, the suspension was washed three times to obtain activated carboxyl magnetic nanoparticles, which were dispersed in 50 mM PH = 5.5 MES buffer to obtain dispersed magnetic microspheres.

[0088] (3) Slowly drop streptavidin into the dispersed magnetic microspheres at a mass ratio of streptavidin: activated carboxyl magnetic nanoparticles = 1:80; fully stir and react at room temperature for 3 hours, add a blocking agent to the system to block the excess sites on the magnetic microspheres, fully stir and block for 1.5 hours, and then wash three times with PBS-T; use MES buffer containing 5% by volume of enzymatically hydrolyzed deep-water fish skin gelatin to a working concentration of 2.5 mg / mL.

[0089] Preparation of reagent L2

[0090] Acridinium ester was dissolved in DMSO to a concentration of 1 mg / ml. According to the molar ratio of β-collagen degradation product specific antibody 1: acridinium ester = 1:1, β-collagen degradation product specific antibody 1 was diluted with 50mM phosphate buffer with pH = 7.6 to a concentration of 1 mg / mL, and then the acridinium ester solution was added to react at 37 degrees Celsius for 1 hour, and the unbound acridinium ester was removed by dialysis to obtain acridinium ester labeled β-collagen degradation product monoclonal antibody 1. Acridinium ester labeled β-collagen degradation product monoclonal antibody 1 was diluted with MES buffer containing 1% hydrolyzed albumin (chicken) by volume to a working concentration of 0.5 μg / mL.

[0091] Preparation of reagent L3

[0092] Biotin was dissolved in DMF to a concentration of 10 mg / ml, and the β-collagen degradation product-specific antibody 2 was diluted to a concentration of 1 mg / mL with 50 mM carbonate buffer at pH 9.0 according to a molar ratio of β-collagen degradation product-specific antibody 2:biotin=1:13, and then the biotin solution was added, and the reaction was carried out at 37 degrees Celsius for 0.5 hours, and the unbound biotin was removed by dialysis to obtain biotin-labeled β-collagen degradation product monoclonal antibody 2. The biotin-labeled β-collagen degradation product monoclonal antibody 2 was diluted to a working concentration of 6 μg / mL with MES buffer containing a volume concentration of 1% protein-free blocking agent FP MAP.

[0093] Comparative Example 1

[0094] Same as Example 1, except that:

[0095] 1. Preparation of reagent L1

[0096] The MES buffer containing 1% BSA by volume was used to replace the MES buffer containing 1% enzymatic hydrolyzed deep-water fish skin gelatin by volume in Example 1, and the working concentration of streptavidin-coated carboxyl magnetic nanoparticles was adjusted.

[0097] 2. Preparation of Reagent L2

[0098] The MES buffer containing 1% BSA by volume was used to replace the MES buffer containing 1% enzymatic hydrolyzed deep-water fish skin gelatin by volume in Example 1, and the working concentration of the acridinium ester-labeled β-collagen degradation product monoclonal antibody 1 was adjusted.

[0099] 3. Preparation of Reagent L3

[0100] The MES buffer containing 1% BSA by volume was used to replace the MES buffer containing 1% by volume of the enzymatic hydrolyzed deep-water fish skin gelatin in Example 1, and the working concentration of the biotin-labeled β-collagen degradation product monoclonal antibody 2 was adjusted. Preparation of chemiluminescent excitation solution A and solution B

[0101] Chemiluminescent excitation liquid A: an aqueous solution containing hydrogen peroxide and nitric acid, wherein the volume concentration of the hydrogen peroxide is 0.5% and the concentration of the nitric acid is 0.2M.

[0102] Chemiluminescent excitation solution B: an aqueous solution containing Tween-20 and sodium hydroxide, wherein the volume concentration of Tween is 1% and the concentration of sodium hydroxide is 0.5M.

[0103] Preparation of cleaning solution

[0104] The cleaning solution is PBS-T buffer, the formula is as follows (taking 1L as an example): pH 6.96

[0105]

[0106] The chemiluminescent detection kit for β-collagen degradation products was used to perform immunoassay detection on the fully automatic chemiluminescence analyzer MAGICL6800 provided by GeneDan Biotechnology:

[0107] In the reaction cup, add 30μl sample, 50μl reagent L2, 50μl reagent L3 and 10μl reagent L1, mix well and incubate at 37℃ for 10min. After the reaction is completed, use PBS-T cleaning solution for magnetic separation and washing three times. The instrument automatically pumps in 100μL of chemiluminescent excitation solution A and 100μL of chemiluminescent excitation solution B to make the complex produce chemiluminescent signals, and measure the luminescence intensity through the photomultiplier. The instrument automatically calculates the test results through the working curve.

[0108] The statistical results of the test sample reaction value and background (specimen concentration is 0), the change of luminescence intensity and the change of gradient are shown in Table 1.

[0109] Table 1 Test data comparison

[0110]

[0111] As can be seen from Table 1, the use of non-mammalian proteins in the buffer of the chemiluminescent detection kit for β-collagen degradation products effectively improves the sensitivity and gradient of the test, wherein the MES buffer to which enzymatic deep-water fish skin gelatin is added at a volume concentration of 1% has the best effect.

[0112] The accelerated stability of the chemiluminescent detection kit for β-collagen degradation products prepared by the present invention was evaluated with reference to ISO-23640-2011. Figure 1 As shown, the buffers of Examples 1 to 5 use no mammalian protein, and the decrease deviation of the accelerated stability can be controlled within 15%, which is better than Comparative Example 1.

[0113] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A buffer, characterized in that The buffer contains one of non-mammalian protein, enzymatic hydrolysate of non-mammalian protein or protein-free blocking agent.

2. The buffer according to claim 1, characterized in that The buffer is selected from one of MES buffer, PB buffer, TRIS buffer and HEPES buffer.

3. The buffer according to claim 1 or 2, characterized in that The non-mammalian protein is selected from enzymatically hydrolyzed deep-water fish skin gelatin or hydrolyzed chicken albumin.

4. A chemiluminescent detection kit for β-collagen degradation products, characterized in that: The method comprises reagent L1, reagent L2 and reagent L3. Reagent L1 is a streptavidin-coated carboxyl magnetic nanoparticle solution containing a buffer solution, reagent L2 is an acridinium ester-labeled β-collagen degradation product monoclonal antibody 1 solution containing a buffer solution, and reagent L3 is a biotin-labeled β-collagen degradation product monoclonal antibody 2 solution containing a buffer solution.

5. The chemiluminescent detection kit according to claim 4, characterized in that: The concentration of streptavidin-coated carboxyl magnetic nanoparticles in the reagent L1 is 0.8-2.5 mg / ml, the concentration of acridinium ester-labeled β-collagen degradation product monoclonal antibody 1 in the reagent L2 is 0.5-1 μg / ml, and the concentration of biotin-labeled β-collagen degradation product monoclonal antibody 2 in the reagent L3 is 5-10 μg / ml.

6. A method for preparing a chemiluminescent detection kit for β-collagen degradation products, characterized in that: The steps include: Preparation of reagent L1: preparing carboxyl magnetic nanoparticles into a carboxyl magnetic nanoparticle suspension; activating the carboxyl magnetic nanoparticles in the carboxyl magnetic nanoparticle suspension to obtain activated carboxyl magnetic nanoparticles; adding streptavidin to the carboxyl magnetic nanoparticle suspension to react with the activated carboxyl magnetic nanoparticles, removing the supernatant to obtain streptavidin-coated carboxyl magnetic nanoparticles, and adjusting the working concentration of the streptavidin-coated carboxyl magnetic nanoparticles with a buffer; Preparation of reagent L2: using acridinium ester to label the β-collagen degradation product specific antibody 1 to obtain acridinium ester labeled β-collagen degradation product monoclonal antibody 1; adjusting the working concentration of acridinium ester labeled β-collagen degradation product monoclonal antibody 1 with buffer. Preparation of reagent L3: using biotin to label the β-collagen degradation product specific antibody 2 to obtain the biotin-labeled β-collagen degradation product monoclonal antibody 2; adjusting the working concentration of the biotin-labeled β-collagen degradation product monoclonal antibody 2 with a buffer solution.

7. The preparation method according to claim 6, characterized in that: When preparing reagent L1, EDC and NHS are used to activate the carboxyl magnetic nanoparticles in the carboxyl magnetic nanoparticle suspension, and the mass ratio of the carboxyl magnetic nanoparticles to EDC and NHS is 1:(0.5-1):(0.5-1).

8. The preparation method according to claim 6, characterized in that: When preparing reagent L1, streptavidin is added to the carboxyl magnetic nanoparticle suspension to react with the activated carboxyl magnetic nanoparticles, and the mass ratio of the added streptavidin to the activated carboxyl magnetic nanoparticles is 1:(10-100); the working concentration of the streptavidin-coated carboxyl magnetic nanoparticles is adjusted to 0.8-2.5 mg / mL with a buffer solution.

9. The preparation method according to claim 6, characterized in that: When preparing reagent L2, the molar ratio of β-collagen degradation product specific antibody 1 to acridinium ester is 1:1-20; the working concentration of acridinium ester labeled β-collagen degradation product monoclonal antibody 1 is adjusted to 0.5-1 μg / mL with buffer.

10. The preparation method according to claim 6, characterized in that: When preparing reagent L3, the molar ratio of β-collagen degradation product specific antibody 2 to biotin is 1:1-20; the working concentration of biotin-labeled β-collagen degradation product monoclonal antibody 2 is adjusted to 5-10 μg / mL with a buffer.

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