An N-MID osteocalcin assay kit and its preparation and detection method

Abstract

This invention provides an N-MID osteocalcin assay kit, comprising reagent R1 and reagent R2. Reagent R1 includes: Tris buffer, trehalose, Genapol X-080, hydroxyethyl cellulose, protein protectant, and preservative. Reagent R2 includes: Tris buffer, trehalose, Genapol X-080, N-MID osteocalcin monoclonal antibody, latex microspheres, protein protectant, and preservative. This invention also provides a method for preparing and detecting the above-mentioned N-MID osteocalcin assay kit. Based on latex-enhanced immunoturbidimetry, this invention enables the detection of N-MID osteocalcin in a fully automated biochemical analyzer, and features simple operation, fast detection speed, high precision, good repeatability, and low cost.

Description

Technical Field

[0001] This invention relates to the field of immunological assay technology, and in particular to an N-MID osteocalcin assay kit and its preparation and detection methods. Background Technology

[0002] Osteocalcin is a non-collagenous bone matrix protein secreted by mature osteoblasts, accounting for 25% of the non-collagenous protein components in the bone matrix and 2% of total bone protein. Complete osteocalcin consists of 49 amino acids with a relative molecular mass of 5800. Serum osteocalcin exhibits diversity, with approximately one-third being complete osteocalcin, one-third being N-MID osteocalcin fragments, and one-third being short amino acid peptides. Osteocalcin is a specific marker of osteoblast function and bone mineralization, and is increasingly being used clinically as a marker of bone turnover.

[0003] Currently, N-MID osteocalcin assay kits on the market are mainly based on immunological methods. However, existing detection methods have certain drawbacks, such as cumbersome detection steps, high operational difficulty, low detection efficiency, poor precision, and the results being affected by the operation. In addition, they are relatively expensive. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an N-MID osteocalcin assay kit based on latex-enhanced immunoturbidimetry and its preparation and detection method, which can realize the detection of N-MID osteocalcin by a fully automated biochemical analyzer, and is simple to operate, fast in detection, highly precise, reproducible and low in cost.

[0005] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0006] An N-MID osteocalcin assay kit, comprising reagent R1 and reagent R2;

[0007] The reagent R1 includes: Tris buffer 10-100 mmol / L, trehalose 10-50 g / L, Genapol X-0801-5 g / L, hydroxyethyl cellulose 10-30 g / L, protein protectant 5-30 g / L, and preservative 0.5-1.5 g / L;

[0008] The reagent R2 comprises: Tris buffer 10–100 mmol / L, trehalose 5–10 g / L, Genapol X-080 0.1–5 g / L, N-MID osteocalcin monoclonal antibody 50–100 mg / L, latex microspheres 0.5%–3%, protein protectant 1–20 g / L, and preservative 0.1–5 g / L.

[0009] As one of the preferred embodiments of the present invention, the protein protectant in reagents R1 and R2 is one or more of glycerol, bovine serum albumin, casein, calf serum, and sucrose.

[0010] As one of the preferred embodiments of the present invention, the preservatives in reagents R1 and R2 are one or more of sodium azide, gentamicin, thimerosal, and Proclin 300.

[0011] As one of the preferred embodiments of the present invention, in reagent R2, the N-MID osteocalcin monoclonal antibody is at least one of mouse anti-human N-MID osteocalcin monoclonal antibody, goat anti-human N-MID osteocalcin monoclonal antibody, and rabbit anti-human N-MID osteocalcin monoclonal antibody.

[0012] As one of the preferred embodiments of the present invention, in reagent R2, the particle size of the latex microspheres is 150-400 nm; the latex microspheres are cross-linked with N-MID osteocalcin monoclonal antibody to form latex microspheres coated with N-MID osteocalcin monoclonal antibody; in this process, Hepes buffer is used as a coupling buffer and Gly buffer is used as a dilution buffer.

[0013] As one of the preferred embodiments of the present invention, the method for preparing the latex microspheres coated with N-MID osteocalcin monoclonal antibody is as follows:

[0014] ① Add latex microspheres to the coupling buffer and mix well. Then place the mixture in a constant temperature stirrer and preheat for 15-25 minutes at a temperature of 30-37℃.

[0015] ② Add activator EDC solution and NHS solution, mix well, and place in a constant temperature stirrer to react for 10-20 minutes at a temperature of 30-37℃;

[0016] ③ Add N-MID osteocalcin monoclonal antibody, mix well, and place in a constant temperature stirrer to react for 100-140 min at a temperature of 30-37℃.

[0017] ④ Add the blocking liquid, mix well, and place in a constant temperature stirrer to react for 25-35 hours at a temperature of 30-37°C.

[0018] ⑤ Centrifuge at 10,000–20,000 rpm for 10–30 min in a high-speed centrifuge at 0–10℃;

[0019] ⑥ Add dilution buffer, place in an ice-water bath, and then place in an ultrasonic cell disruptor to disperse fully for 3-10 minutes at a power of 200-300W.

[0020] A method for preparing an N-MID osteocalcin assay kit includes the following steps:

[0021] (1) Preparation of reagent R1:

[0022] According to the component content of reagent R1, the components are mixed in the same container and mixed evenly to obtain reagent R1;

[0023] (2) Prepare the dilution buffer for reagent R2:

[0024] According to the component content of reagent R2, Tris buffer, trehalose, Genapol X-080, protein protectant and preservative are mixed to obtain the dilution buffer of reagent R2.

[0025] (3) Preparation of reagent R2:

[0026] ① Add latex microspheres to the coupling buffer and mix well. Then place the mixture in a constant temperature stirrer and preheat for 15-25 minutes at a temperature of 30-37℃.

[0027] ② Add activator EDC solution and NHS solution, mix well, and place in a constant temperature stirrer to react for 10-20 minutes at a temperature of 30-37℃;

[0028] ③ Add N-MID osteocalcin monoclonal antibody, mix well, and place in a constant temperature stirrer to react for 100-140 min at a temperature of 30-37℃.

[0029] ④ Add the blocking liquid, mix well, and place in a constant temperature stirrer to react for 25-35 hours at a temperature of 30-37°C.

[0030] ⑤ Centrifuge at 10,000–20,000 rpm for 10–30 min in a high-speed centrifuge at 0–10℃;

[0031] ⑥ Add dilution buffer, place in an ice-water bath, and then place in an ultrasonic cell disruptor to disperse fully for 3-10 minutes at a power of 200-300W to obtain latex microspheres coated with N-MID osteocalcin monoclonal antibody.

[0032] ⑦ Add the latex microspheres coated with N-MID osteocalcin monoclonal antibody to the dilution buffer prepared in step (2), and place them in a constant temperature stirrer to disperse them fully for 30-40 minutes at a temperature of 30-37°C to obtain reagent R2.

[0033] A detection method for an N-MID osteocalcin assay kit includes the following steps:

[0034] (1) Take 5 μL of sample, add 60 μL of reagent R1, and incubate at 37℃ for 3-5 min;

[0035] (2) Add 180 μL of reagent R2 and incubate at 37°C;

[0036] (3) After incubation for 20 seconds, the absorbance value A1 was measured at a wavelength of 570 nm using a fully automated biochemical analyzer; after incubation for another 5 minutes, the absorbance value A2 was measured at the same wavelength.

[0037] (4) Calculate the absorbance change value ΔA according to ΔA=A2-A1, and calculate the N-MID osteocalcin content in the sample based on ΔA.

[0038] This invention relates to an N-MID osteocalcin kit based on latex-enhanced immunoturbidimetric assay. The principle involves chemically conjugating specific antibodies to the surface of latex particles of a specific size. When the cross-linked antibody-coated microspheres bind to the antigen, they rapidly aggregate, altering the absorbance of the reaction solution. N-MID osteocalcin can be detected using a fully automated biochemical analyzer. Based on the increase in absorbance, the turbidity of the immune complex can be measured at a specific wavelength, allowing for the quantitative determination of N-MID osteocalcin content in serum. This method offers advantages such as ease of operation, rapid detection, high precision, good repeatability, and results unaffected by operational procedures, while also being significantly more affordable.

[0039] Key ingredient efficacy:

[0040] Trehalose: Trehalose is a non-reducing disaccharide composed of two glucose molecules. It has a non-specific protective effect on bioactive molecules and can form a unique protective film on the surface of bioactive substances under harsh environmental conditions such as high temperature, high cold, high osmotic pressure and dehydration. It effectively protects the structure of biomolecules such as proteins from being damaged and also has good stability to acids and alkalis, allowing antigens and antibodies to maintain their natural conformation.

[0041] Hydroxyethyl cellulose (HEC): Hydroxyethyl cellulose is a white to pale yellow fibrous or powdery solid. It is non-toxic, odorless, easily soluble in water, and insoluble in common organic solvents. It has properties such as thickening, suspending, binding, emulsifying, dispersing, and water retention. It can be used to prepare solutions with different viscosity ranges. It has exceptionally good salt solubility for electrolytes. In latex reagents, it has good sensitizing effects as a latex thickener and colloidal protectant.

[0042] Genapol X-080: Compared to traditional surfactants used in biochemical reagents, Genapol X-080, as a nonionic surfactant, has better stability, anti-turbidity, dispersibility, and low foaming properties. It does not interfere with the binding reaction of latex and has a strong anti-interference effect.

[0043] The advantages of this invention compared to the prior art are:

[0044] (1) This invention is based on latex-enhanced immunoturbidimetric assay, which can realize the detection of N-MID osteocalcin by a fully automated biochemical analyzer. It is simple to operate, fast to detect, highly precise, good repeatability and low cost.

[0045] (2) The present invention adds trehalose to the reagent formulation, which can effectively protect the structure of biomolecules such as proteins from being destroyed, and also has good stability against acids and bases, so as to maintain the natural conformation of antigens and antibodies, thereby improving the stability of the reagent.

[0046] (3) The present invention adds hydroxyethyl cellulose to the reagent formulation, which has an effective sensitizing effect, thereby greatly improving the detection sensitivity and repeatability of the reagent;

[0047] (4) In this invention, Genapol X-080 is added as a surfactant in the reagent formulation. Compared with traditional surfactants such as Tween-20, Genapol X-080 has better stability, anti-turbidity, dispersibility and low foaming properties. It does not interfere with the binding reaction of latex and has a strong anti-interference effect. Detailed Implementation

[0048] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0049] Example 1

[0050] This embodiment of an N-MID osteocalcin assay kit includes reagent R1 and reagent R2.

[0051] Reagent R1 consists of: Tris buffer 10 mmol / L, trehalose 10 g / L, Genapol X-080 1 g / L, hydroxyethyl cellulose 10 g / L, glycerol 5 g / L, sodium azide 0.5 g / L, pH 7.0.

[0052] Reagent R2 includes: Tris buffer 10 mmol / L, trehalose 5 g / L, Genapol X-080 0.1 g / L, mouse anti-human N-MID osteocalcin monoclonal antibody 50 mg / L, 150 nm latex microspheres 0.5%, glycerol 1 g / L, sodium azide 0.1 g / L, pH 7.0.

[0053] In reagent R2, latex microspheres are cross-linked with N-MID osteocalcin monoclonal antibody to form latex microspheres coated with N-MID osteocalcin monoclonal antibody. In this process, Hepes buffer is used as a coupling buffer and Gly buffer is used as a dilution buffer.

[0054] The specific preparation method of the reagent kit in this embodiment is as follows:

[0055] (1) Preparation of reagent R1:

[0056] According to the component content of reagent R1, the components are mixed in the same container and mixed evenly to obtain reagent R1.

[0057] (2) Prepare the dilution buffer for reagent R2:

[0058] According to the component content of reagent R2, Tris buffer, trehalose, Genapol X-080, glycerol, and sodium azide are mixed to obtain the dilution buffer of reagent R2.

[0059] (3) Preparation of reagent R2:

[0060] ① Take latex microspheres with a particle size of 150 nm and add them to the coupling buffer (10 mmol / L Hepes buffer, pH 7.5) and mix well. Then place them in a constant temperature stirrer to preheat for 15 min at 30℃.

[0061] ② Add equal volumes of activator EDC solution (10 mg / mL) and NHS solution (50 mg / L), mix well, and place in a constant temperature stirrer to react for 10 min at 30℃.

[0062] ③ Add the corresponding N-MID osteocalcin monoclonal antibody, mix well, and place in a constant temperature stirrer for 100 min at 30℃.

[0063] ④ Add blocking solution (100g / L BSA), mix well, and place in a constant temperature stirrer to react for 25h at 30℃;

[0064] ⑤ Centrifuge at 10,000 rpm for 10 minutes in a high-speed centrifuge at 0℃;

[0065] ⑥ Add dilution buffer (30 mmol / L Gly buffer), place in an ice-water bath, and then place in an ultrasonic cell disruptor to disperse fully for 3 min at 200 W to obtain latex microspheres coated with N-MID osteocalcin monoclonal antibody.

[0066] ⑦ Add the latex microspheres coated with N-MID osteocalcin monoclonal antibody to the dilution buffer prepared in step (2), and place them in a constant temperature stirrer to disperse them fully for 30 min at 30℃ to obtain reagent R2.

[0067] Example 2

[0068] This embodiment of an N-MID osteocalcin assay kit includes reagent R1 and reagent R2.

[0069] Reagent R1 includes: Tris buffer 20 mmol / L, trehalose 20 g / L, Genapol X-080 2 g / L, hydroxyethyl cellulose 15 g / L, casein 10 g / L, gentamicin 0.8 g / L, pH 7.4.

[0070] Reagent R2 includes: Tris buffer 20 mmol / L, trehalose 7 g / L, Genapol X-080 2 g / L, goat anti-human N-MID osteocalcin monoclonal antibody 60 mg / L, 200 nm latex microspheres 1.5%, casein 5 g / L, gentamicin 2 g / L, pH 7.4.

[0071] In reagent R2, latex microspheres are cross-linked with N-MID osteocalcin monoclonal antibody to form latex microspheres coated with N-MID osteocalcin monoclonal antibody. In this process, Hepes buffer is used as a coupling buffer and Gly buffer is used as a dilution buffer.

[0072] The specific preparation method of the reagent kit in this embodiment is as follows:

[0073] (1) Preparation of reagent R1:

[0074] According to the component content of reagent R1, the components are mixed in the same container and mixed evenly to obtain reagent R1.

[0075] (2) Prepare the dilution buffer for reagent R2:

[0076] According to the component content of reagent R2, Tris buffer, trehalose, Genapol X-080, casein and gentamicin are mixed to obtain the dilution buffer of reagent R2.

[0077] (3) Preparation of reagent R2:

[0078] ① Take latex microspheres with a particle size of 200 nm and add them to the coupling buffer (10 mmol / L Hepes buffer, pH 7.5) and mix well. Then place them in a constant temperature stirrer to preheat for 20 min at a temperature of 35℃.

[0079] ② Add equal volumes of activator EDC solution (10 mg / mL) and NHS solution (50 mg / L), mix well, and place in a constant temperature stirrer to react for 15 min at 35℃.

[0080] ③ Add the corresponding N-MID osteocalcin monoclonal antibody, mix well, and place in a constant temperature stirrer for 110 min at 35℃.

[0081] ④ Add blocking solution (100g / L BSA), mix well, and place in a constant temperature stirrer to react for 30h at 35℃;

[0082] ⑤ Centrifuge at 12,000 rpm for 15 minutes in a high-speed centrifuge at 4℃;

[0083] ⑥ Add dilution buffer (30 mmol / L Gly buffer), place in an ice-water bath, and then place in an ultrasonic cell disruptor to disperse fully for 5 min at a power of 220 W to obtain latex microspheres coated with N-MID osteocalcin monoclonal antibody.

[0084] ⑦ Add the latex microspheres coated with N-MID osteocalcin monoclonal antibody to the dilution buffer prepared in step (2), and place them in a constant temperature stirrer to disperse them fully for 33 min at 35℃ to obtain reagent R2.

[0085] Example 3

[0086] This embodiment of an N-MID osteocalcin assay kit includes reagent R1 and reagent R2.

[0087] Reagent R1 includes: Tris buffer 30 mmol / L, trehalose 30 g / L, Genapol X-080 5 g / L, hydroxyethyl cellulose 12 g / L, bovine serum albumin 20 g / L, Proclin 300 1.0 g / L, pH 7.5.

[0088] Reagent R2 includes: Tris buffer 30 mmol / L, trehalose 8 g / L, Genapol X-080 5 g / L, mouse anti-human N-MID osteocalcin monoclonal antibody 80 mg / L, 250 nm latex microspheres 2%, bovine serum albumin 10 g / L, Proclin 300 2.5 g / L, pH 7.5.

[0089] In reagent R2, latex microspheres are cross-linked with N-MID osteocalcin monoclonal antibody to form latex microspheres coated with N-MID osteocalcin monoclonal antibody. In this process, Hepes buffer is used as a coupling buffer and Gly buffer is used as a dilution buffer.

[0090] The specific preparation method of the reagent kit in this embodiment is as follows:

[0091] (1) Preparation of reagent R1:

[0092] According to the component content of reagent R1, the components are mixed in the same container and mixed evenly to obtain reagent R1.

[0093] (2) Prepare the dilution buffer for reagent R2:

[0094] According to the component content of reagent R2, Tris buffer, trehalose, Genapol X-080, bovine serum albumin and Proclin 300 are mixed to obtain the dilution buffer of reagent R2.

[0095] (3) Preparation of reagent R2:

[0096] ① Take 250nm latex microspheres and add them to the coupling buffer (10mmol / L Hepes buffer, pH7.5) and mix well. Then place them in a constant temperature stirrer to preheat for 20min at 37℃.

[0097] ② Add equal volumes of activator EDC solution (10 mg / mL) and NHS solution (50 mg / L), mix well, and place in a constant temperature stirrer to react for 15 min at 37℃.

[0098] ③ Add the corresponding N-MID osteocalcin monoclonal antibody, mix well, and place in a constant temperature stirrer for 120 min at 37℃.

[0099] ④ Add blocking solution (100g / L BSA), mix well, and place in a constant temperature stirrer to react for 30h at 37℃;

[0100] ⑤ Centrifuge at 15,000 rpm for 20 minutes in a high-speed centrifuge at 4℃;

[0101] ⑥ Add dilution buffer (30 mmol / L Gly buffer), place in an ice-water bath, and then place in an ultrasonic cell disruptor to disperse fully for 5 min at a power of 250 W to obtain latex microspheres coated with N-MID osteocalcin monoclonal antibody.

[0102] ⑦ Add the latex microspheres coated with N-MID osteocalcin monoclonal antibody to the dilution buffer prepared in step (2), and place them in a constant temperature stirrer to disperse them fully for 35 min at 37℃ to obtain reagent R2.

[0103] Example 4

[0104] This embodiment of an N-MID osteocalcin assay kit includes reagent R1 and reagent R2.

[0105] Reagent R1 includes: Tris buffer 100 mmol / L, trehalose 50 g / L, Genapol X-080 5 g / L, hydroxyethyl cellulose 10–30 g / L, fetal bovine serum 15 g / L, sucrose 15 g / L, thimerosal 1.5 g / L, pH 8.0.

[0106] Reagent R2 includes: Tris buffer 100 mmol / L, trehalose 10 g / L, Genapol X-080 5 g / L, rabbit anti-human N-MID osteocalcin monoclonal antibody 100 mg / L, 400 nm latex microspheres 3%, fetal bovine serum 10 g / L, sucrose 10 g / L, thimerosal 5 g / L, pH 8.0.

[0107] In reagent R2, latex microspheres are cross-linked with N-MID osteocalcin monoclonal antibody to form latex microspheres coated with N-MID osteocalcin monoclonal antibody. In this process, Hepes buffer is used as a coupling buffer and Gly buffer is used as a dilution buffer.

[0108] The specific preparation method of the reagent kit in this embodiment is as follows:

[0109] (1) Preparation of reagent R1:

[0110] According to the component content of reagent R1, each component is mixed in the same container and mixed evenly to obtain reagent R1.

[0111] (2) Prepare the dilution buffer for reagent R2:

[0112] According to the component content of reagent R2, Tris buffer, trehalose, Genapol X-080, fetal bovine serum, sucrose, and thimerosal were mixed to obtain the dilution buffer of reagent R2.

[0113] (3) Preparation of reagent R2:

[0114] ① Take 400nm latex microspheres and add them to the coupling buffer (10mmol / L Hepes buffer, pH 7.5) and mix well. Then place them in a constant temperature stirrer to preheat for 25min at 37℃.

[0115] ② Add equal volumes of activator EDC solution (10 mg / mL) and NHS solution (50 mg / L), mix well, and place in a constant temperature stirrer to react for 20 min at 37℃.

[0116] ③ Add the corresponding N-MID osteocalcin monoclonal antibody, mix well, and place in a constant temperature stirrer for 140 min at 37℃.

[0117] ④ Add blocking solution (100g / L BSA), mix well, and place in a constant temperature stirrer to react for 35h at 37℃;

[0118] ⑤ Centrifuge at 20,000 rpm for 30 minutes in a high-speed centrifuge at 10℃;

[0119] ⑥ Add dilution buffer (30 mmol / L Gly buffer), place in an ice-water bath, and then place in an ultrasonic cell disruptor to disperse fully for 10 min at 300 W to obtain latex microspheres coated with N-MID osteocalcin monoclonal antibody.

[0120] ⑦ Add the latex microspheres coated with N-MID osteocalcin monoclonal antibody to the dilution buffer prepared in step (2), and place them in a constant temperature stirrer to disperse them fully for 40 min at 37℃ to obtain reagent R2.

[0121] Example 5

[0122] The detection method of the above-mentioned N-MID osteocalcin assay kit based on latex-enhanced immunoturbidimetry in this embodiment includes the following steps:

[0123] (1) Take 5 μL of sample, add 60 μL of reagent R1, and incubate at 37℃ for 3-5 min;

[0124] (2) Add 180 μL of reagent R2 and incubate at 37°C;

[0125] (3) After incubation for 20 seconds, the absorbance value A1 was measured at a wavelength of 570 nm using a fully automated biochemical analyzer; after incubation for another 5 minutes, the absorbance value A2 was measured at the same wavelength.

[0126] (4) Calculate the absorbance change value ΔA according to ΔA=A2-A1, and substitute ΔA into the "linear relationship formula between absorbance change value and N-MID osteocalcin concentration" to determine the N-MID osteocalcin content.

[0127] The "linear relationship formula between absorbance change and N-MID osteocalcin concentration" was obtained through the following method:

[0128] (1) The formulation of the calibration buffer is as follows:

[0129]

[0130] The purified N-MID osteocalcin was diluted with calibration buffer to prepare calibrator concentrations of 125 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, and 2000 ng / mL.

[0131] (2) Add 60 μL of reagent R1 to each of the 5 μL N-MID osteocalcin standard obtained, mix well, and incubate at 37℃ for 3-5 min; then add 180 μL of reagent R2 and incubate at 37℃; after incubation for 20 s, use a fully automated biochemical analyzer to measure the absorbance value A1 at a wavelength of 570 nm; incubate for another 5 min and measure the absorbance value A2 at the same wavelength (the reaction method is the endpoint method, the reaction direction is the forward reaction, and the optical path of the cuvette is 1.0 cm); calculate the absorbance change value ΔA = A2 - A1 of the standard respectively, and plot the calibration curve; use multi-point nonlinear fitting to obtain the linear relationship formula between the absorbance change value and the N-MID osteocalcin concentration.

[0132] Comparative Example 1

[0133] This comparative example of an N-MID osteocalcin assay kit is basically the same as the kit in Example 3, except that trehalose is not added to reagents R1 and R2, otherwise it is the same as in Example 3.

[0134] Comparative Example 2

[0135] This comparative example of an N-MID osteocalcin assay kit is basically the same as the kit in Example 3, except that Genapol X-080 is not added to reagents R1 and R2. Otherwise, it is the same as in Example 3.

[0136] Comparative Example 3

[0137] This comparative example of an N-MID osteocalcin assay kit is basically the same as the kit in Example 3, except that Tween-20 is used instead of Genapol X-080 in reagents R1 and R2, otherwise it is the same as in Example 3.

[0138] Comparative Example 4

[0139] This comparative example of an N-MID osteocalcin assay kit is basically the same as the kit in Example 3, except that hydroxyethyl cellulose is not added to reagent R1. Otherwise, it is the same as in Example 3.

[0140] Example 6

[0141] This embodiment is used to evaluate the effectiveness of the N-MID osteocalcin assay kit of the present invention.

[0142] I. Accuracy Analysis

[0143] Experimental instrument: Hitachi 7180 fully automated biochemical analyzer.

[0144] Test samples: 20 serum samples with high N-MID osteocalcin concentrations and no hemolysis, jaundice, or turbidity, and quality control samples with a target value of 160 ng / mL.

[0145] Control reagent kit: N-MID osteocalcin detection kit (latex-enhanced immunoturbidimetric assay) from a certain manufacturer that has been approved for marketing by the State Food and Drug Administration (including reagents R1 and R2, but the composition is different from that of this invention, hereinafter referred to as control reagent).

[0146] Serum samples from individuals exhibiting uniform N-MID osteocalcin concentration distribution and without hemolysis, jaundice, or turbidity were simultaneously measured using both the kit of this invention (taking Example 3 as an example) and the control kit (latex-enhanced immunoturbidimetry). The measurements were repeated three times, and the mean, CV, and bias were calculated. A bias within ±10% was considered non-interference, while a bias exceeding ±10% was considered interference. The results are shown in Table 1.

[0147] Meanwhile, the quality control sample with a target value of 160 ng / mL was measured using the kit of the present invention (taking Example 3 as an example) and the control kit (latex-enhanced immunoturbidimetric assay). The results are shown in Table 2.

[0148] Table 1. Detection results of samples from the present invention and control kit.

[0149]

[0150]

[0151] Table 2. Test results of quality control samples

[0152]

[0153] The results showed that, based on the test results of the reagent of the present invention in Table 1, the average value of the test results of 20 samples was calculated to be 197.18, and the calculated relative deviation was 0.13%. Based on the test results of the present invention and the control reagent in Table 2, the average values ​​of the 10 measurements of the quality control sample with a target value of 160 ng / mL were 159.904 and 160.532, respectively, and the calculated relative deviations were -0.06% and 0.33%, respectively, with coefficients of variation of 1.12% and 4.33%, respectively. This indicates that the test results of the test kit of the present invention are not significantly different from those of the control kit, and have high accuracy (compliance).

[0154] II. Sensitivity Analysis

[0155] Experimental instrument: Hitachi 7180 fully automated biochemical analyzer.

[0156] Test samples: 1 sample of purified water and 1 sample of N-MID osteocalcin antigen at a concentration of 90 ng / mL.

[0157] Each sample was tested 20 times simultaneously using both the kit of this invention (taking Example 3 as an example) and the control kit (latex-enhanced immunoturbidimetry). The absorbance values ​​were recorded, and the mean and standard deviation (SD) were calculated. The average absorbance of water plus 2SD was used as the absorbance value corresponding to the limit of detection (LOD). Since the relationship between absorbance and concentration is essentially linear, the LOD concentration, i.e., sensitivity, can be calculated by comparing it with the average absorbance of a 90 ng / mL sample. Sensitivity = (average absorbance difference of water + 2SD) * sample concentration / average absorbance difference of samples. The detection results are shown in Table 3.

[0158] Table 3. Sensitivity analysis results of the kit and control kit in Example 1 (unit: ng / mL)

[0159]

[0160]

[0161] The results showed that the kit of the present invention had a sensitivity of 7.20 ng / mL for detecting N-MID osteocalcin antigen, while the control reagent had a sensitivity of 19.13 ng / mL, indicating that the kit of the present invention has high sensitivity.

[0162] III. Precision Analysis

[0163] Experimental instrument: Hitachi 7180 fully automated biochemical analyzer.

[0164] Test samples: 2 clinical serum samples (90ng / mL) (low value sample) and (410ng / mL) (high value sample).

[0165] Each sample was tested 10 times using the kit of the present invention (taking Example 3 as an example) and the control kit (latex-enhanced immunoturbidimetric assay). The test results are shown in Table 4.

[0166] Table 4. Precision analysis results of the kit from Example 1 (unit: ng / mL)

[0167]

[0168] The results showed that the precision of the kit of the present invention was: the low CV value was 4.09% and the high CV value was 1.04%, both less than or equal to 10%; while the low CV value of the control kit was 7.97% and the high CV value was 6.64%, indicating that the kit of the present invention has higher precision than the control kit.

[0169] IV. Linear Analysis

[0170] Experimental instrument: Hitachi 7180 fully automated biochemical analyzer.

[0171] Test sample: Serum sample with high N-MID osteocalcin antigen (800 ng / mL).

[0172] The high N-MID osteocalcin antigen serum sample (2000 ng / mL) was diluted with calibrator diluent to six different concentrations: 0 ng / mL, 250 ng / mL, 500 ng / mL, 1000 ng / mL, 1500 ng / mL, and 2000 ng / mL. The kit of this invention (taking Example 3 as an example) was used to detect each concentration of the above samples. Each concentration was detected three times, and the correlation coefficient R value was calculated. The detection results are shown in Table 5.

[0173] Table 5. Results of linearity analysis of the reagent kit

[0174]

[0175] The results showed that the regression equation obtained from the detection results of the kit of the present invention was y = 1.0259x - 11.618, and the correlation coefficient R was 1 / 2. 2 =0.9996, indicating that the kit of the present invention has good linearity in the range of 2000 ng / mL.

[0176] Example 7

[0177] This embodiment is used to verify the effects of trehalose, Genapol X-080, and hydroxyethyl cellulose on the performance of the N-MID osteocalcin assay kit of the present invention.

[0178] I. Effect of Trehalose on Stability

[0179] Stability tests were conducted on the reagents provided in Example 3 and Comparative Example 1. The test protocol was as follows: the reagents provided in Example 3 (with added trehalose) and Comparative Example 1 (without added trehalose) were placed together in a 37°C water bath. The low-value quality control (target value 90 ng / mL), medium-value quality control (target value 200 ng / mL), and high-value quality control (410 ng / mL) were tested daily, and the changes in the measured values ​​of the quality control samples were monitored. The specific test results are shown in Table 6.

[0180] Table 6 Results of reagent thermal stability verification

[0181]

[0182] As shown in Table 6, the reagent in Example 3 provided by this invention showed essentially no change within 12 days under a 37°C water bath, indicating good stability. In contrast, the reagent in Comparative Example 1 showed a significant decrease in detection values ​​compared to Example 3 within 12 days under the same 37°C water bath conditions. In summary, adding trehalose as a stabilizer in R1 effectively improves the stability of the assay kit.

[0183] II. The impact of Genapol X-080 on accuracy

[0184] Experimental instrument: Hitachi 7180 fully automated biochemical analyzer.

[0185] Test samples: 20 serum samples with high N-MID osteocalcin concentrations and no hemolysis, jaundice, or turbidity, and quality control samples with a target value of 160 ng / mL.

[0186] Sensitivity analysis tests of the kit were performed using the reagents provided in Example 3 and Comparative Examples 2 and 3, respectively.

[0187] The experimental protocol was as follows: Human serum samples with relatively uniform N-MID osteocalcin concentration distribution and no hemolysis, jaundice, or turbidity were simultaneously measured using the kits from Example 3 (with Genapol X-080 added as a surfactant), Comparative Example 2 (without any surfactant), and Comparative Example 3 (with Tween-20 added as a surfactant). The assays were repeated three times, and the mean, CV, and bias were calculated. A bias within ±10% was considered non-interference, while a bias exceeding ±10% was considered interference. The results are shown in Table 7.

[0188] Meanwhile, the quality control samples with a target value of 160 ng / mL were measured using the kits of Example 3 (with Genapol X-080 added as a surfactant), Comparative Example 2 (without any surfactant added), and Comparative Example 3 (with Tween-20 added as a surfactant). The results are shown in Table 8.

[0189] Table 7. Detection results of reagent kit samples from Example 3 and Comparative Examples 2 and 3.

[0190]

[0191]

[0192] Table 8. Test results of quality control samples

[0193]

[0194]

[0195] The results in Tables 7 and 8 show that the addition of Genapol X-080 can improve the accuracy of detection, and that Genapol X-080 has a more significant effect than traditional surfactants such as Tween-20.

[0196] III. The Influence of Hydroxyethyl Cellulose on the Sensitivity of Sample Detection Results

[0197] Experimental instrument: Hitachi 7180 fully automated biochemical analyzer.

[0198] Test samples: 1 sample of purified water and 1 sample of N-MID osteocalcin antigen at a concentration of 90 ng / mL.

[0199] Sensitivity analysis tests of the kit were performed using the reagents provided in Example 3 and Comparative Example 4, respectively.

[0200] The experimental protocol was as follows: Each sample was tested 20 times simultaneously using Example 3 (with added hydroxyethyl cellulose) and Comparative Example 4 (without added hydroxyethyl cellulose), and the absorbance values ​​were recorded. The average value and standard deviation (SD) were calculated. The average absorbance of water plus 2SD was used as the absorbance value corresponding to the limit of detection (LOD). Since the relationship between absorbance and concentration is basically linear, the LOD concentration, i.e., the sensitivity, can be calculated by comparing it with the average absorbance of the 90 ng / mL sample. The test results are shown in Table 9.

[0201] Table 9. Sensitivity analysis results of the kits from Example 3 and Comparative Example 4 (unit: ng / mL)

[0202]

[0203]

[0204]

[0205] The results showed that the sensitivity of the kit for detecting N-MID osteocalcin antigen was 6.66 ng / mL, compared to 54.91 ng / mL in Comparative Example 4. This indicates that hydroxyethyl cellulose has a significant impact on improving the sensitivity of the kit.

[0206] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An N-MID osteocalcin assay kit, characterized in that, Including reagent R1 and reagent R2; The reagent R1 includes: Tris buffer 10~100mmol / L, trehalose 10~50g / L, Genapol X-080 1~5g / L, hydroxyethyl cellulose 10~30g / L, protein protectant 5~30g / L, and preservative 0.5~1.5g / L; The reagent R2 comprises: Tris buffer 10~100 mmol / L, trehalose 5~10 g / L, Genapol X-080 0.1~5 g / L, N-MID osteocalcin monoclonal antibody 50~100 mg / L, latex microspheres 0.5%~3%, protein protectant 1~20 g / L, and preservative 0.1~5 g / L; The protein protectant in reagents R1 and R2 is one or more of glycerol, bovine serum albumin, casein, calf serum, and sucrose. In reagent R2, the N-MID osteocalcin monoclonal antibody is at least one of mouse anti-human N-MID osteocalcin monoclonal antibody, goat anti-human N-MID osteocalcin monoclonal antibody, and rabbit anti-human N-MID osteocalcin monoclonal antibody; the latex microspheres have a particle size of 150~400nm; the latex microspheres are cross-linked with the N-MID osteocalcin monoclonal antibody to form latex microspheres coated with N-MID osteocalcin monoclonal antibody. In this process, Hepes buffer is used as a coupling buffer and Gly buffer is used as a dilution buffer.

2. The N-MID osteocalcin assay kit according to claim 1, characterized in that, The preservatives in reagents R1 and R2 are one or more of sodium azide, gentamicin, thimerosal, and Proclin 300.

3. The N-MID osteocalcin assay kit according to claim 1, characterized in that, The method for preparing the latex microspheres coated with N-MID osteocalcin monoclonal antibody is as follows: a. Add latex microspheres to the coupling buffer and mix well. Then place the mixture in a constant temperature stirrer and preheat for 15-25 minutes at a temperature of 30-37°C. b. Add activator EDC solution and NHS solution, mix well, and react in a constant temperature stirrer for 10~20 min at a temperature of 30~37°C; c. Add N-MID osteocalcin monoclonal antibody, mix well, and place in a constant temperature stirrer to react for 100~140 min at a temperature of 30~37°C. d. Add the blocking solution, mix well, and place in a constant temperature stirrer to react for 25~35 hours at a temperature of 30~37°C; e. Centrifuge in a high-speed centrifuge at 10,000-20,000 rpm for 10-30 minutes at a temperature of 0-10°C; f. Add dilution buffer, place in an ice-water bath, and then place in an ultrasonic cell disruptor to disperse thoroughly for 3-10 minutes at a power of 200-300W.

4. A method for preparing an N-MID osteocalcin assay kit as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Preparation of reagent R1: According to the component content of reagent R1, the components are mixed in the same container and mixed evenly to obtain reagent R1; (2) Prepare the dilution buffer for reagent R2: According to the component content of reagent R2, Tris buffer, trehalose, Genapol X-080, protein protectant and preservative are mixed to obtain the dilution buffer of reagent R2; (3) Preparation of reagent R2: a. Add latex microspheres to the coupling buffer and mix well. Then place the mixture in a constant temperature stirrer and preheat for 15-25 minutes at a temperature of 30-37°C. b. Add activator EDC solution and NHS solution, mix well, and react in a constant temperature stirrer for 10~20 min at a temperature of 30~37°C; c. Add N-MID osteocalcin monoclonal antibody, mix well, and place in a constant temperature stirrer to react for 100~140 min at a temperature of 30~37°C. d. Add the blocking solution, mix well, and place in a constant temperature stirrer to react for 25~35 hours at a temperature of 30~37°C; e. Centrifuge in a high-speed centrifuge at 10,000-20,000 rpm for 10-30 minutes at a temperature of 0-10°C; f. Add dilution buffer, place in an ice-water bath, and then place in an ultrasonic cell disruptor to disperse thoroughly for 3-10 minutes at a power of 200-300W to obtain latex microspheres coated with N-MID osteocalcin monoclonal antibody. g. Add latex microspheres coated with N-MID osteocalcin monoclonal antibody to the dilution buffer prepared in step (2), and place them in a constant temperature stirrer to disperse them for 30~40 min at a temperature of 30~37°C to obtain reagent R2.

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