Chemiluminescence detection kit for soluble growth stimulation expression factor 2 and preparation method of chemiluminescence detection kit

Abstract

The invention provides the high-sensitivity and high-stability sST2 chemiluminescence detection kit, by optimizing a magnetic particle coating process and an acridinium ester labeling purification process, the wide linear detection range of 4-1024ng / mL and the excellent performance that the inter-batch CV is less than or equal to 10% are realized, and the kit is suitable for clinical large-scale screening and accurate diagnosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a kit for detecting soluble growth stimulation expression factor 2 (sST2) based on chemiluminescence immunoassay and a preparation method thereof. Background Art

[0002] Cardiovascular diseases are one of the greatest threats to the health of residents in China, ranking first in the composition ratio of disease deaths, reaching more than 47%. Due to the prevalence of unhealthy lifestyles and the acceleration of aging, this proportion is still on the rise. Among them, heart failure is the terminal stage of the development of various cardiovascular diseases, and is also known as the "last battlefield" of cardiovascular diseases, with a 5-year mortality rate of up to 50%. However, in the initial stage of heart function impairment, the reserve capacity of the heart will help to make up for the damage, so the manifestations of early heart failure are generally not obvious, thus it is easy to miss the best treatment opportunity.

[0003] Soluble growth stimulation expression factor 2 (sST2) is an important biomarker for myocardial fibrosis and heart failure, and its accurate detection is of great significance for disease diagnosis and prognosis evaluation. The 2013 ACC / AHA / HFSA heart failure guidelines point out that it has a certain early warning value for heart failure, which helps to identify early heart failure patients and high-risk populations; the 2014 Chinese heart failure guidelines recommend sST2 for the risk stratification of chronic heart failure. The continuous increase in sST2 levels reflects the continuous progression of myocardial fibrosis and myocardial remodeling. It can be seen that sST2 is of great significance for improving the treatment and prognosis of heart failure. Moreover, sST2 is not affected by renal function, and its threshold is not affected by factors such as age and BMI, which is convenient for clinical development and judgment.

[0004] At present, chemiluminescence method is widely used in sST2 detection due to its high sensitivity, but there are still deficiencies in existing kits. The domestic reference value for sST2 used for early identification or risk stratification of heart failure is ≤35 ng / mL, however, its clinical abnormal value can reach 600 ng / mL or even higher, but the linear range of the marketed products is basically within 500 ng / mL, and mainly ≤300 ng / mL. When using such sST2 reagents for detection, when encountering high values beyond the linear range, the results cannot be detected, and the blood sample needs to be diluted multiple times and then detected again, which is not only time-consuming and laborious, increasing the error probability, but also may delay the treatment opportunity in special cases, resulting in irreparable consequences. In addition, the between-batch coefficient of variation (CV) of existing products is relatively high (usually >15%), resulting in poor repeatability of detection results and affecting clinical judgment.

[0005] The above problems are mainly due to low antibody coupling efficiency, insufficient labeling stability and poor control of non-specific binding. Therefore, there is an urgent need to develop a sST2 detection kit with a wide linear range and high stability. Summary of the Invention

[0006] The present invention aims to provide a chemiluminescent kit for detecting sST2. By optimizing the magnetic particle coating process and the acridinium ester labeling and purification process, the following beneficial effects are achieved:

[0007] 1. Broaden the linear range: 4 ng / mL to 1024 ng / mL, covering the common concentrations of clinical samples;

[0008] 2. Improve the inter-batch consistency: the inter-batch coefficient of variation (CV) ≤ 10%, significantly improving the detection reliability.

[0009] The specific technical solutions are as follows:

[0010] The kit of the present invention includes the following core components:

[0011] 1. sST2 antibody solution coated on magnetic particles: prepared by activation, coating, blocking, and resuspension processes;

[0012] 2. sST2 antibody solution labeled with acridinium ester: prepared by labeling, quenching, and purification processes.

[0013] Among them, the specific preparation method of the sST2 antibody solution coated on magnetic particles is as follows:

[0014] ⑴ Activation of magnetic particles: Take 150 μL of magnetic particles. After magnetic separation and discarding the supernatant, add 2 mL of activation buffer to disperse and mix the magnetic particles evenly. Place them on a magnetic separator, let stand for 1.5 - 3 min, discard the supernatant, repeat 2 times, and add 1.5 mL of activation buffer; then add 50 μL of 50 mg / mL EDC solution and 50 μL of 50 mg / mL NHS solution respectively, and react with shaking at room temperature for 20 - 35 min;

[0015] ⑵ Coating: Place the activated solution on a magnetic separator, let stand for 2 - 4 min, discard the supernatant; add 2 mL of coupling buffer to disperse and mix the magnetic particles evenly. Place them on a magnetic separator, let stand for 1.5 - 3 min, discard the supernatant; repeat the washing 2 times; then add 1.5 mL of coupling buffer and 100 μL of 1 mg / mL sST2 coating antibody solution, and react with shaking at room temperature for 15 - 20 min;

[0016] ⑶ Blocking: Add 4.0 mL of magnetic particle blocking solution, and react with shaking at room temperature for 1 - 2 h to block non-specific sites;

[0017] ⑷ Resuspension: Place the blocked solution on a magnetic separator, let stand for 1.5 - 3 min, discard the supernatant; wash repeatedly 3 times with magnetic particle washing solution, and add magnetic particle preservation solution to the precipitate and dilute to 50 mL to obtain.

[0018] The activation buffer is a MES solution with a concentration of 0.025 mol / L and a pH of 6.0 ± 0.1. The EDC solution and NHS solution are both prepared with the activation buffer. The coupling buffer is a phosphate buffer with a concentration of 0.02 mol / L and a pH of 7.0. The blocking solution is a 1.5% bovine serum albumin solution prepared with the coupling buffer. The magnetic particle storage solution is a solution containing 0.5% BSA, 0.8% sucrose, 0.5% trehalose, 0.2% Tween-20 and 0.05% Proclin-300, prepared with 0.05 mol / L Tris-HCl buffer (pH 7.4).

[0019] The specific preparation method of the acridinium ester-labeled sST2 antibody solution is as follows:

[0020] 1) Take 800 μL of the acridinium ester labeling buffer, add 100 μL of the 1 mg / mL sST2 labeled antibody solution, mix well, and then add 100 μL of the acridinium ester stock solution II. Shake in the dark at room temperature for 20 - 30 min;

[0021] 2) Add 300 μL of the acridinium ester quenching solution, shake in the dark at room temperature for 30 min to terminate the reaction, and use it as the sample to be purified;

[0022] 3) Purification:

[0023] ① Equilibrate the desalting column with 15 mL of purified water and 25 mL of the acridinium ester purification buffer in sequence;

[0024] ② Make the acridinium ester purification buffer on the surface of the column bed close to running dry, and slowly load the sample (the sample to be purified) along the column wall;

[0025] ③ After the sample enters the gel, add no less than 15 mL of the acridinium ester purification buffer, collect continuously at 1.0 mL / tube, combine the 5th - 10th tubes and dilute to 70 mL with the acridinium ester storage solution to obtain the product.

[0026] Among them, the acridinium ester purification buffer is a PBS buffer with a concentration of 0.15 mol / L, a pH of 6.5, containing 0.5% BSA and 0.05% Proclin-300. The acridinium ester labeling buffer is a PBS buffer with a concentration of 0.15 mol / L and a pH of 8.0. The acridinium ester storage solution is a solution containing 1% trehalose, 0.2% Tween-20 and 0.05% Proclin-300, prepared with the acridinium ester purification buffer, and this storage solution can enhance the stability of the labeled antibody. The acridinium ester quenching solution is a 3% glycine solution. The acridinium ester stock solution II is: take 1 bottle (1 mg) of acridinium ester, dissolve it in 350 μL of anhydrous DMF, take 10 μL and add it to 90 μL of anhydrous DMF.

[0027] The above-mentioned sST2-coated antibody can also be referred to as an sST2 capture antibody, and the sST2-labeled antibody can also be referred to as an sST2 detection antibody. The sST2 antibody solution coated on magnetic particles and the sST2 antibody solution labeled with acridinium ester obtained by the present invention can be dispensed as needed and used in combination.

[0028] In addition to the aforementioned key reagents such as magnetic micro activation buffer and acridinium ester labeling buffer, the chemiluminescence detection kit usually also includes excitation solution, cleaning solution, etc., but they are all fixed or a few optional solutions, which are conventional technical contents in this field and will not be elaborated here.

[0029] The sST2 chemiluminescence detection kit of the present invention significantly improves the linear range and batch-to-batch consistency of detection by optimizing the magnetic particle antibody coating process and acridinium ester labeling technology, which helps to improve the efficiency and accuracy of clinical detection. The linear range reaches 4 - 1024 ng / mL, covering the sST2 detection values reported clinically at present, and basically no dilution is required for detecting any sample, and the result can be obtained at one time; the batch-to-batch difference (CV) is controlled within 10%. Brief Description of the Drawings

[0030] Figures 1-3 It is a schematic diagram for testing the linear range of reagents in different batches of the present invention. Detailed Description of the Invention

[0031] Example 1 (taking 1000 tests / batch as an example, the same below)

[0032] A chemiluminescence detection kit for soluble growth stimulation expression factor 2. Among them, the preparation method of the sST2 antibody solution coated on magnetic particles is as follows:

[0033] 1) Measure 150 μL of a 10 mg / ml magnetic particle solution into a centrifuge tube, place it on a magnetic bead separator, let it stand at room temperature for 1 min, and discard the supernatant; add 2 mL of activation buffer to disperse and mix the magnetic particles, place it on a magnetic separator, let it stand for 1.5 min, discard the supernatant, repeat 2 times, and add 1.5 mL of activation buffer; then add 50 μL of a 50 mg / mL EDC solution and 50 μL of a 50 mg / mL NHS solution respectively, and react with shaking at room temperature for 20 min;

[0034] 2) Place the solution obtained in step 1) on a magnetic separator, let it stand for 2 min, and discard the supernatant; add 2 mL of coupling buffer to disperse and mix the magnetic particles, place it on a magnetic separator, let it stand for 1.5 min, and discard the supernatant; repeat the washing 2 times; then add 1.5 mL of coupling buffer and 100 μL of a 1 mg / mL sST2-coated antibody solution, and react with shaking at room temperature for 15 min;

[0035] 3) Add 4.0 mL of magnetic particle blocking solution to the solution obtained in step 2) and shake the reaction at room temperature for 1 h;

[0036] 4) Place the sealed solution on a magnetic separator, let it stand for 1.5 minutes, and discard the supernatant; wash it repeatedly with magnetic particle washing solution for 3 times, add magnetic particle preservation solution to the precipitate and dilute it to 50mL, and store it in a sealed container at 2-8℃.

[0037] Example 2

[0038] The chemiluminescent detection kit for soluble growth stimulating expression factor 2, wherein the preparation method of the sST2 antibody solution coated with magnetic particles is:

[0039] 1) Measure 150 μL of 10 mg / ml magnetic particle solution into a centrifuge tube, place it on a magnetic bead separator, let it stand at room temperature for 1 min, and discard the supernatant; add 2 mL of activation buffer to disperse and mix the magnetic particles, place it on a magnetic separator, let it stand for 3 min, discard the supernatant, repeat 2 times, add 1.5 mL of activation buffer; then add 50 μL of 50 mg / mL EDC solution and 50 μL of 50 mg / mL NHS solution respectively, and react at room temperature with shaking for 35 min;

[0040] 2) Place the solution obtained in step 1) on a magnetic separator, let it stand for 4 minutes, and discard the supernatant; add 2 mL of coupling buffer to disperse and mix the magnetic particles, place it on a magnetic separator, let it stand for 3 minutes, and discard the supernatant; repeat the washing twice; then add 1.5 mL of coupling buffer and 100 μL of 1 mg / mL sST2 coating antibody solution, and shake at room temperature for 20 minutes;

[0041] 3) Add 4.0 mL of magnetic particle blocking solution to the solution obtained in step 2) and shake the reaction at room temperature for 2 h;

[0042] 4) Place the sealed solution on a magnetic separator, let it stand for 3 minutes, and discard the supernatant; wash it repeatedly with magnetic particle washing solution for 3 times, add magnetic particle preservation solution to the precipitate and dilute it to 50mL, and store it in a sealed container at 2-8℃.

[0043] Example 3

[0044] A chemiluminescent detection kit for soluble growth stimulating factor 2, wherein the preparation method of the acridinium ester-labeled sST2 antibody solution is:

[0045] 1) Take 800 μL of acridinium ester labeling buffer in a centrifuge tube, add 100 μL of 1 mg / mL sST2 labeled antibody solution, mix well, then add 100 μL of acridinium ester stock solution II, and shake at room temperature in the dark for 20 min;

[0046] 2) Add 300 μL of acridinium ester quenching solution to the solution obtained in step 1), and react with shaking in the dark at room temperature for 30 min to obtain the sample to be purified;

[0047] 3) Purification:

[0048] ① Take out the desalting column from the refrigerator at 2-8 °C and equilibrate it to room temperature;

[0049] ② Equilibrate the desalting column with 15 mL of purified water and 25 mL of acridinium ester purification buffer in sequence;

[0050] ③ When the acridinium ester purification buffer on the surface of the column bed is nearly drained, slowly load the sample (the sample to be purified) along the column wall;

[0051] ④ After the sample enters the gel, add 15 mL of acridinium ester purification buffer, collect continuously at 1.0 mL / tube, combine the 5th - 10th tubes and dilute to 70 mL with acridinium ester preservation solution to obtain the product, which is stored sealed at 2-8 °C.

[0052] Example 4

[0053] Linear range test: Dilute the high-concentration (active) sample close to the upper limit of the linear range to the low-concentration (active) sample close to the lower limit of the linear range by serial dilution, and test with the kit of the present invention (the magnetic particle-coated sST2 antibody solution prepared according to the method of Example 1 and the acridinium ester-labeled sST2 antibody solution prepared according to the method of Example 3). Each dilution concentration is tested 2 times, and the mean value of the detection results is calculated respectively. Taking the dilution concentration as the independent variable and the mean value of the detection results as the dependent variable, the linear regression equation is obtained: y = a + bx, and the correlation coefficient (r) of the linear regression is calculated.

[0054] The test results show that the kit of the present invention has a good linear relationship in the range of 4.0 - 1024.16 ng / mL, and the r value ≥ 0.9999. See Table 1 for details.

[0055] Table 1 Linear range test results

[0056]

[0057] Example 5

[0058] Inter-batch consistency test: Take three batches of kits prepared according to the methods of Example 2 and Example 3 to detect the soluble growth stimulating expression factor 2 sample solutions with concentrations of 16.00 ng / mL and 64.01 ng / mL. Each concentration is detected 10 times for each batch, and the mean value (M) and standard deviation (SD) of the measurement results are calculated respectively. The coefficient of variation is obtained according to the formula CV = SD / M × 100%. The results show that the inter-batch coefficient of variation CV (%) is less than 10%. See Table 2 for details.

[0059] Table 2 Inter-batch difference test results

[0060]

[0061] The above has described the present invention in detail, aiming to enable those skilled in the art to understand the content of the present invention and implement it. However, this should not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A kit for detecting soluble growth-stimulating expressed factor 2 by chemiluminescence method, the kit comprising: Magnetic particle-coated sST2 antibody solution and acridinium ester-labeled sST2 antibody solution, characterized in that the preparation method of the magnetic particle-coated sST2 antibody solution is as follows: ⑴ Activation of magnetic particles: Take 150 μL of magnetic particles. After magnetic separation and discarding the supernatant, add 2 mL of activation buffer to disperse and mix the magnetic particles evenly. Place them on a magnetic separator, let stand for 1.5 - 3 min, discard the supernatant, repeat 2 times, and add 1.5 mL of activation buffer; then add 50 μL of 50 mg / mL EDC solution and 50 μL of 50 mg / mL NHS solution respectively, and react with shaking at room temperature for 20 - 35 min; ⑵ Coating: Place the activated solution on a magnetic separator, let stand for 2 - 4 min, discard the supernatant; add 2 mL of coupling buffer to disperse and mix the magnetic particles evenly. Place them on a magnetic separator, let stand for 1.5 - 3 min, discard the supernatant; repeat the washing 2 times; then add 1.5 mL of coupling buffer and 100 μL of 1 mg / mL sST2 coating antibody solution, and react with shaking at room temperature for 15 - 20 min; ⑶ Blocking: Add 4.0 mL of magnetic particle blocking solution, and react with shaking at room temperature for 1 - 2 h; ⑷ Resuspension: Place the blocked solution on a magnetic separator, let stand for 1.5 - 3 min, discard the supernatant; wash repeatedly 3 times with magnetic particle washing solution, add magnetic particle preservation solution to the precipitate and dilute to 50 mL to obtain.

2. The kit according to claim 1, characterized in that: The preparation method of the acridinium ester-labeled sST2 antibody solution is as follows: 1) Take 800 μL of acridinium ester labeling buffer, add 100 μL of 1 mg / mL sST2 labeling antibody solution, mix evenly, then add 100 μL of acridinium ester stock solution II, and shake in the dark at room temperature for 20 - 30 min; 2) Add 300 μL of acridinium ester quenching solution, and react with shaking in the dark at room temperature for 30 min to obtain the sample to be purified; 3) Purification: ① Equilibrate the desalting column with 15 mL of purified water and 25 mL of acridinium ester purification buffer in sequence; ② Make the acridinium ester purification buffer on the surface of the column bed close to running dry, and slowly load the sample (sample to be purified) along the column wall; ③ After the sample enters the gel, add no less than 15 mL of acridinium ester purification buffer, collect continuously at 1.0 mL / tube, combine the 5th - 10th tubes and dilute to 70 mL with acridinium ester preservation solution to obtain.

3. The kit according to claim 1, wherein: The activation buffer is a MES solution with a concentration of 0.025 mol / L and a pH of 6.0 ± 0.

1.

4. The kit according to claim 1, characterized in that: The coupling buffer is a phosphate buffer with a concentration of 0.02 mol / L and a pH of 7.

0.

5. The kit according to claim 1, characterized in that: The blocking solution is a 1.5% bovine serum albumin solution prepared with coupling buffer.

6. The kit according to claim 1, wherein: Both the EDC solution and the NHS solution are prepared with activation buffer.

7. The kit according to claim 1, characterized in that: The magnetic particle preservation solution is a solution containing 0.5% BSA, 0.8% sucrose, 0.5% trehalose, 0.2% Tween - 20 and 0.05% Proclin - 300 prepared with 0.05 mol / L Tris - HCl buffer (pH 7.4).

8. The kit according to claim 1, characterized in that: The acridinium ester purification buffer is a PBS buffer with a concentration of 0.15 mol / L, a pH of 6.5, containing 0.5% BSA and 0.05% Proclin - 300.

9. The kit according to claim 1, wherein: The acridinium ester preservation solution is a solution containing 1% trehalose, 0.2% Tween-20 and 0.05% Proclin-300 prepared with an acridinium ester purification buffer solution.

10. The kit according to claim 1, characterized in that: The acridinium ester quenching solution is a 3% glycine solution.

Images