Chemiluminescence kit for detecting tumor marker CA72-4

Abstract

The application discloses a chemiluminescence kit for detecting CA72-4 based on an acridinium ester system, which comprises magnetic microspheres connected with anti-CA72-4 monoclonal antibody mAb1, an acridinium ester label of anti-CA72-4 monoclonal antibody mAb2, CA72-4 antigen calibrators, a cleaning solution, an excitation solution and the like. The kit is simple to operate, the whole detection time is 20 min, the detection efficiency is greatly improved, the test precision is relatively high, and the CV is not higher than 5%. In addition, the test results of clinical samples show that the kit has a good clinical coincidence rate, and has important significance for the auxiliary diagnosis of various tumors.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology applications, specifically relating to a chemiluminescent reagent kit for detecting the tumor marker CA72-4, and more particularly to a chemiluminescent reagent kit for detecting CA72-4 based on an acridinium ester system. Background Technology

[0002] CA72-4 (Carbohydrate Antigen 72-4) is a high-molecular-weight mucin-like tumor-associated glycoprotein (TAG72), primarily used as a tumor marker for clinical diagnosis and monitoring. It is widely distributed in the cytoplasm of epithelial cells and malignant tumor cells, and is an important component of the tumor cytoskeleton. When tumor cells proliferate abnormally, CA72-4 is released into the bloodstream via exocrine secretion. The level of CA72-4 in normal individuals is low, but it may increase in benign diseases such as pancreatitis, cirrhosis, and lung disease. Furthermore, as a non-specific tumor marker, CA72-4 can be elevated in various digestive tract tumors and gynecological tumors, including gastric cancer, ovarian cancer, biliary tract tumors, colorectal cancer, and pancreatic cancer. It exhibits high sensitivity in gastric cancer and is an important tumor marker for clinical diagnosis. Currently, clinical diagnostic methods for CA72-4 mainly include ELISA and RIA. These methods generally suffer from cumbersome procedures, long testing times, and low precision, making them difficult to meet the clinical need for rapid and accurate detection. Therefore, developing a CA72-4 detection kit that is easy to operate, has high detection efficiency, and excellent precision is an important research direction for those skilled in the art. Summary of the Invention

[0003] Purpose of the invention To overcome the shortcomings of existing CA72-4 detection methods, this invention provides a chemiluminescent kit for detecting CA72-4 based on an acridinium ester system. This kit is easy to operate, has high detection efficiency, and excellent detection precision and clinical concordance rate, enabling rapid quantitative detection of CA72-4. Technical solution

[0004] A chemiluminescent kit for detecting CA72-4 based on an acridinium ester system includes magnetic microspheres linked to anti-CA72-4 monoclonal antibody mAb1, an acridinium ester label of anti-CA72-4 monoclonal antibody mAb2, CA72-4 antigen calibrator, washing solution, and activation solution.

[0005] 1. CA72-4 antigen calibrators: Using 10mM Tris buffer as the base buffer, add 0.1%-1% bovine serum albumin, 0.1%-1% sucrose, and 0.02%-0.2% preservatives, including but not limited to sodium azide, ProClin300, and BND; prepare 6 calibrators with different concentrations by mixing the base buffer and CA72-4 antigen in a certain ratio, with a concentration range between 0 and 300 U / mL. 2. Magnetic microspheres linked to anti-CA72-4 monoclonal antibody mAb1: The magnetic microspheres are 1.5-3.0 μm carboxyl magnetic microspheres. The preparation method is as follows: Pipette 1 ml of a 10 mg / ml magnetic bead concentrate into a centrifuge tube, magnetically aspirate for 5 min, and then remove the inner solution; add 1 ml of 0.02M-0.1M, pH 5.0 MES buffer and mix for at least 30 s, repeating the washing twice; add 0.1 ml of 2-10 mg / ml EDC (diluted with MES buffer), mix at room temperature for 30 min; add 0.2 ml of 0.2-1 mg / ml anti-CA72-4 monoclonal antibody mAb1, mix at room temperature for 3 h; after magnetic washing, add 1 ml of 1%-5% BSA aqueous solution, mix at room temperature for 6 h; after magnetic washing twice more, obtain 10 mg / ml mAb1-coated magnetic beads, dilute with magnetic microsphere preservation solution to 0.2-0.7 mg / ml to obtain the working suspension. 3. Acridinium ester markers for anti-CA72-4 monoclonal antibody mAb2: Acridinium ester derivatives include acridinium ester (NSP-SA-NHS), acridinium ester (ME-DMAE-NHS), and acridinium ester (NSP-DMAE-NHS), etc.; the preparation method is as follows: weigh an appropriate amount of acridinium ester derivative, dissolve it in anhydrous DMSO to 2 mg / ml; dilute mAb2 to 2 mg / ml with 10-35 mM sodium bicarbonate and 5-15 mM anhydrous sodium carbonate (pH 9.0); according to a ratio of 5:1-20: Mix acridine ester and mAb2 solution at a molar ratio of 1, mix for 30 s, and react at room temperature in the dark for 1 h; add an equal volume of 0.2-1 M glycine buffer, mix for 30 s, and react at room temperature in the dark for 0.5 h; add 200 μl of the above pH 9.0 buffer, and purify using an ultrafiltration tube at 12000 rpm and room temperature for 20 min, repeating three times; collect and measure the final product, add an equal volume of glycerol, mix, and store at -20℃; dilute with label diluent to 0.1-0.5 μg / ml to obtain the working solution. 4. Washing solution: 0.02-0.1M Tris-HCl buffer, pH 8-9, with 0.02%-0.2% Tween-20 added.

[0009] The specific sample addition steps for this kit are as follows: a. Add 10-30 μl of CA72-4 standard or test sample, 50-100 μl of mAb1-linked magnetic microsphere suspension working solution, and 50-100 μl of mAb2 acrid ester labeled working solution to the reaction vessel; b. Incubate at 37℃ for 10-15 min, then perform magnetic separation and wash 3-5 times with washing solution; c. Add pre-excitation solution and excitation solution to the reaction vessel, detect the luminescence signal, and achieve quantitative detection of CA72-4 content.

[0010] Beneficial effects

[0011] This invention's kit, based on an acrid ester chemiluminescence system and utilizing the solid-phase adsorption properties of magnetic microspheres, achieves rapid quantitative detection of CA72-4 in just 20 minutes, significantly improving detection efficiency. The kit exhibits excellent precision, with coefficients of variation (CV) for both low- and high-value samples not exceeding 5%. Furthermore, results from testing 40 clinical samples showed a clinical correlation (R²) of 0.9968 between this kit and a commercially available Roche kit, indicating a high clinical concordance rate. In addition, the kit is easy to use and can quantitatively detect various sample types, including whole blood, serum, and plasma, making it suitable for batch clinical testing. It holds significant clinical value for the auxiliary diagnosis of various tumors, including gastric cancer and ovarian cancer. Attached Figure Description Figure 1 The calibration curve of the chemiluminescence reagent kit for detecting CA72-4 based on the acridinium ester system was established. Figure 2 Clinical relevance of a chemiluminescence assay kit for detecting CA72-4 based on acrid ester system Detailed Implementation The embodiments of the present invention will be described in detail below with reference to specific examples, accompanying drawings, and tables. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The embodiments of the present invention are only used to exemplify the principles and effects of the present invention and are not intended to limit the present invention. Any person skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention, and all equivalent modifications or changes are still covered by the claims of the present invention.

[0012] Example 1: Preparation of a chemiluminescent reagent for detecting CA72-4 based on an acridinium ester system 1. Preparation of magnetic bead coating for anti-CA72-4 monoclonal antibody mAb1 ① Place the concentrated magnetic bead solution on a roller mixer and mix at room temperature for at least 30 minutes; ② Pipette 1 ml of a 10 mg / ml magnetic bead concentrate solution into a centrifuge tube; ③ Press the centrifuge tube wall against the magnet surface for 5 minutes to magnetically attract it and remove the internal solution. Add 1 ml of 0.02M-0.1M, pH 5.0 MES buffer and mix with a vortex mixer for at least 30 seconds. Repeat this step twice. ④ Add 0.1 ml of 2-10 mg / ml EDC (diluted with MES buffer), and continue mixing and reacting at room temperature for 30 min; ⑤ Add 0.2 ml of 0.2-1 mg / ml anti-CA72-4 monoclonal antibody mAb1 and mix at room temperature for 3 h; ⑥ After magnetic aspiration for 5 minutes, remove the internal solution, add 1 ml of 0.02-0.1M, pH 5.0 MES buffer, mix well for at least 30 seconds, and repeat the washing process twice; ⑦ Add 1 ml of 1%-5% BSA aqueous solution and mix well at room temperature for 6 hours; ⑧ After magnetic aspiration for 5 minutes, remove the inner solution, add 1 ml of 0.02-0.1M, pH 5.0 MES buffer, mix for at least 30 seconds, and repeat the washing twice to obtain magnetic beads coated with 10 mg / ml anti-CA72-4 monoclonal antibody mAb1.

[0013] 2. Preparation of acridinium ester-labeled anti-CA72-4 monoclonal antibody mAb2 ① Weigh an appropriate amount of acridinium ester derivative and dissolve it in anhydrous DMSO to a concentration of 2 mg / ml; ② Dilute the anti-CA72-4 monoclonal antibody mAb2 to 2 mg / ml with 10-35 mM sodium bicarbonate and 5-15 mM anhydrous sodium carbonate (pH 9.0); ③ Add 2 mg / ml acridine ester to 2 mg / ml antibody solution. The molar ratio of acridine ester to antibody is 5:1-20:1. Mix well for 30 seconds and then mix and react at room temperature in the dark for 1 hour. ④ Add an equal volume of 0.2-1M glycine buffer, mix for 30 seconds, and then mix and react at room temperature in the dark for 0.5 hours. ⑤ Add 200 μl of buffer solution containing 10-35 mM sodium bicarbonate and 5-15 mM anhydrous sodium carbonate (pH 9.0), and purify for 20 min at 12000 rpm and room temperature using an ultrafiltration tube. Repeat this step three times. ⑥ Collect and measure the final product, add an equal volume of glycerol, mix thoroughly, and store at -20℃; ⑦ Calculate the final concentration of acridine ester-labeled antibody.

[0014] 3. Reagent preparation ① Preparation of R1: Dilute the magnetic beads coated with mAb1 to 0.2-0.7 μg / ml with 10 mM PBS, and denote it as reagent component R1; ② Preparation of R2: Dilute the acridinium ester labeled mAb2 to 0.1-0.5 μg / ml with 10mM PBS containing 1-5% BSA, and denote it as reagent component R2.

[0015] 4. Preparation of calibrators 10 mM PBS diluent was used as the 0 concentration calibrator, and CA72-4 antigen was diluted to 6 different concentration gradients using it. These gradients, together with the 0 concentration calibrator, were designated as S1-S7, covering a concentration range of 0–300 U / mL.

[0016] Example 2: Performance testing of chemiluminescent reagents for detecting CA72-4 based on acridinium ester system The performance of the reagent kit prepared in Example 1 was tested to verify its calibration curve, precision and clinical concordance rate. The specific testing steps were performed according to the sample addition and testing method described in this invention.

[0017] 1. Establishment of calibration curves Take 10-30 μl of CA72-4 calibrator (S1-S7) prepared in Example 1, 50-100 μl of mAb1 magnetic microsphere suspension working solution, and 50-100 μl of mAb2 acridil ester labeled working solution and add them to the reaction vessel. Incubate at 37℃ for 10-15 min. After magnetic separation, wash 3-5 times, add pre-excitation solution and excitation solution, and detect the luminescence signal value (RLU value). Establish a calibration curve with CA72-4 concentration on the x-axis and RLU value on the y-axis. The results are shown in Table 1. Figure 1 .

[0018]

[0019] 2. Precision testing The precision of low-value samples (concentration of 20.12 U / mL) and high-value samples (concentration of 100.5 U / mL) were tested on the instrument. Each concentration sample was tested 10 times. The luminescence signal value was substituted into the calibration curve to obtain the CA72-4 concentration, and the coefficient of variation (CV) was calculated. The results are shown in Tables 2 and 3.

[0020] The results showed that the concentration CV values ​​of the low-value sample and the high-value sample were 2.9% and 1.2%, respectively, both less than 5%, indicating that the kit of the present invention has excellent detection precision.

[0021] 3. Clinical compliance test Forty clinical serum samples were tested using the instrument. The luminescence signal values ​​were substituted into the calibration curve to obtain the detection concentration of the kit of this invention. Simultaneously, the same samples were tested using a Roche detection kit according to its instructions to obtain the detection concentration of the Roche kit. The two sets of data were compared, and a clinical correlation curve was plotted. The results are shown in Table 4. Figure 2 .

[0022] Table 4. Test results of 40 clinical samples

[0023] The results showed that the clinical correlation (R² value) between the chemiluminescent reagent for detecting CA72-4 based on the acridinium ester system of this invention and the Roche detection reagent was 0.9968, indicating that the kit has a high clinical concordance rate.

Claims

1. A chemiluminescent reagent kit for detecting CA72-4 based on an acridinium ester system, characterized in that, The product includes magnetic microspheres linked to anti-CA72-4 monoclonal antibody mAb1, an acrid ester label of anti-CA72-4 monoclonal antibody mAb2, CA72-4 antigen calibrator, washing solution, and activation solution. The method for preparing the magnetic microspheres linked to the anti-CA72-4 monoclonal antibody mAb1 is as follows: Pipette 1 ml of a 10 mg / ml magnetic bead concentrate into a centrifuge tube, press the tube wall against a magnet for 5 min, and remove the internal solution; add 1 ml of 0.02 M-0.1 M, pH 5.0 MES buffer, and vortex mix for at least 30 s, repeating this step twice; add 0.1 ml of 2-10 mg / ml EDC (diluted with MES buffer), and continue mixing at room temperature for 30 min; add 0.2 ml of 0.2-1 mg / ml anti-CA72-4 monoclonal antibody mAb1, and mix at room temperature for 3 h; press the tube wall against a magnet for 5 min, and remove the internal solution. Add 1 ml of 0.02M-0.1M, pH 5.0 MES buffer and vortex for at least 30 seconds. Repeat this step twice. Add 1 ml of 1%-5% BSA aqueous solution and mix at room temperature for 6 hours. Place the centrifuge tube against the magnet surface for 5 minutes and remove the internal solution. Add 1 ml of 0.02M-0.1M, pH 5.0 MES buffer and vortex for at least 30 seconds. Repeat this step twice to obtain magnetic beads with a concentration of 10 mg / ml coated with anti-CA72-4 monoclonal antibody mAb1. Dilute with magnetic microsphere preservation solution to a magnetic bead concentration of 0.2-0.7 mg / ml to obtain the working solution of magnetic microsphere suspension linked to anti-CA72-4 monoclonal antibody mAb1. The preparation method of the acridinium ester label of the anti-CA72-4 monoclonal antibody mAb2 is as follows: Weigh an appropriate amount of acridinium ester derivative and dissolve it in anhydrous DMSO to a concentration of 2 mg / ml; dilute the anti-CA72-4 monoclonal antibody mAb2 to a concentration of 2 mg / ml with a buffer solution containing 10-35 mM sodium bicarbonate, 5-15 mM anhydrous sodium carbonate, and pH 9.0; add 2 mg / ml of acridinium ester to the 2 mg / ml antibody solution, with a molar ratio of acridinium ester to antibody of 5:1-20:1, mix for 30 seconds, and then react at room temperature in the dark for 1 hour; add an equal volume of 0.2-1 M glycine. Add acid buffer, mix for 30 seconds, then mix and react at room temperature in the dark for 0.5 hours; add 200 μl of buffer containing 10-35 mM sodium bicarbonate, 5-15 mM anhydrous sodium carbonate, and pH 9.0, and purify using an ultrafiltration tube at 12000 rpm and room temperature for 20 minutes. Repeat this step three times; collect and measure the final product, add an equal volume of glycerol, mix thoroughly, and store at -20℃; calculate the final concentration of acridine ester labeled antibody, and then dilute with label diluent to 0.1-0.5 μg / ml to obtain the working solution of acridine ester labeled antibody mAb2 against CA72-4 monoclonal antibody.

2. The chemiluminescence reagent kit for detecting CA72-4 based on an acridil ester system according to claim 1, characterized in that, The magnetic microspheres are magnetic microspheres with a particle size of 1μm-3μm and a carboxyl functional group; the acridine ester derivative includes at least one of acridine ester (NSP-SA-NHS), acridine ester (ME-DMAE-NHS), and acridine ester (NSP-DMAE-NHS).

3. The chemiluminescence reagent kit for detecting CA72-4 based on an acrid ester system according to claim 1, characterized in that, This kit can quantitatively detect the CA72-4 content in whole blood, serum and plasma, with a total detection time of 20 minutes.

4. The chemiluminescence reagent kit for detecting CA72-4 based on an acridil ester system according to claim 1, characterized in that, The CA72-4 antigen calibrator uses 10mM Tris buffer as the base buffer, which contains 0.1%-1% bovine serum albumin, 0.1%-1% sucrose, and 0.02%-0.2% preservative. The base buffer and CA72-4 antigen are mixed in a certain proportion to prepare calibrators of 0-300 U / mL.

5. The chemiluminescence reagent kit for detecting CA72-4 based on the acridine ester system according to claim 4, characterized in that, The preservative includes at least one of sodium azide, ProClin300, and BND.

6. The chemiluminescence reagent kit for detecting CA72-4 based on an acridil ester system according to claim 1, characterized in that, The cleaning solution is a 0.02-0.1M Tris-HCl buffer solution with a pH of 8-9 and 0.02%-0.2% Tween-20 added.

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