An immunochromatographic test strip for monitoring the purification efficiency of acridine ester-labeled antibodies

Abstract

This invention belongs to the field of biotechnology detection, and particularly relates to an immunochromatographic test strip for monitoring the purification efficiency of acridine ester-labeled antibodies, comprising test strip A and test strip B; test strip A has a first detection line T1A and a second detection line T2A on its nitrocellulose membrane, as well as a control line CA; the first detection line T1A is coated with an anti-acrididine ester antibody, and the second detection line T2A is coated with an antigen that specifically binds to the antibody to be tested; test strip B has a first detection line T1B and a second detection line T2B on its nitrocellulose membrane, as well as a control line CB; the first detection line T1B is coated with an antigen that specifically binds to the antibody to be tested, and the second detection line T2B is coated with an anti-acrididine ester antibody. The immunochromatographic test strip provided by this invention for monitoring the purification efficiency of acridine ester-labeled antibodies is a simple, intuitive, rapid, and low-cost tool for monitoring purification efficiency.

Description

Technical Field

[0001] This invention relates to the field of biotechnology detection, and more particularly to an immunochromatographic test strip for monitoring the purification efficiency of acridinium ester-labeled antibodies. Background Technology

[0002] Acridinium ester (AE), as a class of highly efficient chemiluminescent labels, has advantages such as high quantum yield, low background signal, and good stability, and has been widely used in fields such as immunoassay, nucleic acid detection, and clinical diagnosis. In the development of chemiluminescent immunoassay reagents, conjugating acridinium ester with an antibody to form a labeled antibody complex is one of the key steps in reagent preparation. After the labeling reaction is completed, an appropriate purification method is needed to remove unreacted free acridinium ester to avoid interfering with subsequent detection. Currently, commonly used purification methods include desalting column chromatography (gel filtration chromatography) and ultrafiltration centrifugation. However, in actual production, the above purification methods have the following technical problems: Purification results are unstable and vary significantly between batches. The purification effect of desalting columns is affected by various factors such as column packing conditions, batch differences, scale of operation, and operator technique, leading to large fluctuations in the amount of free acridinium ester residue in different batches of labeled reagents, ultimately affecting the consistency and reliability of the reagents. Ultrafiltration also suffers from issues such as membrane batch differences and the need for controlled centrifugation conditions. Currently, there is a lack of a rapid and convenient method to evaluate the actual effect of each desalting purification process, making it impossible to accurately determine how much acridinium ester has been successfully bound to the antibody and how much remains in the final product. This is one of the main sources of batch-to-batch variation.

[0003] Existing technology includes a patent document (CN200710008800.8) that discloses a method for detecting acrid ester labels using capillary electrophoresis coupled with chemiluminescence. This method utilizes capillary electrophoresis to separate the sample and then performs chemiluminescence detection. However, this method involves expensive equipment, complex operation, and requires professional personnel, making it difficult to promote its use on production lines.

[0004] Currently, there is a lack of rapid and intuitive detection tools for the content of free acridinium esters and labeled antibodies in purified samples. Existing technologies mostly use sophisticated laboratory instruments (such as HPLC and chemiluminescence analyzers) for analysis. Although these methods offer high accuracy, they have the following drawbacks: ① The equipment is expensive, making it difficult for small and medium-sized enterprises to adopt; ② The operation is complex, requiring specialized technical personnel; ③ The detection cycle is long, making it impossible to provide real-time feedback for process adjustments; ④ It is not suitable as a rapid quality control method for production lines.

[0005] Existing technologies lack a simple, intuitive, rapid, and low-cost purification efficiency monitoring tool, which cannot meet the needs of rapid on-site quality control in the production process of chemiluminescent reagents. Summary of the Invention

[0006] The purpose of this invention is to provide an immunochromatographic test strip for monitoring the purification efficiency of acridinium ester-labeled antibodies, providing a simple, intuitive, rapid, and low-cost purification efficiency monitoring tool to meet the needs of rapid on-site quality control in the production process of chemiluminescent reagents.

[0007] To solve the above-mentioned technical problems, the immunochromatographic test strip for improving the purification efficiency of acridinium ester-labeled antibodies provided by this invention is achieved as follows: An immunochromatographic test strip for monitoring the purification efficiency of acridinium ester-labeled antibodies, comprising test strip A and test strip B; The test strip A has a first detection line T1A and a second detection line T2A on its nitrocellulose membrane, as well as a control line CA; the first detection line T1A is coated with an anti-acridoid ester antibody, and the second detection line T2A is coated with an antigen that specifically binds to the antibody to be tested; The test strip B has a first detection line T1B and a second detection line T2B on its nitrocellulose membrane, as well as a control line CB; the first detection line T1B is coated with an antigen that specifically binds to the antibody to be tested, and the second detection line T2B is coated with an anti-acridone ester antibody.

[0008] Optionally, the concentration of anti-acridoid ester antibody coated on the first detection line T1A and the second detection line T2B is 0.5-0.8 mg / mL; the concentration of antigen coated on the second detection line T2A and the first detection line T1B is 15-25 ng / mL.

[0009] Optionally, the first detection line T1A and the first detection line T1B each consist of 10 parallel detection lines; the second detection line T2A and the second detection line T2B each consist of 5 parallel detection lines.

[0010] Optionally, the anti-acridone ester antibody is an anti-acridone ester monoclonal antibody or a bispecific antibody.

[0011] Optionally, the antigen that specifically binds to the antibody to be tested is the antigen corresponding to the antibody to be tested.

[0012] A method for preparing an immunochromatographic test strip for monitoring the purification efficiency of acridinium ester-labeled antibodies includes the following steps: (1) Preparation of colloidal gold solution; (2) Preparation of gold-labeled antibodies: Anti-immunoglobulin antibodies were labeled with colloidal gold; (3) Spray the gold-labeled antibody onto the gold-labeled pad; (4) Streak anti-acridone ester antibody, antigen and quality control antibody on nitrocellulose membrane to form detection line and quality control line respectively; (5) Assemble the sample pad, gold label pad, nitrocellulose membrane and absorbent pad in sequence on the base plate and cut them into test strips.

[0013] An immunochromatographic test strip for monitoring the purification efficiency of acridine ester-labeled antibodies includes the following steps: (1) Load the sample to be tested onto test strip A and test strip B respectively, and read the colorimetric results after the chromatographic reaction; (2) Record the number of color lines developing at the first detection line T1A of test strip A respectively. Number of colorimetric strips for the second detection line T2A And the number of color development lines on the first detection line T1B of test strip B. Number of T2B colorimetric stripes in the second detection line ; (3) Based on the pre-established concentration-color bar number standard curve, convert the color bar number into the corresponding concentration value, and calculate the content of free acridine ester, free antibody and acridine ester labeled antibody in the sample to be tested.

[0014] Optionally, the free acridine ester content in step (3) is determined according to... Calculations based on the free antibody content The acridinium ester-labeled antibody content was calculated based on... or calculate.

[0015] An immunochromatographic test strip for monitoring the purification efficiency of acridinium ester-labeled antibodies includes a test strip A, wherein a first detection line T1A and a second detection line T2A, and a control line CA are provided on the nitrocellulose membrane of the test strip A; the first detection line T1A is coated with an anti-acridinium ester antibody, and the second detection line T2A is coated with an antigen that specifically binds to the antibody to be tested.

[0016] An immunochromatographic test strip for monitoring the purification efficiency of acridine ester labeled antibodies includes a test strip B, wherein a first detection line T1B and a second detection line T2B, and a control line CB are provided on the nitrocellulose membrane of the test strip B; the first detection line T1B is coated with an antigen that specifically binds to the antibody to be tested, and the second detection line T2B is coated with an anti-acidine ester antibody.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention, based on immunochromatography, utilizes the specific recognition of the "AE-antibody complex" by anti-acrididine ester antibodies. Through a dual-strip design and a multi-line semi-quantitative method, the colorimetric results can be read directly within 15 minutes, enabling semi-quantitative analysis of free acridine ester, free antibody, and acridine ester-labeled antibody. This method fills the technological gap in the production process of chemiluminescent reagents due to the lack of rapid quality control tools.

[0018] This invention innovatively designs two test strips with reversed detection line sequences (A: anti-AE antibody first, antigen second; B: antigen first, anti-AE antibody second). Through simultaneous equation calculations, it can effectively distinguish between free acridine ester, free antibody, and acridine ester-labeled antibody. Results from examples show that this method can clearly distinguish the differences in the content of the three components in the non-desalted group, desalted group, and ultrafiltration group, overcoming the technical difficulty of traditional methods where "the total luminescence value cannot distinguish the signal source."

[0019] This invention features multiple parallel detection lines on a nitrocellulose membrane, with the number of colored lines visually reflecting the concentration range of the analyte. After calibration, each detection line corresponds to an analyte concentration of approximately 50 pg / mL, allowing operators to quickly assess sample concentration without the need for sophisticated instruments. This "band matching method" retains the simplicity of immunochromatography while achieving semi-quantitative functionality, making it an ideal tool for on-site quality control.

[0020] This invention's test strips utilize colloidal gold immunochromatography technology, offering simple operation (results are read directly after sample addition), requiring no instruments or equipment, and can be performed on-site by ordinary operators. The test strips have low production costs, making them suitable for large-scale deployment and use, and can serve as a routine quality control tool for chemiluminescence reagent production lines.

[0021] The results of the embodiments show that the test strip of the present invention can sensitively distinguish the differences in the effects of three processes: non-desalting, desalting column purification, and ultrafiltration purification: Non-desalting group: highest proportion of free AE (6 / 21), lowest proportion of labeled antibody (2 / 3); Desalting group: moderate proportion of free AE (1 / 10), moderate proportion of labeled antibody (17 / 20); Ultrafiltration group: lowest proportion of free AE (1 / 20), highest proportion of labeled antibody (18 / 20). This result indicates that the test strip of the present invention can effectively evaluate the advantages and disadvantages of different purification processes and provide data support for process optimization.

[0022] Compared to affinity chromatography in 1991, this invention requires no expensive equipment or complex operation, making it more suitable for rapid on-site quality control; compared to capillary electrophoresis, this invention requires no professional technicians, significantly reducing costs; compared to HPLC, this invention requires no large instruments, shortening detection time from several hours to 15 minutes; compared to the traditional total luminescence method, this invention can distinguish signal sources and achieve separate quantification. Attached Figure Description

[0023] Figure 1 This invention provides an immunochromatographic test strip A for monitoring the purification efficiency of acridine ester-labeled antibodies; Figure 2 This invention provides an immunochromatographic test strip B for monitoring the purification efficiency of acridine ester-labeled antibodies. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments provide a more detailed description of the invention. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. I. Implementation Examples

[0026] Example 1: An immunochromatographic test strip for monitoring the purification efficiency of acridine ester-labeled MYO antibody and its application. 1. Materials and Reagents Acridine esters (AE): Suzhou Yake Technology Co., Ltd., Product No. Y0266 MYO antibody (mouse anti-human myoglobin monoclonal antibody): Zhejiang Zhunze Biotechnology Co., Ltd., product number ZC2103302 Anti-AE antibody (AE-TSH bispecific antibody): Anhui Qiancheng Biotechnology Co., Ltd., Product No. MC04101E63 MYO antigen (human myoglobin): homemade or commercially available Rabbit anti-mouse IgG antibody: Xibao Biotechnology (Shanghai) Co., Ltd., catalog number DCY0259B Goat anti-rabbit IgG antibody: Shenzhen Heavy Chain Biotechnology Co., Ltd., product number HR213-S1 chloroauric acid Trisodium citrate: analytical grade, Sinopharm Group Nitrocellulose membrane (NC membrane), glass cellulose membrane (sample pad), polyester cellulose membrane (gold standard pad), absorbent pad, PVC base plate: commercially available 2. Preparation of test strips 2.1 Preparation of colloidal gold solution (1) Soak the colloidal gold conical flask and measuring cylinder in aqua regia overnight, rinse them with deionized water six times, and dry them for later use.

[0027] (2) Prepare a 1% sodium citrate solution (reducing solution) and a 1% chloroauric acid solution (oxidizing solution) using ultrapure water.

[0028] (3) Add 500 mL of ultrapure water to an Erlenmeyer flask and heat to boiling with stirring. Add 5 mL of 1% trisodium citrate solution, and after 3 min, add 5 mL of 1% chloroauric acid solution, maintaining a gentle boil for 15 min. After cooling to room temperature, add the evaporated water to a final volume of 500 mL to obtain a wine-red transparent colloidal gold solution. The maximum absorption wavelength was detected by a UV spectrophotometer to be 520 nm, and the colloidal gold particle size was approximately 40 nm.

[0029] 2.2 Preparation of gold-labeled antibodies (1) Take 10 mL of colloidal gold solution and adjust the pH to 8.5 with 0.2 mol / L K2CO3 solution. (2) Add the optimal concentration of rabbit anti-mouse IgG antibody (determined to be 8 μg / mL in preliminary experiments), mix well and let stand for 1 h.

[0030] (3) Add 1 mL of 10% BSA solution and seal for 1 h.

[0031] (4) Centrifuge at 12,000 rpm for 25 min at 4℃, discard the supernatant, resuspend the precipitate in 1 mL of gold standard dilution solution, and store at 4℃ for later use.

[0032] 2.3 Pretreatment of Sample Pads and Gold Label Pads (1) Sample pad treatment: Immerse the glass cellulose membrane in the sample pad treatment solution (containing 0.5% Tris, 0.5% Tween-20, and 1% BSA) for 30 min, remove and drain, dry at 37℃ for 3 h, and seal for storage.

[0033] (2) Gold labeling treatment: Immerse the polyester cellulose membrane in the gold labeling treatment solution (containing 0.5% Tris, 1% Tween-20, and 1% BSA) for 30 min, remove and drain, dry at 37°C overnight, and store in a sealed container.

[0034] 2.4 Marking Spray Film Attach the NC film to the PVC base plate and use a marking sprayer to mark the lines: Test strip A: T1 line: Anti-AE antibody (0.6 mg / mL), draw 10 parallel lines with a line spacing of 1 mm. T2 line: MYO antigen (20 ng / mL), draw 5 parallel lines with a 1 mm spacing between them. Line C: Goat anti-rabbit IgG antibody (1 mg / mL), draw one line. Test strip B: T1 line: MYO antigen (20 ng / mL), draw 10 parallel lines with a 1 mm spacing between them. T2 line: Anti-AE antibody (0.6 mg / mL), draw 5 parallel lines with a 1 mm spacing between them. Line C: Goat anti-rabbit IgG antibody (1 mg / mL), draw one line. After marking, dry at 37°C for 3 hours, then store in a sealed container.

[0035] 2.5 Assembly and Cutting The prepared sample pad, gold-labeled pad (sprayed with gold-labeled antibody), NC membrane, and absorbent pad are sequentially pasted onto a PVC base plate, with each layer overlapping by 2 mm. Use an automatic cutter to cut into 8 mm wide test strips, then seal and dry for storage. Test strip A is shown below. Figure 1 As shown, test strip B is as follows Figure 2 As shown.

[0036] 3. Establishment of a standard curve for concentration-number of colorimetric lines 3.1 Free AE Standard Curve Acridinium ester was dissolved in DMSO to prepare a 4 mg / mL stock solution, which was then diluted with protective buffer to a series of concentrations of 400, 200, 100, 50, 25, 12.5, and 6.25 pg / mL. 200 μL of each concentration of standard was added to test strip B, and after chromatography for 15 min, the number of colored lines at T2 was read to establish a correlation between "free AE concentration and number of colored lines". The results showed that each colored line at T2 corresponds to approximately 50 pg / mL of free AE.

[0037] 3.2 Free antibody standard curve MYO antibodies were diluted with protective buffer to a series of concentrations of 400, 200, 100, 50, 25, 12.5, and 6.25 pg / mL. 200 μL of each concentration of standard was added to test strip A, and after chromatography for 15 min, the number of T2 lines was read to establish a correlation between "free antibody concentration and number of lines." The results showed that each T2 line corresponded to approximately 50 pg / mL of free antibody.

[0038] 4. Sample Testing 4.1 Sample Preparation The MYO antibody was labeled with acridinium ester according to standard procedures, and the labeled samples were divided into three groups: Non-desalting group: No purification process is performed after labeling. Desalting group: Purification was performed using a desalting column (gel filtration chromatography column). Ultrafiltration unit: Purification was performed using ultrafiltration centrifuge tubes (30 kDa). 4.2 Sample Dilution Preliminary experiments revealed that the original sample concentration was too high, causing all lines to show color. After optimization, the optimal dilution factor was determined as follows: Non-desalination group: diluted 10 times Desalination group: diluted 3 times Ultrafiltration unit: diluted 8 times 4.3 Detection and Reading Take 200 μL of the diluted sample and add it to test strip A and test strip B respectively. After chromatography for 15 min, read the number of colored lines for each detection line (half a line is recorded as 0.5). The results are shown in Table 1.

[0039]

[0040] 4.4 Content Calculation Calculate the content of each component (based on a 200 μL sample volume) according to the standard curve (each line corresponds to 50 pg / mL): Non-desalination group: Free AE amount = number of B-T2 cells × 50 pg / mL × 0.2 mL = 3 × 50 × 0.2 = 30 pg Free antibody amount = Number of A-T2 antibodies × 50 pg / mL × 0.2 mL = 0.5 × 50 × 0.2 = 5 pg AE-antibody complex quantity = (Number of A-T1 antibodies - Number of B-T2 antibodies) × 50 × 0.2 = (10 - 3) × 50 × 0.2 = 70 pg Or (Number of B-T1 items - Number of A-T2 items) × 50 × 0.2 = (7.5 - 0.5) × 50 × 0.2 = 70pg Desalination group: Free AE amount = 1 × 50 × 0.2 = 10 pg Free antibody amount = 0.5 × 50 × 0.2 = 5 pg Amount of AE-antibody complex = (9.5 - 1) × 50 × 0.2 = 85 pg Ultrafiltration unit: Free AE amount = 0.5 × 50 × 0.2 = 5 pg Free antibody amount = 0.5 × 50 × 0.2 = 5 pg Amount of AE-antibody complex = (9.5 - 0.5) × 50 × 0.2 = 90 pg 4.5 Proportion Calculation Divide the content of each component by the total amount (free AE + free antibody + complex) to obtain the proportion: Non-desalted group: Free AE 30 / 105 ≈ 6 / 21, Free antibody 5 / 105 ≈ 1 / 21, Complex 70 / 105 ≈ 2 / 3 Desalted group: Free AE 10 / 100 = 1 / 10, Free antibody 5 / 100 = 1 / 20, Complex 85 / 100 = 17 / 20 Ultrafiltration group: Free AE 5 / 100 = 1 / 20, Free antibody 5 / 100 = 1 / 20, Complex 90 / 100 = 18 / 20 The results showed that desalting and ultrafiltration could effectively remove free AE, with ultrafiltration being slightly better than desalting; both purification methods resulted in a small loss of active substances.

[0041] Example 2: Applicability verification of different antibody systems Following the method in Example 1, the MYO antibody was replaced with cardiac troponin I (cTnI) antibody, and the MYO antigen was replaced with cTnI antigen, while other conditions remained unchanged, to prepare a test strip for monitoring the purification efficiency of cTnI antibody labeling. The test results for the three groups of samples showed that the test strip could effectively distinguish between different purification effects, indicating that the method of the present invention has versatility.

[0042] II. Comparison Example Comparison with Example 1: Traditional total luminescence method (direct detection without separation) The desalting sample was taken and added directly (100 μL) to a 96-well plate without any separation. The total luminescence value (RLU) was measured using a chemiluminescence analyzer. The RLU was calculated by substituting the RLU into the free AE standard curve, but it could not distinguish between free AE and labeled AE, nor could it calculate the desalting efficiency or apparent binding rate. The results indicate that this method can only obtain a vague total signal and cannot evaluate the purification effect.

[0043] Comparison Example 2: Ordinary test strips without anti-AE antibodies Plain test strips without anti-AE antibodies (coated only with antigen and secondary antibody) were prepared and used to test desalted samples. Results showed that the T1 line was barely visible due to the inability to recognize the AE-antibody complex, thus no information about the complex could be obtained. This demonstrates that anti-AE antibodies are crucial for the specific recognition of labeled antibodies.

[0044] III. Experimental Examples Test Example 1: Precision Test Take the same batch of ultrafiltration samples and repeat the test 6 times. Record the number of color bars for A-T1, A-T2, B-T1, and B-T2, and calculate the RSD. The results are shown in Table 2.

[0045]

[0046] The results show that the interpretation of the number of colored bars is stable and the method has good precision.

[0047] Experimental Example 2: Accuracy Test (Spiked Recovery Rate) Different amounts of free AE standard were added to labeled antibody samples of known concentration, and the free AE content was determined according to the method in Example 1. The recovery rate was calculated. The results are shown in Table 3.

[0048]

[0049] The results showed that the recovery rate was between 95% and 96%, and the method had good accuracy.

[0050] Experimental Example 3: Linear Range and Sensitivity Analysis of the free AE standard curve showed a good linear relationship between the free AE concentration and the number of colorimetric strips in the range of 6.25-400 pg / mL (R²=0.99), with a detection limit of 6.25 pg / mL. Analysis of the free antibody standard curve also showed a good linear relationship between the free antibody concentration and the number of colorimetric strips in the range of 6.25-400 pg / mL (R²=0.99), with a detection limit of 6.25 pg / mL.

[0051] Experimental Example 4: Application of Inter-batch Difference Monitoring The method of this invention was used to monitor five batches of independently produced labeled antibody samples, and the desalting efficiency and apparent binding rate were calculated. The results are shown in Table 4.

[0052]

[0053] The results show that this method can effectively monitor batch-to-batch differences, providing data support for process optimization and quality control.

[0054] 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 immunochromatographic test strip for monitoring the purification efficiency of acridinium ester-labeled antibodies, characterized in that, Includes test strip A and test strip B; The test strip A has a first detection line T1A and a second detection line T2A on its nitrocellulose membrane, as well as a control line CA; the first detection line T1A is coated with an anti-acridoid ester antibody, and the second detection line T2A is coated with an antigen that specifically binds to the antibody to be tested; The test strip B has a first detection line T1B and a second detection line T2B on its nitrocellulose membrane, as well as a control line CB; the first detection line T1B is coated with an antigen that specifically binds to the antibody to be tested, and the second detection line T2B is coated with an anti-acridone ester antibody.

2. The immunochromatographic test strip according to claim 1, characterized in that, The concentration of anti-acrididine ester antibody coated on the first detection line T1A and the second detection line T2B is 0.5-0.8 mg / mL; the concentration of antigen coated on the second detection line T2A and the first detection line T1B is 15-25 ng / mL.

3. The immunochromatographic test strip according to claim 1, characterized in that, The first detection line T1A and the first detection line T1B each consist of 10 parallel detection lines; the second detection line T2A and the second detection line T2B each consist of 5 parallel detection lines.

4. The immunochromatographic test strip according to claim 1, characterized in that, The anti-acridone ester antibody is an anti-acridone ester monoclonal antibody or a bispecific antibody.

5. The immunochromatographic test strip according to claim 1, characterized in that, The antigen that specifically binds to the antibody to be tested is the antigen corresponding to the antibody to be tested.

6. A method for preparing the immunochromatographic test strip according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Preparation of colloidal gold solution; (2) Preparation of gold-labeled antibodies: Anti-immunoglobulin antibodies were labeled with colloidal gold; (3) Spray the gold-labeled antibody onto the gold-labeled pad; (4) Streak anti-acridone ester antibody, antigen and quality control antibody on nitrocellulose membrane to form detection line and quality control line respectively; (5) Assemble the sample pad, gold label pad, nitrocellulose membrane and absorbent pad in sequence on the base plate and cut them into test strips.

7. A method for monitoring the purification efficiency of acridinium ester-labeled antibodies using the immunochromatographic test strip according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Load the sample to be tested onto test strip A and test strip B respectively, and read the colorimetric results after the chromatographic reaction; (2) Record the number N of the first detection line T1A of test strip A respectively. A1 The second detection line T2A has N colorimetric strips. A2 And the number of color development lines N of the first detection line T1B of test strip B. B1 The number of colorimetric strips (N) for the second detection line T2B B2 ; (3) Based on the pre-established concentration-color bar number standard curve, convert the color bar number into the corresponding concentration value, and calculate the content of free acridine ester, free antibody and acridine ester labeled antibody in the sample to be tested.

8. The method according to claim 7, characterized in that, The free acridine ester content mentioned in step (3) is based on N B2 The free antibody content is calculated based on N. A2 The acridinium ester-labeled antibody content is calculated based on (N) A1 - N B2 ) or (N B1 - N A2 )calculate.

9. An immunochromatographic test strip for monitoring the purification efficiency of acridinium ester-labeled antibodies, characterized in that, The test strip A includes a first detection line T1A and a second detection line T2A on a nitrocellulose membrane, as well as a control line CA. The first detection line T1A is coated with an anti-acridoid ester antibody, and the second detection line T2A is coated with an antigen that specifically binds to the antibody to be tested.

10. An immunochromatographic test strip for monitoring the purification efficiency of acridinium ester-labeled antibodies, characterized in that, The test strip B includes a first detection line T1B and a second detection line T2B on a nitrocellulose membrane, as well as a control line CB. The first detection line T1B is coated with an antigen that specifically binds to the antibody to be tested, and the second detection line T2B is coated with an anti-acridone ester antibody.

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