Method for monitoring the purification efficiency of acridinium ester labeled antibodies and use thereof

Abstract

The application belongs to the field of biological medicine detection, and particularly relates to a monitoring method for purification efficiency of acridinium ester labeled antibody, comprising the following steps: (1) sample separation: separating free acridinium ester from acridinium ester labeled antibody in a to-be-detected sample by using ultrafiltration centrifugation or high performance liquid chromatography; (2) establishing a standard curve; (3) chemiluminescence detection and quantification; and (4) efficiency calculation. The monitoring method for purification efficiency of acridinium ester labeled antibody and the application thereof can distinguish and separately quantify the monitoring method for free acridinium ester and bound acridinium ester, and evaluate the desalting efficiency and apparent binding rate of a purification process.

Description

Technical Field

[0001] This invention relates to the field of biotechnology detection, and in particular to a method for monitoring the purification efficiency of acridinium ester-labeled antibodies and its application. Background Technology

[0002] Acridinium esters (AEs), as a class of highly efficient chemiluminescent labels, possess advantages such as high quantum yield, low background signal, and good stability, and have been widely used in immunoassays, nucleic acid detection, and clinical diagnostics. In the development of chemiluminescent immunoassay reagents, conjugating acridinium esters with antibodies to form labeled antibody complexes is one of the key steps in reagent preparation. The NHS ester group of acridinium ester can form stable amide bonds with the primary amino groups on antibody molecules, thereby achieving covalent labeling.

[0003] After the labeling reaction is complete, appropriate purification methods are needed to remove unreacted free acridine esters to avoid interference with subsequent detection. Commonly used purification methods include desalting column chromatography (gel filtration chromatography) and ultrafiltration centrifugation. Desalting columns utilize molecular sieve principles to separate labeled antibodies from free acridine esters based on molecular weight differences; ultrafiltration uses a filter membrane with a specific molecular weight cutoff to separate small-molecule free acridine esters from large-molecule labeled antibodies through centrifugation. However, the purification effects of these methods are unstable, with significant batch-to-batch variations. The purification effect of desalting columns is affected by various factors such as column packing conditions, batch variations, operational scale, and operator technique, leading to large fluctuations in the residual amount of free acridine esters in different batches of labeled reagents, ultimately affecting the consistency and reliability of the reagents. Ultrafiltration also suffers from issues such as membrane batch variations and the need for controlled centrifugation conditions. Currently, there is a lack of a rapid, quantitative method to evaluate the actual effect of each desalting purification process, making it impossible to accurately determine how much acridine 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 variations.

[0004] Existing patent literature (CN200710008800.8) discloses a method for detecting acrid ester labels using capillary electrophoresis coupled with chemiluminescence. This method utilizes capillary electrophoresis to separate the sample to be tested, followed by chemiluminescence detection to achieve qualitative and quantitative analysis. However, this method has significant limitations: ① Capillary electrophoresis equipment is expensive and complex to operate, making it unsuitable as a quality control method for routine production processes; ② Only the effluent is detected, without collecting fractions to quantify free and bound components separately; ③ It cannot simultaneously obtain the accurate content of free acrid ester and labeled antibody, nor can it calculate process indicators such as desalting efficiency.

[0005] Currently, there is a lack of rapid and accurate quantitative analysis methods for the content of free acridine ester and labeled antibody in purified samples. Existing techniques mostly rely on empirical judgment or indirect assessment, such as roughly estimating the labeling effect through luminescence values, but they cannot distinguish whether the luminescence signal comes from the labeled antibody or the free acridine ester, making it difficult to accurately assess the purification efficiency. Summary of the Invention

[0006] The purpose of this invention is to provide a method for monitoring the purification efficiency of acridine ester-labeled antibodies and its application, which can distinguish and quantify free acridine esters and bound acridine esters, and evaluate the desalting efficiency and apparent binding rate of the purification process.

[0007] To address the aforementioned technical problems, the method for monitoring the purification efficiency of acridine ester-labeled antibodies and its application provided by this invention are implemented as follows:

[0008] A method for monitoring the purification efficiency of acridinium ester-labeled antibodies, comprising the following steps:

[0009] (1) Sample separation: The free acridine ester and the acridine ester-labeled antibody in the sample to be tested were separated by ultrafiltration centrifugation or high performance liquid chromatography. The filtrate containing the free acridine ester and the retentate containing the acridine ester-labeled antibody were collected respectively. (2) Establishment of standard curves: (2a) Establish a standard curve of free acridine ester concentration-luminescence value. (2b) Establish a standard curve of acridine ester-labeled antibody concentration-luminescence value. (3) Chemiluminescence detection and quantification: The filtrate and retentate collected in step (1) were subjected to chemiluminescence detection. The content of free acridine ester in the filtrate was calculated according to the standard curve established in step (2a). The content of bound acridine ester in the retentate was calculated according to the standard curve established in step (2b). (4) Efficiency calculation: The desalting efficiency and / or apparent binding rate were calculated according to the content of free acridine ester and the content of bound acridine ester obtained in step (3).

[0010] Optionally, the ultrafiltration membrane used in the ultrafiltration centrifugation method described in step (1) has a molecular weight cutoff of 10kDa to 30kDa.

[0011] Optionally, the establishment of the acridinium ester-labeled antibody concentration-luminescence value specific standard curve in step (2b) includes the following sub-steps:

[0012] (i) Preparation of acridine ester-labeled antibody standards: Acridine ester is fully bound to the antibody through a labeling reaction;

[0013] (ii) The concentration of acridine ester-labeled antibody in the standard obtained in step (i) was determined by dual-wavelength ultraviolet spectrophotometry;

[0014] (iii) The standard sample quantitatively calibrated in step (ii) is serially diluted, the chemiluminescence value is detected, and a concentration-luminescence value standard curve is established.

[0015] Optionally, the dual-wavelength ultraviolet spectrophotometry in step (ii) includes: measuring the absorbance of the sample at 280 nm and 250 nm, combining the known molar extinction coefficients of the antibody and acridine ester, and solving simultaneous equations to calculate the concentration and molar binding ratio of the acridine ester-labeled antibody.

[0016] Optionally, the desalination efficiency in step (4) is calculated using the following formula: Desalination efficiency = (1 - mass of free acridine ester in the desalted sample / total mass of acridine ester added) × 100%.

[0017] Optionally, the apparent binding rate in step (4) is calculated using the following formula: Apparent binding rate = (mass of acridine ester bound in the retentate / total mass of acridine ester added) × 100%.

[0018] Optionally, the sample to be tested is an acridinium ester-labeled antibody sample purified by ultrafiltration.

[0019] Application of a method for monitoring the purification efficiency of acridinium ester-labeled antibodies in the quality control of chemiluminescent immunoassay reagent production.

[0020] Beneficial effects

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. This invention achieves separate quantification of free and bound acridine esters, solving the problem of signal aliasing. This invention utilizes ultrafiltration centrifugation to physically separate free acridine esters from labeled antibodies based on molecular weight differences. The filtrate and retentate are collected separately, and then quantified using two independent standard curves. This technical solution overcomes the technical bottleneck of existing technologies where "total luminescence value cannot distinguish the signal source," achieving accurate quantification of free and bound acridine esters in the sample, laying the foundation for precise evaluation of purification efficiency.

[0023] 2. This invention establishes a dual standard curve quantitative system to ensure the accuracy and reliability of quantification.

[0024] This invention establishes two independent quantitative curves: a standard curve for free acridine ester concentration versus luminescence value and a specific standard curve for acridine ester-labeled antibody concentration versus luminescence value. The specific standard curve for the labeled antibody was established using dual-wavelength ultraviolet spectrophotometry to accurately calibrate the standard, solving the technical challenge of "not being able to obtain pure labeled antibody standards." Example data show that this method has a wide linear range and high goodness of fit, and can meet the quantitative requirements of samples with different concentrations.

[0025] 3. Define and calculate two key quantitative indicators to transform experience-based control into data-driven control.

[0026] This invention defines two key quantitative indicators that can reflect the purification effect: desalting efficiency: directly reflects the effect of the desalting column or ultrafiltration process in removing free small molecules; apparent binding rate: reflects the efficiency of the overall labeling and purification process.

[0027] The results of Example 1 show that the desalting efficiency reached 98.52% and the apparent binding rate reached 75.47%; the results of Example 2 show that the desalting efficiency reached 99.35% and the apparent binding rate reached 92.34%. Through these two quantitative indicators, the purification process can be upgraded from traditional "experience-based control" to "data-based control," effectively monitoring and reducing batch-to-batch variability.

[0028] 4. It is easy to operate and inexpensive, making it suitable as a routine quality control method.

[0029] The main consumable of this invention is ultrafiltration centrifuge tubes, eliminating the need for large-scale specialized equipment and allowing implementation in a conventional laboratory. The operation is simple, with a short testing cycle, enabling evaluation within hours and guiding process adjustments. It is highly compatible with existing processes and can be directly embedded as a quality control point without disrupting normal production. Compared to complex methods such as capillary electrophoresis, this invention is more suitable for routine quality control in production environments.

[0030] 5. Overcome the limitations of existing technologies and achieve process standardization.

[0031] Compared to the conceptual approach presented in the 1991 literature, this invention provides a complete technical solution and specific calculation methods, transforming the abstract concept of "monitoring labeled antibody yield" into an industrially implementable quality control tool. Compared to the capillary electrophoresis method patented by Fuzhou University, this invention is simpler to operate, lower in cost, and more suitable as a routine monitoring method in the production process. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] I. Raw Materials and Reagents

[0035]

[0036] II. Instruments and Equipment

[0037]

[0038] Example 1: Monitoring the purification efficiency of acridinium ester-labeled antibodies using ultrafiltration centrifugation

[0039] 1. Sample separation

[0040] (1) MYO antibody was labeled with acridine ester according to the labeling and purification process of acridine ester labeled antibody, and the antibody was divided into three groups for non-desalting, desalting and ultrafiltration respectively.

[0041] Acridinium ester-labeled antibody (50 mL):

[0042] Remove the acridine ester, labeling buffer, and antibody from the container and allow them to return to room temperature for later use.

[0043] The lysine solution was prepared with labeling buffer to a concentration of 13.72 mg / ml (prepared fresh for immediate use).

[0044] Take a 0.5 mL centrifuge tube and add (100-200 / antibody concentration-1.98) μL of labeling buffer, 1.98 μL of acridinium ester, and 200 / antibody concentration μL of antibody to make the final antibody concentration 2 mg / mL (2 μg / μL). Mix at 1000 rpm and 25 °C in the dark for 3 h.

[0045] Add 20 μL of the prepared lysine solution and mix at 25 °C for 20 min.

[0046] Desalination: Turn on the computer and ion exchange chromatography analyzer. Place the sample loading end of the tubing into pure water and turn on the constant flow pump to remove air bubbles. After fixing the G-25 desalination column, connect the upper and lower tubing (connect the upper tubing first, then the constant flow pump; connect the lower tubing (with metal connector) to the UV signal collector; when disassembling, connect the lower tubing first, then the upper tubing, ensuring there are no air bubbles in the tubing). (For new columns, use 0.1M...) Wash with NaOH solution at a flow rate of 6 mL / min for 20 min (this step can be omitted for old columns). (The constant flow pump must be paused when changing the wash buffer to prevent air bubbles from entering the tubing, column, and detector.) Then wash with purified water at a flow rate of 6 mL / min for 10 min, followed by washing with desalting buffer at a flow rate of 6 mL / min for 15-20 min until the signal reaches equilibrium. Perform calibration (T: 260%, >A: -0.40 → T: 100%, >A: 0.00). Sample loading: Pour a small beaker of desalting buffer for later use. For samples less than 300 μL, add desalting buffer to bring the total to 300 μL, mix well, and then load the sample. Before loading, rinse the sample outlet with pure water, pause the constant flow pump, and adjust the flow rate to 0.5 mL / min. Load the sample at 3 mL / min, closely monitoring the sample. Stop the constant flow pump the instant the sample is completely absorbed, and place the injection port into the desalting buffer in the small flask. Set the constant flow pump to 3 mL / min for elution. Closely monitor the absorbance peaks and the chromatography analyzer screen (from T: 100%, >A: 0.00 → T: 99%-95%, >A: 0.02). When the peak begins to rise / the analyzer reading begins to change, start collecting the target liquid (in a light-protected centrifuge tube). Stop collecting when the peak reaches a plateau. Dilute the target liquid with acridinium ester preservation solution to a final antibody concentration of 4 μg / mL, then filter through a 0.45 μm aqueous filter membrane. Label the filtered solution and store at 2-8℃.

[0047] After desalting, rinse the injection port with pure water, wipe the surface of the injection port dry with a paper towel, immerse the injection port in 0.1M NaOH solution and wash for 20 min at a flow rate of 6 mL / min, then wash with pure water at the same flow rate for 10 min, and finally wash with 20% ethanol at the same flow rate for 10 min. Disconnect the upper and lower tubing, seal the chromatography column, and store at 2-8℃.

[0048] (2) Pretreatment of ultrafiltration tube: Rinse with 3 mL of buffer 1 for 1 min, centrifuge at 4℃ and 8000×g for 10 min, and remove all liquids in the tube with a pipette; Rinse with 3 mL of buffer 2 for 3 min, centrifuge at 4℃ and 8000×g for 10 min, and remove all liquids in the tube with a pipette.

[0049] (3) Take 3 mL of sample and add it to an ultrafiltration centrifuge tube (Merck, Amicon® Ultra, 30 kDa MWCO), and centrifuge at 4℃ and 8000×g for 10 min. After centrifugation, the lower filtrate collection tube contains the filtrate (containing free acridine ester), which is then transferred to another filtrate collection EP tube.

[0050] (4) Add 2000 μL of buffer solution and gently rinse the ultrafiltration tube by pipetting. Centrifuge at 8000 × g for 10 min at 4 °C. After centrifugation, the lower filtrate collection tube contains the filtrate (containing free acridine ester). Transfer it to the upper filtrate collection EP tube. Repeat this step three times.

[0051] (5) Transfer the upper concentrated sample to the concentrated collection tube to obtain the choke fluid (containing labeled antibody).

[0052] (6) Store the two components in a refrigerator at 2-8℃ away from light.

[0053] 2. Establish a standard curve

[0054] (1) Plotting the standard curve of "acrididium ester concentration-RLU":

[0055] Acridinium ester was dissolved in DMSO to a concentration of 4 mg / mL. This solution was then diluted 1000-fold with acridinium ester-labeled antibody protective buffer to obtain a secondary stock solution with a concentration of 4 μg / mL. This stock solution was subsequently diluted 10-fold downwards to obtain standards with concentrations of 400,000, 40,000, 400, 40, and 4 pg / mL. 100 μL of each standard was added to a 96-well plate, and the results were detected using a microplate chemiluminescence detector. The results are shown in Table 1.

[0056] Table 1. Acridinium ester concentration-RLU standard curve data

[0057]

[0058] Note: Reference 7 exhibits a hook effect; therefore, only the data from the first seven points are used when fitting the curve.

[0059] The fitting equation obtained by using four parameters is: y = (222747507.24434 - 4897.49) / (1+(x / 74010.5)^-0.929106) + 4897.49, R² = 0.9987, which shows that the standard curve has a wide linear range and high fitting degree, and can meet the quantitative requirements of samples with different concentrations.

[0060] (2) Establishment of a standard curve specific to the labeled antibody:

[0061] ① Preparation of standard: Labeling was performed according to the average ratio of 5 acridinium ester molecules bound to one antibody (0.492 μL acridinium ester was added for every 100 μg MYO antibody) to ensure that almost all acridinium esters were bound to the antibody.

[0062] ② Absolute quantification: Using pure antibody solutions and pure acridil ester stock solutions of known concentrations, the absorbance at 280 nm and 250 nm was measured on a spectrophotometer, respectively, and the corresponding εl values ​​were obtained (Table 2). The absorbance of the standard samples at 280 nm and 250 nm was measured, and the results were substituted into Equations 1 and 2 to solve the simultaneous equations and calculate the concentration of the standard (Table 3).

[0063] Table 2. Molar extinction coefficient and optical path product (εl)

[0064]

[0065] Formula 1:

[0066] Formula 2:

[0067] Table 3. Quantitative results of the standard sample using the dual-wavelength method

[0068]

[0069] ③ Establishing a curve: The quantitatively calibrated standard was serially diluted, and the chemiluminescence value was detected to establish a "labeled antibody concentration-RLU" standard curve. The results are shown in Table 4.

[0070] Table 4. Labeled antibody concentration-RLU standard curve data

[0071]

[0072] The fitting equation obtained by using four parameters is: y = (185894827.04804 - 4868.68) / (1+(x / 100884)^-1.04124) + 4868.68, R² = 0.9991, which shows that the standard curve has a wide linear range and high fitting degree, and can meet the quantitative requirements of samples with different concentrations.

[0073] 3. Sample Testing and Data Processing

[0074] The filtrate obtained in step 1 and the retentate diluted 100 times were subjected to chemiluminescence detection, and the RLU values ​​were recorded. The contents were calculated by substituting the values ​​into the corresponding standard curves. The results are shown in Table 5.

[0075] Table 5 Sample Detection and Efficiency Calculation Results

[0076]

[0077] Example 2: Monitoring the purification efficiency of acridine ester-labeled antibody by HPLC-SEC

[0078] 1. Establish a standard curve: consistent with Example 1.

[0079] 2. Sample Analysis and Quantification

[0080] (1) The MYO antibody and acridine ester were labeled and desalted according to the labeling and purification process of acridine ester labeled antibody.

[0081] (2) The desalted sample was injected into the HPLC-SEC system (Agilent 1260 Infinity II Bio-SEC system) under the following chromatographic conditions:

[0082] Column: TSKgel G3000SWXL (7.8 mm × 30 cm)

[0083] Mobile phase: 0.1 mol / L phosphate buffer (pH 7.0)

[0084] Flow rate: 1.0 mL / min

[0085] Detection wavelengths: 280nm, 250nm

[0086] Injection volume: 100 μL

[0087] (3) Chromatographic separation results: Peak 1 (retention time 1 min 08 s) corresponds to the polymer; Peak 2 (retention time 2 min 12 s) corresponds to the antibody-acridine ester conjugate; Peak 3 (retention time 4 min 35 s) corresponds to the free acridine ester.

[0088] (4) Collecting fractions: Collect one tube every 30 seconds, for a total of 20 tubes. Perform chemiluminescence detection on the fractions in each tube. The results are shown in Table 6.

[0089] Table 6. HPLC-SEC fraction luminescence detection results

[0090]

[0091] Note: The sample collection time is calculated from the start of the rise of the first peak, and the flow rate is 1.0 mL / min.

[0092] (5) Quantitative calculation: Substitute the luminescence value of the fraction containing the “AE-antibody complex” peak (6-8) into the labeled antibody standard curve to calculate the labeled acridine ester-antibody concentration; substitute the luminescence value of the fraction containing the “free acridine ester” peak (11-14) into the free acridine ester standard curve to calculate the free acridine ester concentration. The results are shown in Table 7.

[0093] Table 7 Efficiency Calculation Results of HPLC-SEC Method

[0094]

[0095] II. Comparison Example

[0096] Compare with Example 1: Traditional total luminescence value method (without separation)

[0097] Samples were prepared according to the labeling steps in Example 1, but without ultrafiltration separation. The desalted samples were directly subjected to chemiluminescence detection, and the total RLU value was recorded. Substituting the total RLU value into the free acridine ester standard curve to estimate the "total acridine ester equivalent" failed to distinguish between free and bound components, and further prevented the calculation of desalting efficiency and apparent binding rate. The results indicate that this method can only obtain a vague total luminescence signal and cannot evaluate the purification effect.

[0098] Comparison Example 2: Using only the free AE standard curve (label-free antibody-specific curve)

[0099] The sample was processed according to the separation steps in Example 1, but the retentate (containing labeled antibody) was quantified using only the free acridine ester standard curve. The results showed that, due to the difference in luminescent properties between the labeled antibody and the free acridine ester, the calculated "bound acridine ester content" deviated from the true value by more than 30%, making accurate quantification impossible.

[0100] Comparison with Example 3: Ultrafiltration separation only, but without establishing separate standard curves.

[0101] The samples were processed according to the separation steps of Example 1, but instead of establishing separate standard curves for the filtrate and retentate, the same standard curve (free AE standard curve) was used to quantify both components. The results showed that the quantification results for the retentate deviated significantly from the true values, making it impossible to accurately calculate the desalination efficiency and apparent binding rate.

[0102] III. Experimental Examples

[0103] Experimental Example 1: Methodological Validation

[0104] 1. Precision test

[0105] The same batch of samples was used to repeat the measurement 6 times according to the method in Example 1. The relative standard deviation (RSD) of desalination efficiency and apparent binding rate was calculated. The results are shown in Table 8.

[0106] Table 8 Precision test results (n=6)

[0107]

[0108] The results showed that the RSD of both the desalination efficiency and the apparent binding rate was less than 0.2%, indicating that the method had good precision.

[0109] 2. Accuracy test (spiking recovery rate)

[0110] A known amount of free acridine ester standard was added to a labeled antibody sample of known concentration, and the content of free acridine ester in the spiked sample was determined according to the method in Example 1. The recovery rate was calculated, and the results are shown in Table 9.

[0111] Table 9 Results of Spike Recovery Test

[0112]

[0113] The results showed that the spiked recovery rate was between 97.5% and 101.0%, indicating that the method has good accuracy.

[0114] 3. Linear range and sensitivity

[0115] Analysis of the standard curve for free acridine ester showed a linear range of 4–400,000 pg / mL, covering five orders of magnitude; the limit of detection (S / N=3) was 1.2 pg / mL, and the limit of quantitation (S / N=10) was 4.0 pg / mL. Analysis of the standard curve for labeled antibodies showed a linear range of 7.46–746,246 pg / mL, covering five orders of magnitude; the limit of detection was 2.0 pg / mL, and the limit of quantitation was 7.0 pg / mL. The results indicate that this method has a wide linear range and high sensitivity, and can meet the quantitative requirements of samples with different concentrations.

[0116] 4. Ultrafiltration membrane recovery rate test

[0117] Free acridinium ester solution and labeled antibody solution of known concentration were subjected to ultrafiltration and centrifugation, and the recovery rate of the target analyte in the filtrate and retentate was determined. The results are shown in Table 10.

[0118] Table 10 Results of Ultrafiltration Membrane Recovery Test

[0119]

[0120] The results showed that the adsorption loss of free acridinium ester and labeled antibody by the ultrafiltration membrane was less than 2.5%, which did not affect the quantitative accuracy.

[0121] Experimental Example 2: Comparison of the effects of different purification processes

[0122] The method of this invention was used to monitor samples treated by three processes: no desalting, desalting column purification, and ultrafiltration purification. The desalting efficiency and apparent binding rate were compared, and the results are shown in Table 11.

[0123] Table 11 Comparison of the effects of different purification processes

[0124]

[0125] The results showed that the desalting efficiency and apparent binding rate of ultrafiltration purification were higher than those of desalting column purification, which corroborated the results of HPLC-SEC method in Example 2 (99.35% and 92.34%), verifying the reliability of the method of the present invention.

[0126] Experimental Example 3: Application of Inter-batch Difference Monitoring

[0127] The method of this invention was used to monitor five batches of independently produced labeled antibody samples, and the batch-to-batch differences in desalting efficiency and apparent binding rate were calculated. The results are shown in Table 12.

[0128] Table 12 Results of inter-batch difference monitoring

[0129]

[0130] The results show that this method can effectively monitor batch-to-batch differences, providing data support for process optimization and quality control.

[0131] In summary, the dual standard curve system of this invention exhibits a wide linear range, high goodness of fit (R²>0.998), spiked recoveries of 97.5%-101.0%, and precision RSD<0.2%, meeting the accurate quantification requirements for samples of different concentrations. This invention is the first to define and calculate two quantitative indicators: desalination efficiency and apparent binding rate. Example 1 shows a desalination efficiency of 98.52% and an apparent binding rate of 75.47%, while Example 2 shows a desalination efficiency of 99.35% and an apparent binding rate of 92.34%.

[0132] 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. A method for monitoring the purification efficiency of acridinium ester-labeled antibodies, characterized in that, Includes the following steps: (1) Sample separation: The free acridine ester and the acridine ester-labeled antibody in the sample to be tested were separated by ultrafiltration centrifugation or high performance liquid chromatography. The filtrate containing the free acridine ester and the retentate containing the acridine ester-labeled antibody were collected respectively. (2) Establishing a standard curve: (2a) Establish a standard curve of free acridine ester concentration-luminescence value; (2b) Establish a standard curve specific to the concentration of acridinium ester-labeled antibody and its luminescence value; (3) Chemiluminescence detection and quantification: The filtrate and retentate collected in step (1) were subjected to chemiluminescence detection. The content of free acridine ester in the filtrate was calculated according to the standard curve established in step (2a). The content of bound acridine ester in the retentate was calculated according to the standard curve established in step (2b). (4) Efficiency calculation: Based on the free acridine ester content and the bound acridine ester content obtained in step (3), calculate the desalination efficiency and / or apparent binding rate.

2. The method according to claim 1, characterized in that, The ultrafiltration membrane used in step (1) of the ultrafiltration centrifugation method has a molecular weight cutoff of 10kDa to 30kDa.

3. The method according to claim 1, characterized in that, The establishment of the acridinium ester-labeled antibody concentration-luminescence value specific standard curve in step (2b) includes the following sub-steps: (i) Preparation of acridine ester-labeled antibody standards: Acridine ester is fully bound to the antibody through a labeling reaction; (ii) The concentration of acridine ester-labeled antibody in the standard obtained in step (i) was determined by dual-wavelength ultraviolet spectrophotometry; (iii) The standard sample quantitatively calibrated in step (ii) is serially diluted, the chemiluminescence value is detected, and a concentration-luminescence value standard curve is established.

4. The method according to claim 3, characterized in that, The dual-wavelength ultraviolet spectrophotometry method described in step (ii) includes: measuring the absorbance of the sample at 280 nm and 250 nm, combining the known molar extinction coefficients of the antibody and acridine ester, and solving simultaneous equations to calculate the concentration and molar binding ratio of the acridine ester-labeled antibody.

5. The method according to claim 1, characterized in that, The desalination efficiency mentioned in step (4) is calculated according to the following formula: Desalination efficiency = (1 - mass of free acridine ester in the desalted sample / total mass of acridine ester added) × 100%.

6. The method according to claim 1, characterized in that, The apparent binding rate in step (4) is calculated using the following formula: Apparent binding rate = (mass of acridine ester bound in the retentate / total mass of acridine ester added) × 100%.

7. The method according to claim 1, characterized in that, The sample to be tested was an acridinium ester-labeled antibody sample purified by ultrafiltration.

8. The application of the method according to any one of claims 1-7 in the quality control of chemiluminescent immunoassay reagent production.

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