Detection reagents, detection kits, applications and methods of use

Abstract

The present invention relates to the field of biological detection, and in particular to detection reagents, detection kits, applications and methods of use. The present invention provides a detection reagent, including: a coating solution, a washing solution, a blocking solution, an enzyme-labeled secondary antibody, a color developing solution and a stop solution; the coating solution includes: a coating antigen and a coating buffer; the coating antigen includes: 0.5~1µg / unit carrier of recombinant human CD3ε and CD3δ heterodimer proteins. The present invention provides a detection method that is specific for recombinant humanized antibody detection, is simple to operate, has high accuracy and high sensitivity, and can perform large-scale sample detection at a relatively low cost.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to detection reagents, detection kits, applications and use methods. Background Art

[0002] In recent years, with the rapid development of the biomedical field, immunotherapy has gradually become the fourth type of tumor treatment after surgery, radiotherapy, and chemotherapy. Immune cell therapy is to identify and attack tumor cells by activating the patient's own immune cells, and is considered to be a tumor treatment method with great potential. The method is to obtain a large number of T lymphocytes that can recognize tumor-specific antigens through in vitro anti-lymphocyte differentiation antigen antibodies and cytokine combination stimulation. The activated and induced tumor-specific cells can kill tumor cells in the patient's body, thereby achieving the effect of cooperating with radiotherapy and chemotherapy or inhibiting the growth of the primary tumor alone.

[0003] CD3 antibodies play an important role in immune cell therapy. The combined use of CD3 antibodies to stimulate T cells in vitro and simulate the signaling effect of T cell activation is the most widely used method for T cell activation and expansion.

[0004] The preparation technology of CD3 antibodies has gone through four stages of development: the first generation: mouse antibody (momab): hybridoma monoclonal antibody technology, the second generation: human-mouse chimeric antibody (ximab): chimeric antibody and humanized monoclonal antibody technology, the third generation: humanized antibody (zumab): fully human monoclonal antibody technology, the fourth generation: fully humanized antibody (mumab): natural fully humanized monoclonal antibody technology.

[0005] Mouse antibodies can be recognized by the human immune system. Due to their exogenous nature, they can be recognized by the human immune system and trigger human anti-mouse antibody (HAMA) reactions, which not only reduces the efficacy of the drug, but may also cause serious adverse reactions. Humanized antibody modification is to combine the 6 CDRs in the variable region of mouse antibodies with the relatively conservative variable region framework region (FR) of humans. After gene recombination, proteins are expressed, and the degree of humanization can reach 95%. Humanized antibodies reduce the heterologous reactions caused by mouse antibodies in the human body, thereby reducing the risk of anti-mouse antibody effects (HAMA reactions) in the human body. Humanized antibodies retain the high affinity and specificity of the parent mouse monoclonal antibodies, while reducing immunogenicity and possible immune rejection reactions, which helps to improve the therapeutic effect of antibodies.

[0006] The content of CD3 antibodies in immune cell preparations is one of the important parameters for evaluating the quality of cell preparations, and is very important for the safety and quality controllability of cell preparation products. The conventional method currently on the market is the CD3 antibody enzyme-labeled detection kit, which is aimed at natural CD3 antibodies, that is, CD3 molecular antibodies in human serum, plasma and related liquid samples. Its basic principle adopts the double antigen sandwich method, which is to coat the purified CD3 full sequence protein on the enzyme-labeled plate to make a solid phase antigen, add CD3 molecular antibodies (i.e., the sample to be tested) to the coated enzyme-labeled plate, and then combine with the HRP-labeled antigen to form an antigen-antibody-enzyme-labeled antigen complex. After washing, the substrate TMB is added for color development. However, for CD3 antibodies produced by humanized transformation and genetic engineering methods, the affinity and specificity will be reduced, and they cannot be specifically recognized by existing kits, and the sensitivity is low. In addition, the instruments and costs required for high-performance liquid chromatography and chromatography are high, which limits the feasibility of its large-scale application.

[0007] Therefore, it is extremely important to establish an efficient, easy-to-operate and highly accurate method for detecting CD3 antibody content. Summary of the invention

[0008] In view of this, the present invention provides a detection reagent, a detection kit, an application and a method of use. The present invention provides a detection method specifically for recombinant humanized antibody detection, which is simple to operate, highly accurate, highly sensitive, and can perform large-scale sample detection at a relatively low cost.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a detection reagent, including: a coating solution, a washing solution, a blocking solution, an enzyme-labeled secondary antibody, a color developing solution and a stop solution; The coating solution comprises: coating antigen and coating buffer; The coating antigen includes: 0.5-1µg / unit carrier of recombinant human CD3ε and CD3δ heterodimer protein; The enzyme-labeled secondary antibody includes goat anti-human IgG Fc at a dilution multiple of 1:1000 to 1:5000; The coating buffer comprises: 0.05 mol / L carbonate buffer; the washing solution comprises: 0.01 M PBST solution containing 0.05% Tween-20; the blocking solution comprises: 0.01 M PBST solution containing 2% BSA.

[0010] In some embodiments of the present invention, in the above-mentioned detection reagent, the coating antigen includes: 5 μg / mL, 8 μg / mL or 10 μg / mL of recombinant human CD3ε and CD3δ heterodimeric proteins.

[0011] In some embodiments of the present invention, in the above detection reagent, the enzyme-labeled secondary antibody includes goat anti-human IgG Fc at a dilution factor of 1:5000.

[0012] In some embodiments of the present invention, in the above-mentioned detection reagent, the color developing solution includes: TMB color developing solution.

[0013] In some embodiments of the present invention, in the above-mentioned detection reagent, the stop solution includes: 2 mol / L H2SO4 solution.

[0014] The present invention also provides the use of the detection reagent in any of the following items: (1) preparing a product for detecting the content of humanized CD3 antibodies; and / or (2) detecting the content of humanized CD3 antibodies; and / or (3) preparing a product for detecting the residues of humanized CD3 antibodies in immune cell preparations; and / or (4) detecting the residues of humanized CD3 antibodies in immune cell preparations.

[0015] In some embodiments of the present invention, in the above application, the immune cell preparation includes: an immune cell preparation activated in vitro.

[0016] The present invention also provides a detection kit, comprising: the above detection reagent.

[0017] The present invention also provides a method for using the above detection kit, comprising the following steps: S1: Calibration of standard: obtain standard solution; S2: coating: coating the culture carrier with the coating antigen; S3: blocking: taking the washing solution to wash the coated culture carrier, adding the blocking solution, and incubating; S4: adding test samples: taking the washing solution to wash the culture carrier after incubation in S3, adding the standard solution and the test solution, and incubating; S5: adding enzyme-labeled secondary antibody: taking the washing solution to wash the culture carrier after incubation in S4, adding the enzyme-labeled secondary antibody, and incubating; S6: Adding substrate for display: taking the washing solution to wash the culture carrier after incubation in S5, adding the color developing solution, and incubating; S7: Termination: adding the culture carrier after incubation in S6 to the termination solution; S8: OD value determination: detect absorbance value; S9: Curve fitting: According to the OD value and theoretical concentration of the standard curve, fit the standard curve to obtain the regression equation and R value, and calculate the test sample concentration according to the regression equation.

[0018] In some embodiments of the present invention, in the above-mentioned method of use, the test article is obtained by subjecting PBMC cells to a first activation, a second activation, culturing and collecting cells; the first activation uses CD3 monoclonal antibody; the second activation uses: rhIL-2, rhIL-4, rhG-CSF, rhTNF-α and rhIFN-γ.

[0019] In some embodiments of the present invention, in the above-mentioned method of use, the concentration of the CD3 monoclonal antibody is 2 μg / mL; the concentrations of the cytokines are: 600 IU / mL of rhIL-2, 60 IU / mL of rhIL-4, 6000 IU / mL of rhG-CSF, 300 IU / mL of rhTNF-α and 6000 IU / mL of rhIFN-γ.

[0020] In some embodiments of the present invention, in the above-mentioned method of use, the first activation time is 3 hours; the second activation time is 5 hours.

[0021] In some embodiments of the present invention, the incubation time in the above-mentioned method S5 is 30-60 minutes and the temperature is 10-36°C.

[0022] In some embodiments of the present invention, the incubation time in the above method S5 is 60 minutes and the temperature is 36°C.

[0023] In some embodiments of the present invention, the incubation time in the above-mentioned method S6 is 13 to 15 minutes.

[0024] In some embodiments of the present invention, the fitting method described in the above-mentioned method S9 adopts five-parameter fitting.

[0025] In some embodiments of the present invention, the test article in the above-mentioned method of use contains humanized CD3 antibody.

[0026] The detection method and kit for the content of recombinant humanized CD3 antibodies provided by the present invention are suitable for the content detection of cell preparations prepared with recombinant humanized CD3 antibodies as raw materials. Experimental data show that the recombinant humanized CD3 antibodies detected by this method have high accuracy and can accurately reflect the trace residues of recombinant humanized CD3 antibodies in the sample. The detection range can reach 25-1600 pg / mL, and the relative recovery rate (RE%) is between 80% and 120%. The detection method is simple to operate, efficient and accurate, and provides accurate data reference for objectively evaluating the quality of cell therapy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0028] Figure 1 Shows the evolutionary diagram of CD3 antibody;

[0029] Figure 2 The standard curve of four-parameter fitting is shown;

[0030] Figure 3 The standard curve of five-parameter fitting is shown;

[0031] Figure 4 The standard curve of CD3 antibody detection kit (kit 1) is shown;

[0032] Figure 5 The standard curve of CD3 antibody detection enzyme-linked immunosorbent assay kit (Kit 2) is shown;

[0033] Figure 6 It shows the standard curve of the enzyme-linked immunosorbent assay kit (Kit 3) for detecting the content of anti-CD3 antibodies;

[0034] Figure 7 The standard curve of ResDetect CD3 antibody quantitative detection kit (Kit 4) is shown; where: y = (0.0444-11.8) / [1+(x / 3640)^1.09]+11.8, R 2 =1. DETAILED DESCRIPTION

[0035] The invention discloses a detection reagent, a detection kit, an application and a using method.

[0036] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0037] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0038] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0039] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0040] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0041] The test principle of the present invention:

[0042] The content of recombinant humanized CD3 antibody was detected by indirect ELISA. The recombinant human CD3ε and CD3δ heterodimer proteins were coated on the ELISA plate. The recombinant humanized CD3 antibody in the test sample and standard sample could bind to the protein to form an antigen-antibody complex. The free components were removed and the goat anti-human IgG Fc detection antibody (secondary antibody) labeled with horseradish peroxidase (HRP) was added. The antibody bound to the antibody in the complex to form an antigen-antibody-antibody complex. The free components were removed and the color substrate (TMB) was added. The horseradish peroxidase bound to the complex would turn the colorless color developer blue. The color was proportional to the concentration of the recombinant humanized CD3 antibody. It turned yellow after adding the stop solution. The solution had an absorption peak at 450nm. The OD value of 630nm was detected at the same time as the calibration wavelength. By detecting the OD value at 450nm and 630nm, the OD value measured at 450nm minus the OD value measured at 630nm was used as the ordinate for each concentration of the standard, and the theoretical concentration was used as the abscissa. The five-parameter standard curve was fitted to obtain the regression equation and R 2 The CD3 antibody concentration in the test sample was calculated according to the regression equation.

[0043] The present invention comprises the following steps:

[0044] 1) Calibration of recombinant humanized CD3 antibody standards: Conduct purity analysis on candidate recombinant humanized CD3 antibodies, the purity should be above 95%; calibrate them using the double calibration method;

[0045] 2) Coating: Coat a 96-well ELISA plate with recombinant human CD3ε and CD3δ heterodimer proteins, 100 μL per well, and place at 2°C to 8°C overnight;

[0046] 3) Blocking: Wash the ELISA plate with washing solution, pat dry, add blocking solution, and incubate;

[0047] 4) Add test samples: Wash the ELISA plate with detergent, pat dry, add standard solution and test solution, 100µL / well, set up blank wells, set up 2 replicate wells for each sample, seal the plate with a sealing film, and incubate;

[0048] 5) Add enzyme-labeled secondary antibody: Wash the ELISA plate with detergent, pat dry, add goat anti-human IgG Fc (HRP) pre-adsorbed secondary antibody, seal the plate with sealing film, and incubate;

[0049] 6) Add substrate for display: wash the ELISA plate with washing solution, pat dry, add colorimetric solution TMB, and incubate at room temperature;

[0050] 7) Termination: Add 50 µL H2SO4 solution to each well to terminate the reaction;

[0051] 8) OD value determination: measure the absorbance value with an enzyme-labeled instrument;

[0052] 9) Curve fitting: According to the OD value and theoretical concentration of the standard curve, fit the standard curve to obtain the regression equation and R value, and calculate the concentration of the test sample according to the regression equation.

[0053] The recombinant humanized CD3 antibody described in step 1) is a T cell activating antibody produced by genetic engineering methods after humanization modification based on OKT3. The expression host of the antibody is CHO cells, and its production process uses animal-free raw materials and preparations, and is produced in accordance with the "Good Manufacturing Practice for Pharmaceuticals (Revised in 2010)". Before the test, the standard product is first analyzed for purity, and the purity analysis method is carried out with reference to the fifth method of electrophoresis method SDS-polyacrylamide gel electrophoresis (SDS-PAGE method) in Part IV 0541 of the 2020 edition of the Pharmacopoeia of the People's Republic of China; the calibration method is the calibration of its protein content, and is carried out with reference to the second method of protein content determination method Folin phenol method (Lowry method) in Part IV 0731 of the 2020 edition of the Pharmacopoeia of the People's Republic of China.

[0054] The recombinant human CD3ε and CD3δ heterodimer protein described in step 2) is a genetically recombinant protein, which is produced by co-expression of CD3ε and CD4δ, and its protein expression range is Asp 23-Asp 126 and Phe 22-Ala 105, respectively. The C-terminus of the subunit CD3ε is fused with a polyhistidine tag to improve the protein purification efficiency. The CD3 complex is composed of multiple subunits, including CD3ε, CD3γ, CD3δ and CD3ζ, among which the CD3ε subunit is the main target of many CD3 antibodies. Therefore, the recombinant protein can specifically recognize the recombinant humanized CD3 antibody and is used for the detection of the antibody. The coating concentration of the recombinant human CD3ε and CD3δ heterodimer protein is 5-10μg / mL, and the coating solution is 0.05mol / L carbonate buffer, pH9.6.

[0055] In step 3), the washing solution is a 0.01M PBST solution containing 0.05% Tween-20, pH 7.4, and the number of washes is 3 times, 300 μL / well; the blocking solution is the above-mentioned 0.01M PBST solution containing 2% BSA; the amount of the blocking solution added is 300 μl / well, and the incubation conditions are incubation at 36°C for 1 hour.

[0056] The washing solution in step 4) is a 0.01M PBST solution containing 0.05% Tween-20, pH 7.4, and the washing times are 3 times, 300 μL / well; the incubation conditions are incubation at 36°C for 2 hours.

[0057] The washing solution described in step 5) is a 0.01M PBST solution containing 0.05% Tween-20, pH 7.4, and the washing times are 3 times, 300 μL / well; the goat anti-human IgG Fc (HRP) pre-adsorbed secondary antibody has a high specificity. It only reacts specifically with the Fc fragment of the heavy chain of the human IgG molecule, and has no cross-reaction with the Fab fragment of human immunoglobulin. The recombinant humanized CD3 antibody detected by the present invention is Human IgG1 type, so the secondary antibody has a high accuracy and specificity when detecting the recombinant humanized CD3 antibody. The goat anti-human IgG Fc (HRP) pre-adsorbed secondary antibody is diluted 1:1000~1:5000 times, and the addition amount is 100 μL / well; the incubation conditions are incubated at 36°C for 1 hour.

[0058] The washing solution in step 6) is a 0.01M PBST solution containing 0.05% Tween-20, pH 7.4, and the washing times are 3 times, 300 μL / well; the amount of TMB added is 100 μL / well, and the incubation conditions are room temperature in the dark for 13 to 15 minutes.

[0059] The molar concentration of the H2SO4 solution described in step 7) is 2 mol / L.

[0060] The absorbance described in step 8) is the absorbance at a wavelength of 450 nm and a wavelength of 630 nm, wherein the 450 nm is the measurement wavelength, i.e., the main wavelength, and the 630 nm is the reference wavelength, i.e., the secondary wavelength. Measuring the absorbance at these two wavelengths can eliminate the influence of nonspecific absorption and eliminate interference factors during single-wavelength detection, such as sample concentration, interference color, etc. In addition, it can also minimize the errors caused by fingerprints, impurities, and opaque substances to the microplate reader readings, thereby improving the accuracy and reliability of the measurement results.

[0061] The OD value described in step 9) is the OD value measured at 450nm for each sample minus the OD value measured at 630nm. The standard curve is the OD value as the ordinate and the theoretical concentration as the abscissa. The five-parameter fitting standard curve is used to obtain the regression equation and R value, and the concentration of the test sample is calculated according to the regression equation. The advantage of the five-parameter fitting model (5PL) in fitting the standard curve in the low concentration area is mainly reflected in its adaptability to the asymmetry of the curve. Compared with the four-parameter fitting model (4PL), 5PL adds a parameter to describe the asymmetry of the curve, which enables it to more accurately fit the data points with a faster rising rate in the low concentration area, so that more accurate fitting can be used when processing low-concentration samples, which helps to improve the sensitivity and accuracy of the experiment. The test sample is an immune cell preparation containing recombinant humanized CD3 antibodies.

[0062] Flowchart of the present invention:

[0063]

[0064] The specification of recombinant human CD3ε and CD3δ heterodimer proteins is 50μg / tube, the specification of recombinant humanized CD3 antibody is 1mg / 1mL / tube, the specification of horseradish peroxidase (HRP)-labeled goat anti-human IgG Fc detection antibody is 500μg / tube, the specification of TMB is 1L / bottle, and other reagents are of domestic analytical grade.

[0065] In Examples 1 to 3 of the present invention and Comparative Example 1, all the raw materials and reagents used can be purchased from the market.

[0066] The present invention will be further described below in conjunction with embodiments:

[0067] Example 1 Calibration of recombinant humanized CD3 antibody standard

[0068] 1. Purity analysis

[0069] Two recombinant humanized CD3 antibody samples were prepared. After reducing conditions, the solution was prepared, gel was prepared, sample was loaded, and electrophoresis was performed according to the SDS-polyacrylamide gel electrophoresis (SDS-PAGE method) in Part IV, 0541, Electrophoresis Method, Method V of the Pharmacopoeia of the People's Republic of China, 2020 edition. After electrophoresis, the samples were fixed, stained, and decolorized. The gel imaging system Uvitec1D software was used to take pictures and analyze them according to the peak area normalization method to calculate the purity.

[0070] 2. Protein content calibration

[0071] The calibration of the recombinant humanized CD3 antibody standard was performed by two people to ensure the accuracy of the calibration results. The calibration was performed with reference to the second method of the Folin-phenol method (Lowry method) in Part IV 0731 of the 2020 edition of the Pharmacopoeia of the People's Republic of China for the determination of protein content.

[0072] 3. Results

[0073] 3.1 Purity analysis

[0074] The purity of the recombinant humanized CD3 antibody is 95.56%, which is greater than 95%, and can be used as a standard for detection experiments.

[0075] Table 1 Purity analysis results

[0076]

[0077] 3.2. Protein content calibration

[0078] The test results showed that the protein content of the recombinant humanized CD3 antibody calibrated by two people was 1.30 mg / mL, the repeatability was 1.93% and 6.63% respectively, and the intermediate precision was 4.44%. All the results were less than 10%.

[0079] Table 2 Protein content calibration results

[0080]

[0081] Example 2 Establishment of a method for detecting the content of recombinant humanized CD3 antibody

[0082] 1. Screening of coating antigens

[0083] Recombinant Human CD3E (C-Fc), Recombinant Human CD3E (C-mFc), Recombinant Human CD3E, recombinant human CD3ε and CD3δ heterodimer proteins (labeled as A protein, B protein, C protein, D protein, respectively) were coated on the ELISA plate, blocked, washed, and added with different concentration gradients of recombinant humanized CD3 antibodies. After adding enzyme-labeled secondary antibodies, color development and termination were performed, and the OD450nm value was read to study the antigen-antibody binding. The specific method is as follows:

[0084] 1) Dilute protein A, protein B, protein C, and protein D to 1 μg / mL respectively, coat the ELISA plate with 100 μL / well, and place the plate at 2℃~8℃ overnight;

[0085] 2) After washing the plate with washing solution, block it with PBST solution containing 2% BSA at a volume of 300 μL / well and incubate at 36°C for 1 hour;

[0086] 3) After washing the plate with washing buffer, add different concentrations of recombinant humanized CD3 antibody (1600pg / mL, 800pg / mL, 400pg / mL, 200pg / mL, 100pg / mL, 50pg / mL, 25pg / mL), 100μL / well, and incubate at 36℃ for 2 hours;

[0087] 4) After washing the plate with washing buffer, add goat anti-human IgG Fc (HRP) pre-adsorbed secondary antibody, seal the plate with sealing film, and incubate at 36°C for 1 hour;

[0088] 5) Add 100 μL of TMB colorimetric solution to each well and incubate at room temperature in the dark;

[0089] 6) After the H2SO4 reaction is terminated, the OD values ​​at 450nm and 630nm are detected by a microplate reader. The coated antigen is determined based on the data trends of the recombinant humanized CD3 antibodies at different concentrations and their OD450nm values.

[0090] 2. Determination of coating antigen concentration

[0091] Recombinant human CD3ε and CD3δ heterodimer proteins were diluted to 1μg / mL, 5μg / mL, 8μg / mL, and 10μg / mL, respectively, and 100μL / well was coated on the ELISA plate and placed at 2℃~8℃ for overnight coating; recombinant humanized CD3 antibody detection was performed, and the appropriate coating concentration was selected according to the OD450nm value.

[0092] 3. Optimization of enzyme-labeled secondary antibody conditions

[0093] The recombinant humanized CD3 antibody was tested by optimizing the three parameters of washing times (3 times and 6 times), incubation time (36°C and room temperature), and incubation time (30 min and 1 h). The concentrations of CD3 antibody were 5 ng / mL, 2.5 ng / mL, 78 pg / mL, 39 pg / mL, and 0 pg / mL, respectively. The optimal conditions were evaluated by comparing the OD450nm values ​​of recombinant humanized CD3 antibodies of different concentrations.

[0094] 4. Selection of substrate action time

[0095] According to the determined reaction conditions, the substrate action time was selected to be 5 min, 10 min, 13 min and 15 min at room temperature, and the maximum substrate action time was selected according to the OD450nm value.

[0096] 5. Selection of dilution multiples of enzyme-labeled secondary antibody

[0097] According to the determined reaction conditions, the goat anti-human IgG Fc (HRP) pre-adsorbed secondary antibody was diluted at 1:500, 1:1000, 1:2000, and 1:5000, respectively, to determine the optimal dilution multiple of the secondary antibody.

[0098] 6. Selection of standard curve fitting method

[0099] The determined reaction conditions were used to detect the recombinant humanized CD3 antibody, and the four-parameter fitting method and the five-parameter fitting method were used to establish the standard curve. The best fitting method was selected according to the recovery rate of each concentration of the standard curve.

[0100] 7. Confirmation of performance parameters of recombinant humanized CD3 antibody detection method

[0101] 7.1. Establishment of standard curve

[0102] The best coating antigen was selected and coated at the optimal coating concentration. After blocking and washing, the recombinant humanized CD3 antibody standard was added and the optimized indirect ELISA method was used to perform the experiment. The OD value measured at 450nm minus the OD value measured at 630nm was used as the ordinate and the theoretical concentration was used as the abscissa. The five-parameter standard curve was fitted to obtain the regression equation and R. 2 The value was calculated and the range of the standard back-calculated concentration was calculated. Two replicate wells were set for each concentration point and Blank (BL) of the standard curve. Six independent analysis batches were performed and the RE% of each concentration within the batch and the theoretical concentration was statistically analyzed.

[0103] 7.2 Precision and Accuracy

[0104] The ELISA plates coated with the same batch were used to perform parallel determinations of different concentrations of recombinant humanized CD3 antibody standards three times, and the OD values ​​were recorded and the coefficient of variation was calculated to examine the accuracy.

[0105] The ELISA plates coated on different dates were used to perform parallel determinations three times for different concentrations of recombinant humanized CD3 antibody standards, and the OD values ​​were recorded, the coefficient of variation was calculated, and the precision was examined.

[0106] 7.3 Stability

[0107] The ELISA plates coated in the same batch were stored at 4°C and -20°C for 30 days and 180 days, respectively. The recombinant humanized CD3 antibody standard was used for detection, and the recovery rate of the standard curve was calculated to analyze the stability of the antigen after coating under different storage conditions.

[0108] 8. Results

[0109] 8.1. Screening of coating antigens

[0110] The experimental results showed that when coated with D protein (i.e., recombinant human CD3ε and CD3δ heterodimeric protein), the OD450nm results of recombinant humanized CD3 antibodies at various concentrations showed a downward trend as the protein concentration decreased. There was no significant difference in the OD450nm values ​​of recombinant humanized CD3 antibodies at various concentrations after coating with other proteins, indicating that D protein can specifically recognize recombinant humanized CD3 antibodies and can be used as a coating protein to establish a method for detecting the content of recombinant humanized CD3 antibodies.

[0111] Table 3 Coating antigen screening test results (OD450nm value)

[0112]

[0113] 8.2 Determination of coating antigen concentration

[0114] According to the absorbance analysis at 450nm, the result of 10μg / mL coated antigen was equivalent to that of 5μg / mL coated antigen, which was better than that of 1μg / mL coated antigen.

[0115] Table 4 Results of antigen coating at different concentrations

[0116]

[0117] 8.3. Optimization of enzyme-labeled secondary antibody conditions

[0118] The results of the crossover design experiment of incubation temperature and incubation time showed that the OD450nm value of the enzyme-labeled secondary antibody was better when the incubation temperature was 36°C and the incubation time was 1h.

[0119] Table 5 OD450nm values ​​under different incubation conditions

[0120]

[0121] Table 6 compares the effects of different washing times. There is little difference between washing 3 times and washing 6 times. Considering the simplicity of the steps, washing 3 times is selected.

[0122] Table 6 OD450nm values ​​at different washing times

[0123]

[0124] 8.4. Selection of substrate action time

[0125] The changes in OD450nm value at different substrate action times are shown in Table 7. It can be seen that the OD450nm value is at a peak value at 13~15min, so 13~15min is selected as the substrate action time.

[0126] Table 7 Comparison of the effect of substrate action time

[0127]

[0128] 8.5. Selection of dilution multiples for enzyme-labeled secondary antibodies

[0129] According to the determined reaction conditions, the goat anti-human IgG Fc (HRP) pre-adsorbed secondary antibody was diluted at 1:500, 1:1000, 1:2000, and 1:5000, respectively. The results are shown in Table 8. After analysis, it was found that the recovery rate of the standard curve after 1:5000 dilution was higher, so this dilution multiple was selected.

[0130] Table 8 Results of enzyme-labeled secondary antibody dilution multiples

[0131]

[0132] 8.6. Selection of standard curve fitting method

[0133] The four-parameter fitting method and the five-parameter fitting method were respectively used to establish the standard curve, and the recovery rate of each concentration of the standard curve was calculated. The results are shown in Table 9. It can be seen that the recovery rate of the standard curve with five parameters is better than that of the standard curve with four parameters at lower concentrations, which improves the sensitivity and accuracy of the detection data. Therefore, the five-parameter fitting method is selected, and the standard curve is shown in Table 9. Figure 2 , Figure 3 .

[0134] Table 9 Results of different standard curve fitting methods

[0135]

[0136] 8.7. Establishment of the recombinant humanized CD3 antibody detection system

[0137] 1) Coating: Coat the ELISA plate with recombinant human CD3ε and CD3δ heterodimer proteins at 5-10 μg / mL, 100 μL per well, and place at 2℃-8℃ for overnight coating;

[0138] 2) Blocking: Wash the ELISA plate with pH 7.4 washing solution for 3 times, pat dry, add 2% blocking solution, 300 μL / well, and incubate at 36°C for 1 hour;

[0139] 3) Add test samples: Wash the ELISA plate with pH 7.4 washing solution for 3 times, pat dry, add standard solution and test solution, 100 µL / well, set up blank wells, set up 2 replicate wells for each sample, seal the plate with sealing film, and incubate at 36°C for 2 hours;

[0140] 4) Add enzyme-labeled secondary antibody: Wash the ELISA plate with pH 7.4 washing solution for 3 times, pat dry, add 1:1000~1:5000 goat anti-human IgG Fc (HRP) pre-adsorbed secondary antibody, seal the plate with sealing film, and incubate at 36℃ for 1h;

[0141] 5) Add substrate for display: Wash the ELISA plate with pH 7.4 washing solution, pat dry, add colorimetric solution TMB, and incubate at room temperature for 13-15 minutes;

[0142] 6) Termination: Add 50 µL H2SO4 solution to each well to terminate the reaction;

[0143] 7) OD value determination: Use a microplate reader to measure the absorbance at 450nm and 630nm;

[0144] 8) Curve fitting: For each concentration of the standard, the OD value measured at 450nm minus the OD value measured at 630nm was used as the ordinate, and the theoretical concentration was used as the abscissa. The five-parameter standard curve was fitted to obtain the regression equation and R 2 The CD3 antibody concentration in the test sample was calculated according to the regression equation.

[0145] 8.8. Confirmation of performance parameters of recombinant humanized CD3 antibody detection method

[0146] 8.8.1. Establishment of standard curve and range

[0147] The results showed that the recovery rates of the standard curve samples at different concentrations were between 80.54% and 118.78%, and R 2 The above results all meet the requirements of standard curve and range verification. The detection range of this method is 1600~25pg / mL.

[0148] Table 10 Standard curve and range results

[0149]

[0150] 8.8.2 Precision and Accuracy

[0151] The ELISA plates coated with the same batch were used to perform parallel measurements of the recombinant humanized CD3 antibody standards within the linear range three times. The test results showed that the coefficient of variation of the standards at each concentration was between 0.11% and 9.00%, all less than 10%, indicating that the method had good repeatability. The results are shown in Table 11.

[0152] Table 11 Accuracy results

[0153]

[0154] The ELISA plates coated on different dates were used to perform parallel measurements of the recombinant humanized CD3 antibody standards within the linear range three times. The test results showed that the coefficient of variation of the standards at each concentration was between 0.05% and 8.26%, all less than 10%, indicating that the method had good batch-to-batch repeatability. The results are shown in Table 12.

[0155] Table 12 Precision results

[0156]

[0157] 8.8.3 Stability

[0158] The same batch of coated ELISA plates were stored at 4℃ and -20℃ for 30 days and 180 days, respectively, and tested with recombinant humanized CD3 antibody standards to calculate the recovery rate of the standard curve. The experimental results showed that there was no significant difference between the results of the coated plate at 4℃ for 30 days and 4℃ for 0 days; in the results of 4℃ for 180 days, the recovery rate of the low-concentration standard decreased. There was no significant difference between the results of the coated plate at -20℃ for 30 days and -20℃ for 180 days and -20℃ for 0 days. This shows that the antibody has good stability.

[0159] Table 13 Stability results

[0160]

[0161] Example 3 Kit Application

[0162] Based on the determined test parameters, the method was validated to determine whether the kit is suitable for detecting the content of recombinant humanized CD3 antibodies in human in vitro activated immune cell preparations. The method was validated from three aspects: accuracy, precision and specificity.

[0163] 1. Preparation of human in vitro activated immune cell preparations

[0164] 1.1 Methods

[0165] 1.1.1 Isolation of mononuclear cells (PBMC): After single blood or peripheral blood is diluted with compound electrolyte injection, it is separated by density gradient centrifugation using lymphocyte separation solution; after the cells are mixed with serum-free culture medium, the viability of the obtained cells is analyzed by automatic image analysis.

[0166] 1.1.2 Mononuclear cells were cultured in serum-free medium at a concentration of 1×10 6 / mL concentration was inoculated into T25 flasks pre-coated with CD3 monoclonal antibody for the first step of activation for 3 hours, and the CD3 monoclonal antibody concentration was 2μg / mL.

[0167] 1.1.3 Take out the pre-cultured PBMCs and add a combination of cytokines including rhIL-2, rhIL-4, rhGM-CSF, rhTNF-α, and rhIFN-γ for the second step of in vitro activation culture. Continue culturing for 5 hours. The concentrations of rhIL-2, rhIL-4, rhGM-CSF, rhTNF-α, and rhIFN-γ are 6000 IU / mL, 300 IU / mL, and 6000 IU / mL, respectively.

[0168] 1.1.4 Collect the cell suspension and centrifuge it. Mix the cells with serum-free medium and add 3.0×10 5 The cells were inoculated into culture flasks or culture bags at a concentration of 10 viable cells / mL and cultured for 120 h.

[0169] 1.1.5 After collecting the cell suspension and centrifuging it, mix the cells with compound electrolyte injection solution and analyze the viability of the obtained cells using automatic image analysis.

[0170] 2. Accuracy and precision

[0171] In order to confirm the intra-batch and inter-batch precision and accuracy of the method, according to the validation guidelines for quantitative analysis methods of biological samples in Part IV 9012 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, five concentrations of quality control samples were selected for investigation, including the lower limit of quantification (LLOQ), low concentration quality control (LQC), medium concentration quality control (MQC), high concentration quality control (HQC), and upper limit of quantification concentration quality control (LOQ). The inter-batch investigation was carried out in 6 independent analytical batches within a few days, and each batch contained the above 3 sets of quality control samples. The average %CV, %RE within the batch of quality control samples of each concentration, as well as the average %CV, average %RE and total error between batches were calculated.

[0172] The average %RE of each concentration within the batch was within ±20.00% (LOQ and LLOQ were within ±25.00%); the average %CV was within 20.00% (LOQ and LLOQ were within ±25.00%).

[0173] The average %RE between batches was within ±20.00% (LOQ and LLOQ were within ±25.00%); the average %CV was within 20.00% (LOQ and LLOQ were within ±25.00%).

[0174] The total error (%) of the method was within 30.00% (the total error of LOQ and LLOQ was within 40.00%).

[0175] 3. Specificity

[0176] According to the validation guidelines for quantitative analysis methods for biological samples in Part IV, 9012 of the 2020 edition of the Pharmacopoeia of the People's Republic of China, recombinant humanized CD3 antibody standards were added to the same analysis batch to prepare high-concentration and low-concentration quality control samples to examine specificity.

[0177] At least 80% of the quality control samples have an accuracy (RE%) within ±20% (or within ±25% if at the lower limit of quantitation).

[0178] 4. Results

[0179] 4.1 Preparation results of human in vitro activated immune cell preparations

[0180] The results in Table 14 show that the viability of immune cells obtained by the method in 1.1 is as high as 93.7%.

[0181] Table 14 Viability of immune cells activated in vitro

[0182]

[0183] 4.2 Accuracy and Precision

[0184] The results showed that the average RE% within the five concentration quality control samples were 85.34-124.68, 85.71-111.24, 86.44-102.01, 81.19-109.52, and 86.11-103.11, respectively; the average %CV were 4.25-24.29, 1.60-11.72, 1.50-17.98, 0.84-11.91, and 1.63-12.21, respectively; the inter-batch accuracy RE% were 103.10, 95.65, 93.47, 97.59, and 95.87, respectively; the inter-batch precision were 18.18, 11.59, 9.91, 10.40, and 8.66, respectively; and the total method errors were 21.28, 15.94, 16.44, 12.81, and 12.79, respectively. The precision and accuracy of the method met the validation requirements.

[0185] 4.3 Specificity

[0186] The results showed that the accuracy (RE%) of the quality control samples was between 76.80% and 112.26%, and more than 94% of the quality control samples had an accuracy (RE%) within the range of ±20%, meeting the validation requirements.

[0187] 5. Verify the conclusion

[0188] The method for detecting the content of recombinant humanized CD3 antibodies in immune cell preparations was verified, and the verification results showed that the method of the present invention can be used for detecting the content of recombinant humanized CD3 antibodies in immune cell preparations.

[0189] Comparative Example 1 Comparison of the content of recombinant humanized CD3 antibodies detected by 4 CD3 antibody detection kits on the market

[0190] After diluting the recombinant humanized CD3 antibody in multiples, refer to the instructions of the corresponding kit and perform the test according to the steps in the instructions. When the test results show a multiple gradient and are consistent with the dilution multiple gradient, it means that the detection method can be used to detect the content of the recombinant humanized CD3 antibody. The test results are shown in Table 15.

[0191] Table 15 Results of the recombinant humanized CD3 antibody content detection test using the following four kits

[0192]

[0193] Analysis of the results of Comparative Example 1 and Example 2 showed that the kits of the four manufacturers could not specifically identify the recombinant humanized CD3 antibody and could not be used to detect the content of the recombinant humanized CD3 antibody. This patent establishes an enzyme-linked immunosorbent assay method that can be used to detect recombinant humanized CD3 antibodies.

[0194] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A detection reagent, characterized in that: include: Coating solution, washing solution, blocking solution, enzyme-labeled secondary antibody, color development solution and stop solution; The coating solution comprises: coating antigen and coating buffer; The coating antigen includes: 0.5-1µg / unit carrier of recombinant human CD3ε and CD3δ heterodimer protein; The enzyme-labeled secondary antibody includes goat anti-human IgG Fc at a dilution multiple of 1:1000 to 1:5000; The coating buffer comprises: 0.05 mol / L carbonate buffer; the washing solution comprises: 0.01 M PBST solution containing 0.05% Tween-20; the blocking solution comprises: 0.01 M PBST solution containing 2% BSA.

2. Use of the detection reagent according to claim 1 in any of the following items: (1) preparing a product for detecting the content of humanized CD3 antibodies; and / or (2) detecting the content of humanized CD3 antibodies; and / or (3) preparing a product for detecting the residues of humanized CD3 antibodies in immune cell preparations; and / or (4) detecting the residues of humanized CD3 antibodies in immune cell preparations.

3. A detection kit, characterized in that: include: The detection reagent as claimed in claim 1.

4. The method for using the detection kit according to claim 3, characterized in that: The steps include: S1: Calibration of standard: obtain standard solution; S2: coating: coating the culture carrier with the coating antigen; S3: blocking: taking the washing solution to wash the coated culture carrier, adding the blocking solution, and incubating; S4: adding test samples: taking the washing solution to wash the culture carrier after incubation in S3, adding the standard solution and the test solution, and incubating; S5: adding enzyme-labeled secondary antibody: taking the washing solution to wash the culture carrier after incubation in S4, adding the enzyme-labeled secondary antibody, and incubating; S6: Adding substrate for display: taking the washing solution to wash the culture carrier after incubation in S5, adding the color developing solution, and incubating; S7: Termination: adding the culture carrier after incubation in S6 to the termination solution; S8: OD value determination: detect absorbance value; S9: Curve fitting: According to the OD value and theoretical concentration of the standard curve, fit the standard curve to obtain the regression equation and R value, and calculate the test sample concentration according to the regression equation.

5. The method of use according to claim 4, characterized in that: The incubation time described in S5 is 30 to 60 minutes and the temperature is 10 to 36°C.

6. The method of use according to claim 4, characterized in that: The incubation time described in S6 is 13 to 15 minutes.

7. The method of use according to claim 4, characterized in that: The fitting method described in S9 adopts five-parameter fitting.

8. The method of use according to claim 4, characterized in that: The test article contains humanized CD3 antibody.

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