A fluorescence polarization immunoassay method for determining antibody concentration

Abstract

The present invention relates to a fluorescence polarization immunoassay method for determining antibody concentration, the method comprising the following steps: 1). preparing an antibody binding protein with a fluorescent group as an antibody fluorescent tracking molecule; 2). mixing the fluorescent tracking molecule with a sample solution to be tested and incubating the mixture; 3). after the incubation is completed, using an enzyme marker with a fluorescence polarization detection function to detect the light intensity in the horizontal and vertical directions of the incubated solution, which are recorded as I and I respectively. ∥ and I ⊥ ; 4). The relationship between antibody concentration and fluorescence polarization intensity is obtained from the known standard concentration and fluorescence polarization intensity value, and the fluorescence polarization intensity value measured by the sample solution to be tested is substituted into the relationship to obtain the antibody concentration in the sample solution to be tested. The method disclosed in the present invention has the outstanding characteristics of simple and rapid operation, high sample throughput, and cost saving, and is more suitable for the screening of high-yield stable cell lines than the currently commonly used antibody detection method.

Description

Technical Field

[0001] The present invention relates to the field of bioanalysis technology, and in particular to an immunoassay method for measuring antibody concentration based on fluorescence polarization theory. Background Art

[0002] The level of antibody expression is an important indicator for measuring the quality of stable cell lines. Obtaining a cell line with stable and high antibody expression is an important step in bringing therapeutic antibodies from the laboratory to the market and truly applying them to treat patients. Its significance lies not only in providing sufficient experimental drugs for clinical research, but also in reducing production costs as much as possible for mass production of antibody drugs after they are launched on the market. In addition, the determination of antibody concentration runs through almost the entire research and development and production process of antibody drugs.

[0003] Obtaining high-yield stable cell lines usually requires screening from a large number of monoclones. The currently commonly used antibody concentration quantification methods for large-scale screening of high-yield stable cell lines still have defects and need to be improved. The commonly used methods are: high-performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), and biofilm interferometry (BLI). HPLC is considered the "gold standard" for antibody concentration detection, but HPLC has low sample throughput and high requirements for the professional skills of instruments and operators; ELISA is a mature method for protein quantification, but the experimental process is lengthy, with many steps and a long time; BLI has a high throughput, but is expensive and has high economic costs.

[0004] Therefore, there is an urgent need to develop a method with high sample throughput, simple operation and relatively economical for large-scale screening of antibody expression in stable cell lines. Summary of the invention

[0005] In view of the above analysis, the present invention aims to provide a novel antibody concentration detection method based on fluorescence polarization theory, which is used to simplify the detection steps of antibody expression levels in stable cell lines, improve screening throughput and reduce economic costs.

[0006] The present invention is based on the following principles: fluorescent tracking molecules can emit light of different wavelengths from the excitation light when they are irradiated and excited by light of a certain wavelength; the microplate reader with fluorescence polarization function forms polarized excitation light with a specific wavelength and a vibration plane limited to a fixed direction through a filter and a polarizer; if the fluorescent substance is in a stationary state during excitation, the emitted light will maintain the polarization of the original excitation light; if it is in motion, part of the emitted light will lose its polarization, thereby forming an emitted light beam with a polarization different from the excitation light, so that the emitted light passes through two polarizers perpendicular to each other, and the light intensity of the two mutually perpendicular lights is detected for analysis. Free fluorescent tracking molecules are small, and rotate faster in the buffer solution, so that the excitation polarized light loses more polarization and has a smaller polarization; but after combining with the antibody, the molecular weight of the complex becomes larger, the movement rate of the fluorescent marker molecule decreases, the excitation light maintains the original polarization, and the polarization value is larger. The more fluorescent complexes are formed in the solution, the larger the polarization value will be, that is, the solution polarization value has a certain relationship with the antibody concentration, and the antibody concentration is thus analyzed and determined.

[0007] In one aspect, the present invention provides a fluorescence polarization immunoassay method for determining antibody concentration, comprising the following steps:

[0008] 1. Prepare antibody-binding proteins with fluorescent groups as fluorescent tracking molecules;

[0009] 2. Mixing and incubating the fluorescent tracking molecule with the sample solution to be tested, and simultaneously, mixing and incubating the fluorescent tracking molecule with a standard solution with a known antibody concentration, and simultaneously, mixing and incubating the fluorescent tracking molecule with a blank solvent without antibodies to serve as a blank control group;

[0010] 3. After the incubation is completed, the light intensity in the horizontal and vertical directions of the incubated solution obtained in step 2 is detected using an ELISA instrument with a fluorescence polarization detection function, and is recorded as I ∥ and I ⊥ ;

[0011] 4. Data analysis: Use the calculation formula: fluorescence polarization intensity (polarization intensity, PI) = I ∥ -I ⊥ Calculate the fluorescence polarization intensity of the solution, correct the fluorescence polarization intensity value of the standard solution or the sample solution to be tested by deducting the fluorescence polarization intensity value of the blank control group, draw a standard curve with the known standard concentration as the abscissa and the corrected fluorescence polarization intensity value as the ordinate, and obtain a linear fitting relationship between the antibody concentration and the fluorescence polarization intensity. Substitute the corrected fluorescence polarization intensity value measured by the sample solution to be tested into the relationship to obtain the antibody concentration in the sample solution to be tested.

[0012] Wherein, in the method, two replicate wells are set for each concentration of the standard and the sample to be tested, and the mean PI value of the two replicate wells is used for data analysis.

[0013] The above steps are described in detail below.

[0014] Step 1:

[0015] Specifically, in step 1, the fluorescent tracking molecule is prepared by the following process:

[0016] 1.1) Dissolving fluorescein isothiocyanate (FITC) in dimethyl sulfoxide (DMSO) to prepare a FITC preparative solution;

[0017] 1.2) preparing an antibody-binding protein stock solution and ensuring that the protein stock solution does not contain imidazole groups or components containing free amino groups;

[0018] 1.3) Mix the FITC pre-solution with the antibody-binding protein stock solution and perform a molecular coupling reaction;

[0019] 1.4) Terminate the coupling reaction and remove free FITC by purification to obtain a fluorescent tracking molecule solution.

[0020] In a specific embodiment, in step 1.1), the concentration of the obtained FITC preliminary solution is 10 mg / mL or more;

[0021] In a specific embodiment, in step 1.2), the concentration of the prepared antibody-binding protein stock solution is 5 mg / mL or more.

[0022] In a specific embodiment, in step 1.2), the antibody binding protein is recombinant Staphylococcal protein A (SPA).

[0023] In a specific embodiment, in step 1.3), the FITC pre-solution is mixed with the antibody binding protein stock solution at a molar ratio of FITC to SPA of 18-20:1.

[0024] In a specific embodiment, in step 1.3), the concentration of the antibody binding protein is controlled at 2-5 mg / mL using a buffer having a pH in the range of 9.0-10.0, and the mixture is thoroughly mixed and stirred overnight at 4° C. in the dark to perform a coupling reaction.

[0025] In a specific embodiment, in step 1.4), the coupling reaction is terminated by adding NH4Cl solution. After the coupling reaction is terminated, free FITC is removed by using Sepharose-25, and the buffer is replaced with 1×PBS or ddH2O.

[0026] In step 1, step 1.5) is also included: diluting the obtained fluorescent tracking molecule solution, preferably, using 1×PBS, ddH2O or CHO culture medium CD04 to dilute the fluorescent tracking molecule solution to a concentration of 0.40-0.70 mg / L, for example, to 0.42 mg / L.

[0027] The present application optimizes the preparation conditions of the fluorescent tracking molecule in step 1, improves the FITC coupling efficiency of the protein molecule, and obtains a fluorescent tracking molecule that is more suitable for application in the method of the present invention.

[0028] Step 2:

[0029] In a specific embodiment, in step 2, the volume ratio of the fluorescent tracking molecule to the sample solution to be tested is not specifically limited, which depends on the antibody concentration in the sample solution to be tested. In principle, the higher the antibody concentration in the sample solution to be tested, the larger the volume ratio of the fluorescent tracking molecule to the sample solution to be tested. For example, for samples with a higher concentration of the sample to be tested (more than 100 mg / L), a detection system with a fluorescent tracking molecule / sample solution volume ratio of 9:1 (e.g., 180 μL / 20 μL) can be selected. For samples below 100 mg / L, a detection system with a fluorescent tracking molecule / sample solution volume ratio of 4:1 (e.g., 160 μL / 40 μL) can be selected. In the case of cell line screening, since the antibody concentration is usually lower than 100 mg / L, it is preferred to select a detection system with a fluorescent tracking molecule / sample solution volume ratio of 160 μL / 40 μL. In the present application, by adopting the above-mentioned optimized mixing volume ratio, the range of antibody concentration that can be accurately detected is expanded within the acceptable accuracy range of 80%-120%.

[0030] In a specific embodiment, in step 2, the incubation temperature is 20-25° C., and the incubation time is 20-30 minutes.

[0031] In a specific embodiment, in step 2, the sample solution to be tested can be taken from a part of the cell culture supernatant, and the type of antibodies contained therein is not limited as long as they can bind to the antibody-binding protein. Specifically, the antibody types contained therein can be selected from human IgG1, IgG2, IgG4, IgM, IgA, mouse IgG1, IgG2, IgG3, IgM, and antibody Fc-terminal fusion proteins and bispecific antibodies modified on these antibody types.

[0032] In a specific embodiment, in step 2, in addition to the analyte, the sample solution to be tested must not contain other proteins that can specifically or non-specifically bind strongly to SPA. For other known or unknown proteins that can non-specifically bind weakly to SPA that may be contained in the sample solution to be tested, the influence of other components in the solvent can be ignored if the difference in PI value between the 100-fold dilution of the blank solvent and the original blank solvent does not exceed 5%.

[0033] In a specific embodiment, in step 2, mixing is performed by shaking using a pipette or a plate shaker.

[0034] In a specific embodiment, in step 2, the standard with known concentration is obtained by diluting with the same buffer as the sample solution to be tested, preferably, at least 6 concentration gradients are diluted, and these concentrations are within the linear range to ensure the accuracy of the standard curve.

[0035] Step 3:

[0036] In a specific embodiment, in the detection of step 3, the excitation wavelength is set to 485 nm and the emission wavelength is set to 520 nm.

[0037] Step 4:

[0038] In a specific embodiment, in step 4, the data analysis meets the following requirements: 1) the coefficient of variation CV of two parallel wells is less than 2%; 2) the standard curve is determined by at least 6 standard concentration gradients; 3) the linear fit R of the standard curve is greater than 10%; 2 above 0.999; and 4) the concentration of the sample solution to be tested falls between the lowest dilution concentration and the highest dilution concentration of the standard curve.

[0039] For this step, the fluorescence polarization intensity (polarization intensity, PI) proposed and defined for the first time in the present invention has the advantage that, compared with the conventional fluorescence polarization potency (polarization potency, P) calculation formula used in current research reports, the polarization measurement value can be amplified by increasing the labeling rate of the fluorescent tracking molecule fluorescein, thereby increasing the polarization fluorescence intensity. The method established on the basis of PI can obtain a larger detection window and linear analysis range than the previously reported fluorescence polarization immunoassay for detecting antibody concentration (Ben Thompson, Jerry Clifford, Mike Jenns, et al. Anal. Biochem, 2017 (534): 49-55. Based on the P calculation formula, a fluorescent tracking molecule different from the present invention is used, and the maximum analyzable antibody concentration obtained is 100 mg / L).

[0040] On the other hand, the present invention provides a kit for determining antibody concentration, which at least comprises a fluorescent tracking molecule, wherein the fluorescent tracking molecule is an antibody binding protein with a fluorescent group.

[0041] In a specific embodiment, the fluorescent tracking molecule is a molecule in which fluorescein isothiocyanate (FITC) is coupled to an antibody binding protein.

[0042] In a specific embodiment, the antibody binding protein is recombinant Staphylococcus protein A (SPA).

[0043] In a specific embodiment, the antibody binding protein is prepared by the method described above.

[0044] In a specific embodiment, the kit further comprises 1×PBS, CHO culture medium CD04 or deionized water as a diluent for the fluorescent tracking molecule.

[0045] In yet another aspect, the present invention provides use of the above method or the above kit in screening cell lines expressing antibodies.

[0046] Beneficial Effects

[0047] The present application discloses a method for detecting antibody concentration with high sample throughput, simple operation and economy, which can directly detect the antibody concentration in the cell culture supernatant and is particularly suitable for batch screening of high-yield stable cell lines.

[0048] The analytical method disclosed in this application has good linearity, and the PI value is linearly correlated with the antibody concentration. 2 The value is above 0.999; the measured concentration of the antibody is also highly linearly correlated with the theoretical concentration, R 2 The value is also above 0.999.

[0049] For human IgG4 monoclonal antibody, the linear analysis range of the embodiment of the present invention is 5-400 mg / L, and the accuracy is 80%-120%, which meets the requirements of stable cell line screening.

[0050] Compared with HPLC, the method of the present application has the outstanding advantages of high sample throughput and low technical requirements for operators. On the basis of basically meeting the accuracy requirements for stable cell line screening, the sample throughput is greatly improved, and about 100 samples can be detected in 30 minutes; at the same time, compared with the ELISA method that requires more than a dozen steps to operate, the method of the present invention has the outstanding characteristics of being simple and fast, and the detection only requires 3 to 4 steps of experimental operation, which can greatly save detection time and workload and improve detection efficiency; in addition, compared with the BLI method, the method of the present invention does not require the use of expensive consumables, has the advantage of saving economic costs, and its application in the screening of stable cell lines will greatly reduce the cost of antibody detection.

[0051] In summary, the method disclosed in the present invention has the outstanding characteristics of simple and rapid operation, high sample throughput, and reduced detection costs. It is more suitable for screening high-yield stable cell lines than the currently commonly used antibody detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Hereinafter, exemplary embodiments of the present disclosure will be described in more detail with reference to the accompanying drawings.

[0053] Figure 1 A schematic diagram of the steps of determining antibody concentration in an embodiment of the present invention is shown.

[0054] Figure 2 The effect of different coupling pH on coupling efficiency and fluorescence polarization intensity (at 150 mg / L antibody concentration) is shown, where F-SPA is the abbreviation of FITC-SPA fluorescent tracking molecule, and the F / P value is the molar ratio of FITC to protein in the fluorescent protein, reflecting the fluorescence labeling efficiency. In the figure, the left bar represents PI and the right bar represents F / P.

[0055] Figure 3 Curves produced from the fluorescent tracer molecule obtained at different coupling pHs are shown.

[0056] Figure 4 The standard curve (A) and its linear fitting result (B) at different usage amounts of fluorescent tracking molecules are shown.

[0057] Figure 5 The results of the correlation analysis between the measured concentration and the theoretical concentration at different dilutions of the fluorescent tracking molecule are shown.

[0058] Figure 6 The standard curves prepared under different mixing volumes of sample solution and fluorescent tracking molecules in the detection system are shown, wherein the first number (e.g. 160) in the right icon in the figure represents the volume of fluorescent tracking molecules with a concentration of 0.42 mg / L added to the 96-well plate, and the second number (e.g. 40) represents the volume of antibody sample added, both in μL. DETAILED DESCRIPTION

[0059] The technical solutions of the embodiments of the present disclosure are described in more detail and completely with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present disclosure, not all. These embodiments are provided to make the present disclosure more thorough and complete, and to fully convey the scope of the present disclosure to those skilled in the art.

[0060] the term

[0061] In the present invention, "fluorescent tracking molecule" refers to an antibody-binding protein with a fluorescent group, specifically, it refers to FITC-coupled recombinant Staphylococcus protein A. Please give a clearer definition.

[0062] Equipment and Materials

[0063] FITC was purchased from SIGMA; SPA was purchased from Sino Biological Co., Ltd.; M5e microplate reader was from Molecular Devices, Inc.; black 96-well plates were from Greiner bio one; CHO cell culture medium CD04 was purchased from Auprom Biotech Co., Ltd. The 1×PBS used below was obtained by diluting 20×PBS (purchased from Bio-Tech).

[0064] In the following examples, human IgG4 is used as a representative analysis sample, but the scope of the present application is not limited thereto. As described in the claims, the embodiments of the present invention can also detect other proteins that can bind to SPA, which all fall within the scope of protection of the present invention.

[0065] Example 1. Preparation of fluorescent tracking molecule FITC-SPA under different pH coupling conditions

[0066] The preparation steps of the fluorescent tracking molecule FITC-SPA (i.e. FITC-coupled SPA) are as follows:

[0067] 1. Protein solution treatment before coupling: SPA protein concentration is higher than 5 mg / mL, and the protein buffer uses coupling buffer (i.e., carbonate buffer with pH 7.0-10.0). Note that the protein buffer must not contain imidazole or components containing free amino groups (high concentration of imidazole affects FITC coupling efficiency);

[0068] 2. Preparation of FITC solution: Weigh FITC powder and dissolve it in DMSO to prepare a FITC preparatory solution with a FITC concentration of more than 10 mg / mL;

[0069] 3. Coupling: Add the FITC preparatory solution prepared in step 2 and the protein solution prepared in step 1 to a molar ratio of FITC to SPA of 20:1, and dilute the protein solution to 2 mg / mL with carbonate buffer at pH 9.0-10.0. Vortex, seal the tube cap tightly with sealing film, and stir at 4°C in the dark overnight;

[0070] The carbonate buffer solution was prepared as follows: 7.56 g NaHCO3, 1.06 g Na2CO3, and the volume was adjusted to 1 L, that is, the final concentration of NaHCO3 was 0.1 M, and the final concentration of Na2CO3 was 0.01 M. The pH of the carbonate buffer solution was adjusted to between 9.0 and 10.0 using 2 M HCl and 5 M NaOH, respectively.

[0071] 4. Termination of coupling: Add 50 mM / L NH4Cl solution with a molar ratio of NH4Cl to FITC of 1:1 to terminate the coupling, vortex mix, and react at room temperature in the dark for 0.5 hour;

[0072] 5. Purification: Use Sepharose-25 to remove free FITC and replace the buffer with 1× PBS or ddH2O;

[0073] 6. Determination of coupling product concentration: Use Micro Drop to perform full wavelength scanning and read the absorbance at wavelengths of 280nm and 495nm. Use the following formula to calculate the coupling product protein concentration and F / P value.

[0074]

[0075]

[0076] Among them, E 0.1％ represents the extinction coefficient of 1.0 mg / ml SPA at 280 nm, 0.35×A 495 is the correction for the absorbance of FITC at 280 nm, 389 is the molecular weight of FITC, and 195 is the extinction coefficient of the coupled FITC.

[0077] Example 2: Effect of different pH coupling conditions on the coupling efficiency of fluorescent tracking molecules

[0078] 1. Dilute the IgG4 standard to 150 mg / L with 1×PBS. Add 10 μL of the diluted standard solution to each well of the experimental group and 10 μL of 1×PBS to the blank control group;

[0079] 2. Dilute each candidate protein sample to 0.53 mg / L with 1× PBS, and add 190 μL to each well of the experimental group and the blank control group, that is, the amount of fluorescent tracking molecules added is 0.1 μg / well;

[0080] 3. Mix well on an oscillator, incubate at room temperature in the dark for 20 min, use the fluorescence polarization (FP) endpoint detection method of the M5e microplate reader, set the excitation wavelength to 485 nm, the emission wavelength to 520 nm, and read the light intensity in the horizontal and vertical directions;

[0081] 4. Data processing: Calculate the PI value of each well, △PI=PI 实验组 -PI 空白组 , which can be used to preliminarily indicate the size of the analysis window. Plotting with different pH as the horizontal axis, △PI and F / P value as the vertical axis (the data acquisition method has been described in detail in Example 1), as shown in FIG. Figure 2 shown.

[0082] Through this example, the relationship between pH and coupling efficiency during coupling is clarified, that is, the higher the pH, the higher the coupling efficiency of FITC. At the same time, at an antibody concentration of 150 mg / L, the higher the coupling efficiency, the larger the analysis window.

[0083] Example 3: Effect of different pH coupling conditions on PI standard curve

[0084] 1. Dilute the IgG4 standard to 3.6 g / L with 1× PBS, dilute 5 times into 7 concentration gradients, add 20 μL to each well, and set a blank solvent control for each group, i.e., add an equal amount of diluent;

[0085] 2. Dilute F-SPA under different coupling conditions to 0.56 mg / L with 1×PBS, and add 180 μL to each well of the experimental group and the blank control group, that is, the amount of fluorescent tracking molecules added is 0.1 μg / well.

[0086] 3. Mix well on an oscillator, incubate at room temperature in the dark for 20 min, use the fluorescence polarization (FP) endpoint detection method of the M5e microplate reader, set the excitation wavelength to 485 nm, the emission wavelength to 520 nm, and read the light intensity in the horizontal and vertical directions;

[0087] 4. Data processing: Calculate the PI value of each well and correct the PI by deducting the PI value of the blank solvent control group, that is, △PI = PI 实验组 -PI 空白组 , plot the antibody concentration as the horizontal axis and △PI as the vertical axis, such as Figure 3 described.

[0088] Through this example, it was determined that the fluorescent tracking molecule with the largest analysis window was obtained by coupling at pH 9.0 and pH 10.0 (the two are almost equal), so the optimal pH condition range for coupling was determined to be 9.0-10.0.

[0089] Example 4: Effect of different fluorescence tracking molecule concentrations on the standard curve

[0090] 1. Dilute the IgG4 standard to 400 mg / L with 1×PBS, dilute it 3-fold into 7 concentration gradients, add 20 μL to each well, and set a blank solvent control for each group, i.e., add an equal amount of 1×PBS;

[0091] 2. Dilute F-SPA pH 9.0 with 1×PBS to 0.70, 0.56, 0.42, and 0.28 mg / L, respectively. Add 180 μL to each well of the experimental group and the blank control group, that is, the amount of fluorescent tracking molecules added was 0.125, 0.1, 0.075, and 0.05 μg / well;

[0092] 3. The mixing, incubation, plate reading and data processing methods are the same as in Example 3. The standard curve is as follows: Figure 4 A, the fitting result is as follows Figure 4 B.

[0093] This example determined that the optimal amount of the fluorescent tracking molecule to be used should be between 0.075-0.125 μg / well when the fluorescent tracking molecule / test sample solution volume ratio is 180 μL / 20 μL.

[0094] Example 5: Effect of different dilutions on the correlation between measured sample concentration and theoretical concentration

[0095] 1. Dilute the IgG4 standard to 400 mg / L with CHO culture medium CD04, dilute 7 concentration gradients by 3 times, add 20 μL to each well, and set a blank solvent control for each group, that is, add an equal amount of solvent culture medium CD04;

[0096] 2. Dilute the fluorescent tracking molecule to 0.42 mg / L with 1×PBS, culture medium CD04, and deionized water respectively. Add 180 μL to each well of the experimental group and the blank control group, that is, the amount of fluorescent tracking molecule added is 0.75 μg / well.

[0097] 3. The mixing, incubation and plate reading settings are the same as in Example 3;

[0098] 4. Data processing: Calculate the fluorescence polarization intensity PI value of each well by the following formula: PI = I ∥ -I ⊥ ; Correct the PI by deducting the PI of the blank solvent control group; Draw a standard curve with the known standard concentration as the horizontal axis and the corrected PI value as the vertical axis, and obtain the relationship between the concentration of the antibody to be tested and the fluorescence polarization intensity by linear fitting, and substitute the measured corrected PI value of the sample solution to be tested into this relationship to obtain the measured value of the antibody concentration of the sample solution to be tested; Correlation analysis between the measured sample concentration and the theoretical concentration: Draw a curve with the actual theoretical concentration added as the horizontal axis and the measured concentration as the vertical axis, and analyze its linear correlation, such as Figure 5 shown.

[0099] Through Example 5, it is verified that the correlation between the measured concentration and the theoretical concentration is high when using the above different dilutions. Therefore, when testing, the system dilution can preferably be 1×PBS, culture medium CD04 or deionized water.

[0100] Example 6: Effect of the ratio of fluorescent tracking molecules and antibody reactions on the standard curve

[0101] 1. Dilute the IgG4 standard to 400 mg / L with the culture medium supernatant, and dilute it 3-fold into 7 concentration gradients. 180μL+20μL, 160μL+40μL, 140μL+60μL and 60μL+60μL experimental groups (the former volume represents the volume of the fluorescent tracking molecule solution added, and the latter volume represents the volume of the sample solution added). Add 20μL, 40μL, 60μL and 60μL of antibody gradient dilution solution to each well, respectively. Set a blank solvent control for each group, that is, add 20μL, 40μL, 60μL and 60μL of blank cell culture supernatant, respectively.

[0102] 2. Dilute the fluorescent tracking molecule to 0.42 mg / L with the culture medium supernatant, and add 180 μL, 160 μL, 140 μL, and 60 μL of the diluted fluorescent tracking molecule solution to each well of the 180 μL+20 μL, 160 μL+40 μL, 140 μL+60 μL, and 60 μL+60 μL experimental groups, respectively;

[0103] 3. The mixing, incubation and plate reading settings are the same as in Example 3;

[0104] 4. Data processing: Use antibody concentration as the horizontal axis and corrected PI as the vertical axis to draw a standard curve, such as Figure 6 .

[0105] Example 6 reflects the effects of different fluorescent tracing molecules and antibody reaction ratios on the standard curve. For samples with higher concentrations (over 100 mg / L), a detection system with a fluorescent tracing molecule / sample solution volume ratio of 180 μL / 20 μL can be selected. For samples with concentrations below 100 mg / L, a detection system with a fluorescent tracing molecule / sample solution volume ratio of 160 μL / 40 μL can be selected.

[0106] Example 7: Simulation Sampling Accuracy Assessment

[0107] Take the detection system with a fluorescent tracking molecule / test sample solution volume ratio of 160μL / 40μL as an example:

[0108] 1. Dilute the IgG4 standard to 100 mg / L with the culture supernatant, dilute it 2-fold into 6 concentration gradients, add 40 μL to each well, and set up a blank solvent control group, that is, add an equal amount of culture supernatant to each well;

[0109] 2. Prepare simulated samples: dilute the IgG4 standard to 100 mg / L, 50 mg / L, 25 mg / L, and 5 mg / L with culture medium supernatant, 40 μL per well, and set up parallel duplicate wells for standard curves and simulated samples.

[0110] 3. Dilute the fluorescent tracking molecule to 0.42 mg / L with the culture medium supernatant, and add 160 μL of the diluted fluorescent tracking molecule solution to each well;

[0111] 4. The mixing, incubation and plate reading settings are the same as in Example 3;

[0112] 5. Data processing: The calculation method of the concentration of the simulated sample is as described in Example 5. Sample recovery rate = measured concentration / theoretical concentration*100%. The results are shown in Table 1 below.

[0113] Table 1

[0114]

[0115] Embodiment 8:

[0116] The specific implementation of the antibody concentration detection method of the present application is described in detail below:

[0117] 1. Add 40 μL / well of the sample solution to be tested to a black 96-well plate, dilute the standard solution in a certain gradient according to the actual situation, and operate synchronously with the sample solution;

[0118] The sample solution can be directly the cell culture supernatant; the diluent for the standard should be the same buffer as the sample to be tested. If the cell culture supernatant is used directly for testing, the blank cell culture supernatant can be used as the standard diluent.

[0119] 2. Dilute the fluorescent tracking molecule (FITC-SPA) stock solution prepared in Example 1 above to 0.42 mg / L, add 160 μl / well, and mix thoroughly with the above sample solution;

[0120] In this step, the fluorescent tracking molecule diluent can also use 1×PBS, ddH2O, cell culture medium, BSA solution, etc., but it should be noted that the higher the viscosity of the system, the slower the molecular movement speed and the lower the fluorescence polarization intensity;

[0121] The mixture can be evenly mixed by using a pipette or by shaking on a plate shaker for 30 seconds.

[0122] 3. Incubation conditions: room temperature, away from light, incubate for 20 minutes.

[0123] 4. Detection: Use an ELISA reader with fluorescence polarization detection function, select fluorescence polarization detection, set the excitation wavelength to 485nm, and the emission wavelength to 520nm. The instrument will read the light intensity in the horizontal and vertical directions, which are recorded as I ∥ and I ⊥ .

[0124] 5. Data processing: Calculate the fluorescence polarization intensity PI value of each well by the following formula: PI = I ∥ -I ⊥; Correct the fluorescence polarization intensity value of the standard / sample by deducting the fluorescence polarization intensity value of the blank control group, draw a standard curve with the known concentration of the standard as the horizontal axis and the correction value of the fluorescence polarization intensity as the vertical axis, and obtain the relationship between the concentration of the antibody to be tested and the fluorescence polarization intensity by linear fitting. Substitute the measured correction value of the fluorescence polarization intensity of the sample solution to be tested into this relationship to obtain the antibody concentration in the sample solution to be tested. Among them, the coefficient of variation CV of the duplicate well PI difference should be controlled below 2%.

Claims

1. A fluorescence polarization immunoassay method for determining antibody concentration, comprising the following steps: 1). Prepare antibody-binding proteins with fluorescent groups as fluorescent tracking molecules; 2). Mixing and incubating the fluorescent tracking molecule with the sample solution to be tested, simultaneously, mixing and incubating the fluorescent tracking molecule with a standard solution with a known antibody concentration, and simultaneously, mixing and incubating the fluorescent tracking molecule with a blank solvent without antibodies to serve as a blank control group; 3) After the incubation is completed, the light intensity in the horizontal and vertical directions of the incubated solution obtained in step 2) is detected using an ELISA instrument with a fluorescence polarization detection function, and is recorded as I ∥ and I ⊥ ;as well as 4). Data analysis: Use the calculation formula: Fluorescence polarization intensity PI = I ∥ -I ⊥ Calculate the fluorescence polarization intensity of the solution, correct the fluorescence polarization intensity value of the standard solution or the sample solution to be tested by deducting the fluorescence polarization intensity value of the blank control group, draw a standard curve with the known standard concentration as the abscissa and the corrected fluorescence polarization intensity value as the ordinate, and obtain a linear fitting relationship between the antibody concentration and the fluorescence polarization intensity. Substitute the corrected fluorescence polarization intensity value measured by the sample solution to be tested into the relationship to obtain the antibody concentration in the sample solution to be tested. in, In the method, two replicate wells are set for each concentration of the standard and the sample to be tested, and the mean PI value of the two replicate wells is used for data analysis.

2. The method according to claim 1, wherein: In step 1), the fluorescent tracking molecule is prepared by the following process: 1.1) Dissolve fluorescein isothiocyanate (FITC) in dimethyl sulfoxide (DMSO) to prepare FITC stock solution; 1.2) Prepare antibody-binding protein stock solution and ensure that the protein stock solution does not contain imidazole groups or components containing free amino groups; 1.3) mixing the FITC preparative solution with the antibody binding protein stock solution and performing a molecular coupling reaction; and 1.4) Terminate the coupling reaction and remove free FITC by purification to obtain a fluorescent tracking molecule solution.

3. The method according to claim 2, wherein: In step 1.1), the concentration of the obtained FITC preliminary solution is 10 mg / mL or more; In step 1.2), the concentration of the prepared antibody-binding protein stock solution is 5 mg / mL or more; and the antibody-binding protein is recombinant Staphylococcus protein A (SPA); In step 1.3), the FITC pre-solution and the antibody binding protein stock solution are mixed at a molar ratio of FITC to SPA of 18-20:1; In step 1.3), the concentration of the antibody binding protein is controlled at 2-5 mg / mL using a buffer having a pH in the range of 9.0-10.0, the mixture is fully mixed, and stirred overnight at 4° C. in the dark to perform a coupling reaction; In step 1.4), the coupling reaction is terminated by adding NH4Cl solution. After the coupling reaction is terminated, free FITC is removed by using Sepharose-25, and the buffer is replaced with 1×PBS or ddH2O.

4. The method according to claim 2, wherein: Step 1 also includes step 1.5): diluting the obtained fluorescent tracking molecule solution.

5. The method according to claim 4, in step 1.5), the fluorescent tracking molecule solution is diluted to a concentration of 0.40-0.70 mg / L using 1×PBS, ddH2O or CHO culture medium CD04.

6. The method according to claim 4, in step 1.5), the fluorescent tracking molecule solution is diluted to a concentration of 0.42 mg / L using 1×PBS, ddH2O or CHO culture medium CD04.

7. The method according to claim 1, wherein: In step 2), a detection system with a volume ratio of fluorescent tracking molecule / test sample solution of 160 μL / 40 μL is used, the incubation temperature is 20-25° C., and the incubation time is 20-30 minutes; In step 2), the antibody contained in the sample solution to be tested is a protein that can specifically bind to the recombinant Staphylococcus protein A.

8. The method according to claim 1, wherein: In step 2), the antibody types contained in the sample solution to be tested are selected from human IgG1, IgG2, IgG4, IgM, IgA, mouse IgG1, IgG2, IgG3, IgM, and antibody Fc-terminal fusion proteins and bispecific antibodies modified on these antibody types.

9. The method according to claim 1, wherein: In step 3), the excitation wavelength was set to 485 nm and the emission wavelength was set to 520 nm.

10. The method according to claim 1, wherein: In step 4), data analysis meets the following requirements: 1) The coefficient of variation (CV) of two parallel wells is less than 2%; 2) Determine the standard curve using at least 6 standard concentration gradients; 3) Linear fit R of the standard curve 2 Above 0.999; as well as 4) The concentration of the sample solution to be tested falls between the lowest dilution concentration and the highest dilution concentration of the standard curve.

11. Use of the method according to any one of claims 1 to 10 in screening cell lines expressing antibodies.

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