Method for detecting concentration of IL4R monoclonal antibody

Abstract

The invention provides a method for detecting the concentration of an IL4R monoclonal antibody, and belongs to the technical field of biological detection. The method for detecting the concentration of the IL4R monoclonal antibody, provided by the invention, is good in intra-batch and inter-batch accuracy and precision, can be used for preparing a standard curve sample by adopting the IL4R monoclonal antibody to carry out IL4R monoclonal antibody sample detection, and has an excellent application prospect. A verification result of a specific mode of the invention shows that the RLU value sequence of each final concentration point of the IL4R monoclonal antibody obtained in the invention is consistent with the concentration sequence, which shows that no HOOK effect exists, and the method for detecting the concentration of the IL4R monoclonal antibody provided by the invention has the advantages of high accuracy and good reproducibility, and can be used for detecting the concentration of the IL4R monoclonal antibody in a serum sample.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for detecting the concentration of IL4R monoclonal antibody. Background Art

[0002] Interleukin IL-4 is a multi-effect immune cytokine, mainly produced by activated type II helper T cells, basophils and mast cells. IL-4 is involved in the survival and development of immune cells, regulates the development and maturation of B cells, and the proliferation and differentiation of type II helper T cells.

[0003] IL-4 binds to IL4R to exert its biological function. There are two main types of IL4R. Type I receptors are composed of IL-4Rα subunit and γc subunit, and are mainly located on the surface of T cells and NK cells. Type II receptors are composed of IL-4Rα subunit and IL-13Rα1 subunit, and are commonly found on the surface of various solid tumors, such as lung cancer, kidney cancer, melanoma, breast cancer, ovarian cancer, and colon cancer.

[0004] Type II IL-4R contains an IL-13Rα1 subunit. Although IL-4 has a greater affinity for type II IL-4R than IL-13, IL-13 can also bind to this receptor to produce biological effects. IL-13 can bind to the IL-13Rα1 subunit in type II IL-4R on the surface of airway epithelial cells to promote the occurrence of asthma. In addition, it is also involved in the progression of diseases such as allergies and atopic dermatitis. IL-4 has a strong promoting effect on tumor migration and invasion. At the same time, IL-4 is upregulated in the microenvironment of tumors such as breast cancer and colon cancer. At the same time, due to the overexpression of IL-4R on the surface of tumor cells, it can be used as an anti-tumor target molecule to design drugs.

[0005] The IL4R signaling pathway plays an important role in the immune system, mediating allergic and type II inflammatory responses. IL4R is upregulated in various tumors and promotes tumor metastasis through migration, infiltration, survival, proliferation, etc. Currently, the FDA has approved several drugs targeting the IL4R signaling pathway for the treatment of asthma, but these drugs are also used in the development of tumor treatment. Detecting the concentration of IL4R antibodies in serum will have broad application prospects.

[0006] In the prior art, methods capable of accurately detecting the concentration of IL4R antibodies in serum have yet to be developed. Summary of the invention

[0007] The object of the present invention is to provide a method for detecting the concentration of IL4R monoclonal antibody, which provides an effective and reliable means for detecting the concentration of IL4R monoclonal antibody for non-diagnosis and treatment purposes.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A method for detecting the concentration of IL4R monoclonal antibody comprises the following steps:

[0010] S1, coating the captured antigen onto the ELISA plate and blocking it;

[0011] S2, adding the standard curve sample, the quality control sample and the sample to be tested to the ELISA plate, incubating, and obtaining the antigen-antibody complex captured on the plate;

[0012] S3, adding detection antibody and incubating;

[0013] S4. Add DELFIA enhancement solution, incubate and detect, record the RLU value of 334nm excitation light and 620-10nm emission light, and the IL4R monoclonal antibody concentration is positively correlated with the obtained RLU value.

[0014] Preferably, the capture antigen is a recombinant human IL4R / CD124 protein with a His tag.

[0015] Preferably, in S1, the coating solution used for coating is CBS.

[0016] Preferably, the concentration of CBS is 0.03-0.08M.

[0017] Preferably, the capture antigen is diluted and added to the ELISA plate, and the final concentration of the dilution is 0.5-1.5 μg / mL.

[0018] Preferably, in S2, the incubation time is 0.5 to 2 hours.

[0019] Preferably, in S3, the detection antibody is DELFIAEu-N1 Anti-Human IgG.

[0020] Preferably, in S3, the incubation time is 0.5 to 2 hours.

[0021] Preferably, in S4, the amount of the DELFIA enhancement solution in the ELISA plate is 80-120 μL / well.

[0022] Preferably, in S4, the incubation time is 10 to 30 minutes.

[0023] Preferably, the method for detecting the IL4R monoclonal antibody concentration provided by the present invention is for non-diagnostic and therapeutic purposes.

[0024] Beneficial effects of the present invention:

[0025] The detection method of IL4R monoclonal antibody concentration provided by the present invention has good intra-batch and inter-batch accuracy and precision, and can use IL4R monoclonal antibody to prepare standard curve samples for IL4R monoclonal antibody sample detection, which has excellent application prospects. It can be seen from the verification results of the specific method of the present invention that the order of the RLU values ​​of each final concentration point of IL4R monoclonal antibody obtained in the present invention is consistent with the order of concentration, indicating that there is no HOOK effect. The method for detecting the concentration of IL4R monoclonal antibody provided by the present invention has high accuracy and good reproducibility, and can be used for the detection of IL4R monoclonal antibody concentration in serum samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a typical standard curve diagram for detecting the concentration of IL4R monoclonal antibody in mouse serum according to the present invention. DETAILED DESCRIPTION

[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Main instruments and equipment used in the specific implementation of the present invention: Multifunctional microplate reader

[0029] Thermofisher(Thermo)Varioskan LUX

[0030] The main software / data systems used are shown in Table 1 below

[0031] Table 1 Main software / data systems used in the experiment

[0032] Software / Data System Version Number use SkanItDDE 7.0 Edit and control the plate reading program of the multifunctional microplate reader and obtain the RLU value of the sample WatsonLIMS v7.6.1SP1 Fit standard curve, issue standard curve parameters, calculate quality control and sample concentration, etc. Excel 2010 Calculate quality control and sample concentration, data calculation

[0033] Method Materials:

[0034] Reference Material Information

[0035] Name or code: IL4R monoclonal antibody;

[0036] Properties: Colorless, clear liquid (visual properties);

[0037] Specification: 300mg (2mL) / bottle;

[0038] Key reagents are shown in Table 2 below:

[0039] Table 2 Key reagent information

[0040]

[0041] Note: “*” indicates the concentration is 50 ng / μL.

[0042] Self-prepared reagents are shown in Table 3 below:

[0043] Table 3 Self-prepared reagent information

[0044]

[0045]

[0046] Note: The preparation method of Assay buffer is to dissolve 1 bag of Tris Buffered Saline (PH=8.0) in 1L of pure water, and then add 10g BSA and 0.5mL Tween and mix well.

[0047] Blank matrix: Mouse serum was obtained from the blank matrix library of our institution and stored at -15 to -25°C for use as standard curve, quality control samples and sample dilution. It was thawed at room temperature before use.

[0048] Example

[0049] Preparation of standard curve and quality control samples:

[0050] 1. Preparation of standard curve samples

[0051] Prepare the standard curve according to the table below. First, add the corresponding volume of mouse serum to the centrifuge tube, and then take the corresponding volume of IL4R monoclonal antibody (concentration: 154 mg / mL), A, B, STD1~STD7 into the centrifuge tube (see Table 4-1), vortex to mix and set aside.

[0052] Table 4-1 Preparation of standard curve samples

[0053] Solution name Aspirate solution Aspiration volume (μL) Mouse blank serum (μL) Target concentration (ng / mL) A IL4R monoclonal antibody 5 72 10000000 B A 10 90 1000000 STD1 B 5 120 40000 STD2 STD1 50 50 20000 STD3 STD2 50 50 10000 STD4 STD3 50 50 5000 STD5 STD4 50 50 2500 STD6 STD5 50 50 1250 STD7 STD6 50 50 625 STD8* STD7 50 50 313

[0054] Note: “*” is an anchor point, which participates in the standard curve fitting and may not meet the acceptance criteria;

[0055] The target concentration is calculated to three significant digits.

[0056] 2. Preparation of quality control samples

[0057] Prepare quality control samples according to the table below. First, add the corresponding volume of diluent to the centrifuge tube, and then take the corresponding volume of IL4R monoclonal antibody (concentration: 154 mg / mL), A, B, HQC, MQC, and LQC solutions into the centrifuge tube (see Table 4-2), vortex to mix and set aside.

[0058] Table 4-2 Preparation of quality control samples

[0059]

[0060] Note: The target concentration is calculated based on three significant digits.

[0061] 3. Sample Processing

[0062] Before adding samples, standard curve samples, quality control samples, mouse blank serum and samples to be tested were diluted 50 times with Assay buffer. For samples that required high dilution, they were diluted to the required multiple with mouse blank serum and then diluted 50 times with Assay buffer for testing. This ensures that the serum matrix content in all samples is consistent, and all contain 2% mouse blank serum.

[0063] 4. Detection steps

[0064] Coating: Dilute IL4R Protein, Human, Recombinant (ECD, His Tag) with coating solution (0.05M CBS) to a final concentration of 1 μg / mL, add to the ELISA plate, 100 μL / well, cover with film, and incubate at 2-8°C overnight.

[0065] Washing: discard the coating solution, wash three times with washing solution (0.05% PBST), 300 μL / well, and pat dry.

[0066] Blocking: Add Assaybuffer, 300 μL / well, cover with film, and incubate on a shaker at room temperature (about 300 rpm) for 2 h.

[0067] Wash the plate: wash three times with washing solution (0.05% PBST), 300 μL / well, and pat dry.

[0068] Sample addition: Add NSB (2% mouse blank serum), standard curve sample solution, quality control sample solution and test sample solution to the ELISA plate according to the sample arrangement diagram, 50 μL / well, cover with film, and incubate on a shaker at room temperature (about 300 rpm) for 1h±5min.

[0069] Wash the plate: wash three times with washing solution (0.05% PBST), 300 μL / well, and pat dry.

[0070] Add detection antibody: dilute DELFIAEu-N1 Anti-Human IgG to 30 ng / well with Assaybuffer, 50 μL / well, cover with membrane, and incubate on a shaker (about 300 rpm) for 1 h±5 min.

[0071] Wash plate: Wash 5 times with Assaybuffer, 300 μL / well, and pat dry.

[0072] Add enhancement solution: Add 100 μL / well of enhancement solution to the ELISA plate, cover with film, and incubate on a shaker (about 300 rpm) for 15 min.

[0073] Detection: Use an ELISA reader with excitation light at 334 nm and emission light at 620-10 nm to read the RLU value.

[0074] 5. Data Processing and Analysis

[0075] The RLU values ​​of the standard samples obtained by the multifunctional microplate reader are fitted with the standard curve equation using the four-parameter method, and the concentration results of each sample are calculated, and the standard curve parameters are issued.

[0076] average value: (C t is the measured concentration);

[0077] Relative error percentage: (C t is the measured concentration, C n is the theoretical concentration);

[0078] Coefficient of Variation Percent: (C t is the average value of the detected concentration);

[0079] Standard Deviation: ( is the average value of the detected concentration).

[0080] Total allowable error: %Total Error=|%RE|+%CV.

[0081] The OD value was rounded to three decimal places, the concentration result, SD, and standard curve parameters were rounded to three significant figures, and the accuracy (%RE), coefficient of variation (%CV), and total error (%Total Error) were rounded to one decimal place.

[0082] Experimental example

[0083] The aim of this study was to investigate whether the above analytical method can be used to detect the concentration of IL4R monoclonal antibody in mouse serum.

[0084] Standard curve and quantitation range

[0085] A total of 6 independent analysis batches were validated over 2 days, and all analysis batches were valid analysis batches. The quantitative range of the IL4R monoclonal antibody standard curve is: 625-40000ng / mL, the lower limit of quantification is: 625ng / mL, and the anchor point is 313ng / mL. The validation results of each concentration point of the standard curve of each analysis batch of IL4R monoclonal antibody are shown in Table 5:

[0086] Table 5 Validation results of the standard curve for the DELFIA method for detecting the concentration of IL4R monoclonal antibody in mouse serum

[0087]

[0088] Note: “ / ” indicates no calculated data; “#” indicates an anchor point that is not required to meet the standard curve acceptance criteria; “*” indicates that it does not meet the standard curve acceptance criteria and does not participate in the regression calculation.

[0089] Typical standard curves are shown in Figure 1 , the quasi-curve fitting parameters are summarized in Table 6:

[0090] Table 6 Summary of standard curve fitting parameters

[0091]

[0092] Note: Standard curve fitting formula: Resp. = (Min-Max) / (1 + (Conc. / Ed50) ** Slope) + Max, where Resp. is the instrument response value and Conc. is the sample concentration value.

[0093] Accuracy and precision

[0094] A total of 4 independent batches were validated over 2 days, and all of them passed valid analytical batches. In the same analytical batch, IL4R monoclonal antibody was prepared into 3 sets of quality control samples with 5 concentrations of ULOQ, HQC, MQC, LQC and LLOQ, and 2 replicates were set for each set. All quality control samples met the requirements that the average %RE within and between batches was within ±20.0% (the average %RE of ULOQ&LLOQ was within ±25.0%); precision (%CV) ≤20.0% (%CV of ULOQ&LLOQ ≤25.0%); total allowable error (%Total Error) ≤30.0% (the total allowable error of ULOQ&LLOQ ≤40.0%). This indicates that the analytical method has good accuracy and precision within and between batches, and IL4R monoclonal antibody can be used to prepare standard curve samples for IL4R monoclonal antibody sample testing. The specific results are shown in Table 7:

[0095] Table 7 Accuracy and precision of DELFIA method for detecting IL4R monoclonal antibody concentration in mouse serum

[0096]

[0097]

[0098] Dilution linearity and "HOOK" effect

[0099] After dilution, the measured signal values ​​of samples with final concentrations of 2 mg / mL and 500 μg / mL were higher than the upper limit of quantification (ALQ). The average concentration values ​​calculated at each dilution factor (50, 100, 500, 1000) were within ±20.0% of the theoretical value of %RE, and the %CV of the calculated concentrations of all samples was ≤20.0%, indicating that the sample dilutions of 50, 100, 500, and 1000 times were all acceptable dilution factors, with the maximum dilution factor being 1000 times. The order of the RLU values ​​at each final concentration point of the IL4R monoclonal antibody was consistent with the order of the concentrations, indicating that there was no HOOK effect. The specific results are shown in Table 8:

[0100] Table 8 Dilution linearity and "HOOK" effect of DELFIA method for detecting IL4R monoclonal antibody concentration in mouse serum

[0101]

[0102]

[0103] Note: “ / ” indicates no calculated data

[0104] In summary, the method for detecting the concentration of IL4R monoclonal antibody in mouse serum provided by the present invention has high accuracy and good reproducibility, and can be used to detect the concentration of IL4R monoclonal antibody in mouse serum samples.

[0105] 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 method for detecting the concentration of IL4R monoclonal antibody, characterized in that: The following steps are involved: S1, coating the captured antigen onto the ELISA plate and blocking it; S2, adding the standard curve sample, the quality control sample and the sample to be tested to the ELISA plate, incubating, and obtaining the antigen-antibody complex captured on the plate; S3, adding detection antibody and incubating; S4. Add DELFIA enhancement solution, incubate and detect, record the RLU value of 334nm excitation light and 620-10nm emission light, and the IL4R monoclonal antibody concentration is positively correlated with the obtained RLU value.

2. The detection method according to claim 1, characterized in that: The capture antigen is a recombinant human IL4R / CD124 protein with a His tag.

3. The detection method according to claim 1, characterized in that: In S1, the coating solution used for the coating is CBS.

4. The detection method according to claim 3, characterized in that: The concentration of the CBS is 0.03-0.08M.

5. The detection method according to claim 1, characterized in that: The capture antigen is diluted and added to the ELISA plate, and the final concentration of the dilution is 0.5-1.5 μg / mL.

6. The detection method according to claim 5, characterized in that: In S2, the incubation time is 0.5 to 2 hours.

7. The detection method according to claim 1, characterized in that: In S3, the detection antibody is DELFIA Eu-N1 Anti-Human IgG.

8. The detection method according to claim 7, characterized in that: In S3, the incubation time is 0.5 to 2 hours.

9. The detection method according to claim 1, characterized in that: In S4, the amount of the DELFIA enhancement solution in the ELISA plate is 80-120 μL / well.

10. The detection method according to claim 9, characterized in that: In S4, the incubation time is 10 to 30 minutes.

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