A method for detecting antibodies against recombinant human GLP-1-Fc fusion protein

Abstract

The present invention relates to the field of pharmaceutical detection technology. The present invention provides a method for detecting anti-recombinant human GLP-1-Fc fusion protein antibodies, comprising: (1) acidifying and incubating a sample to be tested, and then neutralizing it; (2) adding a working solution containing a biotin-labeled GLP-1 fusion protein and a GLP-1 fusion protein labeled with an electrochemiluminescent substance to the reaction solution after neutralization in step (1), and incubating the mixture; (3) adding the reaction solution after incubation in step (2) to a microplate coated with streptavidin, sealing the plate, incubating the plate, and then washing the plate; (4) adding a plate reading buffer to the microplate after washing in step (3), and performing electrochemiluminescence detection; the method has high sensitivity and a high drug tolerance level.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a method for detecting antibodies against recombinant human GLP-1-Fc fusion protein. Background Art

[0002] Recombinant human GLP-1-Fc fusion protein is a fusion protein composed of two recombinant human glucagon-like peptide-1 analogs, the C-termini of which are connected to IgG4 Fc (CH2) and Fc (CH3) through short peptides. Its half-life is extended to nearly 4 days, and 90% of its amino acid sequence is homologous to endogenous human GLP-1. It activates the GLP-1 receptor, promotes insulin secretion, and lowers the patient's fasting blood sugar; inhibits the secretion of glucagon after meals, and lowers the patient's postprandial blood sugar; prolongs gastric elimination time, reduces appetite, and improves pancreatic β-cell function, thereby producing a hypoglycemic effect.

[0003] As a biotherapeutic agent, recombinant human GLP-1-Fc fusion protein is a foreign antigen that may potentially induce an immune response, leading to the formation of anti-drug antibodies, which may cause undesirable effects such as loss of drug exposure, loss of efficacy, and serious adverse events. Therefore, immunogenicity assessment is an important component of biotherapeutic drug safety testing. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a method for detecting anti-recombinant human GLP-1-Fc fusion protein antibodies, which has high sensitivity and high drug tolerance level.

[0005] To this end, the present invention provides the following technical solutions:

[0006] A method for detecting antibodies against recombinant human GLP-1-Fc fusion protein, comprising:

[0007] (1) Acidifying and incubating the sample to be tested, and then neutralizing it;

[0008] (2) adding a working solution containing a biotin-labeled GLP-1 fusion protein and a GLP-1 fusion protein labeled with an electrochemiluminescent substance to the reaction solution after neutralization in step (1), and incubating;

[0009] (3) adding the reaction solution after incubation in step (2) to a microplate coated with streptavidin, sealing the plate, incubating the plate, and then washing the plate;

[0010] (4) Add plate reading buffer to the microplate after washing in step (3) and perform electrochemiluminescence detection.

[0011] Optionally, in step (1), in the acidification step, the sample to be tested is mixed with the acidic solution in a volume ratio of 1:4; and / or

[0012] In step (1), in the acidification step, the acidic solution used is a 300 mM acetic acid solution.

[0013] Optionally, in step (1), in the neutralization step, a Tris-HCl solution with a concentration of 1 M and a pH of 9.5 is used as a neutralizing solution for neutralization;

[0014] Optionally, the volume ratio of the sample to be tested, the acidic solution and the neutralizing solution is 20:80:30.

[0015] Optionally, in step (2), in the working solution containing biotin-labeled GLP-1 fusion protein and electrochemiluminescent substance-labeled GLP-1 fusion protein, the concentration of biotin-labeled GLP-1 fusion protein is 0.5 μg / mL, and the concentration of electrochemiluminescent substance-labeled GLP-1 fusion protein is 0.5 μg / mL.

[0016] Optionally, in step (2), the volume ratio of the sample to be tested to the working solution containing the biotin-labeled GLP-1 fusion protein and the electrochemiluminescent substance-labeled GLP-1 fusion protein is 1:5; and / or

[0017] In step (2), the electrochemiluminescent substance in the GLP-1 fusion protein labeled with an electrochemiluminescent substance is ruthenium.

[0018] Optionally, in the incubation step, the incubation conditions are at room temperature and 500 rpm for 50-70 minutes.

[0019] Optionally, in the plate washing step, the plate is washed three times at 300 μL / well using a washing solution; and / or

[0020] The washing solution is 1×PBS containing 0.05 v / v% Tween 20.

[0021] Optionally, in step (3), before adding the reaction solution incubated in step (2) to the streptavidin-coated microplate, the process further includes blocking the streptavidin-coated microplate, adding an assay buffer at 150 μL / well, sealing the plate, and then incubating the plate at room temperature with shaking at 500 rpm for 1-3 hours, followed by washing the plate three times at 300 μL / well with a washing solution; and / or

[0022] In step (3), the reaction solution after incubation in step (2) is added to the microplate coated with streptavidin at a rate of 100 μL / well.

[0023] Optionally, in step (4), a plate reading buffer is added to the microplate after washing in step (3) at a rate of 150 μL / well, a voltage is applied, the plate is read within 15 minutes, and the anti-recombinant human GLP-1-Fc fusion protein antibody is qualitatively detected based on the obtained electrochemiluminescence signal.

[0024] Optionally, the qualitative detection is carried out by testing a batch of individual sera to determine the threshold value, and if the electrochemiluminescence signal is detected above the threshold value, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is considered to be positive; if the electrochemiluminescence signal is detected below the threshold value, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is considered to be negative.

[0025] Optionally, the threshold includes a screening threshold, a confirmation threshold, or an endogenous GLP-1 confirmation threshold;

[0026] Optionally, the sample is judged by a preliminary screening threshold value, and the preliminary screening threshold value is determined by testing a batch of individual sera. When the S / N value of the sample to be tested is detected to be ≥ the preliminary screening threshold factor, the anti-recombinant human GLP-1-Fc fusion protein antibody in the sample to be tested is considered to be positive in the preliminary screening; when the S / N value of the sample to be tested is detected to be less than the preliminary screening threshold factor, the anti-recombinant human GLP-1-Fc fusion protein antibody in the sample to be tested is negative in the preliminary screening; the S / N value of the sample = the signal value of the sample / the signal value of the negative control sample NC;

[0027] Optionally, the sample is determined by the confirmation threshold, and the working solution containing biotin-labeled GLP-1 fusion protein and electrochemiluminescent substance-labeled GLP-1 fusion protein added in step (2) is replaced with an inhibition working solution containing biotin-labeled GLP-1 fusion protein, ruthenium-labeled GLP-1 fusion protein and recombinant human GLP-1 fusion protein, and then the confirmation threshold is determined by testing a batch of individual sera. When the detected signal inhibition rate is ≥ the confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the sample to be tested is confirmed positive; when the detected signal inhibition rate is < the confirmation threshold, the anti-recombinant human GLP-1-Fc fusion protein antibody in the sample to be tested is confirmed negative, and the signal inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%; the average signal value without drug addition is the average signal value of the negative control sample NC, and the average signal value with drug addition is the average signal value of the sample;

[0028] Optionally, the sample is determined by the endogenous GLP-1 confirmation threshold, and the working solution containing biotin-labeled GLP-1 fusion protein and electrochemiluminescent substance-labeled GLP-1 fusion protein added in step (2) is replaced with a working solution containing biotin-labeled GLP-1 fusion protein, ruthenium-labeled GLP-1 fusion protein and endogenous GLP-1, and then the endogenous GLP-1 confirmation threshold is determined by testing a batch of individual sera. When the signal inhibition rate is detected to be ≥ the endogenous GLP-1 confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample confirms the GLP-1 cross-reaction is positive; when the signal inhibition rate is detected to be < the confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample confirms the GLP-1 cross-reaction is negative; signal inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%; the average signal value without drug addition is the average signal value of the negative control sample NC, and the average signal value with drug addition is the average signal value of the sample.

[0029] The technical solution of the present invention has the following advantages:

[0030] 1. The present invention provides a method for detecting anti-recombinant human GLP-1-Fc fusion protein antibodies, comprising: (1) acidifying and incubating a sample to be tested, and then neutralizing it; (2) adding a working solution containing a biotin-labeled GLP-1 fusion protein and a GLP-1 fusion protein labeled with an electrochemiluminescent substance to the reaction solution after neutralization in step (1), and incubating; (3) adding the reaction solution after incubation in step (2) to a microplate coated with streptavidin, sealing the plate, incubating, and then washing the plate; (4) adding a plate reading buffer to the microplate after washing in step (3), and performing electrochemiluminescence detection; in the above method, based on the bivalency of the antibody, the sample to be tested (such as a serum sample) is treated with acid, and the anti-recombinant human GLP-1-Fc fusion protein antibody is separated from the recombinant human GLP-1-Fc fusion protein that may be contained in the sample to be tested. The sample to be tested is then neutralized with Tris-HCl, and biotin-labeled recombinant human GLP-1 fusion protein and recombinant human GLP-1 fusion protein labeled with an electrochemiluminescent substance are added to form a bridge complex. The incubated and bound sample to be tested is then added to a microplate pre-coated with streptavidin. The complex binds to the streptavidin on the plate through the biotin in the biotin-labeled recombinant human GLP-1 fusion protein, and the unbound substances are washed away. In the plate reading buffer, after applying voltage, an electrochemiluminescent substance such as ruthenium produces an electrochemiluminescent signal value. Only antibodies that bind to both the biotin-labeled recombinant human GLP-1 fusion protein and the electrochemiluminescent substance-labeled recombinant human GLP-1 fusion protein can produce a signal. Therefore, qualitative detection of anti-recombinant human GLP-1-Fc fusion protein antibodies can be performed;

[0031] Furthermore, the method has high sensitivity, with a screening sensitivity of 8.3 ng / mL, a confirmation sensitivity of 13.6 ng / mL, and an endogenous GLP-1 confirmation sensitivity of 3.1 ng / mL. It also has a high drug tolerance level. Under the condition of 100 ng / mL positive control antibody, the method of the present invention can tolerate recombinant human GLP-1-Fc protein concentrations between 500-1000 ng / mL. Under the condition of 500 ng / mL positive control antibody, the method can tolerate recombinant human GLP-1-Fc protein concentrations of 5857 ng / mL. DETAILED DESCRIPTION

[0032] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0033] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. If the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional reagents.

[0034] In the following embodiments:

[0035] 1. Abbreviations

[0036]

[0037]

[0038] 2. Data processing

[0039] Data acquisition system, MSD Discovery Workbench 4.0.

[0040] Data processing system, Microsoft Excel 2016.

[0041] 3. Reagents

[0042] Table 2. Reagents

[0043]

[0044]

[0045] 4. Instruments, equipment and consumables

[0046] Table 3. Instruments, Equipment and Consumables

[0047]

[0048] 5. Solution preparation

[0049] Washing solution: 1×PBS containing 0.05v / v% Tween 20.

[0050] Assay buffer: 1×PBS containing 1 wt% BSA and 0.05 v / v% Tween 20.

[0051] 300mM acetic acid solution: Add glacial acetic acid to pure water and mix thoroughly to make the final concentration of acetic acid 300mM.

[0052] Working solution containing biotin-labeled GLP-1 fusion protein and ruthenium-labeled GLP-1 fusion protein: Add Bio-GLP-1 fusion protein (Biotin-CRB1501) and Ru-GLP-1 fusion protein (Ru-CRB1501) to the assay buffer and mix thoroughly to make the final concentration of Bio-GLP-1 fusion protein and Ru-GLP-1 fusion protein reach 0.5 μg / mL. Store at room temperature away from light and use on the day of preparation.

[0053] Inhibition working solution: Add 120 μL of CRB1501 (recombinant human GLP-1 fusion protein) stock solution to 4662 μL of working solution containing biotin-labeled GLP-1 fusion protein and ruthenium-labeled GLP-1 fusion protein, mix thoroughly to make the CRB1501 concentration reach 40 μg / mL, store at room temperature in the dark, and use on the day of preparation.

[0054] Working solution containing biotin-labeled GLP-1 fusion protein, ruthenium-labeled GLP-1 fusion protein and endogenous GLP-1: Add the endogenous GLP-1 stock solution to the working solution containing biotin-labeled GLP-1 fusion protein and ruthenium-labeled GLP-1 fusion protein, mix thoroughly to make the concentration of endogenous GLP-1 reach 40μg / mL, the concentration of biotin-labeled GLP-1 fusion protein reach approximately 0.5μg / mL, and the concentration of ruthenium-labeled GLP-1 fusion protein reach approximately 0.5μg / mL. Store at room temperature away from light and use on the day of preparation.

[0055] Reading buffer: Mix equal volumes of MSD Read Buffer T (4X) With Surfactant with pure water and store at room temperature for 6 months.

[0056] The room temperature in the following examples is in the range of 20-25°C.

[0057] Example 1

[0058] This embodiment provides a method for detecting antibodies against recombinant human GLP-1-Fc fusion protein, comprising the following steps:

[0059] (1) Add 20 μL / well of the sample to be tested to dilution plate 1, then add 80 μL / well of 300 mM acetic acid solution, incubate at room temperature and 500 rpm for 60 minutes, then add 30 μL / well of Tris-HCl solution (1 M, pH 9.5) to neutralize, and mix on a shaker for about 1 minute;

[0060] (2) Add 100 μL / well of a working solution containing biotin-labeled GLP-1 fusion protein and ruthenium-labeled GLP-1 fusion protein (the concentration of biotin-labeled GLP-1 fusion protein is 0.5 μg / mL, and the concentration of ruthenium-labeled GLP-1 fusion protein is 0.5 μg / mL) to the reaction solution after neutralization in step (1), and incubate at room temperature and 500 rpm for 60 minutes;

[0061] (3) First, the microplate coated with streptavidin (MSD-SA plate) was blocked, and the analysis buffer was added at 150 μL / well, and the plate was sealed with a sealing film. Then, the plate was incubated at room temperature and shaken at 500 rpm for 2 h. Then, the plate was washed three times with a washing solution at 300 μL / well and patted dry on absorbent paper. Then, the reaction solution after incubation in step (2) was added to the microplate coated with streptavidin at 100 μL / well, and the plate was sealed with a sealing film. The plate was incubated at room temperature and 500 rpm for 60 minutes. Then, the plate was washed three times with a washing solution at 300 μL / well and patted dry on absorbent paper.

[0062] (4) adding plate reading buffer to the microplate after washing in step (3) at a rate of 150 μL / well, applying voltage, reading the plate within 15 minutes, and performing qualitative detection of anti-recombinant human GLP-1-Fc fusion protein antibodies based on the obtained electrochemiluminescence signal;

[0063] The qualitative test:

[0064] Determine the sample by the initial screening threshold: According to the above method, the initial screening threshold is determined by testing a batch of individual sera. When the S / N value of the sample to be tested is detected to be ≥ the initial screening threshold factor, the anti-recombinant human GLP-1-Fc fusion protein antibody in the sample to be tested is considered to be positive in the initial screening; if the S / N value of the sample to be tested is detected to be < the initial screening threshold factor, the anti-recombinant human GLP-1-Fc fusion protein antibody in the sample to be tested is negative in the initial screening; S / N = sample signal value / negative quality control sample NC signal value.

[0065] Determine the sample by the confirmation threshold: According to the above method, the difference is that in the above step (2), an inhibition working solution containing biotin-labeled GLP-1 fusion protein, ruthenium-labeled GLP-1 fusion protein and recombinant human GLP-1 fusion protein is added to the reaction solution after neutralization in step (1) (the concentration of biotin-labeled GLP-1 fusion protein is about 0.5 μg / mL, the concentration of ruthenium-labeled GLP-1 fusion protein is about 0.5 μg / mL, and the concentration of recombinant human GLP-1 fusion protein is 40 μg / mL). Then, the confirmation threshold is determined by testing a batch of individual sera. When the detected signal inhibition rate is ≥ the confirmation threshold, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is considered to be confirmed positive; when the detected signal inhibition rate is < the confirmation threshold, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is considered to be negative. Signal inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%. The average signal value without adding drugs is the average signal value of the negative control sample NC, and the average signal value with adding drugs is the average signal value of the sample.

[0066] Determine the sample by the endogenous GLP-1 confirmation threshold: According to the above method, the difference is that in the above step (2), a working solution containing biotin-labeled GLP-1 fusion protein, ruthenium-labeled GLP-1 fusion protein and endogenous GLP-1 is added to the reaction solution neutralized in step (1) (the concentration of biotin-labeled GLP-1 fusion protein is about 0.5 μg / mL, the concentration of ruthenium-labeled GLP-1 fusion protein is about 0.5 μg / mL, and the concentration of endogenous GLP-1 is 40 μg / mL), and then the endogenous GLP-1 confirmation threshold is determined by testing a batch of individual sera. When the signal inhibition rate is detected to be ≥ the endogenous GLP-1 confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample confirms that the GLP-1 cross-reaction is positive; when the signal inhibition rate is detected to be < the endogenous GLP-1 confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample confirms that the GLP-1 cross-reaction is negative. Signal inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%. The average signal value without drug addition is the average signal value of the negative control sample NC, and the average signal value with drug addition is the average signal value of the sample.

[0067] Example 2

[0068] This embodiment provides a method for detecting antibodies against recombinant human GLP-1-Fc fusion protein, comprising the following steps:

[0069] (1) In dilution plate 1, take 20 μL / well of the sample to be tested, add 80 μL / well of 300 mM acetic acid solution, incubate at room temperature and 500 rpm for 50 minutes, then add 30 μL / well of Tris-HCl solution (1 M, pH 9.5) to neutralize, and mix on a shaker for about 1 minute;

[0070] (2) Add 100 μL / well of biotin-labeled GLP-1 fusion protein and ruthenium-labeled GLP-1 fusion protein working solution (biotin-labeled GLP-1 fusion protein concentration is 0.5 μg / mL, ruthenium-labeled GLP-1 fusion protein concentration is 0.5 μg / mL) to the reaction solution after neutralization in step (1), and incubate at room temperature and 500 rpm for 50 minutes;

[0071] (3) First, the microplate coated with streptavidin was blocked, and the assay buffer was added at 150 μL / well, and the plate was sealed with a sealing film. Then, the plate was incubated at room temperature with shaking at 500 rpm for 1 h, and then the plate was washed three times with a washing solution at 300 μL / well, and patted dry on absorbent paper. Then, the reaction solution after incubation in step (2) was added to the microplate coated with streptavidin at 100 μL / well, and the plate was sealed with a sealing film. The plate was incubated at room temperature at 500 rpm for 50 minutes, and then the plate was washed three times with a washing solution at 300 μL / well, and patted dry on absorbent paper.

[0072] (4) adding plate reading buffer to the microplate after washing in step (3) at a rate of 150 μL / well, applying voltage, reading the plate within 15 minutes, and performing qualitative or quantitative detection of the anti-recombinant human GLP-1-Fc fusion protein antibody based on the obtained electrochemiluminescence signal;

[0073] The qualitative test:

[0074] Determine the sample by the initial screening threshold: According to the above method, the initial screening threshold is determined by testing a batch of individual sera. When the detected S / N value is ≥ the initial screening threshold factor, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is considered to be positive in the initial screening; if the detected S / N value is < the initial screening threshold factor, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is negative in the initial screening; S / N = sample signal value / negative quality control sample NC signal value.

[0075] Determine the sample by the confirmation threshold: According to the above method, the difference is that in the above step (2), an inhibition working solution containing biotin-labeled GLP-1 fusion protein, ruthenium-labeled GLP-1 fusion protein and recombinant human GLP-1 fusion protein is added to the reaction solution after neutralization in step (1) (the concentration of biotin-labeled GLP-1 fusion protein is about 0.5 μg / mL, the concentration of ruthenium-labeled GLP-1 fusion protein is about 0.5 μg / mL, and the concentration of recombinant human GLP-1 fusion protein is 40 μg / mL). Then, the confirmation threshold is determined by testing a batch of individual sera. When the detected signal inhibition rate is ≥ the confirmation threshold, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is confirmed positive; when the detected signal inhibition rate is < the confirmation threshold, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is confirmed negative. Signal inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%. The average signal value without adding drugs is the average signal value of the negative control sample NC, and the average signal value with adding drugs is the average signal value of the sample.

[0076] Determine the sample by the endogenous GLP-1 confirmation threshold: According to the above method, the difference is that in the above step (2), a working solution containing biotin-labeled GLP-1 fusion protein, ruthenium-labeled GLP-1 fusion protein and endogenous GLP-1 is added to the reaction solution neutralized in step (1) (the concentration of biotin-labeled GLP-1 fusion protein is about 0.5 μg / mL, the concentration of ruthenium-labeled GLP-1 fusion protein is about 0.5 μg / mL, and the concentration of endogenous GLP-1 is 40 μg / mL), and then the endogenous GLP-1 confirmation threshold is determined by testing a batch of individual sera. When the signal inhibition rate is detected to be ≥ the endogenous GLP-1 confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample confirms that the GLP-1 cross-reaction is positive; when the signal inhibition rate is detected to be < the endogenous GLP-1 confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample confirms that the GLP-1 cross-reaction is negative. Signal inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%. The average signal value without drug addition is the average signal value of the negative control sample NC, and the average signal value with drug addition is the average signal value of the sample.

[0077] Example 3

[0078] This embodiment provides a method for detecting antibodies against recombinant human GLP-1-Fc fusion protein, comprising the following steps:

[0079] (1) In dilution plate 1, take 20 μL / well of the sample to be tested, add 80 μL / well of 300 mM acetic acid solution, incubate at room temperature and 500 rpm for 70 minutes, then add 30 μL / well of Tris-HCl solution (1 M, pH 9.5) to neutralize, and mix on a shaker for about 1 minute;

[0080] (2) Add 100 μL / well of a working solution containing biotin-labeled GLP-1 fusion protein and ruthenium-labeled GLP-1 fusion protein (the concentration of biotin-labeled GLP-1 fusion protein is 0.5 μg / mL, and the concentration of ruthenium-labeled GLP-1 fusion protein is 0.5 μg / mL) to the reaction solution after neutralization in step (1), and incubate at room temperature and 500 rpm for 70 minutes;

[0081] (3) First, the microplate coated with streptavidin was blocked, and the assay buffer was added at 150 μL / well, and the plate was sealed with a sealing film. Then, the plate was incubated at room temperature with shaking at 500 rpm for 3 h, and then the plate was washed three times with a washing solution at 300 μL / well, and patted dry on absorbent paper. Then, the reaction solution after incubation in step (2) was added to the microplate coated with streptavidin at 100 μL / well, and the plate was sealed with a sealing film. The plate was incubated at room temperature at 500 rpm for 70 minutes, and then the plate was washed three times with a washing solution at 300 μL / well, and patted dry on absorbent paper.

[0082] (4) adding plate reading buffer to the microplate after washing in step (3) at a rate of 150 μL / well, applying voltage, reading the plate within 15 minutes, and performing qualitative or quantitative detection of the anti-recombinant human GLP-1-Fc fusion protein antibody based on the obtained electrochemiluminescence signal;

[0083] The qualitative test: Determine the sample by the initial screening threshold: According to the above method, the initial screening threshold is determined by testing a batch of individual sera. When the detected S / N value is ≥ the initial screening threshold factor, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is considered to be positive in the initial screening; if the detected S / N value is < the initial screening threshold factor, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is negative in the initial screening; S / N = sample signal value / negative quality control sample NC signal value.

[0084] Determine the sample by the confirmation threshold: According to the above method, the difference is that in the above step (2), an inhibition working solution containing biotin-labeled GLP-1 fusion protein, ruthenium-labeled GLP-1 fusion protein and recombinant human GLP-1 fusion protein is added to the reaction solution after neutralization in step (1) (the concentration of biotin-labeled GLP-1 fusion protein is about 0.5 μg / mL, the concentration of ruthenium-labeled GLP-1 fusion protein is about 0.5 μg / mL, and the concentration of recombinant human GLP-1 fusion protein is 40 μg / mL). Then, the confirmation threshold is determined by testing a batch of individual sera. When the detected signal inhibition rate is ≥ the confirmation threshold, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is confirmed positive; when the detected signal inhibition rate is < the confirmation threshold, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is confirmed negative. Signal inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%. The average signal value without adding drugs is the average signal value of the negative control sample NC, and the average signal value with adding drugs is the average signal value of the sample.

[0085] Determine the sample by the endogenous GLP-1 confirmation threshold: According to the above method, the difference is that in the above step (2), a working solution containing biotin-labeled GLP-1 fusion protein, ruthenium-labeled GLP-1 fusion protein and endogenous GLP-1 is added to the reaction solution neutralized in step (1) (the concentration of biotin-labeled GLP-1 fusion protein is about 0.5 μg / mL, the concentration of ruthenium-labeled GLP-1 fusion protein is about 0.5 μg / mL, and the concentration of endogenous GLP-1 is 40 μg / mL), and then the endogenous GLP-1 confirmation threshold is determined by testing a batch of individual sera. When the signal inhibition rate is detected to be ≥ the endogenous GLP-1 confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample confirms that the GLP-1 cross-reaction is positive; when the signal inhibition rate is detected to be < the endogenous GLP-1 confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample confirms that the GLP-1 cross-reaction is negative. Signal inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%. The average signal value without drug addition is the average signal value of the negative control sample NC, and the average signal value with drug addition is the average signal value of the sample.

[0086] Experimental example

[0087] 1. Sample preparation

[0088] Sensitivity curve samples and drug tolerance samples were prepared according to the following table.

[0089] Table 4. Sensitivity curve sample preparation

[0090]

[0091] Table 5. Preparation of drug-tolerant samples

[0092]

[0093]

[0094] Mix 30 μL of 2*PC1, 2*PC2, and 30 μL of 2×D and incubate at room temperature and 600 rpm for 30 minutes.

[0095] 2. Experimental methods and results

[0096] (1) Confirmation of threshold

[0097] 1) Initial screening threshold

[0098] Method for detecting the primary screening threshold factor: The samples in Table 6 were tested according to the method of Example 1. At the same time, a negative control sample NC (not containing a positive control antibody) was set up, the signal values ​​obtained by the test were recorded, and the S / N ratio was calculated, where S / N = the average signal value of the sample / the signal value of NC.

[0099] Initial screening threshold = initial screening threshold factor × NC signal value.

[0100] Outliers are eliminated by the box plot method. The upper limit of the S / N value = 75th percentile + M×(75th percentile-25th percentile), and the lower limit = 25th percentile-M×(75th percentile-25th percentile). M can be used with a value between 1 and 3 (usually 1.5, 1.5 is taken in the present invention, see Table 6) to calculate and eliminate outliers. If there are more outliers (greater than 10%), it is necessary to investigate the experiment, research population and statistical methods to determine whether the elimination of these outliers is reasonable. The 95th percentile of the data after eliminating the outliers is taken to define the primary screening threshold factor SCPF. The formula is as follows:

[0101] SCPF=PERCENTILE(A1,A2…An,0.95);

[0102] Where A1, A2…An refer to all data after removing outliers.

[0103] Table 6. Initial screening threshold factors

[0104]

[0105]

[0106] 2) Confirmation Threshold Detection Method: The confirmation threshold is calculated based on the signal inhibition rate. The samples in Table 7 were tested according to the method in Example 1. A negative control sample (NC) (not containing the positive control antibody) was also tested. The signal values ​​obtained were recorded and the signal inhibition rate of the samples was calculated. The average signal value without drug addition was the average signal value of the negative control sample NC, and the average signal value with drug addition was the average signal value of the samples.

[0107] Inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%.

[0108] First, outliers are removed by the box plot method. The upper limit of the signal suppression rate = 75th percentile + M × (75th percentile - 25th percentile), and the lower limit = 25th percentile - M × (75th percentile - 25th percentile). M can be used with a value between 1 and 3 (usually 1.5, 1.5 was selected in this experiment, see Table 7) to calculate and remove outliers. If there are many outliers (greater than 10%), it is necessary to investigate the experiment, research population, and statistical methods to determine whether the removal of these outliers is reasonable. The 99th percentile of the data after removing the outliers is used to define the confirmation threshold CCP. Calculate according to the following formula:

[0109] CCP=PERCENTILE(A1,A2…An,0.99)

[0110] Where A1, A2…An refer to all data after removing outliers.

[0111] Table 7. Confirmation thresholds

[0112]

[0113]

[0114] 3) Endogenous GLP-1 Confirmation Threshold Detection Method: The GLP-1 confirmation threshold is calculated based on the signal inhibition rate. The samples listed in Table 8 were tested according to the method in Example 1. The signal values ​​obtained were recorded and the signal inhibition rate was calculated. The average signal value without drug addition was the average signal value of the negative control sample (NC). The average signal value with drug addition was the average signal value of the sample.

[0115] Inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%.

[0116] First, outliers are removed using a boxplot. The upper limit of the signal inhibition rate = 75th percentile + M × (75th percentile - 25th percentile), and the lower limit = 25th percentile - M × (75th percentile - 25th percentile). M can be a value between 1 and 3 (usually 1.5, selected in this experiment, see Table 8) to calculate and remove outliers. If there are a large number of outliers (greater than 10%), the experiment, study population, and statistical methods need to be investigated to determine whether the removal of these outliers is reasonable. The 99th percentile of the data after removing outliers is used to define the endogenous GLP-1 confirmation threshold nCCP. Calculate according to the following formula:

[0117] nCCP=PERCENTILE(A1,A2…An,0.99)

[0118] Where A1, A2…An refer to all data after removing outliers.

[0119] Table 8. Endogenous GLP-1 Confirmation Thresholds

[0120]

[0121]

[0122] (2) Sensitivity analysis

[0123] Sensitivity analysis method: Take the sensitivity curve samples in Table 4 (select no drug addition, addition of final concentration of 40 μg recombinant human GLP-1 fusion protein, and addition of final concentration of 40 μg endogenous GLP-1 fusion protein), and implement according to the method of Example 1. At the same time, set a negative quality control sample NC (without Rabbit PAb to human GLP-1-Fc protein), record the detected electrochemiluminescence signal value, and calculate the inhibition rate (%) = (no drug addition group signal value - drug addition group signal value) / no drug addition group signal value × 100%. The drug addition group is the above-mentioned addition of 40 μg recombinant human GLP-1 fusion protein or 40 μg endogenous GLP-1 fusion protein. S / N = sample signal value / NC signal value, CV (%) = standard deviation / average value × 100. The results are shown in the table below.

[0124] Screening sensitivity = FORECAST(x,known_y's,known_x's);

[0125] Among them, in the formula:

[0126] x:SCPF

[0127] Known_y's: The two sample concentrations across SCPF;

[0128] known_x's: is the S / N of the two samples across SCPF.

[0129] ConfirmSensitivity = FORECAST(x,known_y's,known_x's);

[0130] Among them, in the formula:

[0131] x:CPP

[0132] Known_y's: two sample concentrations across the CPP;

[0133] Known_x's: The signal suppression ratio across the two samples of the CPP. Endogenous GLP-1 Confirmation Sensitivity = FORECAST(x, known_y's, known_x's); where:

[0134] x:nCPP

[0135] Known_y's: the two sample concentrations across nCPP;

[0136] Known_x's: The signal suppression ratio across the two samples of nCPP.

[0137] Table 9 Sensitivity analysis results

[0138]

[0139] *The lowest concentration of the positive control antibody sample (3.125 ng / mL) still showed a signal inhibition rate greater than nCCP after the addition of 40 μg / mL endogenous GLP-1. This lowest concentration was directly used as the endogenous GLP-1 confirmation sensitivity.

[0140] (3) Drug tolerance analysis

[0141] 30 μL of each of samples 2*D1, 2*D2, 2*D3, and 2*D4 from Table 5 was selected and then mixed with 2*PC1 (30 μL) or 2*PC2 (30 μL) from Table 4, respectively. The mixture was incubated at room temperature at 600 rpm for 30 minutes. The positive control antibody concentrations and drug (recombinant human GLP-1-Fc fusion protein) concentrations in the prepared drug-tolerant samples DT1, DT2, DT3, and DT4 are shown in the table below. The drug-tolerant samples obtained above were treated according to the method of Example 1. A negative control sample NC (not containing Rabbit PAb to human GLP-1-Fc protein) was also prepared. The detected electrochemiluminescence signal values ​​were recorded: S / N = sample signal value / NC signal value, CV (%) = standard deviation / mean × 100; drug tolerance concentration = FORECAST(x, known_y's, known_x's).

[0142] Among them, in the formula:

[0143] x:SCPF

[0144] known_y's: drug concentrations across the two samples of the SCPF

[0145] Known_x's: S / N of two samples across SCPF...; see the table below for the results.

[0146] Table 10 Drug tolerance analysis results

[0147]

[0148] DT: drug-resistant samples

[0149] In summary, the screening sensitivity of the method of the present invention is 8.3 ng / mL, the confirmation sensitivity is 13.6 ng / mL, and the confirmation sensitivity of endogenous GLP-1 is 3.1 ng / mL;

[0150] Under the condition of 100 ng / mL positive control antibody, the method of the present invention can tolerate a recombinant human GLP-1-Fc protein concentration between 500-1000 ng / mL. Under the condition of 500 ng / mL positive control antibody, the method can tolerate a recombinant human GLP-1-Fc protein concentration of 5857 ng / mL.

[0151] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for detecting anti-recombinant human GLP-1-Fc fusion protein antibodies, characterized in that: include: (1) Acidify and incubate the sample to be tested, and then neutralize it; In the acidification step, the sample to be tested is mixed with an acidic solution in a volume ratio of 1:4; in the acidification step, the acidic solution used is a 300 mM acetic acid solution; and a 1 M Tris-HCl solution with a pH of 9.5 is used as a neutralizing solution for neutralization; The volume ratio of the sample to be tested, the acid solution and the neutralizing solution is 20:80:30; (2) adding a working solution containing a biotin-labeled GLP-1 fusion protein and a GLP-1 fusion protein labeled with an electrochemiluminescent substance to the reaction solution neutralized in step (1) and incubating; (3) Add the reaction solution after incubation in step (2) to the microplate coated with streptavidin, seal the plate, incubate, and then wash the plate; (4) adding plate reading buffer to the microplate after washing in step (3) and performing electrochemiluminescence detection; Qualitative detection: A threshold value is determined by testing a batch of individual sera. If the electrochemiluminescence signal is detected above the threshold value, the sample is considered positive for anti-recombinant human GLP-1-Fc fusion protein antibodies; if the electrochemiluminescence signal is detected below the threshold value, the sample is considered negative for anti-recombinant human GLP-1-Fc fusion protein antibodies. The threshold includes a screening threshold, a confirmation threshold or an endogenous GLP-1 confirmation threshold; The sample is judged by the initial screening threshold, which is determined by testing a batch of individual sera. When the S / N value of the sample to be tested is ≥ the initial screening threshold factor, the anti-recombinant human GLP-1-Fc fusion protein antibody in the sample to be tested is considered to be positive in the initial screening; when the S / N value of the sample to be tested is < the initial screening threshold factor, the anti-recombinant human GLP-1-Fc fusion protein antibody in the sample to be tested is negative in the initial screening; the S / N value of the sample = the sample signal value / the signal value of the negative quality control sample NC; The sample is judged by the confirmation threshold, and the working solution containing biotin-labeled GLP-1 fusion protein and electrochemiluminescent substance-labeled GLP-1 fusion protein added in step (2) is replaced with an inhibition working solution containing biotin-labeled GLP-1 fusion protein, ruthenium-labeled GLP-1 fusion protein and recombinant human GLP-1 fusion protein, and then the confirmation threshold is determined by testing a batch of individual sera. When the detected signal inhibition rate is ≥ the confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is confirmed positive; when the detected signal inhibition rate is < the confirmation threshold, the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample is confirmed negative, and the signal inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%; the average signal value without drug addition is the average signal value of the negative control sample NC, and the average signal value with drug addition is the average signal value of the sample; The sample is determined by the endogenous GLP-1 confirmation threshold. The working solution containing biotin-labeled GLP-1 fusion protein and electrochemiluminescent substance-labeled GLP-1 fusion protein added in step (2) is replaced with a working solution containing biotin-labeled GLP-1 fusion protein, ruthenium-labeled GLP-1 fusion protein and endogenous GLP-1. Then, the endogenous GLP-1 confirmation threshold is determined by testing a batch of individual sera. When the signal inhibition rate is detected to be ≥ the endogenous GLP-1 confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample confirms the GLP-1 cross-reaction is positive; when the signal inhibition rate is detected to be less than the confirmation threshold, it is considered that the anti-recombinant human GLP-1-Fc fusion protein antibody in the test sample confirms the GLP-1 cross-reaction is negative; signal inhibition rate = (average signal value without drug addition - average signal value with drug addition) / average signal value without drug addition × 100%; the average signal value without drug addition is the average signal value of the negative control sample NC, and the average signal value with drug addition is the average signal value of the sample.

2. The method for detecting anti-recombinant human GLP-1-Fc fusion protein antibodies according to claim 1, characterized in that: In step (2), in the working solution containing the biotin-labeled GLP-1 fusion protein and the electrochemiluminescent substance-labeled GLP-1 fusion protein, the concentration of the biotin-labeled GLP-1 fusion protein is 0.5 μg / mL, and the concentration of the electrochemiluminescent substance-labeled GLP-1 fusion protein is 0.5 μg / mL.

3. The method for detecting anti-recombinant human GLP-1-Fc fusion protein antibodies according to any one of claims 1 to 2, characterized in that: In step (2), the volume ratio of the sample to be tested to the working solution containing the biotin-labeled GLP-1 fusion protein and the electrochemiluminescent substance-labeled GLP-1 fusion protein is 1:5; and / or In step (2), the electrochemiluminescent substance in the GLP-1 fusion protein labeled with an electrochemiluminescent substance is ruthenium.

4. The method for detecting anti-recombinant human GLP-1-Fc fusion protein antibodies according to any one of claims 1 to 2, characterized in that: In the incubation step, the incubation condition is at room temperature and 500 rpm for 50-70 minutes.

5. The method for detecting anti-recombinant human GLP-1-Fc fusion protein antibodies according to any one of claims 1 to 2, characterized in that: In the plate washing step, the plate is washed three times at 300 μL / well using a washing solution; and / or The washing solution is 1×PBS containing 0.05 v / v% Tween 20.

6. The method for detecting anti-recombinant human GLP-1-Fc fusion protein antibodies according to any one of claims 1 to 2, characterized in that: In step (3), before adding the reaction solution incubated in step (2) to the streptavidin-coated microplate, the method further includes blocking the streptavidin-coated microplate by adding an assay buffer at 150 μL / well, sealing the plate, and then incubating the plate at room temperature with shaking at 500 rpm for 1-3 hours, followed by washing the plate three times at 300 μL / well with a washing solution; and / or In step (3), the reaction solution after incubation in step (2) is added to the microplate coated with streptavidin at a rate of 100 μL / well.

7. The method for detecting anti-recombinant human GLP-1-Fc fusion protein antibodies according to any one of claims 1 to 2, characterized in that: In step (4), a plate reading buffer is added to the microplate after washing in step (3) at a rate of 150 μL / well, a voltage is applied, and the plate is read within 15 minutes. The anti-recombinant human GLP-1-Fc fusion protein antibody is qualitatively detected based on the obtained electrochemiluminescence signal.

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