Method for qualitative detection of peg-rhifn alpha-2b antibodies and peg antibodies in plasma samples

Abstract

The application belongs to the technical field of biological medicine, and relates to a method for qualitatively detecting PEG-rhIFN alpha-2b antibodies and PEG antibodies in a plasma sample, which comprises the following steps: coating streptavidin on an enzyme-labeled plate, adding PEG-rhIFN alpha-2b-Biotin into the coated enzyme-labeled plate to incubate and combine, adding a to-be-detected sample and PEG-rhIFN alpha-2b-Digoxigenin into the above enzyme-labeled plate to co-incubate and combine, adding anti-digoxin-POD into the enzyme-labeled plate to incubate and combine, and forming a complex; terminating the reaction, measuring an OD value, and calculating the to-be-detected sample. The application adopts two sets of classical biological affinity recognition systems, significantly improves the detection performance, has good specificity and precision, high sensitivity, good drug resistance, and the characteristics of easy high-throughput detection, standardized operation and verification, and can simultaneously detect PEG antibodies and PEG-rhIFN alpha-2b antibodies.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for qualitative detection of PEG-rhIFNα-2b antibody and PEG antibody in plasma samples. Background Technology

[0002] Interferon alpha-2b is an interferon drug prepared through gene recombination technology. It belongs to the class of biological response modifiers and possesses broad-spectrum antiviral, antitumor, and immunomodulatory effects. Clinically, it is used to treat diseases such as chronic hepatitis B, hepatitis C, condyloma acuminata, hairy cell leukemia, and malignant melanoma. To prolong its half-life, long-acting recombinant interferon alpha-2b drugs have become the mainstream of current biotechnology drug development.

[0003] PEG-rhIFNα-2b, officially known as PEG-modified recombinant human interferon α-2b, is a long-acting interferon injection. In clinical treatment, it may elicit an immune response, leading to adverse immune reactions and the formation of anti-therapeutic antibodies (ADAs), indicating immunogenicity. Currently, there is no immunoquantitative method for detecting antibodies against the complete PEG-rhIFNα-2b molecule. Commercially available kits only detect rhIFNα antibodies and cannot distinguish the multiple domains of PEG-rhIFNα-2b. Furthermore, the presence of endogenous IFN and IFN antibodies in plasma interferes with the detection of antibodies against the entire PEG-rhIFNα-2b molecule. Therefore, selecting appropriate coating antigens and detection antibodies to specifically detect antibodies that recognize the complete PEG-rhIFNα-2b molecule and reflect its metabolism in vivo is a significant challenge in this field. Thus, there is an urgent need to establish a sensitive and specific method for detecting PEG-rhIFNα-2b antibodies to meet the needs of clinical analysis and evaluation of this type of drug.

[0004] Antibody detection typically employs a multi-stage analytical approach. First, all samples undergo screening tests. Then, specificity confirmation tests are performed on suspected antibody-positive samples. For confirmed antibody-positive samples, titer tests are conducted, and antibody neutralizing activity is assessed. Enzyme-linked immunosorbent assay (ELISA) is the preferred method for initial antibody screening / confirmation due to its speed, accuracy, and readily available reagents. However, in practice, factors such as reagents, materials, and the environment, especially interfering substances in the sample, can cause non-specific binding, leading to false positives and affecting the reliability of the results. The key to antibody detection methods lies in their specificity, sensitivity, and throughput. There is an urgent need to develop a method that improves specificity and sensitivity while reducing false positive and false negative rates to meet regulatory requirements and practical testing needs. Summary of the Invention

[0005] Due to dual-antigen interference, both anti-interferon antibodies and anti-PEG antibodies coexist in the body. Existing methods cannot simultaneously detect PEG antibodies and interferon antibodies; the steric hindrance and bivalent binding structure caused by the repeating epoxy units of PEG lead to false negatives and low signal-to-noise ratios; the heterogeneity of recombinant human interferon antibodies, due to the presence of IgM and IgG, makes it difficult for indirect methods to simultaneously cover subtypes. Based on the ELISA dual-antigen sandwich method, utilizing the specific binding of "streptavidin and biotin" and "digoxigenin and digoxigenin antibody," this invention provides a method for qualitatively detecting PEG-rhIFNα-2b antibodies and PEG antibodies in human plasma samples.

[0006] This invention employs two classic biological affinity recognition systems: a streptavidin-biotin interaction system and a digoxigenin-anti-digoxigenin antibody immunorecognition system. Through systematic optimization of antigen capture, reporter antibody, and reaction concentration conditions, detection performance is significantly improved, achieving faster detection, increased throughput, and cost optimization. Methodological evaluation of this invention shows good specificity and precision, high sensitivity, and good drug resistance. It is easily adapted for high-throughput detection, and its standardized operation and validation characteristics provide a technical means for meeting the clinical analysis and evaluation needs of long-acting interferon drugs. The method of this invention can simultaneously detect PEG-rhIFNα-2b antibody and PEG antibody by using biotinylated PEG-rhIFNα-2b and digoxigenin-labeled PEG-rhIFNα-2b.

[0007] Firstly, a method for qualitatively detecting PEG-rhIFNα-2b antibody and PEG antibody in plasma samples is provided, comprising the following steps: S1: Coat the ELISA plate with streptavidin to obtain the coated ELISA plate; S2: Biotin-labeled PEG-rhIFNα-2b (PEG-rhIFNα-2b-Biotin) is added to the coated ELISA plate and incubated to form a streptavidin-PEG-rhIFNα-2b-Biotin complex (antigen-biotin-labeled antigen complex, denoted as Streptavidin / PEG-rhIFNα-2b-Biotin). S3: Add the sample to be tested and digoxigenin-labeled PEG-rhIFNα-2b (PEG-rhIFNα-2b-Digoxigenin) to the ELISA plate obtained in S2 for co-incubation to form a streptavidin-biotin-labeled PEGylated human interferon α-2b-detection antibody-digoxigenin-labeled antigen complex (capture antigen-detection antibody-reporter antigen complex, denoted as Streptavidin / PEG-rhIFNα-2b-Biotin / PEG-rhIFNα-2b-ADA / PEG-rhIFNα-2b-Digoxigenin); S4: Add anti-digoxigenin-POD antibody to the ELISA plate obtained in S3 and incubate for binding. The complex is called Streptavidin / PEG-rhIFNα-2b-Biotin / PEG-rhIFNα-2b-ADA / PEG-rhIFNα-2b-Digoxigenin / anti-Digoxigenin-POD. S5: Add chromogenic substrate for color development, add stop reaction solution for reaction, measure OD value, and determine whether the sample contains PEG-rhIFNα-2b antibody and PEG antibody.

[0008] In some embodiments, the streptavidin is diluted with coating buffer to prepare a solution with a concentration of 1 to 10 μg / mL, preferably any one of the following ranges: 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, or any two of the above values.

[0009] In some embodiments, the coating buffer is prepared by thoroughly mixing 0.5M Na2CO3 and 0.5M NaHCO3 at a ratio of 1:(15~20) (V / V) and then diluting it 10~15 times with ultrapure water. Preferably, the volume ratio is 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, or any two of the above values. More preferably, the dilution is 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, or any two of the above values.

[0010] In some embodiments, in S1, streptavidin is coated at 100 μL / well and incubated overnight at 2-8°C and 600-800 rpm for 12-16 h, preferably any one of any two values ​​from 12 h, 13 h, 14 h, 15 h, 16 h, or above, preferably any one of any two values ​​from 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, or above.

[0011] In some embodiments, after coating in S1, the plate is washed with washing buffer, and then incubated with blocking buffer at room temperature to obtain the coated ELISA plate. In some embodiments, after coating in S1, the plate is washed with washing buffer, and then incubated with blocking buffer at room temperature (600-800 rpm) for 1-3 hours to obtain the coated ELISA plate. In this invention, room temperature refers to 20-30°C, preferably 25°C. In some embodiments, the washing buffer in S1 is 0.1-0.5 wt% Chaps-PBS solution. In some embodiments, the blocking buffer in S1 is 1-5 wt% BSA-Tris-HCl solution.

[0012] In some embodiments, the biotin-labeled human interferon α-2b is PEG-rhIFNα-2b-Biotin.

[0013] In some embodiments, the concentration of the biotin-labeled human interferon α-2b is 100-150 ng / mL, preferably 100 ng / mL, 110 ng / mL, 120 ng / mL, 130 ng / mL, 140 ng / mL, 150 ng / mL, or any two of the above values. In some embodiments, the incubation in S2 is carried out at 140-180 μL / well, preferably 140 μL / well, 150 μL / well, 160 μL / well, 170 μL / well, 180 μL / well, or any two of the above values, at room temperature, 600-800 rpm, for 1-3 h, preferably 1 h, 1.5 h, 2 h, 3 h, or any two of the above values.

[0014] In some embodiments, the preparation process of the PEG-rhIFNα-2b-Biotin includes: reconstituted a Sulfo-NHS-LC-Biotin solution, added PEG-rhIFNα-2b and boric acid, performing a biotinylation reaction, incubation, desalting via a desalting column, and collection. In some embodiments, the molar ratio of Sulfo-NHS-LC-Biotin to PEG-rhIFNα-2b is (7~9):1, preferably 7:1, 8:1, 9:1, or any two of the above values ​​forming a range. In some embodiments, the concentration of PEG-rhIFNα-2b is 5~5.5 mg / mL, preferably 5 mg / mL, 5.1 mg / mL, 5.2 mg / mL, 5.3 mg / mL, 5.4 mg / mL, 5.5 mg / mL, or any two of the above values ​​forming a range. In some embodiments, PEG-rhIFNα-2b accounts for 15-50% of the total reaction volume, preferably any two values ​​within the range of 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, etc. In some embodiments, the boric acid concentration is 100-120 mM, and the pH is 8-8.5. In some embodiments, boric acid accounts for 46.5-82.5% of the total reaction volume, preferably any two values ​​within the range of 46.5%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 82.1%, 82.2%, 82.4%, 82.5%. In some embodiments, the incubation is performed at room temperature for 60-80 minutes, preferably 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, etc. In some embodiments, Sulfo-NHS-LC-Biotin accounts for 1 to 5% of the total reaction volume, preferably 1%, 1.1%, 1.2%, 1.3%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, and any two of the above values ​​forming a range.

[0015] In some embodiments, in S3, the amount of the sample to be tested added is 200~250 μL / well, preferably 200 μL / well, 210 μL / well, 220 μL / well, 230 μL / well, 240 μL / well, 250 μL / well, or any two of the above values ​​forming a range.

[0016] In some embodiments, in S3, after the sample to be tested is added, digoxigenin-labeled antigen is added and co-incubated.

[0017] In some embodiments, in step S3, the digoxigenin-labeled antigen is PEG-rhIFNα-2b-Digoxigenin. In some embodiments, in step S3, the concentration of PEG-rhIFNα-2b-Digoxigenin is 100.0~150 ng / mL, preferably 100.0 ng / mL, 110.0 ng / mL, 120.0 ng / mL, 130.0 ng / mL, 140.0 ng / mL, 150.0 ng / mL, or any two of the above values ​​forming a range. In some embodiments, in step S3, the amount of PEG-rhIFNα-2b-Digoxigenin added is 50~100 μL / well, preferably 50 μL / well, 60 μL / well, 70 μL / well, 80 μL / well, 90 μL / well, 100 μL / well, or any two of the above values ​​forming a range. In some embodiments, after the PEG-rhIFNα-2b-Digoxigenin is added in S3, it is incubated at room temperature and 600-800 rpm for 1-5 hours, preferably 1 hour, 2 hours, 2.5 hours, 3 hours, 4 hours, or 5 hours.

[0018] In some embodiments, the preparation process of the PEG-rhIFNα-2b-Digoxigenin includes: reconstituted a DIG-X-NHS solution, added PEG-rhIFNα-2b and boric acid, performing a digoxigenin labeling reaction, incubation, desalting via a desalting column, and collection. In some embodiments, the molar ratio of DIG-X-NHS to PEG-rhIFNα-2b is (7~9):1, preferably 7:1, 8:1, 9:1, or any two of the above values ​​forming a range. In some embodiments, the concentration of PEG-rhIFNα-2b is 5~5.5 mg / mL, preferably 5 mg / mL, 5.1 mg / mL, 5.2 mg / mL, 5.3 mg / mL, 5.4 mg / mL, 5.5 mg / mL, or any two of the above values ​​forming a range. In some embodiments, the boric acid concentration is 100~120 mM, and the pH is 8~8.5. In some embodiments, boric acid accounts for 45-75% of the total reaction volume, preferably 45%, 46%, 46.5%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 73.5%, 74%, 74.5%, 75%, or any two of the above values ​​forming a range. In some embodiments, the concentration of the DIG-X-NHS solution is 10-15 mM, accounting for 1.5-3.5% of the reaction volume, preferably 1.5%, 1.6%, 1.7%, 1.8%, 2%, 2.5%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, or any two of the above values ​​forming a range. In some embodiments, PEG-rhIFNα-2b accounts for 25-50% of the total reaction volume, preferably 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, or any two of the above values ​​forming a range.

[0019] In some embodiments, in step S4, the amount of anti-digoxin-POD added is 50-100 μL / well, preferably 50 μL / well, 60 μL / well, 70 μL / well, 80 μL / well, 90 μL / well, 100 μL / well, or any two of the above values ​​forming a range. In some embodiments, in step S4, the concentration of anti-digoxin-POD is 50-100.0 ng / mL, preferably 50.0 ng / mL, 60.0 ng / mL, 70.0 ng / mL, 80.0 ng / mL, 90.0 ng / mL, 100.0 ng / mL, or any two of the above values ​​forming a range. In some embodiments, in step S3, after adding the anti-digoxin-POD, incubate at room temperature and 600-800 rpm for 1-5 hours. In some embodiments, the POD is HRP (horseradish peroxidase).

[0020] In some embodiments, the test sample is prepared by diluting plasma containing the test sample by 10 to 15 times with a diluent, preferably by 10 times.

[0021] In some embodiments, the diluent is at least one of 0.5-1 wt% BSA-PBS, 0.5-1 wt% BSA-0.05-0.1 wt% T-PBS, and 1-1.5 wt% BSA-0.1-0.5 wt% chaps-PBS, preferably at least one of 0.5 wt% BSA-PBS, 0.5 wt% BSA-0.05 wt% T-PBS, and 1 wt% BSA-0.1 wt% chaps-PBS.

[0022] In some embodiments, the chromogenic substrate is TMB.

[0023] In some embodiments, the amount of the chromogenic substrate added is 150~180 μL / well, preferably 150 μL / well, 160 μL / well, 170 μL / well, 180 μL / well, or any two of the above values ​​forming a range. In some embodiments, the chromogenic reaction is carried out at room temperature in the dark for 5~100 min, preferably 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, or any two of the above values ​​forming a range.

[0024] In some embodiments, the terminating reaction solution is 2-2.5M H3PO4. Preferably, it is 2.5M H3PO4.

[0025] In some embodiments, the OD value is measured at dual wavelengths of 450 nm and 620 nm. 620 nm is a reference wavelength, which is an auxiliary wavelength used to correct for non-specific background interference in the detection signal. It is equivalent to a non-absorbed background signal used to correct for instrument interference or systematic errors in the ELISA plate material.

[0026] In some embodiments, the OD value of the sample to be tested is calculated using the formula: sample OD = [(OD 450nm -OD 620nm ) 样品 - (OD) 450nm -OD 620nm ) 空白 ].

[0027] In some implementations, SNR = Mean (OD - sample to be tested) / Median (OD) NCs ).

[0028] In some implementations, during the screening phase, the screening threshold SNR for the PEG-rhIFNα-2b antibody and the PEG antibody is... SCP Based on a 5% false positive rate, the SNRSCP is calculated to be 1.298.

[0029] In some implementations, the sample detection value (SNR) is used. 样品 ) and SCP (i.e., SNR) SCP Compare and judge: SNR 样品 / SNR SCP ≥1.00; This indicates a positive result for PEG-rhIFNα-2b antibody or PEG antibody. SNR 样品 / SNR SCP <1.00; identified as PEG-rhIFNα-2b antibody or PEG antibody, negative.

[0030] In some implementations, positive samples are screened for confirmation. The inhibition rate of the sample to be tested is calculated using the formula: Inhibition rate % = 100% × (1 – SNR value of the drug-treated sample / SNR value of the untreated sample). The confirmation threshold for the PEG-rhIFNα-2b antibody is CCP (calculated based on a 1% false positive rate), CCP-PEG-rhIFNα-2b = 31.85%, CCP-PEG = 35.10%.

[0031] In some implementations, the test value of the sample is compared and determined with the confirmatory cutoff value (CCP): If the inhibition rate of the sample with the inhibitor is less than CCP, it is determined to be PEG-rhIFNα-2b antibody or PEG antibody, and the confirmation is negative; If the inhibition rate of the sample with the inhibitor is ≥ CCP, it is determined to be PEG-rhIFNα-2b antibody or PEG antibody, and confirmed as positive.

[0032] Compared with the prior art, one of the above technical solutions has the following advantages or beneficial effects: This invention employs two classic bioaffinity recognition systems: a streptavidin-biotin interaction system and a digoxigenin-anti-digoxigenin antibody immunorecognition system. Through systematic optimization of antigen capture, reporter antibody, and reaction concentration conditions, detection performance is significantly improved. This reduces the risk of false positives and false negatives while achieving faster detection speed, increased throughput, and cost optimization. Methodological evaluation results show that this invention exhibits good specificity, accuracy, and precision, high sensitivity, good drug resistance, and ease of high-throughput detection. Its standardized operation and validation characteristics provide a technical means for meeting the clinical analysis and evaluation needs of long-acting interferon drugs. This method can simultaneously detect PEG-rhIFNα-2b antibody and PEG antibody by using biotinylated PEG-rhIFNα-2b and digoxigenin-labeled PEG-rhIFNα-2b.

[0033] In this invention, ① human interferon α antibody is used as a reference: the sensitivity is 45.8 ng / mL; ② PEG antibody is used as a reference: the sensitivity is 71.5 ng / mL. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the double-antigen sandwich ELISA method for detecting PEG-rhIFNα-2b antibody.

[0035] Figure 2 Distribution of data for screening critical values ​​(SCP) and confirmatory critical values ​​(CCP).

[0036] Unless otherwise stated, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. All patents and publications related to this invention are incorporated herein by reference in their entirety. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0038] Table 1. Glossary of Terms

[0039] All reagents used in this invention can be purchased commercially or prepared by the methods described in this invention.

[0040] Table 2 Main Reagents

[0041] Example 1 1. Reagent Preparation (1) Preparation process of PEG-rhIFNα-2b-Biotin ①Biotin modification: According to the EZ-Link® Sulfo-NHS-LC-Biotinylation Kit instructions, the 10mM Sulfo-NHS-LC-Biotin solution was reconstituted, the volume of the reaction system added was calculated according to the modification molar ratio, and biotin modification was performed on each component according to Table 3.

[0042] Table 3. Components of the biotin modification reaction (Sulfo-NHS-LC-Biotin)

[0043] ② Desalting: Collect the permeate according to the instructions; this is the protein sample PEG-rhIFNα-2b-Biotin.

[0044] ③ Protein content measurement: The protein concentration was determined using a UV-spectrum spectrophotometer and was 0.690 mg / mL.

[0045] (2) Preparation process of PEG-rhIFNα-2b-Digoxigenin ① Digoxin modification: Digoxin was reconstituted according to the DIG-X-NHS instructions. The volume of the reaction system to be added was calculated according to the modification molar ratio. The components were reacted according to Table 4 to carry out digoxin modification.

[0046] Table 4. Components of the digoxin-modified reaction (DIG-X-NHS)

[0047] ② Desalting: Collect the permeate according to the instructions; this is the protein sample PEG-rhIFNα-2b-Digoxigenin.

[0048] ③ Protein content measurement: The protein concentration was determined using a UV-spectrum spectrophotometer and was 0.585 mg / mL.

[0049] (3) Preparation of standard products and samples (A) Experimental preparation for the initial screening stage: ① System Applicability Samples: HPC, LPC, NC: Detection of PEG-rhIFNα-2b antibody (using human interferon antibody as reference): HPC-P 150 ng / mL, LPC-P 5 ng / mL, NC (blank mixed plasma); Detection of PEG antibodies (using PEG antibody as a reference): HPC-A 400 ng / mL, LPC-A 10 ng / mL, NC (blank mixed plasma); ② Sample: Human plasma to be tested, diluted 10 times with 0.5wt% BSA-PBS.

[0050] (B) Experimental preparation for the confirmatory phase: ① System Applicability Samples: HPC, LPC, NC: Detection of PEG-rhIFNα-2b antibody (using human interferon antibody as reference): HPC-P 300 ng / mL, LPC-P 10 ng / mL, NC (blank mixed plasma); then diluted 50 / 5 with the inhibitor (PEG-rhIFNα-2b); Detection of PEG antibodies (using PEG antibody as a reference): HPC-A 400 ng / mL, LPC-A 10 ng / mL, NC (blank mixed plasma); ② Sample: Human plasma to be tested, diluted 5 times with 0.5wt% BSA-PBS. Then diluted 50 / 50 with the inhibitor (PEG-rhIFNα-2b).

[0051] 2. Operating Steps (1) Preparation of coating solution: Mix 0.5M Na2CO3 and 0.5M NaHCO3 at a ratio of 1:16 (v / v), dilute with ultrapure water 10 times, and wash the plate twice at 360μL / well.

[0052] (2) Wrapping: Use a wrapping blanket Liquid Dilute streptavidin to 5.0 μg / mL, apply 100 μL / well to a plate, and incubate overnight at 2-8℃ and 600 rpm for 12-16 h.

[0053] (3) Blocking: Discard the supernatant, wash the plate 3 times with 360 μL / well, add blocking solution, 200 μL / well, incubate at room temperature and 600 rpm for 1 h.

[0054] (4) Capture: Discard the supernatant, wash the plate 3 times with 360 μL / well, add PEG-rhIFNα-2b-Biotin (100.0 ng / mL), 150 μL / well, and incubate at room temperature for 1 h at 600 rpm.

[0055] (5) Plate loading: The system suitability samples (HPC, LPC / NC) and the test samples (with or without drugs) are added to the 96-well cell culture plate in advance according to the loading order on the ELISA plate at 230 μL / well (Tables 5 and 6 are the sample arrangements for the screening and confirmation stages, respectively).

[0056] (6) Sample loading: Discard the PEG-rhIFNα-2b-Biotin supernatant, wash the plate 3 times with 360 μL / well, and then load the sample in the cell plate row by row with a pipette at 100 μL / well, repeating the loading in duplicate. Then add PEG-rhIFNα-2b-Digoxigenin (100.0 ng / mL) at 50 μL / well; incubate at room temperature and 600 rpm for 1 h.

[0057] (7) Report: Discard the supernatant, wash the plate 3 times with 360 μL / well, add Anti-Digoxigenin-POD (0.05 U / mL) at 150 μL / well, and incubate at room temperature for 1 h at 600 rpm. POD is HRP.

[0058] (8) Color development: Discard the supernatant, wash the plate 3 times with 360 μL / well, add TMB at 150 μL / well, and develop the color at room temperature in the dark.

[0059] (9) Termination: Add 2.5M H3PO4 at 50µL / well to terminate the reaction, and measure the absorbance at dual wavelengths of 450nm / 620nm.

[0060] 3. Data Analysis The absorbance (OD) of a sample is measured using an ELISA reader. Sample OD = [(OD...] 450nm -OD 620nm ) 样品 - (OD) 450nm -OD 620nm ) 空白 To avoid the influence of external environmental factors on the analytical batch test values, SNR (Signal to Noise Ratio) is used instead of OD value, and the NC value is determined using the median method. SNR = Mean (OD validation sample or test sample) / Median (OD) NCs ).

[0061] In the confirmation phase (positive samples are screened to enter the confirmation phase), the inhibition rate of the sample to be tested is calculated using the following formula: Inhibition rate % = 100% × (1 – SNR value of the drug-treated sample / SNR value of the untreated sample).

[0062] Table 5 Sample arrangement during the screening stage

[0063] Note: The samples to be tested are designated as 1P and 2P, respectively, and so on; HPC-P and LPC-P represent high-concentration positive control and low-concentration positive control of PEG-IFNα-2b antibody, respectively; HPC-A and LPC-A represent high-concentration positive control and low-concentration positive control of PEG antibody, respectively; NC is blank mixed plasma from healthy sources.

[0064] Table 6. Sample arrangement during the confirmation phase

[0065] Note: ① The samples to be tested are represented by 1P and 2P respectively, and so on; 1P+PEG-rhIFNα-2b means that PEG-rhIFNα-2b has been added to sample 1 to be tested. ②HPC-P and LPC-P represent high and low concentration positive controls of PEG-rhIFNα-2b antibody; HPC-P+PEG-rhIFNα-2b and LPC-P+PEG-rhIFNα-2b represent high and low concentration confirmatory positive controls of PEG-rhIFNα-2b antibody with added PEG-rhIFNα-2b. ③HPC-A and LPC-A represent high and low concentration positive controls of PEG antibody; ④NC represents blank mixed serum from healthy sources, and NC+PEG-rhIFNα-2b represents negative control sample with added PEG-rhIFNα-2b.

[0066] 3.1 Determination of Critical Values ​​(SCP and CCP) At least 50 blank individual matrices were used, and six analytical batches were tested by two analysts over three days. Screening and confirmatory tests were performed in the same analytical batch. Screening cutoff values ​​(SCP) and confirmatory cutoff values ​​(CCP) were determined by adding inhibitors (PEG-rhIFNα-2b, rhIFNα-2b, and PEG) and without adding inhibitors. The sample testing arrangement is shown in Table 5. Results are as follows: Figure 2 The data distribution chart shown is used as a reference for auxiliary evaluation.

[0067] The screening critical value (SCP) is represented by the sample NR. The calculation process is as follows: The SNR values ​​of the screening samples are logarithmically transformed and outlier analyzed. The SCP value is calculated using a 5% false positive rate. That is: when the data is normally distributed, Log(SCP) = Mean + 1.645 × SD; when the data is not normally distributed but not highly biased, Log(SCP) = Median + 1.645 × (1.4826 × MAD) (MAD is the absolute median difference); when the data is not normally distributed and highly biased, (LogSCP) = 95th percentile. The antilogarithm of Log(SCP) is the screening critical value.

[0068] The confirmatory cutoff (CCP) is represented by the inhibition rate. The calculation process is as follows: Inhibition rate (Inhibition rate % = (1 – SNR value of samples with inhibitor / SNR value of samples without inhibitor) × 100%). After outlier analysis, a 1% false positive rate is used to calculate the CCP. That is: when the data is normally distributed, CCP = Mean + 2.33 × SD; when the data is not normally distributed but not highly biased, CCP = Mean + 2.33 × (1.4826 × MAD); when the data is not normally distributed and highly biased, CCP = 99th percentile.

[0069] Table 7. Critical values ​​(SCP and CCP) determine sample arrangement

[0070] Note: ① Individual plasma is represented by 1P, 2P, etc.; this is plate 1, the rest are omitted; 1P+PEG-IFNα-2b represents the sample of healthy individual 1 with PEG-IFNα-2b inhibitor added; 1P+IFNα-2b represents the sample of healthy individual 1 with IFNα-2b inhibitor added; 1P+PEG represents the sample of healthy individual 1 with PEG inhibitor added; NC is blank mixed plasma from healthy sources.

[0071] ②HPC-P and LPC-P represent high and low concentrations of PEG-IFNα-2b antibody positive controls; HPC-P+PEG-IFNα-2b and HPC-P+IFNα-2b represent high concentrations of PEG-IFNα-2b antibody with added PEG-IFNα-2b and IFNα-2b as confirmatory positive controls; LPC-P+PEG-IFNα-2b and LPC-P+IFNα-2b represent low concentrations of PEG-IFNα-2b antibody with added PEG-IFNα-2b and IFNα-2b as confirmatory positive controls. ③ HPC-A and LPC-A represent positive controls with high and low concentrations of PEG antibody; HPC-A+PEG and LPC-A+PEG represent positive controls with high and low concentrations of PEG antibody and added PEG. 3.3 Criteria for Judging Results in the Screening Phase (1) System suitability determination: LPC and HPC were determined to be positive; and SNR SCP ≤SNRLPC <SNRHPC That is, SNR SCP ≤SNR LPC-P <SNR HPC-P SNR SCP ≤SNR LPC-A <SNR HPC-A (2) Sample result determination SNR 样品 / SNR SCP ≥1.00; identified as PEG-IFNα-2b antibody or PEG antibody, screening positive.

[0072] SNR 样品 / SNR SCP <1.00; identified as PEG-IFNα-2b antibody or PEG antibody, screening negative.

[0073] 3.4 Criteria for Determining Results in the Confirmation Stage (1) System suitability determination: LPC and HPC were determined to be positive; and the inhibition rate was [missing information]. CCP ≤Inhibition rate LPC Inhibition rate HPC That is, inhibition rate CCP ≤Inhibition rate LPC-P Inhibition rate HPC-P Inhibition rate CCP ≤Inhibition rate LPC-A Inhibition rate HPC-A ; (2) Sample result determination If the inhibition rate of the sample with the inhibitor is less than that of CCP, it is determined to be PEG-IFNα-2b antibody or PEG antibody, and the confirmation is negative; If the inhibition rate of the sample with the inhibitor is ≥ CCP, it is determined to be PEG-IFNα-2b antibody or PEG antibody, and confirmed as positive.

[0074] The final test result for the sample shall be a confirmed positive result; 4. Methodological Research Results 4.1 Sensitivity Sensitivity was calculated by serially diluting PEG-IFNα-2b antibody samples in mixed plasma to 2000 ng / mL, 1000 ng / mL, 800 ng / mL, 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, and 13 ng / mL; and serially diluting PEG antibody samples to 5000 ng / mL, 2500 ng / mL, 1000 ng / mL, 800 ng / mL, 400 ng / mL, 200 ng / mL, 100 ng / mL, 50 ng / mL, and 25 ng / mL. The sensitivity was calculated using the intercept method across two points spanning the screening / confirmatory cutoff value. ① Using human interferon α antibody as a reference: the sensitivity of PEG-IFNα-2b antibody is 45.8 ng / mL; ② Using PEG antibody as a reference: PEG antibody with a sensitivity of 71.5 ng / mL. The results are shown in Table 8.

[0075] Table 8. Summary of Sensitivity Results

[0076] Note: Sensitivity calculation = Mean + t 0.05，df ×SD,t 0.05，df(5) =2.02.

[0077] 4.2 Hook effect PEG-IFNα-2b antibody and PEG antibody were diluted to specific concentrations (PEG-IFNα-2b antibody: 8000 ng / mL, 4000 ng / mL, 2000 ng / mL, 400 ng / mL, 200 ng / mL, 20 ng / mL; PEG antibody: 20000 ng / mL, 10000 ng / mL, 5000 ng / mL, 1000 ng / mL, 500 ng / mL, 100 ng / mL); the hook effect was investigated. The results showed that the SNR value did not decrease significantly with increasing positive reference concentration, and was therefore considered to have no hook effect.

[0078] ①PEG-IFNα-2b antibody: No hook effect in human whole plasma in the range of 20.0 ng / mL to 8000.0 ng / mL.

[0079] ②PEG antibody: No hook effect in human whole plasma in the range of 100.0 ng / mL to 20000.0 ng / mL.

[0080] The test results are shown in Tables 9 and 10.

[0081] Table 9 Summary of HOOK effect results of PEG-IFNα-2b antibody (with PEG-rhIFNα-2b as the inhibitor)

[0082] Note: "+" indicates a positive result in screening / confirmation, and "-" indicates a negative result in screening / confirmation. SNR1 represents the SNR value of a sample without inhibitor, and SNR2 represents the SNR value of a sample with inhibitor.

[0083] Table 10 Summary of PEG antibody hook effect results (PEG was added as the inhibitor)

[0084] Note: *The OD value of the sample is greater than 4.0; "+" indicates a positive result in screening / confirmation, "-" indicates a negative result in screening / confirmation. SNR1 represents the SNR value of the sample without the inhibitor, and SNR2 represents the SNR value of the sample with the inhibitor.

[0085] 4.3 Precision Intra-batch precision: assessed by the coefficient of variation (%CV) of the test values ​​from at least 6 sets of system suitability samples (NC, LPC, HPC).

[0086] Inter-batch precision: Examines the coefficient of variation (%CV) for all reportable analytical batches and system suitability samples.

[0087] Six different batches of validation samples showed: ①Precision of PEG-IFNα-2b antibody – Screening stage: Intra-assay %CV and inter-assay %CV are in the range of 1.8%-27.2% (of which the intra-assay %CV and inter-assay %CV of quality control samples are in the range of 1.8%-19.1%); ②Precision of PEG-IFNα-2b antibody – confirmatory stage: intra-assay %CV and inter-assay %CV are in the range of 0.1%-19.4%.

[0088] ③ PEG antibody precision – screening stage: intra-batch %CV and inter-batch %CV are in the range of 1.7%-23.7% (of which the intra-batch %CV and inter-batch %CV of quality control samples are in the range of 1.7%-11.5%); ④ PEG antibody precision – confirmatory stage: intra-assay %CV and inter-assay %CV are within the range of 0.0%-5.8%.

[0089] During the screening and validation phases of PEG-IFNα-2b antibody and PEG antibody quality control samples, the intra- and inter-batch %CV was no greater than 20%, and during the screening and validation phases of NC samples, the intra- and inter-batch %CV was no greater than 30%. The method of this invention demonstrates good precision in sample detection and meets acceptance criteria.

[0090] 4.4 Specificity Other antibodies (Anti-EPO, Anti-GH, etc.) were added at 150.0 ng / mL to PEG-IFNα-2b antibody (NC, LPC) samples for method specificity testing. Results showed that all LPC samples were positive, and all NC samples were negative, indicating that the specificity test met the acceptance criteria.

[0091] 4.5 Drug resistance Drug tolerance was assessed by adding drugs (PEG-IFNα-2b concentrations of 100,000, 50,000, 10,000, 5,000, 2,500, 1,000, 500, and 100 ng / mL) to PEG-IFNα-2b antibodies (LPC and HPC).

[0092] ① In human whole plasma, the maximum tolerable concentration of PEG-rhIFNα-2b is 5000.0 ng / mL when the PEG-IFNα-2b antibody is at 1500.0 ng / mL.

[0093] ② In human whole plasma, the maximum tolerable concentration of PEG-rhIFNα-2b at a PEG-IFNα-2b antibody concentration of 50.0 ng / mL was 1000.0 ng / mL. The test results are shown in Table 11.

[0094] Table 11. Results of human interferon α antibody drug tolerability (with PEG-rhIFNα-2b as the inhibitor).

[0095] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0096] Example 2 Samples were tested using the method described in Example 1. Ten individual samples were tested, and the selectivity results met the requirements (at least 80% of the low-concentration samples were positive, and at least 80% of the blank samples were screened and confirmed to be negative).

[0097] With the addition of the inhibitor PEG-rhIFNα-2b, 100% of the selective negative samples were screened and confirmed to be negative; 100% of the low-concentration positive samples were screened and confirmed to be positive, meeting the requirements. See Table 12.

[0098] With the addition of PEG as the inhibitor, the screening results were as follows: 80% of the negative selective samples were negative, and 100% of the low-concentration positive samples were positive. The confirmation results were as follows: 100% of the negative selective samples were confirmed negative, and 90% of the low-concentration positive samples were positive. (See Table 13)

[0099] Table 12 Summary of selectivity results for 10 plasma samples (with PEG-rhIFNα-2b as the inhibitor)

[0100] Note: "+" indicates a positive result in screening / confirmation, and "-" indicates a negative result in screening / confirmation. SNR1 represents the SNR value of a sample without inhibitor, and SNR2 represents the SNR value of a sample with inhibitor.

[0101] Table 13 Summary of selectivity results for 10 plasma samples (with PEG as the inhibitor)

[0102] Note: "+" indicates a positive result in screening / confirmation, and "-" indicates a negative result in screening / confirmation. SNR1 represents the SNR value of the sample without the inhibitor, and SNR2 represents the SNR value of the sample with the inhibitor. " / " indicates the removal of samples with a high %CV.

[0103] Example 3 The difference from Example 1 is that INFα-2b was used instead of SA for coating. The rest of the process is the same as in Example 1, and the double antigen sandwich method is used for detection to obtain rhIFN-α2b / PEG-IFNα-2b antibody / rhIFN-α2b-Biotin / Streptavindin-HRP.

[0104] The results showed that the OD values ​​of the individual blank matrix coated with INFα-2b were all low. Using the 95th percentile method, the calculated CP OD value was 0.011, which is lower than the standard curve quantitation limit OD value of 0.043, outside the linear range, and therefore cannot be assigned a value or is inaccurate. Furthermore, the antigen used in the method lacks a PEG-modified structure and cannot identify ADA specific to the anti-PEG backbone, potentially leading to missed detections.

[0105] Using SA coating (Example 1), 50 individual blank matrices were converted to Log(SNR), and the CP value was calculated using the 95th percentile method. Furthermore, the method in Example 1 can simultaneously detect PEG-rhIFNα-2b antibody and PEG antibody.

[0106] Example 4 The difference from Example 1 is that INFα-2b was used instead of SA for coating. The remaining components and processes were the same as in Example 1. A double-antigen sandwich assay was used to obtain rhIFN-α2b / PEG-IFNα-2b antibody / rhIFN-α2b-Biotin / Streptavindin-HRP. Simultaneously, the sample was acidified (diluted 10-fold with 0.3M acetic acid, acidified at 28°C for 0.5 h, and then neutralized with 1M Tris HCl) to complete the detection.

[0107] The results showed that the OD signal values ​​detected in the individual blank plasma matrix were uniform, concentrated between 0.009 and 0.011, consistent with the blank control signal value of 0.011. Both values ​​were too low to be calculated using the 95th percentile method. Furthermore, acidification during plate transfer often resulted in unsuccessful plate coating and abnormal color development.

[0108] Example 5 The difference from Example 1 is that the appropriate concentration range of the antibody was investigated.

[0109] The results showed that the PEG-rhIFNα-2b antibody had a good linear range of 2~250 ng / mL. The high-concentration quality control at 200 ng / mL showed normal color development and a high color value. The HPC was appropriately reduced to 150 ng / mL, which met the color development requirements. The LPC was confirmed by sensitivity gradient test.

[0110] Example 6 The difference from Example 1 is that the color development of different PEG antibodies was examined.

[0111] The results showed that the OD values ​​of several PEG antibodies from GenScript (A01795), abcam (ab190652), abcam (ab133471), abcam (ab170969), IBMS (rAGP6), and IBMS (rAGP6.3) at 250 ng / mL were approximately 0.83, 0.69, 0.05, 0.05, 0.05, and 0.05, respectively. Except for abcam (ab190652) and GenScript (A01795), which showed color development, the other antibodies from abcam and IBMS showed slow or almost no color development. GenScript (A01795) showed the best color development, making it the preferred positive control for PEG antibodies.

[0112] Example 7 The difference from Example 1 is that in step 5 of Example 1, the concentration of the added inhibitor was examined. The concentrations of the inhibitors (PEG-rhIFNα-2b, rhIFNα-2b, and PEG) were confirmed by adding a gradient of PEG-rhIFNα-2b, IFNα-2b, and PEG. Specifically, the concentrations of PEG-rhIFNα-2b and IFNα-2b were 1 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL, while the concentrations of PEG were 0.02 μg / mL, 0.2 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL.

[0113] The results showed that adding 50 μg / mL of PEG-rhIFNα-2b, 50 μg / mL of rhIFNα-2b, and 100 μg / mL of PEG could all suppress the colorimetric signal to the Blank level.

[0114] Example 8 The difference from Example 1 is that PEG-G-CSF was used instead of SA for coating, and the PEG antibody was detected by an indirect method to obtain PEG-rhG-CSF / ADA / Rabbit anti-human IgG-HRP.

[0115] The results showed that the sensitivity was 100 ng / mL, and the effect of Example 8 was inferior to that of Example 1.

[0116] The method of this invention has been described through preferred embodiments. Those skilled in the art will readily be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and context of this invention to implement and apply the technology of this invention. Those skilled in the art can refer to the content herein to appropriately improve process parameters. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included within the scope of this invention.

Claims

1. A method for qualitative detection of PEG-rhIFNα-2b antibody and PEG antibody in plasma samples, comprising the following steps: S1: Coat the ELISA plate with streptavidin to obtain the coated ELISA plate; S2: PEG-rhIFNα-2b-Biotin is added to the coated ELISA plate and incubated to form a streptavidin-PEG-rhIFNα-2b-Biotin complex. S3: Add the sample to be tested and PEG-rhIFNα-2b-Digoxigenin into the enzyme-labeled plate obtained in S2 and incubate together to form a complex of streptavidin-biotin-labeled PEGylated human interferon α-2b-detection antibody-digoxigenin-labeled PEGylated human interferon α-2b-. S4: Add anti-digoxigenin-POD to the enzyme-labeled plate obtained in S3 and incubate to form a complex of streptavidin-biotin-labeled PEGylated human interferon α-2b-detection antibody-digoxigenin-labeled PEGylated human interferon α-2b-anti-digoxigenin-POD. S5: Add chromogenic substrate for color development, add stop reaction solution for reaction, measure OD value, and determine whether the sample to be tested contains PEG-rhIFNα-2b antibody and PEG antibody.

2. The method according to claim 1, characterized in that, The streptavidin was diluted with coating buffer to prepare a solution with a concentration of 1~10 μg / mL; Alternatively, the coating buffer is prepared by thoroughly mixing 0.5M Na2CO3 and 0.5M NaHCO3 at a volume ratio of 1:(15~20), and then diluting it 10~15 times with ultrapure water. Alternatively, in S1, coat the plate with 100 μL / well of streptavidin and incubate overnight at 2-8°C and 600-800 rpm for 12-16 h. Alternatively, after coating with S1, wash the plate with washing buffer, add blocking buffer, and incubate at room temperature to obtain the coated microplate; Alternatively, after coating with S1, wash the plate with washing buffer, add blocking buffer, and incubate at room temperature (600-800 rpm) for 1-3 hours to obtain the coated microplate. Alternatively, the washing solution described in S1 is a 0.1~0.5wt% Chaps-PBS solution; Alternatively, the blocking solution described in S1 is a 1~5wt% BSA-Tris-HCl solution.

3. The method according to claim 1, characterized in that, The preparation process of PEG-rhIFNα-2b-Biotin includes: redissolving Sulfo-NHS-LC-Biotin solution, adding PEG-rhIFNα-2b and boric acid, performing biotin labeling reaction, incubation, desalting via a desalting column, and collecting the product; Alternatively, the concentration of the PEG-rhIFNα-2b-Biotin is 100~150 ng / mL; Alternatively, as described in S2, incubate at 140-180 μL / well, room temperature, 600-800 rpm, for 1-3 hours.

4. The method according to claim 3, characterized in that, The molar ratio of Sulfo-NHS-LC-Biotin to PEG-rhIFNα-2b is (7~9):1; or, the concentration of PEG-rhIFNα-2b is 5~5.5 mg / mL; or, PEG-rhIFNα-2b accounts for 15~50% of the total reaction volume; or, the boric acid concentration is 10~15 mM, and the pH is 8~8.5; or, boric acid accounts for 46.5~82.5% of the total reaction volume; or, the incubation is carried out at room temperature for 60~80 minutes; or, Sulfo-NHS-LC-Biotin accounts for 1~5% of the total reaction volume.

5. The method according to claim 1, characterized in that, In S3, the amount of the sample to be tested added is 200~250 μL / well; Alternatively, in S3, after the sample to be tested is added, PEG-rhIFNα-2b-Digoxigenin is added for co-incubation; Alternatively, in S3, the concentration of PEG-rhIFNα-2b-Digoxigenin is 100~150 ng / mL; Alternatively, in S3, the amount of PEG-rhIFNα-2b-Digoxigenin added is 50~100 μL / well; Alternatively, in S3, after adding the PEG-rhIFNα-2b-Digoxigenin, incubate at room temperature and 600-800 rpm for 1-5 hours.

6. The method according to claim 5, characterized in that, The preparation process of PEG-rhIFNα-2b-Digoxigenin includes: reconstituted DIG-X-NHS solution, added PEG-rhIFNα-2b and boric acid, performing digoxigenin labeling reaction, incubation, desalting via a desalting column, and collection; or, the molar ratio of DIG-X-NHS and PEG-rhIFNα-2b is (7~9):1; or, the concentration of PEG-rhIFNα-2b is 5~5.5 mg / mL; or, the concentration of boric acid is 100~120 mM, and the pH is 8~8.5; or, boric acid accounts for 45~75% of the total reaction volume; or, the concentration of DIG-X-NHS solution is 10~15 mM, accounting for 1.5~3.5% of the reaction volume; or, PEG-rhIFNα-2b accounts for 25~50% of the total reaction volume.

7. The method according to claim 1, characterized in that, In S4, the amount of anti-digoxigenin-POD antibody added is 50~100μL / well; Alternatively, in S4, the concentration of the anti-digoxigenin-POD antibody is 0.05~0.1U / mL; Alternatively, in S4, after the anti-digoxigenin-POD antibody is added, it is incubated at room temperature and 600-800 rpm for 1-5 hours; or, the POD is HRP.

8. The method according to claim 1, characterized in that, The test sample is prepared by diluting plasma containing the test sample 10 times with a diluent. Alternatively, the diluent is at least one of 0.5-1 wt% BSA-PBS, 0.5-1 wt% BSA-0.05-0.1 wt% T-PBS, and 1-1.5 wt% BSA-0.1-0.5 wt% chaps-PBS; Alternatively, the chromogenic substrate is TMB; Alternatively, the amount of the chromogenic substrate added is 140~180 μL / well; Alternatively, the colorimetric reaction can be performed at room temperature in the dark for 5-100 minutes. Alternatively, the terminating reaction solution is 2~2.5M H3PO4; Alternatively, the OD value is measured at dual wavelengths of 450 nm and 620 nm.

9. The method according to claim 1, characterized in that, The formula for calculating the OD value of the sample to be tested is: Sample OD = [(OD 450nm -OD 620nm ) 样品 - (OD) 450nm -OD 620nm ) 空白 SNR = Mean (OD - Sample Size) / Median (OD) NCs ).

10. The method according to claim 1, in the screening stage, characterized in that, The screening threshold SNR for PEG-rhIFNα-2b antibody and PEG antibody SCP Based on a 5% false positive rate, the SNR is calculated. SCP =1.298, or, the measured value of the sample (SNR) 样品 ) and SCP (i.e., SNR) SCP Compare and judge: SNR 样品 / SNR SCP ≥1.00; This indicates a positive result for PEG-rhIFNα-2b antibody or PEG antibody. SNR 样品 / SNR SCP <1.00; identified as PEG-rhIFNα-2b antibody or PEG antibody, negative; Alternatively, during the confirmation phase, the inhibition rate of the sample to be tested is calculated as follows: Inhibition rate % = 100% × (1 – SNR value of the drug-treated sample / SNR value of the untreated sample). Alternatively, during the confirmatory phase, the confirmatory threshold (CCP) for the PEG-rhIFNα-2b antibody and the PEG antibody (calculated with a 1% false positive rate) is: CCP-PEG-rhIFNα-2b = 31.85%, CCP-PEG = 35.10%; or, the test value of the sample to be tested is compared and determined with the confirmatory threshold (CCP). If the inhibition rate of the sample with the inhibitor is less than CCP, it is determined to be PEG-rhIFNα-2b antibody or PEG antibody, and the confirmation is negative; If the inhibition rate of the sample with the inhibitor is ≥ CCP, it is determined to be PEG-rhIFNα-2b antibody or PEG antibody, and confirmed as positive.

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