Detection reagent for determining biomacromolecular drug immunogenicity and application thereof

Abstract

The invention provides a detection reagent for determining biomacromolecular drug immunogenicity and application thereof, and belongs to the technical field of therapeutic drug detection. The invention provides a detection reagent for determining biomacromolecule drug immunogenicity. The detection reagent comprises fusion protein, biotin modified biomacromolecule drug, magnetic beads, dissociation liquid and AMPPD luminescent liquid, the fusion protein comprises a Fab fragment of a monoclonal antibody of a biomacromolecular drug and alkaline phosphatase. The detection reagent disclosed by the invention can be used for determining the immunogenicity of the biological macromolecular medicine by a double-antibody sandwich method and a fixed-point coupling ligand, and has the advantages of high specificity, high sensitivity and relatively high anti-interference capability.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug detection, and particularly relates to a detection reagent for determining the immunogenicity of biologic drugs and its application. Background Art

[0002] The detection of the immunogenicity of biologic drugs is an important topic in the field of biopharmaceuticals. Immunogenicity refers to the potential of biologic drugs to trigger an immune response in the human body, and such a response may lead to drug ineffectiveness or adverse reactions. Therefore, the development of effective immunogenicity detection reagents is crucial for ensuring drug safety and effectiveness.

[0003] The double-antibody sandwich capture of autoantibodies against biologic drugs (Anti-Drug Antibody, ADA) is the current gold standard detection strategy. An immunogenicity detection reagent for a biologic drug usually includes a capture ligand antibody and a detection label. First, such a reagent needs to contain a specific ligand (usually a biologic drug or its derivative fragment) for capturing and binding autoantibodies (ADA) in body fluids. The selection and preparation of the capture ligand are the core of reagent development and determine its specificity and sensitivity. The design of the detection reagent needs to consider factors such as sensitivity, specificity, and stability. A reagent with high sensitivity can detect low concentrations of anti-drug antibodies, and specificity ensures that the reagent is not interfered by other non-specific antibodies. Stability guarantees the effectiveness and consistency of the reagent in different environments.

[0004] Currently, the main problems faced in the development of high-sensitivity and high-specificity ADA reagents are as follows: 1. Interference from biologic drugs. Usually, the content of biologic drugs in blood is in the range of μg / mL, while the content of ADA is usually at the ng / mL level. Therefore, a large amount of biologic drugs and ADA form immune complexes, competitively affecting the binding of the capture ligand to ADA. 2. Specifically capturing ADA. Usually, the labeled ligand is coupled to the tracer and the capture ligand by chemical coupling. The commonly used coupling chemistry through amino groups lacks site specificity, thus destroying the Fab region epitope of the capture ligand (biologic drug) or generating new epitopes, which may lead to false positive results.

[0005] Therefore, there is currently a lack of immunogenicity detection reagents for biologic drugs with high specificity, high sensitivity, and high anti-interference ability. Summary of the Invention

[0006] The purpose of the present invention is to provide a detection reagent for determining the immunogenicity of biologic drugs and its application. The detection reagent of the present invention is used for determining the immunogenicity of biologic drugs, and has strong specificity, high sensitivity, and high anti-interference ability.

[0007] The present invention provides a detection reagent for determining the immunogenicity of biologic macromolecule drugs, comprising a fusion protein, a biotinylated biologic macromolecule drug, magnetic beads, a dissociation solution, and an AMPPD luminescent solution;

[0008] The fusion protein comprises a Fab fragment of a monoclonal antibody against a biologic macromolecule drug and alkaline phosphatase; the Fab fragment of the monoclonal antibody against the biologic macromolecule drug comprises a heavy chain and a light chain; the alkaline phosphatase is connected to the downstream of the variable region of the heavy chain through a flexible amino acid linker.

[0009] Preferably, the amino acid sequence of the alkaline phosphatase is as shown in SEQ ID NO.1.

[0010] Preferably, the biologic macromolecule drug comprises adalimumab, rituximab, and bevacizumab;

[0011] The amino acid sequence of the light chain of the Fab fragment of adalimumab is as shown in SEQ ID NO.2;

[0012] The amino acid sequence of the heavy chain of the Fab fragment of adalimumab is as shown in SEQ ID NO.3;

[0013] The amino acid sequence of the light chain of the Fab fragment of rituximab is as shown in SEQ ID NO.4;

[0014] The amino acid sequence of the heavy chain of the Fab fragment of rituximab is as shown in SEQ ID NO.5;

[0015] The amino acid sequence of the light chain of the Fab fragment of bevacizumab is as shown in SEQ ID NO.6;

[0016] The amino acid sequence of the heavy chain of the Fab fragment of bevacizumab is as shown in SEQ ID NO.7;

[0017] The amino acid sequence of the flexible amino acid linker is as shown in SEQ ID NO.8.

[0018] Preferably, it further comprises a neutralization solution; the neutralization solution is based on Tris with a concentration of 0.5M and further comprises calf serum with a volume concentration of 1%; the pH of the neutralization solution is 8.

[0019] Preferably, the dissociation solution is an acetic acid aqueous solution; the concentration of acetic acid in the acetic acid aqueous solution is 300 mM; the pH of the dissociation solution is 2.5.

[0020] Preferably, the fusion protein is a fusion protein solution; the concentration of the fusion protein in the fusion protein solution is 4 μg / mL; the solvent of the fusion protein solution is a first buffer; the first buffer is based on a MES buffer with a concentration of 50 mM and a pH of 6.5, and further includes the following components at the following concentrations: 0.9% (m / v) NaCl, 5 mg / mL BSA, 1 mM MgCl2, and 0.1 mM ZnCl2;

[0021] The biotinylated biologic macromolecule drug is a biotinylated biologic macromolecule drug solution; the concentration of the biotinylated biologic macromolecule drug in the biotinylated biologic macromolecule drug solution is 4 μg / mL; the solvent of the biotinylated biologic macromolecule drug solution is a second buffer; the second buffer is based on a MES buffer with a concentration of 50 mM and a pH of 6.5, and further includes the following components at the following concentrations: 0.9% (m / v) NaCl and 5 mg / mL BSA;

[0022] The magnetic beads are a magnetic bead suspension; the concentration of the magnetic beads in the magnetic bead suspension is 0.5 mg / mL; the suspending agent of the magnetic bead suspension is the second buffer.

[0023] Preferably, the magnetic beads include SA magnetic beads.

[0024] The present invention provides a test strip or kit for determining the immunogenicity of a biologic macromolecule drug, and the test strip or kit includes the detection reagent according to the above technical solution.

[0025] The present invention provides the use of the detection reagent according to the above technical solution or the test strip or kit according to the above technical solution in determining the immunogenicity of a biologic macromolecule drug.

[0026] The present invention provides a method for determining the immunogenicity of a biologic macromolecule drug based on the detection reagent according to the above technical solution, including the following steps:

[0027] 1) Mix the sample to be tested with a dissociation solution, and perform a dissociation reaction under acidic conditions to obtain a dissociation product;

[0028] 2) Mix the dissociation product, the fusion protein, and the biotinylated biologic macromolecule drug, and perform an immune reaction to obtain an immune reaction product;

[0029] 3) Mix the immune reaction product with the magnetic beads to obtain a complex;

[0030] 4) After washing the complex, mix it with an AMPPD luminescent solution and perform a color reaction.

[0031] The present invention provides a detection reagent for determining the immunogenicity of biologic drugs, comprising a fusion protein, a biotinylated biologic drug, magnetic beads, a dissociation solution, and an AMPPD luminescent solution; the fusion protein comprises a Fab fragment of a monoclonal antibody against the biologic drug and alkaline phosphatase; the Fab fragment of the monoclonal antibody against the biologic drug comprises a heavy chain and a light chain; the alkaline phosphatase is linked to the downstream of the variable region of the heavy chain through a flexible amino acid linker arm. The fusion protein of the present invention fuses alkaline phosphatase and the Fab fragment of the drug antibody at a fixed point, completely retaining the integrity of the Fab structure, not generating additional epitopes, having better specificity, and removing the Fc region, thus greatly reducing the interference of endogenous RF and HAMA. In addition, the intact Fab structure in the fusion protein of the present invention has a higher affinity for endogenous ADA than the allosteric biologic drug ligand produced by chemical conjugation, showing higher sensitivity. Therefore, the detection reagent of the present invention can be used for sandwich immunoassay and ligand determination at a fixed point to determine the immunogenicity of biologic drugs, has strong specificity, high sensitivity, and high anti-interference ability, and can be used for the development of clinical diagnostic reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings based on these drawings without creative efforts.

[0033] Figure 1 It is a schematic diagram of the detection method for the blood drug concentration of anti-drug antibody by magnetic particle luminescence method;

[0034] Figure 2 It is an SDS-PAGE electrophoresis diagram. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The present invention provides a detection reagent for determining the immunogenicity of biologic drugs, comprising a fusion protein, a biotinylated biologic drug, magnetic beads, a dissociation solution, and an AMPPD luminescent solution; the fusion protein comprises a Fab fragment of a monoclonal antibody against the biologic drug and alkaline phosphatase; the Fab fragment of the monoclonal antibody against the biologic drug comprises a heavy chain and a light chain; the alkaline phosphatase is linked to the downstream of the variable region of the heavy chain through a flexible amino acid linker arm.

[0036] In the specific implementation process of the present invention, the amino acid sequence of the alkaline phosphatase is as shown in SEQ ID NO.1; the alkaline phosphatase is calf intestinal alkaline phosphatase, from Uniprot P19111 SEQ4, and the amino acid sequence shown in SEQ ID NO.1 is specifically:

[0037] LVPVEEEDPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQCKTTRGNEVTSVMNRAKKAGKSVGVVTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQMNGCQDIAAQLVNNMDIDVILGGGRKYMFPVGTPDPEYPDDASVNGVRKRKQNLVQAWQAKHQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLQEMTEVALRVVSRNPRGFYLFVEGGRIDHGHHDDKAYMALTEAGMFDNAIAKANELTSELDTLILVTADHSHVFSFGGYTLRGTSIFGLAPSKALDSKSYTSILYGNGPGYALGGGSRPDVNDSTSEDPSYQQQAAVPQASETHGGEDVAVFARGPQAHLVHGVEEETFVAHIMAFAGCVEPYTDCNLPAPTTAT。

[0038] In the specific implementation process of the present invention, the biologic drugs include adalimumab, rituximab, and bevacizumab.

[0039] In the specific implementation process of the present invention, the amino acid sequence of the Fab fragment of adalimumab is obtained by querying the Drug Bank database; the amino acid sequence of the light chain of the Fab fragment of adalimumab is shown in SEQ ID NO.2, specifically: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; the nucleotide sequence of the coding gene of the light chain of the Fab fragment of adalimumab is shown in SEQ ID NO.12, specifically:

[0040] gatatccaaatgactcaaagtccaagtagtctctccgcctctgtgggcgacagagtgacaatcacctgtagggcctctcagggcatccggaactacctggcctggtaccagcagaagcctggcaaggctcctaagctgctgatctacgctgcttctacactgcagtccggcgtgccttctagattctccggctctggatccggcaccgacttcaccctgaccatctccagcctgcagcctgaggacgtggctacctactactgccagcggtacaacagagctccctacaccttcggccagggaacaaaagtggaaatcaagcggaccgtggccgctccatccgtgttcatctttcctcctagcgatgaacagctgaagtctggcaccgcctctgtcgtgtgcctgctgaacaacttctaccccagagaggccaaagtgcagtggaaggtggacaatgccctgcaaagcggcaactcccaagagtccgtcaccgagcaggatagcaaggactccacctattccctgagctctaccctgaccctgtctaaggccgactacgagaagcacaaggtgtacgcctgcgaagtgactcaccagggcctgtcctctcctgtgaccaagtccttcaacagaggcgagtgc;

[0041] The amino acid sequence of the heavy chain of the Fab fragment of adalimumab is shown in SEQ ID NO.3, specifically: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV。

[0042] In the specific implementation process of the present invention, the amino acid sequence of the light chain of the Fab fragment of rituximab is as shown in SEQ ID NO.4, specifically:

[0043] QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; The nucleotide sequence of the coding gene of the light chain of the Fab fragment of rituximab is as shown in SEQ ID NO.14, specifically:

[0044] caaatcgtgctaagtcaaagtccagcaatcctctctgcttcccctggcgaaaaagtcaccatgacctgcagagcctcgtccagcgtgtcctacatccattggttccagcagaagcccggctcttctcctaagccttggatctacgctacctccaacctggcttctggcgtgcccgtgcggttctccggcagcggatctggaacatcttactccctgacaatctccagagtggaagccgaggacgccgccacctactactgccagcagtggaccagcaatcctcctacctttggcggcggcaccaagctggaaatcaagcgcaccgtggctgctccttccgtgttcatcttccctccatctgatgaacagctgaagtctggcacagcttctgtggtgtgcctgctgaacaacttctaccctagagaggccaaggtgcagtggaaggtggacaacgccctgcagtctggcaactcccaagagtccgtgaccgagcaggattccaaggactccacttattctctgagctccaccctgaccctgagcaaggccgactacgagaagcacaaagtgtacgcctgcgaggtcacccaccagggcctgtcctcccccgtgaccaagtccttcaaccggggcgagtgt; The amino acid sequence of the heavy chain of the Fab fragment of rituximab is shown in SEQ ID NO.5, specifically:

[0045] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK.

[0046] In the specific implementation process of the present invention, the amino acid sequence of the light chain of the Fab fragment of bevacizumab is shown in SEQ ID NO.6, specifically:

[0047] DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; The nucleotide sequence of the coding gene of the light chain of the Fab fragment of bevacizumab is shown in SEQ ID NO.16, specifically:

[0048] gatatccaaatgactcaaagtccaagtagtctgagcgcctccgtgggcgatagagtgaccatcacctgttctgcttctcaggacatcagcaactacctgaattggtaccagcagaagcctggcaaggctcctaaagtgctgatctacttcacctcctccctgcattctggcgtgccatctcggttctccggctctggaagcggaaccgacttcacactgacaatctccagcctgcagcctgaggattttgctacctactactgccagcagtactccaccgtgccttggaccttcggccagggcaccaaagtggaaatcaagcggacagtggccgctccctccgtgttcatctttcctccttccgacgagcagctgaagtctggcaccgcctctgtggtgtgcctgctgaacaacttctaccccagagaagccaaggtgcagtggaaggtggacaacgccctgcagtccggcaactcccaagagtccgtcacagaacaggattctaaggactccacctatagcctctcttctaccctgaccctgtccaaggccgactacgagaagcacaaggtgtacgcctgcgaggtcacccaccaaggcctgtcctctcctgtgaccaagtccttcaacagaggcgagtgc; The amino acid sequence of the heavy chain of the Fab fragment of bevacizumab is shown in SEQ ID NO.7, specifically:

[0049] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV。

[0050] In the specific implementation process of the present invention, the amino acid sequence of the flexible amino acid linker is as shown in SEQ ID NO.8, specifically: (GGGS)4, that is: GGGSGGGSGGGSGGGS.

[0051] In the specific implementation process of the present invention, the alkaline phosphatase is connected downstream of the heavy chain variable region through a flexible amino acid linker; the heavy chain and alkaline phosphatase are expressed by fusion.

[0052] In one embodiment of the present invention, the amino acid sequence of the heavy chain-alkaline phosphatase fusion protein of the Fab fragment of adalimumab is as shown in SEQ ID NO.9, specifically:

[0053] EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSLVPVEEEDPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQCKTTRGNEVTSVMNRAKKAGKSVGVVTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQMNGCQDIAAQLVNNMDIDVILGGGRKYMFPVGTPDPEYPDDASVNGVRKRKQNLVQAWQAKHQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLQEMTEVALRVVSRNPRGFYLFVEGGRIDHGHHDDKAYMALTEAGMFDNAIAKANELTSELDTLILVTADHSHVFSFGGYTLRGTSIFGLAPSKALDSKSYTSILYGNGPGYALGGGSRPDVNDSTSEDPSYQQQAAVPQASETHGGEDVAVFARGPQAHLVHGVEEETFVAHIMAFAGCVEPYTDCNLPAPTTAT; The nucleotide sequence of the coding gene of the heavy chain-alkaline phosphatase fusion protein of the Fab fragment of adalimumab is shown in SEQ ID NO. 13, specifically:

[0054]

[0055] In another embodiment of the present invention, the amino acid sequence of the heavy chain-alkaline phosphatase fusion protein of the Fab fragment of rituximab is as shown in SEQ ID NO.10, specifically:

[0056] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKGGGSGGGSGGGSGGGSLVPVEEEDPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQCKTTRGNEVTSVMNRAKKAGKSVGVVTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQMNGCQDIAAQLVNNMDIDVILGGGRKYMFPVGTPDPEYPDDASVNGVRKRKQNLVQAWQAKHQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLQEMTEVALRVVSRNPRGFYLFVEGGRIDHGHHDDKAYMALTEAGMFDNAIAKANELTSELDTLILVTADHSHVFSFGGYTLRGTSIFGLAPSKALDSKSYTSILYGNGPGYALGGGSRPDVNDSTSEDPSYQQQAAVPQASETHGGEDVAVFARGPQAHLVHGVEEETFVAHIMAFAGCVEPYTDCNLPAPTTAT; the nucleotide sequence of the coding gene of the heavy chain-alkaline phosphatase fusion protein of the Fab fragment of rituximab is as shown in SEQ ID NO.15, specifically:

[0057]

[0058] In another embodiment of the present invention, the amino acid sequence of the heavy chain-alkaline phosphatase fusion protein of the Fab fragment of bevacizumab is as shown in SEQ ID NO.11, specifically:

[0059] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVGGGSGGGSGGGSGGGSLVPVEEEDPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSAAARYNQCKTTRGNEVTSVMNRAKKAGKSVGVVTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQMNGCQDIAAQLVNNMDIDVILGGGRKYMFPVGTPDPEYPDDASVNGVRKRKQNLVQAWQAKHQGAQYVWNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLQEMTEVALRVVSRNPRGFYLFVEGGRIDHGHHDDKAYMALTEAGMFDNAIAKANELTSELDTLILVTADHSHVFSFGGYTLRGTSIFGLAPSKALDSKSYTSILYGNGPGYALGGGSRPDVNDSTSEDPSYQQQAAVPQASETHGGEDVAVFARGPQAHLVHGVEEETFVAHIMAFAGCVEPYTDCNLPAPTTAT; the nucleotide sequence of the coding gene of the heavy chain-alkaline phosphatase fusion protein of the Fab fragment of bevacizumab is as shown in SEQ ID NO.17, specifically:

[0060]

[0061] In one embodiment of the present invention, the fusion protein is an adalimumab Fab-alkaline phosphatase fusion protein; the preparation steps of the adalimumab Fab-alkaline phosphatase fusion protein are as follows: The coding genes (SEQ ID NO.12) of the amino acid sequence shown in SEQ ID NO.2 and the coding genes (SEQ ID NO.13) of the amino acid sequence shown in SEQ ID NO.9 are respectively constructed into the pCDNA3.4 vector using homologous recombination, and the amino acid sequence shown in SEQ ID NO.2 and the amino acid sequence shown in SEQ ID NO.9 are co-expressed using the ExpiCHO transient transfection system of Thermo Fisher, and the target protein is obtained through ProteinG purification.

[0062] In another embodiment of the present invention, the fusion protein is a rituximab Fab-alkaline phosphatase fusion protein; the preparation steps of the rituximab Fab-alkaline phosphatase fusion protein are as follows: The coding genes (SEQ ID NO.14) of the amino acid sequence shown in SEQ ID NO.4 and the coding genes (SEQ ID NO.15) of the amino acid sequence shown in SEQ ID NO.10 are respectively constructed into the pCDNA3.4 vector using homologous recombination, and the amino acid sequence shown in SEQ ID NO.4 and the amino acid sequence shown in SEQ ID NO.10 are co-expressed using the ExpiCHO transient transfection system of Thermo Fisher, and the target protein is obtained through ProteinG purification.

[0063] In another embodiment of the present invention, the fusion protein is a bevacizumab Fab-alkaline phosphatase fusion protein; the preparation steps of the bevacizumab Fab-alkaline phosphatase fusion protein are as follows: The coding genes (SEQ ID NO.16) of the amino acid sequence shown in SEQ ID NO.6 and the coding genes (SEQ ID NO.17) of the amino acid sequence shown in SEQ ID NO.11 are respectively constructed into the pCDNA3.4 vector using homologous recombination, and the amino acid sequence shown in SEQ ID NO.6 and the amino acid sequence shown in SEQ ID NO.11 are co-expressed using the ExpiCHO transient transfection system of Thermo Fisher, and the target protein is obtained through ProteinG purification.

[0064] The gene sequences in the present invention are synthesized by Beijing Deaoping Biotechnology Co., Ltd.

[0065] The purity of the fusion protein prepared by the above method of the present invention can reach more than 90%.

[0066] In the specific implementation process of the present invention, a neutralizing solution is further included; the neutralizing solution is based on Tris with a concentration of 0.5M, and further includes calf serum with a volume concentration of 1%; the pH of the neutralizing solution is 8; the neutralizing solution is added after the sample and the dissociation solution are mixed for dissociation reaction, and its function is to correct the pH value of the acidic dissociation system back to near neutrality.

[0067] In the specific implementation process of the present invention, the dissociation solution is an acetic acid aqueous solution; the concentration of acetic acid in the acetic acid aqueous solution is 300mM; the pH of the dissociation solution is 2.5.

[0068] In the specific implementation process of the present invention, the fusion protein is a fusion protein solution; the concentration of the fusion protein in the fusion protein solution is 4μg / mL; the solvent of the fusion protein solution is the first buffer solution; the first buffer solution is based on MES buffer solution with a concentration of 50mM and a pH of 6.5, and further includes components with the following concentrations: NaCl with a mass-volume concentration of 0.9%, 5mg / mL BSA, 1mM MgCl2, and 0.1mM ZnCl2.

[0069] In the specific implementation process of the present invention, the biotinylated biologic macromolecule drug is a biotinylated biologic macromolecule drug solution; the concentration of the biotinylated biologic macromolecule drug in the biotinylated biologic macromolecule drug solution is 4μg / mL; the solvent of the biotinylated biologic macromolecule drug solution is the second buffer solution; the second buffer solution is based on MES buffer solution with a concentration of 50mM and a pH of 6.5, and further includes components with the following concentrations: NaCl with a mass-volume concentration of 0.9% and 5mg / mL BSA. In the specific implementation process of the present invention, the preparation method of the biotinylated biologic macromolecule drug is as follows:

[0070] Mix the dilution of the biologic macromolecule drug and Biotin-PEG8-NHS for amidation reaction, and change the solution to PBS buffer solution using a G25 desalting column to obtain the biotinylated biologic macromolecule drug; the concentration of the biologic macromolecule drug in the dilution of the biologic macromolecule drug is 5mg / mL; the addition amount of Biotin-PEG8-NHS is 1 / 10 of the mass of the biologic macromolecule drug; the mixing includes vortex mixing; the temperature of the amidation reaction is 25°C; the rotation speed of the amidation reaction is 1000rpm; the time of the amidation reaction is 1h; the storage temperature of the biologic macromolecule drug is -20°C.

[0071] In the specific implementation process of the present invention, the magnetic beads are a magnetic bead suspension; the concentration of the magnetic beads in the magnetic bead suspension is 0.5mg / mL; the suspending agent of the magnetic bead suspension is the second buffer solution. In the specific implementation process of the present invention, the magnetic beads include SA magnetic beads.

[0072] The present invention also provides a test strip or kit for determining the immunogenicity of a biologic drug, and the test strip or kit includes the detection reagent described in the above solution.

[0073] The present invention also provides the use of the detection reagent described in the above solution or the test strip or kit in determining the immunogenicity of a biologic drug.

[0074] The present invention also provides the use of the detection reagent described in the above solution in determining the immunogenicity of a biologic drug.

[0075] The present invention also provides a method for determining the immunogenicity of a biologic drug based on the detection reagent described in the above solution, including the following steps:

[0076] 1) Mix the sample to be tested with the dissociation solution, and perform a dissociation reaction under acidic conditions to obtain a dissociation product;

[0077] 2) Mix the dissociation product, the fusion protein, and the biotinylated biologic drug, and perform an immune reaction to obtain an immune reaction product;

[0078] 3) Mix the immune reaction product with magnetic beads to obtain a complex;

[0079] 4) After washing the complex, mix it with AMPPD luminescent solution and perform a color development reaction.

[0080] The present invention first mixes the sample to be tested with the dissociation solution, and performs a dissociation reaction under acidic conditions to obtain a dissociation product.

[0081] In the specific implementation process of the present invention, the volume ratio of the sample to be tested to the dissociation solution is 1:(2-20), further 1:5; the temperature of the dissociation reaction is 37°C; the time of the dissociation reaction is 1-20 min, further 5 min; the function of the dissociation solution is to dissociate the macromolecular biologic drug-autoantibody complex in the sample to be tested (clinical sample), and the dissociation product is free anti-drug autoantibody.

[0082] In the specific implementation process of the present invention, taking the test sample as 5 μL, the volumes of the fusion protein and the biotinylated macromolecular drug are each 50 μL; the time of the immune reaction is 4-30 min, further 10 min; the temperature of the immune reaction is 36-38°C.

[0083] After the dissociation reaction, the present invention further includes mixing the product after the dissociation reaction with a neutralization solution; taking the test sample as 5 μL, the dosage of the neutralization solution is 50 μL.

[0084] After obtaining the dissociation products, the present invention mixes the dissociation products, the fusion protein, and the biotinylated biologic macromolecule drug to conduct an immune reaction, thereby obtaining an immune reaction product.

[0085] The immune reaction product is a biotinylated biologic macromolecule drug - anti-drug autoantibody - fusion protein complex.

[0086] After obtaining the immune reaction product, the present invention mixes the immune reaction product with magnetic beads to obtain a complex.

[0087] In a specific implementation process of the present invention, taking the test sample as 5 μL, the volume of the magnetic beads is 50 μL; the temperature for mixing the immune reaction product and the magnetic beads is 37 °C; the time for mixing the immune reaction product and the magnetic beads is 5 min; during the mixing process, biotin reacts with the streptavidin protein on the magnetic beads; the complex is a solid-phase biotinylated biologic macromolecule drug - anti-drug autoantibody - fusion protein complex.

[0088] After obtaining the complex, the present invention washes the complex and then mixes it with AMPPD luminescent solution to conduct a color reaction.

[0089] In the present invention, the reagent used for washing is based on 20 mM Tris-Hcl with a pH of 8.0, and further includes 0.9% NaCl, 0.1% TW20, and 0.1% P300 in terms of mass-volume concentration.

[0090] In the present invention, the method reflects the level of immunogenicity by measuring the concentration of the autoantibody against the biologic macromolecule drug; the method can achieve quantitative determination of the immunogenicity of the biologic macromolecule drug. Taking adalimumab as an example, a specific monoclonal antibody against adalimumab itself is used as a calibrator to calculate the content of autoantibodies in the sample.

[0091] To further illustrate the present invention, the following describes in detail a detection reagent for measuring the immunogenicity of a biologic macromolecule drug provided by the present invention and its application in combination with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0092] Example 1

[0093] The detection principle diagram is shown in Figure 1 .

[0094] 1. Preparation of adalimumab Fab - alkaline phosphatase fusion protein

[0095] The heavy chain downstream of the Fab fragment of adalimumab was fused and expressed with alkaline phosphatase through a flexible amino acid linker. The amino acid sequence of the light chain of the Fab fragment of adalimumab is shown in SEQ ID NO.2; the amino acid sequence of the heavy chain of the Fab fragment of adalimumab is shown in SEQ ID NO.3; the amino acid sequence of the linker is shown in SEQ ID NO.8; the alkaline phosphatase is calf intestinal alkaline phosphatase, from Uniprot P19111, and the amino acid sequence is shown in SEQ ID NO.1; the amino acid sequence of the heavy chain-alkaline phosphatase fusion protein of the Fab fragment of adalimumab is shown in SEQ ID NO.9.

[0096] The coding genes of the amino acid sequences shown in SEQ ID NO.2 (SEQ ID NO.12) and the amino acid sequence shown in SEQ ID NO.9 (SEQ ID NO.13) were respectively homologously recombined and constructed into the pCDNA3.4 vector. The ExpiCHO transient transfection system of Thermo Fisher was used to co-express the amino acid sequence shown in SEQ ID NO.2 and the amino acid sequence shown in SEQ ID NO.9, and the target protein was obtained by ProteinG purification. After confirmation by SDS-PAGE electrophoresis, a fusion protein with a purity of more than 90% was obtained, as Figure 2 shown, indicating that the adalimumab Fab-alkaline phosphatase fusion protein was successfully prepared.

[0097] 2. Preparation of biotinylated biologic macromolecule drug (biotinylated macromolecule drug, Biotin-adalimumab)

[0098] Take 2 mg of biologic macromolecule drug, dilute it to 5 mg / mL with PBS, add 0.2 mg of Biotin-PEG8-NHS, vortex and mix well, react at 25 °C and 1000 rpm for 1 h, change the solution to PBS buffer using a G25 desalting column, and store it at -20 °C for later use.

[0099] 3. Construction of ADA detection reagent

[0100] a. Preparation of fusion protein (enzyme-labeled component) solution: The drug Fab-ALP conjugate prepared above was diluted to 4 μg / mL with a buffer of 50 mM MES-pH6.5, 0.9% (m / v) NaCl, 5 mg / mL BSA, 1 mM MgCl2, and 0.1 mM ZnCl2.

[0101] b. Preparation of biotinylated biologic macromolecule drug solution: The Biotin-biological macromolecule drug prepared above was diluted to 4 μg / mL with a buffer of 50 mM MES-pH6.5, 0.9% (m / v) NaCl, and 5 mg / mL BSA.

[0102] c. Preparation of magnetic bead suspension: The SA magnetic beads were purchased from Deaoping Biotechnology Co., Ltd. and diluted to 0.5 mg / mL with a buffer solution containing 50 mM MES-pH6.5, 0.9% NaCl (mass / volume concentration), and 5 mg / mL BSA.

[0103] d. Dissociation solution: 300 mM acetic acid, pH 2.5.

[0104] e. Neutralization solution: 0.5 M Tris, 1% calf serum, pH 8.0.

[0105] 4. Reaction procedure:

[0106] The samples were prepared by adding idiotypic antibodies (purchased from Beijing Deaoping) at different concentrations to normal human serum without biopharmaceuticals, and configured to 0 - 1 - 5 - 50 - 200 ng / mL.

[0107] 5 μL of the sample to be tested + 45 μL of the dissociation solution, react at 37°C for 10 min, add 50 μL of the neutralization solution, 50 μL of Biotin-biological macromolecule drug, and 50 μL of the fusion protein, react for 10 min, add 50 μL of the magnetic bead component, react at 37°C for 5 min, wash, and add AMPPD luminescent solution for color development.

[0108] Example 2

[0109] 1. Preparation of Rituximab Fab-alkaline phosphatase fusion protein

[0110] The heavy chain downstream of the Fab fragment of rituximab was fused and expressed with alkaline phosphatase through a flexible amino acid linker. The amino acid sequence of the light chain of the Fab fragment of rituximab is shown in SEQ ID NO.4; the amino acid sequence of the heavy chain of the Fab fragment of rituximab is shown in SEQ ID NO.5; the amino acid sequence of the heavy chain-alkaline phosphatase fusion protein of the Fab fragment of rituximab is shown in SEQ ID NO.10.

[0111] The coding genes of the amino acid sequences shown in SEQ ID NO.4 (SEQ ID NO.14) and the amino acid sequence shown in SEQ ID NO.10 (SEQ ID NO.15) were respectively homologously recombined and constructed into the pCDNA3.4 vector. The amino acid sequences shown in SEQ ID NO.4 and the amino acid sequence shown in SEQ ID NO.10 were co-expressed using the ExpiCHO transient transfection system of Thermo Fisher, and the target protein was obtained through ProteinG purification. After confirmation by SDS-PAGE electrophoresis, a fusion protein with a purity of more than 90% was obtained, as Figure 2 shown, indicating that the Rituximab Fab-alkaline phosphatase fusion protein was successfully prepared.

[0112] 2. The preparation of biotinylated biologic macromolecule drug (Biotin-rituximab) refers to Example 1.

[0113] 3. The construction of the ADA detection reagent is the same as that in Example 1.

[0114] 4. The reaction procedure is the same as that in Example 1. The samples are prepared by adding different concentrations of idiotypic antibodies (purchased from Beijing Deaoping) to normal human serum without biologic drugs, and configured into 0 - 1 - 5 - 50 - 200 ng / mL.

[0115] Example 3

[0116] 1. Preparation of bevacizumab Fab - alkaline phosphatase fusion protein

[0117] The heavy chain downstream of the Fab fragment of bevacizumab is fused and expressed with alkaline phosphatase through a flexible amino acid linker. The amino acid sequence of the light chain of the Fab fragment of bevacizumab is shown in SEQ ID NO.6; the amino acid sequence of the heavy chain of the Fab fragment of bevacizumab is shown in SEQ ID NO.7; the amino acid sequence of the heavy chain - alkaline phosphatase fusion protein of the Fab fragment of bevacizumab is shown in SEQ ID NO.11.

[0118] The coding genes of the amino acid sequences shown in SEQ ID NO.6 (SEQ ID NO.16) and the amino acid sequence shown in SEQ ID NO.11 (SEQ ID NO.17) are respectively homologously recombined and constructed into the pCDNA3.4 vector. The ExpiCHO transient transfection system of Thermo Fisher is used to co - express the amino acid sequences shown in SEQ ID NO.6 and the amino acid sequence shown in SEQ ID NO.11, and the target protein is obtained through ProteinG purification. After confirmation by SDS - PAGE electrophoresis, a fusion protein with a purity of more than 90% is obtained, as Figure 2 shown, indicating the successful preparation of bevacizumab Fab - alkaline phosphatase fusion protein.

[0119] 2. The preparation of biotinylated biologic macromolecule drug (Biotin - bevacizumab) refers to Example 1.

[0120] 3. The construction of the ADA detection reagent is the same as that in Example 1.

[0121] 4. The reaction procedure is the same as that in Example 1. The samples are prepared by adding different concentrations of idiotypic antibodies (purchased from Beijing Deaoping) to normal human serum without biologic drugs, and configured into 0 - 1 - 5 - 50 - 200 ng / mL.

[0122] Comparative Example 1 Adalimumab free ADA

[0123] The samples are the same as those in Example 1.

[0124] Preparation of the fusion protein component (enzyme-labeled component):

[0125] Dissolve 1 mg of ALP in 1 mL of PBS, add 0.5 mg of adalimumab, mix well, add 10 μL of 50% glutaraldehyde solution, and mix evenly at room temperature for 2 h. Dialyze into PBS, dilute with a buffer of 50 mM MES - pH 6.5, 0.9% (m / v) NaCl, 5 mg / mL BSA, 1 mM MgCl2, and 0.1 mM ZnCl2 to 4 μg / mL.

[0126] Other components are exactly the same as those in Example 1.

[0127] Reaction procedure: Add 5 μL of the sample, 50 μL of Biotin - adalimumab and 50 μL of the fusion protein, react for 10 min, add 50 μL of the magnetic bead component, react at 37 °C for 5 min, wash, and add AMPPD luminescent solution for color development.

[0128] Comparative Example 2 Rituximab free ADA

[0129] The sample is the same as that in Example 2.

[0130] Preparation of the fusion protein component (enzyme-labeled component):

[0131] Dissolve 1 mg of ALP in 1 mL of PBS, add 0.5 mg of rituximab, mix well, add 10 μL of 50% glutaraldehyde solution, and mix evenly at room temperature for 2 h. Dialyze into PBS, dilute with a buffer of 50 mM MES - pH 6.5, 0.9% (m / v) NaCl, 5 mg / mL BSA, 1 mM MgCl2, and 0.1 mM ZnCl2 to 4 μg / mL.

[0132] Other components are exactly the same as those in Example 2.

[0133] Reaction procedure: Add 5 μL of the sample, 50 μL of Biotin - rituximab and 50 μL of the fusion protein, react for 30 min, add 50 μL of the magnetic bead component, react at 37 °C for 5 min, wash, and add AMPPD luminescent solution for color development.

[0134] Comparative Example 3 Bevacizumab free ADA

[0135] The sample is the same as that in Example 3.

[0136] Preparation of the fusion protein component (enzyme-labeled component):

[0137] 1 mg of ALP was dissolved in 1 mL of PBS, 0.5 mg of bevacizumab was added, and the mixture was homogenized. Then 10 μL of 50% glutaraldehyde solution was added and mixed evenly at room temperature for 2 h. It was dialyzed into PBS, diluted to 4 μg / mL with a buffer solution of 50 mM MES - pH 6.5, 0.9% (mass / volume) NaCl, 5 mg / mL BSA, 1 mM MgCl2, and 0.1 mM ZnCl2.

[0138] Other components were exactly the same as those in Example 3.

[0139] Reaction procedure: 5 μL of the sample was added with 50 μL of Biotin - bevacizumab and 50 μL of the fusion protein and reacted for 10 min. Then 50 μL of the magnetic bead component was added and reacted at 37 °C for 5 min. After washing, AMPPD luminescent solution was added for color development.

[0140] Test Example 1 Comparison of the performance of different free ADA reagents

[0141] The Fab2 - ALP conjugate prepared by the present invention was compared with the drug - ALP conjugate prepared using glutaraldehyde. The results are shown in Table 1. In the three items of adalimumab, rituximab, and bevacizumab, the S1 / S0 signal - to - noise ratio of the present invention was significantly better than that of Comparative Example 6, indicating a significant improvement in sensitivity.

[0142] Table 1 Comparison of the sensitivity of different free ADA

[0143]

[0144]

[0145] Test Example 2

[0146] Random sera from 200 cases of patients who had not used the corresponding biologic drugs were measured with different reagents to evaluate the specificity of different reagents. Samples with a detected concentration > 0.1 ng / mL were defined as false - positive samples. The statistical results are shown in Table 2.

[0147] Table 2 Results of measuring random sera from 200 cases of patients who had not used the corresponding biologic drugs with different reagents

[0148]

[0149] The results showed that the false - positive ratio of the examples of the present invention was significantly lower than that of the comparative examples. This was related to the fact that the marker in the examples did not contain the Fc (Fragment, crystallizable) fragment of the antibody and was less interfered with. Moreover, the comparative examples used a non - site - specific coupling scheme, which might produce some coupling aggregates and cause specificity problems.

[0150] Test Example 3 Comparison of the performance of different total ADA reagents

[0151] The samples were prepared by adding normal human serum without biopharmaceuticals, idiotype antibodies at concentrations of 0 and 5 ng / mL (purchased from Beijing DeaoPing Biotechnology Co., Ltd.), and biomacromolecule drugs at 30 μg / mL to 5 ng / mL to simulate interference samples.

[0152] Anti-interference ability = signal value of the sample with drug interference / signal value of the sample without drug. The closer this ratio is to 1, the closer the influence of the drug on the detection interference is to 0. The results are shown in Table 3. From the results in Table 3, it can be seen that the methods of Examples 1 to 3 all showed good anti-interference ability in the three items, significantly superior to Comparative Examples 1 to 3.

[0153] Table 3 Comparison results of the performance of different total ADA reagents

[0154]

[0155] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A detection reagent for determining the immunogenicity of a biologic drug, characterized in that, It includes a fusion protein, a biotinylated biologic macromolecule drug, magnetic beads, a dissociation solution, and an AMPPD luminescent solution; The fusion protein includes the Fab fragment of a monoclonal antibody of a biologic macromolecule drug and alkaline phosphatase; the Fab fragment of the monoclonal antibody of the biologic macromolecule drug includes a heavy chain and a light chain; the alkaline phosphatase is connected downstream of the heavy chain variable region through a flexible amino acid linker.

2. The detection reagent according to claim 1, characterized in that The amino acid sequence of the alkaline phosphatase is as shown in SEQ ID NO.

1.

3. The detection reagent according to claim 1, characterized in that, The biologic macromolecule drug includes adalimumab, rituximab, and bevacizumab; The amino acid sequence of the light chain of the Fab fragment of adalimumab is as shown in SEQ ID NO.2; The amino acid sequence of the heavy chain of the Fab fragment of adalimumab is as shown in SEQ ID NO.3; The amino acid sequence of the light chain of the Fab fragment of rituximab is as shown in SEQ ID NO.4; The amino acid sequence of the heavy chain of the Fab fragment of rituximab is as shown in SEQ ID NO.5; The amino acid sequence of the light chain of the Fab fragment of bevacizumab is as shown in SEQ ID NO.6; The amino acid sequence of the heavy chain of the Fab fragment of bevacizumab is as shown in SEQ ID NO.7; The amino acid sequence of the flexible amino acid linker is as shown in SEQ ID NO.

8.

4. The detection reagent according to claim 1, wherein It further includes a neutralizing solution; the neutralizing solution is based on Tris with a concentration of 0.5 M and further includes calf serum with a volume concentration of 1%; the pH of the neutralizing solution is 8.

5. The detection reagent according to claim 1, wherein The dissociation solution is an acetic acid aqueous solution; the concentration of acetic acid in the acetic acid aqueous solution is 300 mM; the pH of the dissociation solution is 2.

5.

6. The detection reagent according to claim 1, characterized in that, The fusion protein is a fusion protein solution; the concentration of the fusion protein in the fusion protein solution is 4 μg / mL; the solvent of the fusion protein solution is a first buffer solution; the first buffer solution is based on MES buffer solution with a concentration of 50 mM and a pH of 6.5, and further includes the following components at the following concentrations: NaCl with a mass-volume concentration of 0.9%, BSA at 5 mg / mL, 1 mM MgCl2, and 0.1 mM ZnCl2; The biotinylated biologic macromolecule drug is a biotinylated biologic macromolecule drug solution; the concentration of the biotinylated biologic macromolecule drug in the biotinylated biologic macromolecule drug solution is 4 μg / mL; the solvent of the biotinylated biologic macromolecule drug solution is a second buffer solution; the second buffer solution is based on MES buffer solution with a concentration of 50 mM and a pH of 6.5, and further includes the following components at the following concentrations: NaCl with a mass-volume concentration of 0.9% and BSA at 5 mg / mL; The magnetic beads are a magnetic bead suspension; the concentration of the magnetic beads in the magnetic bead suspension is 0.5 mg / mL; the suspending agent of the magnetic bead suspension is the second buffer solution.

7. The detection reagent according to claim 1, characterized in that, The magnetic beads include SA magnetic beads.

8. A test strip or kit for determining the immunogenicity of a biologic macromolecule drug, characterized in that, The test strip or kit includes the detection reagent according to any one of claims 1 to 7.

9. Use of the detection reagent according to any one of claims 1 to 7 or the test strip or kit according to claim 8 in determining the immunogenicity of a biologic macromolecule drug.

10. A method for determining the immunogenicity of a biological macromolecule drug based on the detection reagent according to any one of claims 1 to 7, characterized in that, It includes the following steps: 1) Mix the sample to be tested with the dissociation solution and carry out a dissociation reaction under acidic conditions to obtain a dissociation product; 2) Mix the dissociation product, the fusion protein, and the biotinylated biopolymer drug to carry out an immune reaction to obtain an immune reaction product; 3) Mix the immune reaction product with magnetic beads to obtain a complex; 4) After washing the complex, mix it with the AMPPD luminescent solution to carry out a color reaction.

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