Weakly acidic immunoassay for detecting an analyte

Abstract

The present invention provides an immunoassay that reduces the influence of interference in the detection and quantification of analytes including, but not limited to, cytokines, enzymes, antibodies, etc., by acidifying a sample and enabling the binding of analytes and corresponding capture reagents in the acidified sample without neutralization.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 388,839, filed Jul. 13, 2022, the entire content of which is incorporated herein by reference.

[0002] The present invention relates to immunoassays for the detection and quantification of analytes including, but not limited to, cytokines, enzymes, antibodies, etc.

Background Art

[0003] Immunoassays are widely used bioanalytical methods for measuring the presence or concentration of analytes ranging from small molecules to macromolecules in solution based on the interaction between analytes and antibodies. Immunoassays play important roles in life science research and drug product analysis, such as disease diagnosis, monitoring of therapeutic drugs, and clinical pharmacokinetics and bioequivalence.

[0004] The performance of ligand - binding assays in complex matrices such as serum can be affected by specific endogenous components that interfere with the assay (Zhong, Z.D., et al, AAPS J, 19:1564 (2017)). Interference in immunoassays can lead to misinterpretation of patient results by laboratories and implementation of incorrect treatment regimens by physicians (Tate, J., et al, Clin Biochem Rev. 2004, 25(2):105 - 120). For some analytes, the problems are even greater due to the unique properties of the analytes and the complex interactions of endogenous and exogenous components within biological samples. Commercially available methods are limited by complex procedures, intolerance to interference, and low sensitivity.

[0005] Therefore, there is a need for more sensitive, reliable, and efficient assays and methods for detecting and quantifying analytes while avoiding or minimizing interference.

Summary of the Invention

[0006] Acidic buffers, particularly buffers with a very low pH, are used to dissociate molecules from complexes. For example, antibodies with affinity tags are always eluted from purification columns using an elution buffer with a pH of about 2.0. However, under low pH conditions, even after the buffer containing the protein is neutralized, the structure of the protein may change and their functions may be lost. The invention in the present disclosure is based on the surprising discovery that the analyte of interest can be dissociated from other interfering molecules in a sample under weakly acidic conditions, and that a capture reagent, such as an antibody, can still bind to the analyte under weakly acidic conditions, thereby enabling the detection of the analyte in an assay without neutralizing the sample (in other words, without adding a basic solution to the acidified sample to raise the pH of the sample to, for example, neutral pH).

[0007] In some embodiments, the present invention provides a method for detecting an analyte in a sample, the method comprising: (i) diluting a sample containing the analyte with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent capable of binding to the analyte; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to the analyte captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of the analyte in the sample. These methods use a solid support, and the solid support is pre-coated with a capture reagent.

[0008] The method can further comprise washing the solid support after removing the acidified sample from the solid support. The method can comprise washing the solid support after removing the detection reagent from the solid support.

[0009] In some embodiments, the present invention provides a method for detecting an analyte in a sample, the method comprising: (i) diluting a sample containing the analyte with a weak acid to produce an acidified sample; (ii) adding a capture reagent to the acidified sample without neutralizing the acidified sample, wherein the capture reagent specifically binds to the analyte; (iii) adding a mixture comprising the acidified sample and the capture reagent to a solid support, wherein the solid support specifically binds to the capture reagent; (iv) after a first incubation period, removing the mixture of the acidified sample and the capture reagent from the solid support; (vi) directly adding a detection reagent to the solid support; (vii) after a second incubation period, removing the detection reagent from the solid support; and (viii) detecting the detection reagent bound to the analyte captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of the analyte in the sample. These methods use a solid support that is not pre-coated with the capture reagent, and the capture reagent binds to the solid support after binding to the analyte in the acidified sample.

[0010] The analyte can be IL2Rγ, EGFR, a human IgG4 antibody (AbA) specific for the natriuretic peptide receptor 1 (NPR1), a human monovalent monoclonal antibody (AbB) against the anti-NPR1 antibody AbA, or factor XI.

[0011] The sample can be a body fluid selected from the group consisting of blood, serum, plasma, cerebrospinal fluid (CSF), urine, and saliva.

[0012] The sample is derived from a subject having a disease or disorder. The sample can be derived from a subject having a disease or disorder.

[0013] The sample can be derived from a subject administered with a substance and / or a drug product.

[0014] The sample can be diluted at least 3-fold.

[0015] The acidified sample has a pH of about 3.0 to 6.5. The acidified sample can have a pH of about 4.1, about 4.4, or about 4.5.

[0016] The weak acid can be an acid that does not completely dissociate into its ions in an aqueous solution and is selected from the group consisting of acetic acid, citric acid, formic acid, lactic acid, phosphoric acid, and PIPES.

[0017] The sample can be acidified for 5 to 120 minutes.

[0018] The capture reagent can be an antibody. The capture reagent antibody can be selected from the group consisting of polyclonal antibodies, monoclonal antibodies, bispecific antibodies, Fab fragments, F(ab’)2 fragments, monospecific F(ab’)2 fragments, bispecific F(ab’)2, trispecific F(ab’)2, monovalent antibodies, scFv fragments, diabodies, bispecific diabodies, trispecific diabodies, scFv-Fc, minibodies, IgNAR, v-NAR, hcIgG, and vhH.

[0019] The capture reagent can bind to the analyte with a dissociation constant (K D ) value of ≤1 μΜ, ≤100 nM, ≤50 nM, ≤25 nM, ≤20 nM, ≤15 nM, ≤10 nM, ≤5 nM, ≤2 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.

[0020] The method can further include the step of identifying a capture reagent that can bind to the analyte at the pH of the acidified sample.

[0021] The capture reagent can be biotinylated, and the solid support is coated with streptavidin or avidin.

[0022] The capture reagent can be directly bound to the solid support.

[0023] The detection reagent can be an antibody. The detection reagent antibody can be a polyclonal antibody, a monoclonal antibody, a bispecific antibody, a Fab fragment, an F(ab’)2 fragment, a monospecific F(ab’)2 fragment, a bispecific F(ab’)2, a trispecific F(ab’)2, a monovalent antibody, a scFv fragment, a diabody, a bispecific diabody, a trispecific diabody, an scFv-Fc, a minibody, an IgNAR, a v-NAR, an hcIgG, or a vhH.

[0024] The detection reagent can be bound to a detectable label.

[0025] The detectable label can be selected from the group consisting of rare transition metal particles, fluorophores, chromophores, enzymes, quantum dots, and noble metal nanoparticles.

[0026] The detectable label can be horseradish peroxidase.

[0027] The detectable label can be ruthenium.

[0028] The solid support can be an electrochemiluminescence platform.

[0029] The sample can contain an interferent that binds to the analyte.

[0030] The method can further include determining the working pH of an acidified sample in which the interferent dissociates partially or completely from the analyte.

[0031] This method can recover at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the signal of the analyte in the sample containing the interfering agent, as compared to a sample without the interfering agent. The amount of the analyte can be determined by correlating the amount of the detected detection reagent with a predetermined reference standard.

[0032] In another aspect, the present invention provides a kit for detecting an analyte in a sample, the kit comprising a capture reagent, a detection reagent, and a dilution buffer containing a weak acid.

[0033] In a particular aspect, the present invention also provides a method for detecting IL2Rγ in a sample, the method comprising: (i) diluting a sample containing IL2Rγ with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent capable of binding to IL2Rγ; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to IL2Rγ captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of IL2Rγ in the sample.

[0034] The method can further comprise washing the solid support after removing the acidified sample from the solid support. The method can include washing the solid support after removing the detection reagent from the solid support.

[0035] The sample can be a body fluid such as blood, serum, plasma, CSF, urine, or saliva. In some embodiments, the sample is derived from a subject administered an IL2Rγ drug product.

[0036] The IL2Rγ pharmaceutical product can include an antibody that binds to IL2Rγ.

[0037] The sample can be derived from a subject diagnosed with or suspected of having an IL2Rγ-related disease or disorder.

[0038] IL2Rγ can non-covalently bind to an interfering agent in the sample. The interfering agent can be a human anti-IL2Rγ antibody (e.g., Int-IL2Rγ-Ab1). The interfering antibody can be its IL2Rγ-binding fragment.

[0039] The acidified sample can have a pH of about 3.4 - 4.5, for example, 4.4 or 4.5.

[0040] The weak acid can be an acid that does not completely dissociate into its ions in an aqueous solution, including but not limited to acetic acid, citric acid, formic acid, lactic acid, phosphoric acid, and PIPES.

[0041] The sample can be acidified for 5 - 120 minutes.

[0042] The capture reagent can be an IL2Rγ-specific antibody. The capture reagent antibody can be a polyclonal antibody, monoclonal antibody, bispecific antibody, Fab fragment, F(ab’)2 fragment, monospecific F(ab’)2 fragment, bispecific F(ab’)2, trispecific F(ab’)2, monovalent antibody, scFv fragment, diabody, bispecific diabody, trispecific diabody, scFv-Fc, minibody, IgNAR, v-NAR, hcIgG, or vhH. The capture reagent is biotinylated and the solid support is coated with streptavidin. The capture reagent can bind directly to the solid support.

[0043] The detection reagent can be an antibody. The detection reagent antibody can be a polyclonal antibody, a monoclonal antibody, a bispecific antibody, a Fab fragment, an F(ab’)2 fragment, a monospecific F(ab’)2 fragment, a bispecific F(ab’)2, a trispecific F(ab’)2, a monovalent antibody, a scFv fragment, a diabody, a bispecific diabody, a trispecific diabody, an scFv-Fc, a minibody, an IgNAR, a v-NAR, an hcIgG, or a vhH.

[0044] The detection reagent can bind to a detectable label. The detectable label can be selected from the group consisting of rare transition metal particles, fluorophores, chromophores, enzymes, quantum dots, and noble metal nanoparticles.

[0045] The detectable label can be horseradish peroxidase. The detectable label can be ruthenium.

[0046] The solid support can be an electrochemiluminescence platform.

[0047] The capture reagent and / or the detection reagent can be selected from human anti-IL2Rγ monoclonal antibodies (anti-IL2Rγ-Ab2 and anti-IL2Rγ-Ab1).

[0048] This method can recover at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the signal of the analyte in the sample containing the interferent as compared to a sample without the interferent. This method can recover at least 70% - 100% of the signal of the analyte in the sample containing the interferent as compared to a sample without the interferent.

[0049] The amount of the analyte can be determined by correlating the amount of the detected detection reagent with a predetermined reference standard.

[0050] In another aspect, the present invention provides a kit for detecting IL2Rγ in a sample, the kit comprising a capture reagent, a detection reagent, and a dilution buffer containing a weak acid.

[0051] In some embodiments, a method for detecting IL2Rγ in a sample comprises: (i) diluting a sample containing IL2Rγ with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent, and the capture reagent is a first IL2Rγ-specific antibody; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support, wherein the detection reagent is a second IL2Rγ-specific antibody; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to IL2Rγ captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of IL2Rγ in the sample.

[0052] The capture reagent and / or the detection reagent in the kit can be selected from human anti-IL2Rγ monoclonal antibodies (anti-IL2Rγ-Ab2 and anti-IL2Rγ-Ab1).

[0053] The acidified sample can have a pH of about 3.4 to 4.5, for example, 4.4 or 4.5.

[0054] The assay can have an analyte recovery rate of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the presence of an interferent (e.g., human monoclonal anti-IL2Rγ antibody, Int-IL2Rγ-Ab1) at 100 to 1000 μg / mL.

[0055] In certain embodiments, the present invention provides a method for detecting EGFR in a sample, the method comprising: (i) diluting a sample containing EGFR with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent capable of binding to EGFR; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to EGFR captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of EGFR in the sample.

[0056] The method can further comprise washing the solid support after removing the acidified sample from the solid support. The method can include washing the solid support after removing the detection reagent from the solid support.

[0057] The sample can be a body fluid such as blood, serum, plasma, CSF, urine, or saliva.

[0058] The sample can be derived from a subject administered an EGFR drug product.

[0059] The EGFR drug product can include an antibody that binds to EGFR.

[0060] The sample can be derived from a subject diagnosed with or suspected of having an EGFR-related disease or disorder.

[0061] EGFR can non-covalently bind to an interfering agent in the sample, wherein the interfering agent is an antibody.

[0062] The interfering antibody can be Int-EGFR-Ab1 or its EGFR-binding fragment.

[0063] The acidified sample can have a pH of about 4.0 to 6.0, for example, 4.5 or 5.0.

[0064] The weak acid can be an acid that does not completely dissociate into its ions in an aqueous solution, including, but not limited to, acetic acid, citric acid, formic acid, lactic acid, phosphoric acid, and PIPES.

[0065] The sample can be acidified for 5 to 120 minutes.

[0066] The capture reagent can be an EGFR-specific antibody. The capture reagent antibody can be a polyclonal antibody, monoclonal antibody, bispecific antibody, Fab fragment, F(ab’)2 fragment, monospecific F(ab’)2 fragment, bispecific F(ab’)2, trispecific F(ab’)2, monovalent antibody, scFv fragment, diabody, bispecific diabody, trispecific diabody, scFv-Fc, minibody, IgNAR, v-NAR, hcIgG, or vhH.

[0067] The capture reagent can be biotinylated, and the solid support is coated with streptavidin. The capture reagent can be directly bound to the solid support.

[0068] The detection reagent can be an antibody. The detection reagent antibody can be a polyclonal antibody, monoclonal antibody, bispecific antibody, Fab fragment, F(ab’)2 fragment, monospecific F(ab’)2 fragment, bispecific F(ab’)2, trispecific F(ab’)2, monovalent antibody, scFv fragment, diabody, bispecific diabody, trispecific diabody, scFv-Fc, minibody, IgNAR, v-NAR, hcIgG, or vhH.

[0069] The detection reagent can be bound to a detectable label. The detectable label can be selected from the group consisting of rare transition metal particles, fluorophores, chromophores, enzymes, quantum dots, and noble metal nanoparticles.

[0070] The detectable label can be horseradish peroxidase. The detectable label can be ruthenium.

[0071] The solid support can be an electrochemiluminescence platform.

[0072] The capture reagent and / or the detection reagent can be selected from the EGFR antibodies disclosed in WO2014 / 004427 and the EGFR antibodies in the R&D Systems Human EGFR Quantikine Kit DEGFR0.

[0073] This method can recover at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the signal of the analyte in the sample containing the interferent as compared to a sample not containing the interferent.

[0074] The amount of the analyte can be determined by correlating the amount of the detected detection reagent with a predetermined reference standard.

[0075] In another aspect, the present invention provides a kit for detecting EGFR in a sample, the kit comprising a capture reagent, a detection reagent, and a dilution buffer containing a weak acid.

[0076] In some embodiments, a method for detecting EGFR in a sample comprises: (i) diluting a sample containing EGFR with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent, and the capture reagent is a first EGFR-specific antibody; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support, wherein the detection reagent is a second EGFR-specific antibody; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to the EGFR captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of EGFR in the sample.

[0077] The capture reagent and / or the detection reagent can be selected from the EGFR antibodies disclosed in WO2014 / 004427 and the EGFR antibodies in the R&D Systems Human EGFR Quantikine Kit DEGFR0.

[0078] The acidified sample can have a pH of about 4.0 to 6.0, such as 4.5 or 5.0.

[0079] The assay can have an analyte recovery rate of at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the presence of at least 0 - 2000 μg / mL of an interferent (e.g., Int-EGFR-Ab1).

[0080] The assay can detect EGFR at a low concentration of 0.31 ng / mL in the presence of 2% human serum or 15.6 ng / mL in undiluted human serum.

[0081] In certain embodiments, the present invention provides a method for detecting AbA, a human monoclonal antibody of the IgG4 subclass that is specific for natriuretic peptide receptor 1 in a sample, the method comprising: (i) diluting a sample containing AbA with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent capable of binding to AbA; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to AbA captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of AbA in the sample.

[0082] The method can further comprise washing the solid support after removing the acidified sample from the solid support. The method can comprise washing the solid support after removing the detection reagent from the solid support.

[0083] The sample can be a body fluid such as blood, serum, plasma, CSF, urine, or saliva.

[0084] The sample can be derived from a subject diagnosed with or suspected of having a natriuretic peptide receptor 1-related disease or disorder.

[0085] The sample can be derived from a subject to whom AbA has been administered.

[0086] AbA can non-covalently bind to interferents in the sample.

[0087] The interference agent can be an antibody. The interference antibody can include one or more AbA antagonists that are human anti-AbA monoclonal antibodies (Int-AbA-Ab1, Int-AbA-Ab2, AbB, Int-AbA-Ab3), or antigen-binding fragments thereof.

[0088] The acidified sample can have a pH of about 4.0 to 6.0, for example, about 4.5 or 5.0.

[0089] The weak acid can be acetic acid, citric acid, formic acid, lactic acid, phosphoric acid, and PIPES, etc., and is not limited thereto, and can be an acid that does not completely dissociate into its ions in an aqueous solution.

[0090] The sample can be acidified for 5 to 120 minutes.

[0091] The capture reagent can be an AbA-specific antibody. The capture reagent antibody can be a polyclonal antibody, monoclonal antibody, bispecific antibody, Fab fragment, F(ab’)2 fragment, monospecific F(ab’)2 fragment, bispecific F(ab’)2, trispecific F(ab’)2, monovalent antibody, scFv fragment, diabody, bispecific diabody, trispecific diabody, scFv-Fc, minibody, IgNAR, v-NAR, hcIgG, or vhH.

[0092] The capture reagent can be biotinylated, and the solid support is coated with avidin or streptavidin. The capture reagent can be directly bound to the solid support.

[0093] The detection reagent can be an antibody. The detection reagent antibody can be a polyclonal antibody, a monoclonal antibody, a bispecific antibody, a Fab fragment, an F(ab’)2 fragment, a monospecific F(ab’)2 fragment, a bispecific F(ab’)2, a trispecific F(ab’)2, a monovalent antibody, a scFv fragment, a diabody, a bispecific diabody, a trispecific diabody, an scFv-Fc, a minibody, an IgNAR, a v-NAR, an hcIgG, or a vhH.

[0094] The detection reagent can bind to a detectable label.

[0095] The detectable label can be selected from the group consisting of rare transition metal particles, fluorophores, chromophores, enzymes, quantum dots, and noble metal nanoparticles.

[0096] The detectable label can be horseradish peroxidase. The detectable label can be ruthenium.

[0097] The solid support can be an electrochemiluminescence platform.

[0098] The capture reagent can be a mouse anti-AbA monoclonal antibody (anti-AbA-Ab1), and / or the detection reagent can be selected from a mouse anti-AbA antibody (anti-AbA-Ab2) and a mouse anti-human IgG4 Fc-specific monoclonal antibody (anti-hIgG4Fc).

[0099] This method can recover at least 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, or 100% of the signal of the analyte in the sample containing the interferent compared to a sample not containing the interferent.

[0100] The amount of the analyte can be determined by correlating the amount of the detected detection reagent with a predetermined reference standard.

[0101] In another aspect, the present invention provides a kit for detecting AbA in a sample, the kit comprising a capture reagent, a detection reagent, and a dilution buffer containing a weak acid.

[0102] In some embodiments, a method for detecting AbA in a sample comprises: (i) diluting a sample containing AbA with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent, and the capture reagent is a first AbA-specific antibody; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support, wherein the detection reagent is a second AbA-specific antibody; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to the AbA captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of AbA in the sample.

[0103] The assay can have an analyte recovery rate of at least about 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, or 100% in the presence of at least 0 - 2 mg / mL of an interferent (e.g., a human monoclonal antibody to AbA).

[0104] The assay can detect AbA at a low concentration of 0.27 ng / mL in the presence of 2% human serum, or at a concentration of 13.7 ng / mL in undiluted human serum.

[0105] In certain embodiments, the present invention provides a method for detecting human monoclonal antibody AbB against AbA in a sample, the method comprising: (i) diluting the sample containing AbB with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent capable of binding to AbB; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to AbB captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of AbB in the sample.

[0106] The method can further comprise washing the solid support after removing the acidified sample from the solid support. The method can comprise washing the solid support after removing the detection reagent from the solid support.

[0107] The sample can be a body fluid such as blood, serum, plasma, CSF, urine, or saliva.

[0108] The sample can be derived from a subject administered with an AbB drug product.

[0109] The AbB drug product can comprise an antibody that binds to AbB.

[0110] The sample can be derived from a subject diagnosed with or suspected of having a disease or disorder.

[0111] AbB can non-covalently bind to an interferent in the sample.

[0112] The interference agent can be an antibody.

[0113] The interfering antibody can be AbA or its AbB-binding fragment.

[0114] The acidified sample can have a pH of about 4.0 to 6.0, for example, about 4.5 or 5.0.

[0115] The weak acid can be acetic acid, citric acid, formic acid, lactic acid, phosphoric acid, and PIPES, etc., and is not limited thereto. It can be an acid that does not completely dissociate into its ions in an aqueous solution.

[0116] The sample can be acidified for 5 to 120 minutes.

[0117] The capture reagent can be an AbB-specific antibody.

[0118] The capture reagent antibody can be a polyclonal antibody, monoclonal antibody, bispecific antibody, Fab fragment, F(ab’)2 fragment, monospecific F(ab’)2 fragment, bispecific F(ab’)2, trispecific F(ab’)2, monovalent antibody, scFv fragment, diabody, bispecific diabody, trispecific diabody, scFv-Fc, minibody, IgNAR, v-NAR, hcIgG, or vhH.

[0119] The capture reagent can be biotinylated, and the solid support is coated with streptavidin.

[0120] The capture reagent can be directly bound to the solid support.

[0121] The detection reagent can be an antibody.

[0122] The detection reagent antibody can be a polyclonal antibody, monoclonal antibody, bispecific antibody, Fab fragment, F(ab’)2 fragment, monospecific F(ab’)2 fragment, bispecific F(ab’)2, trispecific F(ab’)2, monovalent antibody, scFv fragment, diabody, bispecific diabody, trispecific diabody, scFv-Fc, minibody, IgNAR, v-NAR, hcIgG, or vhH.

[0123] The detection reagent can bind to a detectable label.

[0124] The detectable label can be selected from the group consisting of rare transition metal particles, fluorophores, chromophores, enzymes, quantum dots, and noble metal nanoparticles.

[0125] The detectable label can be horseradish peroxidase. The detectable label can be ruthenium.

[0126] The solid support can be an electrochemiluminescence platform.

[0127] The capture reagent and / or the detection reagent can be selected from mouse anti-AbB antibodies (e.g., anti-AbB-Ab1 and anti-AbB-Ab2).

[0128] This method can recover at least 50%, 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, or 100% of the signal of the analyte in a sample containing the interferent compared to a sample not containing the interferent.

[0129] The amount of the analyte can be determined by correlating the amount of the detected detection reagent with a predetermined reference standard.

[0130] In another aspect, the present invention provides a kit for detecting AbB in a sample, the kit comprising a capture reagent, a detection reagent, and a dilution buffer containing a weak acid.

[0131] In some embodiments, a method for detecting AbB in a sample comprises: (i) diluting a sample containing AbB with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent, and the capture reagent is a first AbB-specific antibody; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support, wherein the detection reagent is a second AbB-specific antibody; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to AbB captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of AbB in the sample.

[0132] The capture reagent and / or the detection reagent can be selected from mouse anti-AbB antibodies (anti-AbB-Ab1 and anti-AbB-Ab2).

[0133] The assay can have an analyte recovery rate of at least about 75%, 80%, 85%, 90% or 95% in the presence of at least 0 - 2000 μg / mL of an interferent (e.g., AbA).

[0134] The assay can detect AbB at a low concentration of 1.56 ng / mL in 2% human serum or 78 ng / mL in pure human serum.

[0135] In certain embodiments, the present invention provides a method for detecting Factor XI in a sample, the method comprising: (i) diluting a sample containing Factor XI with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent capable of binding to Factor XI; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to Factor XI captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of Factor XI in the sample.

[0136] The method can further comprise washing the solid support after removing the acidified sample from the solid support. The method can comprise washing the solid support after removing the detection reagent from the solid support.

[0137] The sample can be a body fluid such as blood, serum, plasma, CSF, urine, or saliva.

[0138] The sample can be derived from a subject administered with a Factor XI drug product.

[0139] The Factor XI drug product can comprise an antibody that binds to Factor XI.

[0140] The sample can be derived from a subject diagnosed with or suspected of having a Factor XI-related disease or disorder.

[0141] Factor XI can non-covalently bind to an interferent in the sample.

[0142] The interference agent can be an antibody.

[0143] The interference antibody can be a human anti-factor XI antibody (for example, Int-FXI-Ab1) or its factor XI-binding fragment.

[0144] The acidified sample can have a pH of about 3.0 to 5.0, for example, about 4.1.

[0145] The weak acid can be an acid that does not completely dissociate into its ions in an aqueous solution, including but not limited to acetic acid, citric acid, formic acid, lactic acid, phosphoric acid, and PIPES.

[0146] The sample can be acidified for 5 to 120 minutes.

[0147] The capture reagent can be a factor XI-specific antibody.

[0148] The capture reagent antibody can be a polyclonal antibody, monoclonal antibody, bispecific antibody, Fab fragment, F(ab’)2 fragment, monospecific F(ab’)2 fragment, bispecific F(ab’)2, trispecific F(ab’)2, monovalent antibody, scFv fragment, diabody, bispecific diabody, trispecific diabody, scFv-Fc, minibody, IgNAR, v-NAR, hcIgG, or vhH.

[0149] The capture reagent can be biotinylated, and the solid support is coated with streptavidin.

[0150] The capture reagent can be directly bound to the solid support.

[0151] The detection reagent can be an antibody.

[0152] The detection reagent antibody can be a polyclonal antibody, monoclonal antibody, bispecific antibody, Fab fragment, F(ab’)2 fragment, monospecific F(ab’)2 fragment, bispecific F(ab’)2, trispecific F(ab’)2, monovalent antibody, scFv fragment, diabody, bispecific diabody, trispecific diabody, scFv-Fc, minibody, IgNAR, v-NAR, hcIgG, or vhH.

[0153] The detection reagent can bind to a detectable label.

[0154] The detectable label can be selected from the group consisting of rare transition metal particles, fluorophores, chromophores, enzymes, quantum dots, and noble metal nanoparticles.

[0155] The detectable label can be horseradish peroxidase. The detectable label can be ruthenium.

[0156] The solid support can be an electrochemiluminescence platform.

[0157] The capture reagent and / or the detection reagent can be a polyclonal antibody.

[0158] This method can recover at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the signal of the analyte in a sample containing the interferent compared to a sample not containing the interferent.

[0159] The amount of the analyte can be determined by correlating the amount of the detected detection reagent with a predetermined reference standard.

[0160] In another aspect, the present invention provides a kit for detecting Factor XI in a sample, the kit comprising a capture reagent, a detection reagent, and a dilution buffer containing a weak acid.

[0161] In some embodiments, a method for detecting factor XI in a sample comprises: (i) diluting a sample containing factor XI with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent, and the capture reagent is a first factor XI-specific antibody; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support, wherein the detection reagent is a second factor XI-specific antibody; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to factor XI captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of factor XI in the sample.

[0162] The assay can have an analyte recovery rate of at least 70%, 75%, 80%, 85%, 90%, or 95% in the presence of an interferent (e.g., a human anti-factor XI antibody, e.g., Int-FXI-Ab1) at 0 - 1500 μg / mL.

[0163] The assay can detect factor XI at a low concentration of 1.56 ng / mL in the presence of 2% monkey plasma or 0.078 μg / mL in undiluted monkey plasma.

[0164] In summary, the invention disclosed herein is advantageous over methods currently used in the field of analyte detection because the step of neutralizing the acidic sample is not required for the binding of the analyte to the capture reagent. A further advantage is that the method of the invention enables sufficient direct detection and quantification of the analyte of interest in samples that are subject to interference.

[0165] Non-limiting aspects and examples of the present invention will be described with reference to the drawings appended hereto, which are listed after this paragraph. Identical features that appear in multiple figures are generally given the same label in all figures in which they appear.

Brief Description of the Drawings

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Best Mode for Carrying Out the Invention

[0183] A. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. However, any compositions, methods, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications mentioned are hereby incorporated by reference in their entirety.

[0184] All numerical limits and ranges shown in this specification include all numbers or values around or between the numbers of the range or limit. The ranges and limits described in this specification are defined by the range or limit and explicitly represent and indicate all integers, decimals, and fractional values included and encompassed. Accordingly, the recitation of a range of values in this specification is merely intended to function as a way of referring individually to each separate value within that range, and each individual value is incorporated into the specification as if it were individually recited herein.

[0185] The term "about" in the context of numerical values and ranges refers to a value or range that approximates or is close to the recited value or range, as is apparent from the teachings contained herein, such that the present invention can be practiced as intended, for example, having a desired speed, amount, density, degree, increase, decrease, percentage, value, purity, pH, concentration, form or presence of a variant, temperature or amount of time. For example, "about" can indicate a value that is either above or below the recited value by approximately + / - 10% or less, depending on the ability to perform. Accordingly, this term encompasses only values other than those resulting from systematic error.

[0186] As used herein, the term "antibody" refers to an example of a binding molecule and typically refers to an immunoglobulin comprising four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains. Each heavy chain comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (C L 1). The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.

[0187] The term "antigen-binding portion" or "antibody-binding fragment" (or simply "antibody protein" or "antibody fragment") of an antibody as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IL2Rγ, EGFR, FXI). It has been shown that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Binding fragments included in the term "antigen-binding portion" of an antibody include, for example, (i) the Fab fragment, which is a monovalent fragment containing the VL, VH, CT1, and Cn1 domains; (ii) the F(ab')2 fragment, which is a divalent fragment containing two F(ab') fragments linked by a disulfide bridge in the hinge region; (iii) the Fc fragment containing the VH and C111 domains; (iv) the Fv fragment containing the VL and VH domains of a single arm of the antibody; (v) the dAb fragment containing the VH domain (Ward, E.S., et al., Nature 241:544-546 (1989)); and (vi) the CDR. Further, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be linked by a synthetic linker that allows them to be generated as a single continuous chain in which the VL region pairs with the VH region to form a monovalent molecule (known as a single-chain Fv (scFv); see, for example, Bird, R.E., et al., Science 242:423-426 (1988), and Huston, J.S., et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). Such single-chain antibodies are also intended to be included within the scope of the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also included (see, for example, Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)).

[0188] As used herein, the term "binding molecule" is intended to refer to a molecule that specifically interacts with and binds to a particular target. The target can include biological molecules or small (chemical) molecules. The target molecule may define an antigen or antigenic moiety. Binding molecules include, but are not limited to, antibodies (including polyclonal antibodies, monoclonal antibodies, bispecific antibodies, and antibody fragments), fusion proteins, and other antigen-binding molecules known to those of skill in the art. The binding molecule can be used as a capture reagent, a detection reagent, or both in the assays of the present invention.

[0189] A "CDR" or complementarity-determining region is a hypervariable region interspersed within a more conserved region termed the "framework region" (FR). The FR can be identical to the human germline sequence or may be naturally or artificially modified.

[0190] The term "epitope" is an antigenic determinant that interacts with the specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have two or more epitopes. The epitope can be either conformational or linear. A conformational epitope is produced by amino acids that are spatially juxtaposed from different segments of a linear polypeptide chain. A linear epitope is produced by adjacent amino acid residues in a polypeptide chain. In certain circumstances, an epitope may include a portion of a carbohydrate, phosphoryl group, or sulfonyl group on the antigen.

[0191] The terms "individual", "subject", and "patient" are used interchangeably herein and refer to mammals including, but not limited to, humans, rodents such as mice and rats, and other laboratory animals.

[0192] The terms "interferent" and "assay interference" refer to endogenous and / or exogenous components in an assay that block or inhibit the detection of an analyte, such as human monoclonal antibodies against IL2Rγ, EGFR, NPR1 (e.g., AbA), human monovalent monoclonal antibodies against AbA (AbB), and FXI, or anti-IL2Rγ, EGFR, AbA, AbB, and FXI antibodies. Substances that alter the measurable concentration of an analyte or the binding of an antibody may cause interference in immunoassays. An interferent may bind directly to the analyte or be part of a complex containing the analyte. The complex containing the analyte and the interferent is referred to herein as an "interferent:analyte complex" or "analyte:interferent complex". Assay interference may or may not be analyte-dependent. Interference that is not analyte-dependent refers to common interferences due to hemolysis, lipemia, and the effects of anticoagulants and sample storage, independent of the concentration of the analyte. Analyte-dependent interference in immunoassays refers to direct or indirect interactions between the interferent and the analyte that block or inhibit the detection of the analyte, or interactions between the interferent and the analyte-specific antibody. An interferent may be a compound that is chemically different but has structural similarity that cross-reacts with the antibody. Interfering and endogenous substances that are natural, polyreactive antibodies or autoantibodies (heterophilic), human anti-animal antibodies, or anti-drug antibodies (ADA), along with other unexpected binding proteins specific to an individual, can interfere with the reaction between the analyte and the reagent antibody in immunoassays.

[0193] Interference can be caused by the soluble binding target of the analyte, the endogenous ligand of the analyte including but not limited to the soluble receptor of the analyte, the soluble ligand of the analyte, the released receptor of the analyte, or serum factors such as rheumatoid factors and biotin. Interference can be caused by antibodies against the analyte.

[0194] The term "weak acid" refers to an acid that does not completely dissociate into ions in aqueous solution. Most organic acids are weak acids. The strength of an acid is its acid dissociation constant, K aIt can be quantified by a value. Some examples of weak acids include acetic acid, ascorbic acid, benzoic acid, boric acid, citric acid, formic acid, hydrazoic acid, hydrocyanic acid, hydrofluoric acid, hypochlorous acid, lactic acid, nitrous acid, oxalic acid, phenolic acid, propanoic acid, sulfurous acid, uric acid, phosphoric acid, and PIPES (piperazine - N,N - bis(2 - ethanesulfonic acid)).

[0195] The term "analyte" refers to a substance whose presence is intended to be quantitatively analyzed. The analyte can be a ligand that specifically binds or couples to both an immobilized capture agent and a detection agent. The analyte can be substances such as peptides, proteins, antibodies, and hormones present in body fluids that play important roles in biological functions.

[0196] The term "analyte recovery rate (%AR)" or "recovery rate" refers to the proportion of the analyte detected in a sample (e.g., a sample containing an interferent) compared to the actual amount or concentration of the analyte in the sample. In other words, the analyte recovery rate is the percentage of the measured analyte concentration (concentration or average concentration) as a proportion of the nominal (spike) concentration.

[0197] B. Detection and Quantification Assays, Kits, and Methods of Using the Same a. Method The present invention relates to assays and methods for the detection and quantification of analytes. The disclosed assays and methods can be used to detect and quantify protein analytes (e.g., human monoclonal antibodies (AbA) against IL2Rγ, EGFR, NPR1, human monovalent monoclonal antibodies (AbB) against AbA, FXI) in a sample. The sample can be obtained from an individual being treated with a drug product or an individual undergoing a medical treatment.

[0198] The disclosed assay reduced assay interference as compared to commercially available assays and / or control assays. The interference may or may not depend on the analyte, or both. The method can reduce or inhibit assay interference caused by endogenous soluble analyte binding molecules present in the sample. Endogenous soluble analyte binding molecules include, but are not limited to, serum components, anti-target analyte antibodies, and target analyte receptors.

[0199] The methods and assays of the present invention are for detecting an analyte in a sample by acidifying the sample to a pH sufficient to dissociate the analyte from its complex in the sample. A capture reagent and a detection reagent that specifically bind to the analyte are used to detect and quantify the analyte in the sample, e.g., to determine the amount or concentration of the analyte in the sample.

[0200] The methods and assays of the present invention are also useful for reducing assay interference by acidifying the sample to a pH sufficient to dissociate the analyte from its complex in the sample. The method includes acidifying the capture reagent and the sample mixture to a pH sufficient to dissociate the interferent from the analyte and allow binding of the analyte to the capture reagent, immobilizing the capture reagent on a solid support, adding a detection reagent, optionally wherein the detection reagent comprises a detectable label, and allowing the detection reagent to bind to the analyte captured by the immobilized capture reagent. The amount of the detection reagent correlates with the amount of the captured analyte in the sample.

[0201] In certain embodiments, the methods and assays of the invention are provided for reducing interference from an analyte in a sample by acidifying the sample to a pH sufficient to dissociate the analyte from its complex in the sample. The method includes adding the acidified sample to a capture reagent immobilized on a solid support, and adding a detection reagent, optionally wherein the detection reagent comprises a detectable label, and enabling the detection reagent to bind to the analyte captured by the capture reagent, wherein the amount of the detected detectable label correlates with the amount of the analyte in the sample.

[0202] In certain embodiments, the methods and assays of the invention are provided for quantifying an analyte in a sample by acidifying the sample to a pH sufficient to dissociate the analyte from its complex in the sample. The method includes adding the acidified sample to a capture reagent immobilized on a solid support, and adding a detection reagent, optionally wherein the detection reagent comprises a detectable label, and enabling the detection reagent to bind to the analyte captured by the capture reagent, wherein the amount of the detected detectable label correlates with the amount of the analyte in the sample.

[0203] The methods and assays can be used to determine the concentration of an analyte in a human serum sample using an electrochemiluminescence immunoassay. The method includes an acid pretreatment of the serum sample that dissociates soluble analytes that are interfering agent complexes present in the sample and improves detection of the analyte in the presence of the interfering agent, thereby quantitatively measuring the level of the analyte.

[0204] The amount of label detected in the sample can be compared to a reference standard calibrated with known concentrations of the analyte and the corresponding amounts of the detection label for those concentrations. The amount of the analyte in the sample can be determined by comparing the amount of the detection label in the sample to the reference standard and matching the amount of the detection label to the concentration indicated by the reference standard for that amount of the detection label.

[0205] In this method and assay, plates coated with streptavidin can be used with biotinylated antibodies as capture reagents, and recombinant analytes can be utilized as standards. Standards, controls, and samples are diluted in a dilution buffer containing acetic acid. The detection reagent is an antibody labeled with ruthenium. The analyte captured on the plate is measured by the chemiluminescence signal generated by the ruthenium label when a voltage is applied to the plate by a plate reader. The resulting electrochemiluminescence signal (e.g., counts) is proportional to the amount of analyte present in the sample.

[0206] The capture reagent can be pre-coated on a solid support, such as a solid support spotted with streptavidin, to capture the analyte of interest in the acidified sample. The capture antibody can be mixed with a sample containing the analyte of interest first and acidified for a certain period of time, e.g., about 1 hour, before being added to the blocking assay plate for further detection.

[0207] The analyte can be treated with an assay dilution buffer (ADB) containing various concentrations of weak acids (e.g., 20 mM, 30 mM, 60 mM, 80 mM, 100 mM, 120 mM, or 150 mM acetic acid) to dissociate the interferent, which is an analyte complex, efficiently and adjust the pH level to 6.5 - 3.0.

[0208] The interferent can be completely dissociated from the analyte after acidification of the sample. The interferent can be partially dissociated from the analyte after acidification of the sample.

[0209] About 50% - 100% of the analyte in the sample can be dissociated from the interferent, which is an analyte complex. At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the analyte in the analyte can be dissociated from the interferent, which is an analyte complex.

[0210] The method can further include determining whether the interfering agent dissociates from the analyte after acidification of the sample. The method can include testing different pH levels to determine at which buffered acid pH the interfering agent dissociates from the analyte. The method can include testing different pH levels to determine at which buffered acid pH the interfering agent dissociates from the analyte. The method can include testing different pH levels to determine at which buffered acid pH the interfering agent does not bind to the analyte or the binding to the analyte is decreased as compared to the binding at the original pH of the sample. Methods for measuring the interaction between two molecules are well known in the art (e.g., surface plasmon resonance) and can be used to determine the binding and dissociation of the interfering agent and the analyte under different pH conditions.

[0211] The working pH useful for the methods and assays of the present invention is the pH level at which the interfering agent dissociates partially or completely from the analyte. At least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the interfering agent bound to the analyte in the non-acidified sample can dissociate from the analyte after acidification of the sample.

[0212] The method can further include identifying a capture agent that can bind to the analyte at the pH of the acidified sample. The method can include screening for a capture agent that can bind to the analyte at a pH at which the interfering agent dissociates or the binding to the analyte is decreased. Methods for measuring the interaction between two molecules are well known in the art (e.g., surface plasmon resonance) and can be used to determine the binding and dissociation of the candidate capture reagent and the analyte under different pH conditions.

[0213] The sensitivity of the assay, as determined by the lower limit of quantification (i.e., limit of detection) of the analyte, can be determined under various sample acidification conditions. The resistance of the analyte to one or more interfering agents can also be determined under various sample acidification conditions.

[0214] The lower limit of quantification (LLOQ) (i.e., the minimum amount of analyte that must be present in the sample in order to be detected by the method) of this method can be 0.5 - 20 pg / mL, 1 - 50 pg / mL, 5 - 200 pg / mL, 20 - 400 pg / mL, 0.1 - 0.5 ng / mL, 0.2 - 0.8 ng / mL, 0.1 - 1 ng / mL, 0.5 - 2 ng / mL, 1 - 10 ng / mL, 5 - 50 ng / mL, 20 - 60 ng / mL, 40 - 100 ng / mL, 50 - 150 ng / mL, or 100 - 200 ng / mL. The lower limit of quantification (LLOQ) of this method can be 0.5 pg / mL, 1 pg / mL, 5 pg / mL, 10 pg / mL, 15 pg / mL, 20 pg / mL, 40 pg / mL, 60 pg / mL, 80 pg / mL, 0.1 ng / mL, 0.5 ng / mL, 0.8 ng / mL, 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, 300 ng / mL, 500 ng / mL, 750 ng / mL, 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, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, or 50 μg / mL, less than 60 μg / mL, 70 μg / mL.

[0215] The assay can be performed in the absence of serum. The assay can be performed in the presence of serum (e.g., human serum, bovine serum, monkey serum, or horse serum). The assay can be performed with undiluted (e.g., neat) serum.

[0216] This method can recover at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the signal of the analyte in the sample containing the interfering agent as compared to the sample without the interfering agent.

[0217] This method can be carried out at about 4 to 37 °C, for example, about 4 °C. This method can be carried out at about 20 to 37 °C, for example, 20 to 25 °C, 25 to 30 °C, or 30 to 37 °C. For example, this method can be carried out at about 20 °C, 25 °C, 30 °C, or 37 °C.

[0218] The acidified sample can be added directly to the capture reagent without being neutralized with a buffered base solution.

[0219] The blocker can be further added to the sample to reduce or prevent the reformation of interfering agents that are analyte complexes after acidification of the sample.

[0220] b. Sample The sample used in the disclosed method is typically a biological sample such as a body fluid containing the analyte to be detected. Body fluids include, but are not limited to, blood, plasma, serum, saliva, cerebrospinal fluid (CSF), and urine.

[0221] The sample can be obtained from a subject having or suspected of having a disorder or disease.

[0222] The sample can be derived from a subject to whom a drug product has been administered.

[0223] The sample can also be derived from a subject who abuses drugs or is dependent on alcohol and tobacco products.

[0224] The sample can be derived from a subject who has been exposed or is suspected of having been exposed to toxins, allergens, or irritants.

[0225] The analyte to be detected is typically a component of serum taken from a subject, for example, a human subject. Representative analytes detected and / or quantified in the sample include, but are not limited to, cytokines, protein drug products, and their metabolites or fragments.

[0226] Representative protein drug products include, but are not limited to, recombinant proteins, antibodies, and fusion proteins. Antibodies can be polyreactive antibodies or autoantibodies (heterophilic), human anti-animal antibodies, or antidrug antibodies.

[0227] c. Acidification of the sample The sample in the disclosed method can be acidified for 1 to 120 minutes. The sample can be acidified for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 minutes. The sample can be acidified for about 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 minutes.

[0228] Acids that can be used in the acidification step include, but are not limited to, acetic acid, citric acid, formic acid, lactic acid, phosphoric acid, and PIPES.

[0229] The sample can be acidified to a certain pH, for example, pH 3.0 to 6.5, to dissociate the analyte from its complex with one or more interferents. The pH can be about 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. The analyte can be completely dissociated from the complex with the interferent at the pH of the acidified sample. The analyte can be partially dissociated from the complex with the interferent at the pH of the acidified sample.

[0230] d. Capture reagent The capture reagents or capturers described in this specification include antagonists or inhibitors of analytes, such as molecules that bind to analytes, for example, antibodies or antigen-binding fragments of antibodies that specifically bind to analytes. The antibodies can be monoclonal, polyclonal, or humanized. Terms such as "specifically bind" mean that the antibody or its antigen-binding fragment forms a relatively stable complex with the antigen. The capture reagents of the present invention bind to the corresponding analyte at the pH of the acidified sample, that is, the pH of the acidified sample does not significantly reduce the binding affinity of the capture reagent for the analyte. These capture reagents can be either monoclonal antibodies or polyclonal antibodies produced in various animal species.

[0231] The capture reagent can be biotinylated. The capture reagent can be immobilized or linked to a solid support. The solid support can be a microplate, for example, a microplate coated with streptavidin.

[0232] The capture reagent can bind to the analyte with a dissociation constant (K -8 ) value of ≦1 μM, ≦100 nM, ≦50 nM, ≦25 nM, ≦20 nM, ≦15 nM, ≦10 nM, ≦5 nM, ≦2 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (for example, 10 -8 M or less, for example, 10 -13 M to 10 -9 M, for example, 10 -13 M). D

[0233] The capture reagent can bind to IL2Rγ, EGFR, human monoclonal antibodies (AbA) against NPR1, human monovalent monoclonal antibodies (AbB) against AbA, or FXI for use in methods or assays for measuring IL2Rγ, EGFR, AbA, AbB, or FXI, respectively, in a sample.

[0234] The capture reagents in the disclosed methods and assays can be biotinylated anti-AbA-Ab1, a mouse anti-AbA monoclonal antibody. The capture reagent can be an EGFR antibody or a biotinylated anti-EGFR antibody, a mouse anti-AbB antibody (anti-AbB-Ab2), or a goat anti-human FXI polyclonal antibody.

[0235] e. Detection reagent The detection reagents and detection agents described herein include molecules that bind to the analyte, such as an analyte antagonist or inhibitor, such as an analyte antibody or an antigen-binding fragment of an antibody that specifically binds to the analyte. The antibody can be monoclonal, polyclonal, or humanized.

[0236] The detection reagent can be labeled with a detectable label. Detectable labels are known in the art and include, but are not limited to, rare transition metal particles, fluorophores, chromophores, quantum dots, noble metal nanoparticles, radioactive moieties, enzymes, biotin / avidin labels, and chemiluminescent labels. The detection reagent can be ruthenylated anti-AbA-Ab2, which is a mouse anti-AbA monoclonal antibody. The detection reagent can be a biotinylated mouse anti-AbB antibody (anti-AbB-Ab1), a goat anti-human FXI polyclonal antibody conjugated to peroxidase (HRP), an HRP-conjugated anti-EGFR antibody, or a ruthenium-labeled anti-EGFR antibody.

[0237] The detection reagent can bind to the analyte with a dissociation constant (K -8 ) value of ≤ 1 μM, ≤ 100 nM, ≤ 50 nM, ≤ 25 nM, ≤ 20 nM, ≤ 15 nM, ≤ 10 nM, ≤ 5 nM, ≤ 2 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -13 M to 10 -9 M, e.g., 10 -13 M to 10 D M).

[0238] The detection reagent can bind to IL2Rγ, EGFR, AbA, AbB, or FXI and is used in a method or assay for measuring IL2Rγ, EGFR, AbA, AbB, or FXI, respectively, in a sample.

[0239] f. Kit There is also provided a kit for assaying a test sample for the presence, amount, or concentration of an analyte (or a fragment thereof) in a sample. The kit includes at least one component for assaying a sample of the analyte (or a fragment thereof) and instructions for assaying a sample of the analyte (or a fragment thereof).

[0240] The kit of the present invention includes the above detection reagent and the above capture reagent.

[0241] The detection reagent and the capture reagent can be the same or different analyte-binding molecules as described above, such as a monoclonal antibody (or a fragment thereof, a variant thereof, or a fragment of a variant thereof), a fusion protein, or an aptamer, optionally immobilized on a solid phase.

[0242] After the capture reagent binds to the analyte, the binding site and epitope of the detection reagent are still available for the detection reagent to bind to the analyte.

[0243] The capture reagent and the detection reagent can bind to different epitopes of the analyte.

[0244] Binding of the capture reagent to the analyte may not prevent binding of the detection reagent to the analyte. For example, the epitopes of the capture reagent and the detection reagent are far apart, so binding of one reagent does not sterically prevent binding of the other reagent.

[0245] The detection reagent is typically labeled with a detectable label such as a chemiluminescent label. The detection reagent can incorporate a detectable label described herein such as a fluorophore, a radioactive moiety, an enzyme, a biotin / avidin label, a chromophore or a chemiluminescent label, or alternatively the kit can include reagents for performing a detectable label. Antibodies, calibrators and / or controls can be provided in separate containers or can be pre-dispensed into a suitable assay format, such as a microtiter plate. The analyte detection reagent can be labeled with ruthenium. The detection reagent can bind to HRP.

[0246] The kit can contain a solid support, such as a microtiter plate or an electrochemiluminescence platform, coated with a biotinylated capture reagent and streptavidin. The capture reagent and the microtiter plate or electrochemiluminescence platform can be provided in separate containers. The kit can contain a microtiter or electrochemiluminescence platform plate coated with the capture reagent.

[0247] The kit can also contain the acidic solution and buffer for processing the sample.

[0248] The kit can include a calibrator or control, such as an isolated native and / or recombinant analyte. The kit can include at least one container for performing the assay (e.g., a tube, a microtiter plate or strip, or an electrochemiluminescence platform), and / or a buffer such as an assay buffer or a wash buffer that can be provided as either a concentrated solution, a substrate solution for a detectable label (e.g., an enzyme label), or a stop solution. Preferably, the kit includes all of the components necessary to perform the assay, i.e., reagents, standards, buffers, diluents, etc. The instructions can be in paper or computer-readable form.

[0249] Optionally, the kit includes quality control components (e.g., sensitivity panels, calibrators, and positive controls). The preparation of quality control reagents is well known in the art and is described in the insert sheets of various immunoassay products. Sensitivity panel members are optionally used to establish assay performance characteristics and, further optionally, are useful indicators of the integrity of immunoassay kit reagents and assay standardization.

[0250] The kit can also optionally include other reagents necessary to perform a diagnostic assay or to facilitate quality control evaluation, such as buffers, salts, enzymes, enzyme cofactors, enzyme substrates, and detection reagents. The kit can include other components such as buffers and solutions (e.g., pretreatment reagents) for isolation and / or processing of test samples. The kit can additionally include one or more other controls. One or more of the components of the kit can be lyophilized, in which case the kit can further include reagents suitable for reconstitution of the lyophilized components.

[0251] The various components of the kit are optionally provided, as needed, in suitable containers, e.g., microtiter plates. The kit can further include a container for holding or storing a sample (e.g., a container or cartridge for urine samples). Where appropriate, the kit can optionally include reaction vessels, mixing vessels, and other components that facilitate the preparation of reagents or test samples. The kit can also include one or more instruments for assisting in the acquisition of a test sample, such as syringes, pipettes, forceps, or measuring spoons.

[0252] C. Methods and Assays for Detection and Quantification of IL2Rγ a. Interleukin 2 Receptor γ-Chain IL2Rγ or "interleukin-2 receptor gamma chain" refers to a 64 kDa IL2Rγ transmembrane protein having 347 amino acids, 84 of which are cytoplasmic among the 347 amino acids. IL2Rγ plays a very important role in the formation of a bona fide IL-2 receptor that is important for T cell differentiation, activation, proliferation, and survival. IL2Rγ, together with the beta chain, is involved in the increase of IL-2 binding affinity and intracellular signal transduction. IL2Rγ is also a common component of the receptor complexes for cytokines IL-2, IL-4, IL-7, IL-9, and IL-15. Multiple pathways downstream of these receptors are essential for the differentiation and proliferation of T cells and NK cells, and since they collectively have effects related to the control of cancer, autoimmune diseases, and immunodeficiency, they attract great clinical interest. IL2Rγ mutations are associated with X-linked severe combined immunodeficiency in humans, a disease characterized by the virtual absence of T cells.

[0253] b. Methods and kits The methods and kits described herein are intended to quantitatively determine IL2Rγ or a fragment thereof, such as native and / or recombinant IL2Rγ, in a sample by acidifying the sample to a pH sufficient to dissociate IL2Rγ from its complex in the sample. Capture reagents and detection reagents specific for IL2Rγ are used in methods and kits for detecting and determining the amount or concentration of IL2Rγ in a sample.

[0254] The methods and kits described herein also provide a combination of capture reagents and detection reagents for the detection and quantification of IL2Rγ, and after the capture reagent binds to IL2Rγ, the binding sites and epitopes of the detection reagent are available for the detection reagent to bind to IL2Rγ.

[0255] The capture reagent can be an anti-IL2Rγ antibody or an antigen-binding fragment thereof. The detection reagent can be an anti-IL2Rγ antibody or an antigen-binding fragment thereof.

[0256] Anti-IL2Rγ antibodies, as well as methods for generating anti-IL2Rγ antibodies, are known in the art. For example, descriptions of multiple anti-IL2Rγ antibodies may be found in Patent Publication US2020 / 0247894A1.

[0257] The capture reagent can be the anti-IL2Rγ antibody disclosed in US2020 / 0247894A1. The capture reagent can be anti-IL2Rγ-Ab2 or anti-IL2Rγ-Ab1. The detection reagent can be the anti-IL2Rγ antibody disclosed in US2020 / 0247894A1. The detection reagent can be anti-IL2Rγ-Ab2 or anti-IL2Rγ-Ab1.

[0258] The method disclosed herein can recover at least 70-100% of the signal of IL2Rγ, that is, the recovery rate is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0259] The sample can be acidified to a pH of about 4.0-5.0, for example, a pH of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0260] In some exemplary and non-limiting assays, as described herein, IL2Rγ is detected and quantified in biological samples including serum, plasma, saliva, CSF, and urine. The assay uses streptavidin microplates coated with biotinylated anti-IL2Rγ antibody (bio-anti-IL2Rγ-Ab1) as a capture agent and utilizes IL2Rγ as a standard. Standards, controls, and samples are diluted with assay dilution buffer (ADB) before addition to the assay plate. The captured IL2Rγ is then detected using ruthenium-labeled IL2Rγ detection antibody (Ru-anti-IL2Rγ-Ab2). When a voltage is applied to the plate by a reader, an electrochemiluminescence signal is generated by the ruthenium label. The measured electrochemiluminescence (i.e., counts) is proportional to the concentration of total IL2Rγ in the sample. The sample may contain interfering reagents such as human anti-IL2Rγ antibody, which is Int-IL2Rγ-Ab1.

[0261] The present invention also provides a method for evaluating the effect of sample acidification on assay sensitivity in the quantification of IL2Rγ. The method includes incubating an IL2Rγ-containing sample with a neutral or acidic assay dilution buffer (ADB) to acidify the sample, for example, lowering the pH of the sample to 4.4 - 4.0, detecting IL2Rγ using the methods and assays of the present invention as described in this disclosure, and comparing the detected value of IL2Rγ in the acidic sample with the value in the neutral sample.

[0262] The present invention also provides a method for evaluating the signal recovery rate of IL2Rγ in the presence of an interfering agent, for example, Int-IL2Rγ-Ab1. Samples containing only IL2Rγ or IL2Rγ and one or more interfering agents (e.g., Int-IL2Rγ-Ab1) are diluted with a neutral or acidic assay dilution buffer (ADB) to acidify the sample, for example, lowering the pH of the sample to 4.4 - 4.0. The IL2Rγ signal detected from the sample containing the interfering agent is compared with the signal detected from a sample without the interfering agent to determine the analyte recovery rate.

[0263] Methods and assays for the detection and quantification of D.EGFR a. Epidermal growth factor receptor (EGFR) EGFR, "human epidermal growth factor receptor", ErbB1 or HER1 refers to a 170 kDa type I transmembrane glycoprotein encoded by the EGFR gene located on chromosome 7. EGFR plays an essential role in the regulation of cell growth, survival, differentiation and migration. The human epidermal growth factor receptor of receptor tyrosine kinase (RTK) consists of four members: EGFR (HER1), HER2, HER3, and HER4. EGFR is activated by binding its cognate ligands such as EGF (epidermal growth factor) and TGF alpha (transforming growth factor alpha) to the extracellular domain, which causes EGFR dimerization and subsequent autophosphorylation of tyrosine residues in the cytoplasmic domain. Phosphorylation of EGFR at specific residues is mediated by Src non-receptor kinase. EGFR activation signals to multiple downstream signaling cascades including the Ras / MARK, PLCγ1 / PCK, PI3K / AKT, and STAT pathways that assist cell growth and proliferation. Phosphorylation of EGFR at Y1086 specifically enables the binding of the adapter protein GRB2, causing activation of the MAPK pathway. By receptor activation and signaling, EGFR is endocytosed and targeted for degradation or recycling. Soluble EGFR consists of the extracellular domain of EGFR and can be directly measured in body fluids such as serum or plasma. Overexpression of EGFR is associated with uncontrolled tumor growth in head and neck, brain, bladder, stomach, breast, lung, endometrium, cervix, vulva, ovary, esophagus, stomach, and squamous cell carcinoma. Therefore, the detection and measurement of EGFR in samples such as human serum provide an important method for the diagnosis of diseases and the monitoring of therapeutic drugs.

[0264] b. Methods The assays described herein are intended to quantitatively determine soluble EGFR or fragments thereof in a sample, such as a sample containing native and / or recombinant EGFR, by acidifying the sample to a pH sufficient to dissociate EGFR from its complex. Capture reagents and detection reagents specific for EGFR are used in methods and kits for detecting and determining the amount or concentration of EGFR in a sample.

[0265] The methods and kits described herein also provide a combination of capture reagents and detection reagents for the detection and quantification of EGFR, and after the capture reagent binds to EGFR, the binding sites and epitopes of the detection reagent are available for the detection reagent to bind to EGFR.

[0266] The capture reagent can be an anti-EGFR antibody or an antigen-binding fragment thereof. The detection reagent can be an anti-EGFR antibody or an antigen-binding fragment thereof.

[0267] Anti-EGFR antibodies and methods for generating anti-EGFR antibodies are known in the art. For example, descriptions of multiple anti-EGFR antibodies or antigen-binding fragments thereof are disclosed in Patent Publications WO2014 / 004427 and WO2020 / 198009.

[0268] Interfering agents may be present in the sample. The interfering agent may be a molecule administered to a subject as a drug targeting EGFR, such as the human anti-EGFR antibody disclosed in WO2020 / 198009.

[0269] The method can recover at least 50% - 100% of the signal of EGFR, that is, the recovery rate is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0270] The sample can be acidified to a pH of about 4.0 - 5.0, for example, a pH of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0271] Sample acidification can be achieved by adding an assay dilution buffer containing 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, or 5 mM acetic acid.

[0272] In some exemplary and non - limiting assays for determining the concentration of total soluble EGFR in a sample, an EGFR capture antibody is pre - coated on a solid support to capture soluble EGFR in the sample. An HRP - conjugated EGFR detection antibody is used to detect the captured EGFR. Then, a peroxidase - specific substrate is added to generate a signal intensity proportional to the amount and concentration of EGFR in the sample.

[0273] In other exemplary and non - limiting assays, streptavidin microplates are coated with biotinylated EGFR capture antibody, and the EGFR captured on the plate is detected using a ruthenium - labeled EGFR detection antibody. Recombinant EGFR is used as a standard. The standard, control, and samples are diluted with an acidic buffer before adding to the assay plate. When a voltage is applied to the plate by a reader, an electrochemiluminescence signal is generated by the ruthenium label. The measured electrochemiluminescence (i.e., counts) is proportional to the amount and concentration of EGFR in the sample.

[0274] It is understood that one or both assay formats can be used in any of the examples disclosed herein.

[0275] The recovery rate of EGFR in the presence of an interfering agent, such as a bispecific human anti - EGFR antibody like Int - EGFR - Ab1, can be evaluated in a neutral or acidic assay dilution buffer using the above - mentioned method.

[0276] The present invention also provides a method for evaluating the effect of sample acidification on assay sensitivity in the quantification of EGFR. The method includes incubating a sample containing EGFR with a neutral or acidic assay dilution buffer (ADB) to acidify the sample, for example, lowering the pH of the sample to 4.4 - 4.0, detecting EGFR using the method and assay of the present invention as described in this disclosure, and comparing the detected value of EGFR in the acidic sample with the value in a neutral sample (e.g., pH 7.4).

[0277] The present invention also provides a method for evaluating the signal recovery rate of EGFR in the presence of an interfering agent, for example, a bispecific human anti - EGFR antibody such as Int - EGFR - Ab1. Samples containing only EGFR or EGFR and one or more interfering agents (e.g., Int - EGFR - Ab1) are diluted with a neutral or acidic assay dilution buffer (ADB) to acidify the sample, for example, lowering the pH of the sample to 4.4 - 4.0. The EGFR signal detected from the sample containing the interfering agent is compared with the signal detected from the sample without the interfering agent to determine the analyte recovery rate.

[0278] Methods and assays for the detection and quantification of an antibody targeting a human monoclonal antibody (IgG4 subclass) (AbA) specific for E.NPR1 a. A human monoclonal antibody (IgG4 subclass) (AbA) specific for a.NPR1 The natriuretic peptide receptor 1 (NPR1) is the main receptor for the natriuretic peptides ANP and BNP. Binding of ANP to the extracellular ligand - binding domain and ATP to the intracellular kinase - homology domain activates cytoplasmic guanylate cyclase. AbA is a human antibody (IgG4 subclass) specific for the natriuretic peptide receptor 1 (NPR1) and is a drug candidate.

[0279] b. Method The assays described herein are intended to quantitatively measure AbA in a sample by acidifying the sample to a pH sufficient to dissociate AbA from its complex. Capture and detection reagents specific for AbA are used in methods and kits for detecting and determining the amount or concentration of AbA in a sample.

[0280] The methods and kits described herein also provide a combination of capture and detection reagents for the detection and quantification of AbA, and after the capture reagent binds to AbA, the binding site and epitope of the detection reagent are available for the detection reagent to bind to AbA.

[0281] The capture reagent can be an anti-AbA antibody or an antigen-binding fragment thereof. The detection reagent can be an anti-AbA antibody or an antigen-binding fragment thereof.

[0282] The capture agent can be a mouse anti-AbA monoclonal antibody (e.g., anti-AbA-Ab1). The detection reagent can be selected from an anti-human IgG4 Fc monoclonal antibody (e.g., anti-hIgG4Fc) or a mouse anti-AbA monoclonal antibody (e.g., anti-AbA-Ab2). Examples of anti-human IgG4 Fc antibodies can be found in Mol Cancer Ther (2019) 18(11):2051-2062.

[0283] One or more interferents may be present in the assay, e.g., AbA antagonists that are human anti-AbA monoclonal antibodies (Int-AbA-Ab1, Int-AbA-Ab2, AbB, and Int-AbA-Ab3).

[0284] Sample acidification can recover at least 50%, 55%, 60%, 65%, 70% - 100% of the signal of the analyte, i.e., the recovery rate is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0285] The sample can be acidified to a pH of about 4.0 to 5.0, for example, a pH of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0286] Some exemplary and non-limiting assay formats relate to determining the concentration of total AbA in a sample by immobilizing a biotinylated AbA capture reagent on an avidin-coated plate to capture total AbA and detecting the captured AbA with a ruthenium-conjugated anti-human IgG4 Fc antibody (anti-hIgG4Fc). Then, when a voltage is applied to the plate by a reader, an electrochemiluminescence signal is generated by the ruthenium label. The measured electrochemiluminescence is proportional to the concentration of total AbA in the sample.

[0287] In another exemplary and non-limiting assay format, streptavidin microplates are coated with a biotinylated mouse anti-AbA monoclonal antibody that is anti-AbA-Ab1 as a capture agent, and a ruthenylated mouse anti-AbA monoclonal antibody that is REGN1049 is used as a detection reagent. The concentration of AbA is determined as described above.

[0288] It is understood that one or more assay formats can be used in any of the inventions disclosed herein.

[0289] The present invention also provides a method for evaluating the effect of sample acidification on assay sensitivity in the quantification of AbA. The method includes incubating an AbA-containing sample with a neutral or acidic assay dilution buffer (ADB) to acidify the sample, for example, lowering the pH of the sample to 5.0 to 4.0, and detecting AbA using the methods and assays of the present invention as described in this disclosure, and comparing the detected value in the acidic sample with the value in a neutral sample (e.g., pH 7.4) for EGFR.

[0290] The present invention also provides a method for evaluating the signal recovery rate of AbA in the presence of one or more interfering agents, such as Int-AbA-Ab1, Int-AbA-Ab2, AbB, or Int-AbA-Ab3. Samples containing only AbA or AbA and one or more interfering agents (e.g., Int-AbA-Ab1, Int-AbA-Ab2, AbB, or Int-AbA-Ab3) are diluted with a neutral or acidic assay dilution buffer (ADB) to acidify the sample, e.g., to lower the pH of the sample to 5.0 - 4.0. The analyte recovery rate is determined by comparing the AbA signal detected from the sample containing the interfering agent with the signal detected from the sample without the interfering agent.

[0291] In some exemplary and non-limiting examples, the recovery rate of AbA in the presence of one or more of the interfering agents, such as Int-AbA-Ab1, Int-AbA-Ab2, AbB, and Int-AbA-Ab3, is measured in both a neutral assay dilution buffer and an acidic assay dilution buffer, e.g., an assay dilution buffer containing 30 mM acetic acid. The AbA tolerance to the interfering agent is evaluated by adding various amounts of the interfering agent such that the interfering agent:AbA ratio ranges from 0 - 3,300:1.

[0292] The recovery rate of AbA at concentrations of 13.7 ng / mL to 7.5 μg / mL, e.g., 13.7 ng / mL and 7.5 μg / mL, in the presence of various amounts of AbB can be at least 55 - 100%, e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0293] Methods and assays for the detection and quantification of antagonists of human monoclonal IgG4 antibodies against F.NPR1 a. An antagonist (AbB) of a human monoclonal IgG4 antibody against NPR1 AbB is a human monovalent monoclonal antibody against AbA, which is a human monoclonal antibody (IgG4 subclass) specific for NPR1.

[0294] b. Method The assays described herein are intended to quantitatively measure AbB in a sample by acidifying the sample to a pH sufficient to dissociate AbB from its complex. Capture reagents and detection reagents specific for AbB are used in methods and kits for detecting and determining the amount or concentration of AbB in a sample.

[0295] The methods and kits described herein also provide a combination of capture reagents and detection reagents for the detection and quantification of AbB, and after the capture reagent binds to AbB, the binding site and epitope of the detection reagent are available for the detection reagent to bind to AbB.

[0296] The capture reagent can be an anti-AbB antibody or an antigen-binding fragment thereof. The detection reagent can be an anti-AbB antibody or an antigen-binding fragment thereof.

[0297] The capture agent can be a mouse anti-AbB antibody (anti-AbB-Ab1 or anti-AbB-Ab2). The detection reagent can be a mouse anti-AbB antibody (anti-AbB-Ab1 or anti-AbB-Ab2).

[0298] An interfering agent may be present in the assay, for example, AbA.

[0299] By sample acidification, at least 70% - 100% of the signal of the analyte can be recovered, that is, the recovery rate is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0300] The sample can be acidified to a pH of about 4.0 - 5.0, for example, a pH of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0301] Sample acidification can be achieved by adding an assay dilution buffer containing 30 mM acetic acid.

[0302] In some exemplary and non-limiting assays, AbB in a biological sample, such as human serum, is quantified by using a mouse anti-AbB antibody (anti-AbB-Ab2) as a capture agent and different biotinylated mouse anti-AbB antibodies (anti-AbB-Ab1) as detection agents. The amount of AbB is proportional to the signal intensity detected by the detection agent.

[0303] The present invention also provides a method for evaluating the effect of sample acidification on assay sensitivity in the quantification of AbB. This method includes incubating a sample containing AbB with a neutral or acidic assay dilution buffer (ADB) to acidify the sample, for example, lowering the pH of the sample to 5.0 - 4.0, detecting AbB using the methods and assays of the present invention as described in this disclosure, and comparing the detected value of AbB in the acidic sample with the value in a neutral sample (e.g., pH 7.4).

[0304] The present invention also provides a method for evaluating the signal recovery rate of AbB in the presence of an interfering agent, such as AbA. Samples containing only AbB or AbB and one or more interfering agents (e.g., AbA) are diluted with a neutral or acidic assay dilution buffer (ADB) to acidify the sample, for example, lowering the pH of the sample to 5.0 - 4.0. The AbB signal detected from the sample containing the interfering agent is compared with the signal detected from the sample without the interfering agent to determine the analyte recovery rate.

[0305] The recovery rate of 0.078 ng / mL of AbB in the presence of various amounts of AbA can be at least 55 - 100%, for example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0306] G. Methods and Assays for the Detection and Quantification of Factor XI a. Factor XI Factor XI or FXI is a 160 kDa disulfide - linked dimer of identical 607 - amino - acid subunits, each containing four 90 or 91 - amino - acid repeats and a C - terminal trypsin - like catalytic domain. FXI encodes coagulation factor XI, which triggers the mid - stage of the intrinsic pathway of the blood - coagulation cascade by activating factor IX protein. Factor XI deficiency, also known as hemophilia C, plasma thromboplastin antecedent deficiency, or Rosenthal syndrome, is a disorder that can cause abnormal bleeding, especially after trauma or surgery. FXI deficiency is an autosomal recessive disorder that commonly occurs in patients of Ashkenazi Jewish descent.

[0307] b. Method The assays described herein are intended to quantitatively determine total factor XI antigen in a sample, e.g., native and / or recombinant factor XI, by acidifying the sample to a pH sufficient to dissociate FXI from its complex. Capture reagents and detection reagents specific for FXI are used in methods and kits for detecting and determining the amount or concentration of FXI in a sample.

[0308] The methods and kits described herein also provide combinations of capture reagents and detection reagents for the detection and quantification of FXI, and after the capture reagent has bound to FXI, the binding sites and epitopes of the detection reagent are available for the detection reagent to bind to FXI.

[0309] The capture reagent can be an anti - FXI antibody or an antigen - binding fragment thereof. The detection reagent can be an anti - FXI antibody or an antigen - binding fragment thereof.

[0310] Anti - FXI antibodies and methods for generating anti - FXI antibodies are known in the art. Anti - FXI antibodies are also commercially available (e.g., Affinity Biologicals, catalog number FXI - AG).

[0311] The interfering agent may be present in the assay and is, for example, a human anti-EGFR monoclonal antibody (e.g., Int-FXI-Ab1).

[0312] By acidifying the sample, at least 70% - 100% of the signal of FXI can be restored, i.e., the recovery rate is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0313] The sample can be acidified to a pH of about 2.0 - 5.0, for example, a pH of about 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

[0314] Acidification of the sample can be achieved by adding an assay dilution buffer containing 30 - 90 mM acetic acid, for example, 90 mM, 60 mM, 40 mM, or 30 mM acetic acid.

[0315] In some exemplary and non-limiting assays, as described herein, FXI is detected and quantified in samples including serum, plasma, saliva, CSF, and urine. In the assay procedure, a microplate coated with a goat anti-human FXI polyclonal antibody is used, and recombinant monkey FXI is utilized as a standard. The FXI captured on the plate is detected with a goat anti-human FXI polyclonal antibody (HPA) conjugated to peroxidase. Next, a peroxidase-specific substrate is added to achieve a signal intensity proportional to the concentration of total FXI.

[0316] The present invention also provides a method for evaluating the effect of sample acidification on assay sensitivity in the quantification of FXI. The method includes incubating a sample containing FXI with a neutral or acidic assay dilution buffer (ADB) to acidify the sample, for example, lowering the pH of the sample to 5.0 - 2.0, detecting FXI using the methods and assays of the present invention as described in this disclosure, and comparing the detected value of FXI in the acidic sample with the value in a neutral sample (e.g., pH 7.4).

[0317] The present invention also provides a method for evaluating the signal recovery rate of FXI in the presence of an interfering agent, for example, an anti - FXI antibody, for example, Int - FXI - Ab1. A sample containing only FXI or FXI and one or more interfering agents (e.g., Int - FXI - Ab1) is diluted with a neutral or acidic assay dilution buffer (ADB) to acidify the sample, for example, lowering the pH of the sample to 5.0 - 2.0. The analyte recovery rate is determined by comparing the FXI signal detected from the sample containing the interfering agent with the signal detected from the sample without the interfering agent.

[0318] A schematic diagram of an exemplary assay approach for determining the concentration of factor XI (FXI) in a sample is shown in FIG. 7. The anti - FXI antibody binds to a solid plate to capture FXI. The captured FXI is detected using an HRP - conjugated anti - factor XI antibody. [Table 1] [Examples]

[0319] The present invention will be further illustrated by the following examples, which do not limit the present invention in any way. The order of implementation of the following examples can be changed or combined as determined by those skilled in the art considering the teachings and data included in this specification.

[0320] Example 1: Assay for Detecting IL2Rγ This study was conducted to improve the assay sensitivity for the detection and quantification of IL2Rγ by optimizing procedures and pH conditions that minimize potential drug interference in the samples. This study also examined whether neutralization of the acidified samples was necessary for the capture reagent to capture IL2Rγ in the samples. Briefly, anti-IL2Rγ-Ab1, a biotinylated human anti-IL2Rγ antibody, was bound to a solid support spotted with streptavidin to capture IL2Rγ, and anti-IL2Rγ-Ab2, a ruthenium-labeled human anti-IL2Rγ antibody, was used to detect the captured human IL2Rγ. The signal intensity from the ruthenium label (Figure 1A), which is proportional to the concentration of total IL2Rγ in the sample, was generated using the MSD read buffer. Interferents, such as human anti-IL2Rγ antibody, Int-IL2Rγ-Ab1, may be present in the sample.

[0321] Materials, Methods, and Results Microplates coated with streptavidin were blocked by adding 300 μL / well of 5% BSA blocking buffer and incubated at room temperature for 1 to 4 hours. Biotinylated anti-IL2Rγ antibody (Bio-anti-IL2Rγ-Ab1) was added to the microplates at a concentration of 2 μg / mL and incubated for approximately 1 hour. Samples were diluted at a ratio of 1:4 in neutral assay dilution buffer (ADB) or ADB containing 80 mM to 150 mM acetic acid, and both buffers contained 5% bovine serum albumin (BSA). Without neutralization, 50 μL of the diluted sample was incubated in the antibody-coated microplates for 1 hour. After removing the diluted sample from each well, 50 μL of ruthenium-labeled anti-IL2Rγ antibody (Ru-anti-IL2Rγ-Ab2) at a concentration of 200 ng / mL was added to the microplates and incubated for approximately 1 hour. At the end of the experiment, 150 μL / well of 2-fold MSD GOLD read buffer was added to the assay plates. Data were obtained with an MSD plate reader at OD450 - 540 nm within 10 minutes after the addition of the read buffer.

[0322] Data Analysis The signal counts of each sample generated by ruthenium labeling and read by an MSD reader were plotted against an approximate calibration curve using a 4PLV equation based on the nominal concentration of each calibrator. The concentration of IL2Rγ was extrapolated based on the count values corresponding to known concentrations of the IL2Rγ standard.

[0323] Results Diluting the sample with ADB containing 80 - 150 mM acetic acid decreased the overall signal, which resulted in the pH of the sample decreasing from 7.4 to the range of 4.4 - 4.0 (see Figure 1B). The decrease in signal increased the lower limit of quantification (LLOQ), which was approximately 5 - 10 times greater than the baseline sample at neutral pH. In other words, when the sample was acidified, the sensitivity of the assay decreased.

[0324] In the presence of the interferent Int-IL2Rγ-Ab1 (Int-IL2Rγ-Ab1), the signal from IL2Rγ decreased to less than 60% in the neutral assay dilution buffer. Decreasing the pH of the sample by adding ADB that lowers the pH increased the signal recovery, and a pH of approximately 4.2 provided acceptable tolerance to the interferent (see Figure 1C).

[0325] Surprisingly, acidification resulted in the dissociation of Int-IL2Rγ-Ab1 from IL2Rγ, reducing the interference from Int-IL2Rγ-Ab1, but not affecting the binding efficiency between Bio-anti-IL2Rγ-Ab1 and IL2Rγ at a low pH of 4.0, which was demonstrated by a signal recovery of up to 90 - 100%. This suggests that neutralization of the acidified sample before incubating the sample with the capture reagent is not necessary, thus simplifying the assay.

[0326] Example 2: Assay for Detecting EGFR In an exemplary assay for determining the concentration of total soluble EGFR in a sample, an EGFR capture antibody was pre-coated on a solid support to capture soluble EGFR in the sample. A HRP-conjugated EGFR detection antibody was used to detect the captured EGFR (Figure 2A).

[0327] Materials, Methods and Results Preparation of 50-fold EGFR reference standards: Prior to the experiment, 50-fold EGFR standards used for the standard curve were prepared in a calibrator diluent by serially diluting a 1000 ng / mL EGFR solution 1:2 to obtain standards containing 1000, 500, 250, 125, 62.5, 31.3, 15.6, and 0 ng / mL of EGFR.

[0328] Preparation of 50-fold EGFR quality control samples: Samples with known concentrations of EGFR were used as quality control samples to verify the performance of the assay. 50-fold high quality control (HQC), medium quality control (MQC), and low quality control (LQC) samples were prepared in a calibrator diluent at concentrations of 750 ng / mL, 120 ng / mL, and 45 ng / mL, respectively.

[0329] Preparation of standards, quality control samples and samples in assay buffer: Each of the 50 standards, 50-fold QCs and samples was diluted 1:50 in a calibrator diluent with 20 mM acetic acid.

[0330] Preparation of assay plate: Microplates coated with anti-EGFR polyclonal antibody and detection antibody are part of the Human EGFR Quantikine ELISA Kit (R&D Systems Quantikine Kit catalog number DEGFR0). After adding 100 μL / well of acidic assay diluent to the anti-EGFR microplate coated with anti-EGFR antibody (R&D Systems, catalog / part number 893730), another 50 μL / well of calibrator, QC, and research acidified samples (without neutralization) were added to double-load the microplate. The plate was covered and incubated at room temperature for 120 ± 10 minutes with shaking at 200 rpm. After washing 5 times with 300 μL / well of 1-fold wash buffer, the detection antibody (horseradish peroxidase-conjugated anti-EGFR polyclonal antibody, R&D Systems, catalog / part number 893731) was added to the microplate at 200 μL / well. The plate was covered and incubated for an additional 120 ± 10 minutes at room temperature in the dark with shaking at 200 rpm. At the end of the experiment, 50 μL / well of stop solution was added to the microplate, and the absorbance was measured at OD450 - 540 nm using a microplate reader within 30 minutes after the addition of the stop solution.

[0331] Data analysis The average optical density (OD) of each sample was plotted against an approximate calibration curve using a 4PLV equation based on the nominal concentration of each calibrator. The concentration of EGFR was extrapolated based on the OD values corresponding to known concentrations of EGFR standards.

[0332] Results The effect of sample acidification on Int-EGFR-Ab1 drug resistance was evaluated using the methods disclosed herein. More specifically, the interfering agent Int-EGFR-Ab1 at 0 - 2 mg / mL was spiked into EGFR-containing samples to test the effect of pH on Int-EGFR-Ab1:EGFR dissociation. Samples were diluted 1:50 in RD5L assay buffer with or without 30 mM acetic acid. 100 μL of the diluent of the RD1-72 kit assay buffer was added to each well of a coated microplate, and 50 μL of the diluted sample was mixed with the RD1-72 diluent in each well. The diluted samples were incubated in the wells for approximately 2 hours, followed by incubation with the HRP-conjugated anti-EGFR antibody for approximately 2 hours. After washing 5 times with wash buffer, 200 μL of TMB substrate was added to each well, incubated in the dark for approximately 25 minutes, and 50 μL of stop solution (2N sulfuric acid) was added to stop the color development. Absorbance was measured at OD 450~540 (see Figure 2A for a schematic of the assay).

[0333] The results in Figure 2B show that detection of EGFR was inhibited at neutral ADB (pH = 7.4), but dissociation of the EGFR complex and detection of EGFR were significantly increased by sample acidification. Furthermore, the assay dilution buffer with 30 mM acetic acid (pH = 4.5) greatly improved the EGFR assay resistance to Int-EGFR-Ab1. Specifically, the analyte recovery rate was approximately 100% in the presence of up to 2 mg / mL of Int-EGFR-Ab1.

[0334] Similar improvements in analyte detection were observed in a similar assay using a biotinylated EGFR antibody as the capture reagent and a ruthenium-labeled EGFR antibody as the detection reagent (Figure 3A). These antibodies are from the MSD R-PLEX human EGFR antibody set (catalog number F21N5), and both capture and detection are goat polyclonal antibodies. After sample acidification, the analyte recovery rate was approximately 100% at an Int-EGFR-Ab1 concentration of up to 1 mg / mL, and the analyte recovery rate was approximately 75% in the presence of 2 mg / ML of Int-EGFR-Ab1 (Figure 3B).

[0335] In mildly acidic samples, the detection of EGFR was slightly lower than in neutral ADB buffer (Figure 3C), suggesting that the binding of EGFR to the anti-EGFR antibody in the assay of Figure 3A is somewhat pH-sensitive, and acidic ADB slightly decreased the detection of EGFR compared to samples treated with neutral ADB.

[0336] In Figure 3D, the effect of sample acidification to various pHs on the recovery rate of EGFR was evaluated using the R-PLEX acid titer assay in EGFR-containing samples spiked with 0 μg / mL, 31.3 μg / mL, 250 μg / mL, and 2000 μg / mL of pure Int-EGFR-Ab1. The data show that decreasing the pH of the sample increased the resistance of EGFR to the interfering agent Int-EGFR-Ab1. Notably, while the recovery rate of EGFR was adversely affected by the sample dilution buffer containing 5 - 15 mM acetic acid from high concentrations of Int-EGFR-Ab1 (2000 μg / mL), ADB with 20 - 30 mM acetic acid (sample pH of 5 - 4.5) stabilized the resistance of EGFR to up to 2 mg / mL of Int-EGFR-Ab1, with a recovery rate exceeding 90%. The overall data also showed that the assay was able to significantly and effectively recover signals from EGFR in acidified samples even when the acidified samples were not neutralized prior to the binding step with the capture antibody.

[0337] The assay was tested using samples containing various amounts of serum to determine its lower limit of detection or lower limit of quantification. At a pH of approximately 5.0, in the presence of 2% human serum, an EGFR as low as 0.31 ng / mL was detectable. In the presence of undiluted human serum, an EGFR as low as 15.6 ng / mL was detectable.

[0338] Example 3: Immunoassay for Detecting AbA In an exemplary assay for determining the concentration of AbA, a human IgG4 monoclonal antibody against NPR1, in a sample, the biotinylated mouse anti-AbA monoclonal antibody (anti-AbA-Ab1) was immobilized as a capture agent on a plate coated with avidin, and the captured AbA was detected using a ruthenium-conjugated mouse anti-human IgG4 Fc antibody (anti-hIgG4Fc). Then, when a voltage is applied to the plate by an MSD reader, an electrochemiluminescence signal is generated by the ruthenium label (Figure 4A). In another exemplary assay, a ruthenylated anti-AbA-Ab2, a mouse anti-AbA monoclonal antibody, was used as a detector. (Figure 5A).

[0339] Materials and Methods Preparation of 50-fold AbA standards: Prior to the experiment, 50-fold AbA standards used for the standard curve were prepared in human serum by serially diluting a 1 mg / mL AbA placebo at a 1:3 dilution to obtain standards containing 10000, 3333, 1111, 370, 123, 41.2, 13.7, and 0 ng / mL of AbA. Frozen standards may be used for up to 90 days from the date of storage.

[0340] Preparation of 50-fold AbA quality control samples: Samples with known concentrations of AbA were used as quality control samples to verify the performance of the assay. 50-fold high quality control (HQC), medium quality control (MQC), and low quality control (LQC) samples were prepared in human serum at concentrations of 7500 ng / mL, 400 ng / mL, and 40 ng / mL, respectively.

[0341] Preparation of Standards, Quality Controls, and Samples in Assay Buffer: 30 mM acidic assay dilution buffer (ADB), prepared by diluting 300 mM acetic acid with ADB, was then used to prepare assay buffer containing 10 μg / mL mouse IgG. For the assay procedure, 50 each of standards, 50-fold QCs, and samples were diluted 1:50 in assay buffer.

[0342] Preparation of Assay Plates: Microplates coated with streptavidin were blocked by adding 300 μL / well of 5% BSA blocking buffer and incubated at room temperature for 1 - 2 hours if used on the same day. The blocked microplates were washed three times with 300 μL / well of 1X wash buffer using the MSD_PLATE_3X wash program. The capture antibody (biotin anti-AbA-Ab1) was prepared at 1 μg / mL in ADB and then added to the microplates at 50 μL / well. Next, the microplates were coated and incubated at room temperature for 1 - 2 hours with shaking at 400 rpm during the incubation. Next, the microplates were washed three times with 300 μL / well of 1X wash buffer using the MSD_PLATE_3X wash program.

[0343] Assay Procedure: The prepared standards, QC, and samples (without neutralization) were added in duplicate to the microplate at 50 μL / well. The microplate was coated and incubated at room temperature for 60 ± 10 minutes while shaking at 400 rpm during the incubation. Next, the microplate was washed three times with 1× wash buffer at 300 μL / well using the MSD_PLATE_3X wash program. The detection antibody (Ru-anti-AbA-Ab2) was prepared in ADB at 400 ng / mL and added to the plate at 50 μL / well, and incubated for 60 ± 10 minutes while shaking at 400 rpm. After washing three times with 1× wash buffer at 300 μL / well using the MSD_PLATE_3X wash program, 150 μL / well of MSD GOLD read buffer (MSD, catalog number R92TG) was added to the assay plate. The data was acquired with an MSD plate reader within 10 minutes after the addition of the read buffer.

[0344] Data Analysis The average count (signal) for each sample was plotted against an approximate calibration curve using a 4PLV equation based on the nominal concentration of each calibrator. The concentration of AbA was extrapolated based on the count values corresponding to the AbA standards of known concentration.

[0345] Results In the assays shown in FIGS. 5A-5B, biotinylated anti-AbA-Ab1 was used as the capture agent and ruthenium-labeled anti-AbA-Ab2 was used as the detection agent. Both anti-AbA-Ab1 and anti-AbA-Ab2 bind to the CDR region of AbA (FIG. 5A). The resistance of AbA to various levels of interfering agent AbB was evaluated in both neutral (pH = 7.4) and acidic (pH = 4.5) ADBs. That is, the high-quality control sample (HQC) contained 7.5 μg / mL of AbA, and the lower limit of quantification sample (LLOQ) included 13.7 ng / mL. Each sample was diluted 1:50 in either a neutral buffer (pH 7.4) containing 10 μg / mL of mouse IgG or a weakly acidic buffer containing 30 mM acetic acid and 10 μg / mL of mouse IgG. The AbA recovery rate was measured in the presence of up to 1 mg / mL of interfering agent AbB.

[0346] As a result, both the HQC sample and the LLOQ sample had similar AbA recovery rates of approximately 95% in the presence of interfering agent AbB (FIG. 5B). Furthermore, moderate acidification of the sample effectively dissociated the AbB:AbA complex, resulting in acceptable resistance to at least 1 mg / mL of AbB at pH 4.5.

[0347] In a similar assay, ruthenylated anti-hIgG4Fc was used as the detection agent (FIG. 4A). To evaluate the effect of sample acidification on AbA recovery rate and assay sensitivity, a blocker selected from among interfering agents Int-AbA-Ab1, Int-AbA-Ab2, AbB, and Int-AbA-Ab3 (an AbA antagonist that is a human anti-AbA monoclonal antibody) was added to the AbA-containing sample to reach a blocker:AbA ratio of up to approximately 3300:1. The AbA recovery rate was evaluated by 1:50 sample dilution in either neutral ADB (pH = 7.4) containing 10 μg / mL of mouse IgG (FIG. 4B) or acidic ADB (pH = approximately 4.5) containing 30 mM acetic acid and having 10 μg / mL of mouse IgG (FIG. 4C). The sandwich immunoassay was performed by the same procedure as above.

[0348] The results in Figure 4B suggest that after dilution in a neutral buffer, the presence of the interferents Int-AbA-Ab1, Int-AbA-Ab2, AbB, and Int-AbA-Ab3 inhibited the detection of AbA. This was reflected by the decrease in the recovery rate of AbA when the interferent:AbA ratio (blocker:AbA ratio) was greater than 10:1. More specifically, when the ratios of Int-AbA-Ab1:AbA and Int-AbA-Ab2:AbA were greater than 10:1 and the ratios of AbB:AbA and Int-AbA-Ab3:AbA were greater than 100:1, the recovery rate of AbA decreased to less than 75%. When the ratios of Int-AbA-Ab1:AbA and Int-AbA-Ab2:AbA were 100:1 or more, the recovery rate of AbA decreased to less than 10%.

[0349] Figure 4C demonstrated that sample acidification effectively prevented interference by Int-AbA-Ab1 and AbB, rather than interference by Int-AbA-Ab2 and Int-AbA-Ab3, in the detection of AbA when the blocker:AbA ratio was greater than 10:1. In an assay dilution buffer containing 30 mM acetic acid, the AbA signal was fully recovered when the Int-AbA-Ab1:AbA and AbB:AbA ratios were up to approximately 3300:1. However, the recovery rate of AbA remained low at 6% for Int-AbA-Ab2 and 14% for Int-AbA-Ab3 when the blocker:AbA ratio was approximately 3300:1.

[0350] These data suggest that sample acidification selectively increased the detection of AbA in the presence of interferents.

[0351] The data presented in Figures 4B, 4C, and 5B demonstrated that sample acidification in an immunoassay served to prevent drug interference caused by Int-AbA-Ab1 and AbB without affecting the sensitivity of AbA quantification. The overall data also showed that the assay very effectively recovered the signal from AbA in the acidified sample even when the acidified sample was not neutralized prior to the binding step with the capture antibody.

[0352] The assay was tested using samples containing various amounts of serum to determine its lower limit of detection or lower limit of quantification. At a pH of about 4.5, in the presence of 2% human serum, as low as 0.27 ng / mL of AbA was detectable. In the presence of undiluted human serum, as low as 13.7 ng / mL of AbA was detectable.

[0353] Example 4: Assay for detecting an antagonist of a human monoclonal IgG4 antibody (AbB) against NPR1 This experiment relates to an assay similar to the above assay for determination of the concentration of total AbB in a sample. In one exemplary assay, an anti-AbB antibody was immobilized on a solid plate as a capture agent, and different biotinylated mouse anti-AbB antibodies (biotin-anti-AbB-Ab1) were used to detect the captured AbB. NeutrAvidin-HRP (ThermoFisher, catalog number 31001) and a pico chemiluminescent substrate were used to read out the amount of detection reagent bound to the captured AbB, as well as the amount and concentration of AbB (Figure 6A). The sample may contain the interfering reagent AbA.

[0354] Results To evaluate sample acidification regarding the resistance of AbB to the interfering agent AbA, the recovery of 0.078 μg / mL (LLOQ) of AbB was measured at pH = 7.4 and pH = 4.5 in the presence of 31.25 μg / mL to 2 mg / mL of AbA (Figure 6B). Compared to the neutral assay binding buffer (ADB), acidic ADB dramatically increased the recovery rate to 97 - 105%, which demonstrated the effectiveness of sample acidification in improving the sensitivity of the AbB quantification assay and its resistance to AbA (see Figure 6B). The data also showed that the assay effectively recovered a significant signal from AbB in the acidified sample even when the acidified sample was not neutralized prior to the binding step with the capture antibody.

Claims

**Claim 1** A method for detecting an analyte in a sample, comprising: (i) diluting the sample containing the analyte with a weak acid to produce an acidified sample; (ii) directly adding the acidified sample to a solid support without neutralizing the acidified sample, wherein the solid support is coated with a capture reagent capable of binding to the analyte; (iii) removing the acidified sample from the solid support after a first incubation period; (iv) directly adding a detection reagent to the solid support; (v) removing the detection reagent from the solid support after a second incubation period; and (vi) detecting the detection reagent bound to the analyte captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of the analyte in the sample. **Claim 2** The method according to claim 1, further comprising washing the solid support after step (iii). **Claim 3** The method according to claim 1 or 2, further comprising washing the solid support after step (v). **Claim 4** The method according to any one of the preceding claims, wherein the acidified sample has a pH of about 3.0 to 6.

5. **Claim 5** The method according to claim 4, wherein the acidified sample has a pH of about 4.1, about 4.4, or about 4.

5. **Claim 6** The method according to any one of the preceding claims, wherein the analyte is IL2Rγ, EGFR, an antibody specific for natriuretic peptide receptor 1, a human monovalent monoclonal antibody against the anti-NPR1 antibody, or factor XI. **Claim 7** The method according to any one of the preceding claims, wherein the sample is a body fluid selected from the group consisting of blood, serum, plasma, cerebrospinal fluid (CSF), urine, and saliva. **Claim 8** The method according to any one of the preceding claims, wherein the sample is derived from a subject having a disease or disorder. **Claim 9** The method according to any one of claims 1 to 7, wherein the sample is derived from a subject suspected of having a disease or disorder. **Claim 10** The method according to any one of the preceding claims, wherein the sample is derived from a subject to whom a substance and / or a drug product has been administered. **Claim 11** The method according to any one of the preceding claims, wherein the sample is diluted at least three-fold. **Claim 12** The method according to any one of the preceding claims, wherein the weak acid is an acid that does not completely dissociate into its ions in an aqueous solution and is selected from the group consisting of acetic acid, citric acid, formic acid, lactic acid, phosphoric acid, and PIPES.

13. The method according to any one of the preceding claims, wherein the sample is acidified for 5 to 120 minutes.

14. The method according to any one of the preceding claims, wherein the capture reagent is an antibody.

15. The method according to claim 14, wherein the capture reagent antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a bispecific antibody, a Fab fragment, an F(ab')2 fragment, a monospecific F(ab')2 fragment, a bispecific F(ab')2, a trispecific F(ab')2, a monovalent antibody, a scFv fragment, a diabody, a bispecific diabody, a trispecific diabody, a scFv-Fc, a minibody, an IgNAR, a v-NAR, an hcIgG, and a vhH.

16. The capture reagent binds to the analyte with a dissociation constant (K Ｄ ) value of ≤ 1 μM, ≤ 100 nM, ≤ 50 nM, ≤ 25 nM, ≤ 20 nM, ≤ 15 nM, ≤ 10 nM, ≤ 5 nM, ≤ 2 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM, the method according to claim 14 or 15.

17. The method according to any one of the preceding claims, further comprising the step of identifying a capture reagent capable of binding to the analyte at the pH of the acidified sample.

18. The method according to any one of the preceding claims, wherein the capture reagent is biotinylated and the solid support is coated with streptavidin or avidin.

19. The method according to any one of the preceding claims, wherein the capture reagent binds directly to the solid support.

20. The method according to any one of the preceding claims, wherein the detection reagent is an antibody.

21. The method according to claim 20, wherein the detection reagent antibody is a polyclonal antibody, a monoclonal antibody, a bispecific antibody, a Fab fragment, an F(ab')2 fragment, a monospecific F(ab')2 fragment, a bispecific F(ab')2, a trispecific F(ab')2, a monovalent antibody, a scFv fragment, a diabody, a bispecific diabody, a trispecific diabody, a scFv-Fc, a minibody, an IgNAR, a v-NAR, an hcIgG, or a vhH.

22. The method according to any one of the preceding claims, wherein the detection reagent binds to a detectable label.

23. The method according to claim 22, wherein the detectable label is selected from the group consisting of rare transition metal particles, fluorophores, chromophores, enzymes, quantum dots, and noble metal nanoparticles.

24. The method according to claim 22, wherein the detectable label is horseradish peroxidase.

25. The method according to claim 22, wherein the detectable label is ruthenium.

26. The method according to any one of the preceding claims, wherein the solid support is an electrochemiluminescence platform.

27. The method according to any one of the preceding claims, wherein the sample comprises an interferent that binds to the analyte.

28. The method according to claim 27, further comprising determining the working pH of the acidified sample in which the interferent dissociates partially or completely from the analyte.

29. The method according to claim 27 or 28, wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of the signal of the analyte is recovered in the sample containing the interferent as compared to a sample without the interferent.

30. The method according to claim 29, wherein at least 70% to 100% of the signal of the analyte is recovered in the sample containing the interferent as compared to a sample without the interferent.

31. The method according to any one of the preceding claims, wherein the amount of the analyte is determined by correlating the amount of the detected detection reagent with a predetermined reference standard.

32. A kit for use in the method according to any one of the preceding claims, the kit comprising the capture reagent, the detection reagent, and a dilution buffer containing the weak acid.

33. A method for detecting an analyte in a sample, comprising: (i) diluting a sample containing the analyte with a weak acid to produce an acidified sample; (ii) adding a capture reagent to the acidified sample without neutralizing the acidified sample, the capture reagent specifically binding to the analyte; (iii) adding a mixture containing the acidified sample and the capture reagent to a solid support, the solid support specifically binding to the capture reagent; (iv) removing the mixture of the acidified sample and the capture reagent from the solid support after a first incubation period; (vi) adding a detection reagent directly to the solid support; (vii) removing the detection reagent from the solid support after a second incubation period. (viii)Detecting the detection reagent bound to the analyte captured by the capture reagent, wherein the amount of the detected detection reagent correlates with the amount of the analyte in the sample, and a step, a method.

34. The method according to claim 33, further comprising the step of washing the solid support after step (iv).

35. The method according to any one of claims 33 to 34, further comprising the step of washing the solid support after step (vi).

36. The method according to any one of claims 33 to 34, wherein the acidified sample has a pH of about 3.0 to 6.

5.

37. The method according to claim 36, wherein the acidified sample has a pH of about 4.1, about 4.4, or about 4.

5.

38. The analyte is IL2Rγ, EGFR, an antibody specific for natriuretic peptide receptor 1 (AbA), a human monovalent monoclonal antibody against the anti-NPR1 antibody (AbB), or factor XI, according to any one of claims 33 to 37. The method described in the item.

39. The method according to any one of claims 33 to 38, wherein the sample is a body fluid selected from the group consisting of blood, serum, plasma, cerebrospinal fluid (CSF), urine, and saliva.

40. The method according to any one of claims 33 to 39, wherein the sample is derived from a subject having a disease or disorder.

41. The method according to any one of claims 33 to 39, wherein the sample is derived from a subject suspected of having a disease or disorder.

42. The method according to any one of claims 33 to 41, wherein the sample is derived from a subject to whom a substance and / or a drug product has been administered.

43. The method according to any one of claims 33 to 42, wherein the sample is diluted at least three-fold.

44. The weak acid is an acid that does not completely dissociate into its ions in an aqueous solution and is selected from the group consisting of acetic acid, citric acid, formic acid, lactic acid, phosphoric acid, and PIPES. The method described in the item.

45. The method according to any one of claims 33 to 44, wherein the sample is acidified for 5 to 120 minutes.

46. The method according to any one of claims 33 to 45, wherein the capture reagent is an antibody.

47. The capture reagent antibody according to the method of claim 46, which is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a bispecific antibody, a Fab fragment, an F(ab')2 fragment, a monospecific F(ab')2 fragment, a bispecific F(ab')2, a trispecific F(ab')2, a monovalent antibody, a scFv fragment, a diabody, a bispecific diabody, a trispecific diabody, an scFv-Fc, a minibody, an IgNAR, a v-NAR, an hcIgG, and a vhH.

48. The capture reagent binds to the analyte with a dissociation constant (K Ｄ ) value of ≤ 1 μM, ≤ 100 nM, ≤ 50 nM, ≤ 25 nM, ≤ 20 nM, ≤ 15 nM, ≤ 10 nM, ≤ 5 nM, ≤ 2 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM, the method according to claim 46 or 47.

49. The method according to any one of claims 33 to 48, further comprising the step of identifying a capture reagent capable of binding to the analyte at the pH of the acidified sample.

50. The method according to any one of claims 33 to 49, wherein the capture reagent is biotinylated and the solid support is coated with streptavidin or avidin.

51. The method according to any one of claims 33 to 50, wherein the capture reagent binds directly to the solid support.

52. The method according to any one of claims 33 to 51, wherein the detection reagent is an antibody.

53. The detection reagent antibody according to the method of claim 52, which is a polyclonal antibody, a monoclonal antibody, a bispecific antibody, a Fab fragment, an F(ab')2 fragment, a monospecific F(ab')2 fragment, a bispecific F(ab')2, a trispecific F(ab')2, a monovalent antibody, a scFv fragment, a diabody, a bispecific diabody, a trispecific diabody, an scFv-Fc, a minibody, an IgNAR, a v-NAR, an hcIgG, or a vhH.

54. The method according to any one of claims 33 to 53, wherein the detection reagent binds to a detectable label.

55. The method according to claim 54, wherein the detectable label is selected from the group consisting of rare transition metal particles, fluorophores, chromophores, enzymes, quantum dots, and noble metal nanoparticles.

56. The method according to claim 54, wherein the detectable label is horseradish peroxidase.

57. The method according to claim 54, wherein the detectable label is ruthenium.

58. The method according to any one of claims 33 to 57, wherein the solid support is an electrochemiluminescence platform.

59. The method according to any one of claims 33 to 58, wherein the sample contains an interferent that binds to the analyte. **Claim 60** The method according to claim 59, further comprising the step of determining the working pH of the acidified sample in which the interferent dissociates partially or completely from the analyte. **Claim 61** The method according to claims 59 to 60, wherein, when compared with a sample not containing the interferent, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the signal of the analyte is recovered in the sample containing the interferent. **Claim 62** The method according to claim 61, wherein, when compared with a sample not containing the interferent, at least 70% to 100% of the signal of the analyte is recovered in the sample containing the interferent. **Claim 63** The method according to any one of claims 33 to 62, wherein the amount of the analyte is determined by correlating the amount of the detected detection reagent with a predetermined reference standard. **Claim 64** A kit for use in the method according to any one of claims 33 to 63, comprising the capture reagent, the detection reagent, and a dilution buffer containing the weak acid.

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