METHOD FOR DETECTING ANTI-DRUG ANTIBODIES (ADAs) AGAINST ANTI-

Abstract

The present technology includes a method for detecting anti-drug antibodies (ADAs) that bind to anti-TNF [alpha] antibodies (target antibodies) in a sample, the method comprising incubating the sample with a composition comprising an affinity antibody and a detection antibody, and isolating and detecting the ADAs that form complexes with the affinity antibody and the detection antibody. The techniques also include methods for determining the concentration of ADA in a sample and confirming that the sample contains ADA. The technology also includes a kit for performing any of the methods of the technology.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 460,821, filed April 20, 2023; U.S. Provisional Patent Application No. 63 / 461,131, filed April 21, 2023; and U.S. Provisional Patent Application No. 63 / 508,436, filed June 15, 2023. The contents of each of these provisional applications are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application contains a sequence list conforming to ST.26, which has been submitted by the Patent Center in XML format and is hereby incorporated by full reference. The XML copy created on April 16, 2024, is named 140505_8005_WO00_SL.xml and has a size of 20,939 bytes. Technical Field

[0005] This technology includes an in vitro assay method for recognizing antibodies, such as antidrug antibodies (ADAs) that bind to anti-TNFα antibodies. Summary of the Invention

[0006] In some embodiments, this technology generally relates to a method for detecting an antidrug antibody (ADA) in a sample that binds a target antibody including an anti-TNFα antibody, the method comprising: (i) incubating the sample together with a composition comprising: (a) a first affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label, and (b) a detection antibody comprising an anti-TNFα antibody conjugated to a detection label; (ii) isolating the ADA that forms a complex with both the first affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label that binds to the first affinity label; and (iii) detecting the ADA by measuring the level of the detection label above a predetermined cutoff point.

[0007] In some embodiments, separating the ADA includes: (a) providing a solid phase containing the second affinity marker; (b) contacting the sample and the composition with the solid phase; and (c) washing the solid phase.

[0008] In some embodiments, the target antibody, the affinity antibody, and / or the detection antibody comprises a chimeric human-mouse monoclonal antibody IgG1-κ. In some embodiments, the target antibody, the affinity antibody, and / or the detection antibody comprises SEQ ID NO: 1 and SEQ ID NO: 4.

[0009] In some embodiments, the solid phase is a plate.

[0010] In some embodiments, the first affinity label is biotin. In some embodiments, the second affinity label is streptavidin.

[0011] In some embodiments, the detection antibody includes a sulfonyl-tagged anti-TNFα antibody.

[0012] In some embodiments, measuring the level of the detection marker includes: (1) contacting the solid phase with a marker configured to generate a chemiluminescence or electrochemiluminescence signal when the marker is near the detection marker; and (2) detecting the chemiluminescence or electrochemiluminescence signal.

[0013] In some embodiments, the marker is tripropylamine (TPA), and measuring the level of the detection marker further includes applying an electric current to the marker to generate a chemiluminescent or electrochemiluminescent signal.

[0014] In some embodiments, the solid phase is blocked with a blocking buffer before the sample and the composition are brought into contact with the solid phase.

[0015] In some embodiments, the method further includes performing steps (i) to (iii) on a control sample. In some embodiments, the control sample is incubated together with the composition and contacted with the second affinity marker.

[0016] In some embodiments, the predetermined cutoff point corresponds to a level with a 5% false positive rate. In some embodiments, the 5% false positive rate is determined by measuring the detection marker in multiple control samples and calculating the 5% false positive rate.

[0017] In some embodiments, the chemiluminescence or electrochemiluminescence signal is measured as electrochemiluminescence (ECL).

[0018] In some embodiments, the predetermined cutoff point is an ECL value of at least 1. In some embodiments, the predetermined cutoff point is an ECL value of approximately 1.10.

[0019] In some embodiments, the method has a sensitivity of less than 6 ng / mL. In some embodiments, the sensitivity is less than 4 ng / mL. In some embodiments, the method exhibits resistance of approximately 100 ng / mL.

[0020] In some embodiments, this technology generally relates to a method for detecting ADA in a sample by binding a target antibody including an anti-TNFα antibody, the method comprising: (i) contacting the sample with one or more anti-TNFα conjugates to form a complex between the ADA and the anti-TNFα conjugate; and (ii) measuring the level of the anti-TNFα conjugate in the complex; wherein the method detects the ADA with a sensitivity of less than 5 ng / mL.

[0021] In some embodiments, the one or more anti-TNFα conjugates comprise an affinity antibody comprising an anti-TNFα antibody conjugated to an affinity label.

[0022] In some embodiments, the one or more anti-TNFα conjugates comprise a detection antibody comprising an anti-TNFα antibody conjugated to a detection marker.

[0023] In some embodiments, the affinity label comprises biotin. In some embodiments, the detection label comprises a sulfonate tag.

[0024] In some embodiments, the method detects the ADA with a sensitivity of less than 3 ng / mL. In some embodiments, the method detects the ADA with a sensitivity of 2.77 ng / mL or lower.

[0025] In some embodiments, this technology generally relates to a method for estimating the amount of ADA in a sample that binds to a target antibody including an anti-TNFα antibody, the method comprising: (i) incubating the sample together with a composition comprising: (a) an affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label, and (b) a detection antibody comprising an anti-TNFα antibody conjugated to a detection label; (ii) isolating ADA that forms a complex with both the affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label conjugated to the first affinity label; (iii) detecting the ADA by measuring the level of the detection label above a predetermined cutoff point; (iv) performing steps (i) to (iii) on a known sample having a known concentration of ADA; and (v) comparing the level of the detection label in the sample with the level of the detection label in the known sample.

[0026] In some embodiments, the known sample is continuously diluted into multiple titer samples, wherein comparing the level of the detection marker in the sample with the level of the detection marker in the known sample includes generating a curve of the level of the detection marker for each titer sample and comparing the detection marker in the sample with the curve.

[0027] In some embodiments, this technology generally relates to a method for confirming that ADA in a sample binds to a target antibody including an anti-TNFα antibody by performing a competitive binding assay, the method comprising: (i) incubating the sample together with a composition comprising: (a) a first affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label, and (b) a detection antibody comprising an anti-TNFα antibody conjugated to a detection label; (ii) isolating ADA that forms a complex with both the first affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label that binds to the first affinity label; and (iii) detecting the ADA by measuring the level of the detection label above a predetermined cutoff point, wherein the composition further comprises an unlabeled anti-TNFα antibody.

[0028] In some embodiments, the presence of the ADA in the sample is confirmed by measuring the level of the detection marker, the level of which is lower by a predetermined factor than the level of the detection marker in the absence of the unlabeled anti-TNFα antibody.

[0029] In some embodiments, this technology generally relates to a kit for detecting ADA in a sample by binding a target antibody including an anti-TNFα antibody, the kit comprising: (i) an affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label; (ii) a detection antibody comprising an anti-TNFα antibody conjugated to a detection label; and (iii) a solid phase comprising a second affinity label, wherein the second affinity label binds to the first affinity label.

[0030] In some embodiments, the solid phase is a plate. In some embodiments, the second affinity label is streptavidin. In some embodiments, the first affinity label is biotin. In some embodiments, the detection antibody comprises a sulfonyl-tagged anti-TNFα antibody.

[0031] In some embodiments, the kit further includes a biomarker configured to generate a chemiluminescent or electrochemiluminescent signal in the presence of the biomarker near the detection marker. In some embodiments, the biomarker is tripropylamine (TPA).

[0032] In some embodiments, the kit further includes a blocking buffer. In some embodiments, the kit further includes a washing buffer.

[0033] In some embodiments, this technology generally relates to a kit for estimating the amount of ADA bound to a target antibody including an anti-TNFα antibody in a sample, the kit comprising: (i) an affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label; (ii) a detection antibody comprising an anti-TNFα antibody conjugated to a detection label; (iii) a solid phase comprising a second affinity label, wherein the second affinity label binds to the first affinity label; and (iv) a positive control. In some embodiments, the positive control is a plurality of titer samples.

[0034] In some embodiments, the present technology generally relates to a kit for confirming ADA binding to a target antibody including an anti-TNFα antibody, the kit comprising: (i) an affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label; (ii) a detection antibody comprising an anti-TNFα antibody conjugated to a detection label; (iii) an unlabeled anti-TNFα antibody; and (iv) a solid phase comprising a second affinity label, wherein the second affinity label binds to the first affinity label. Background Technology

[0035] Anti-TNFα (tumor necrosis factor alpha) antibodies are novel biological agents that can be used to treat various diseases. One obstacle to the development and implementation of anti-TNFα antibodies as a treatment is that subjects receiving such antibodies may develop an immune response. The immunogenicity of anti-TNFα antibodies in subjects, particularly the production of anti-drug antibodies (ADAs) that bind to anti-TNFα antibodies, can reduce the efficacy of anti-TNFα antibody treatment for the subject's disease. To advance the development of treatments using anti-TNFα antibodies, reliable methods are needed to determine the presence and amount of ADAs in subjects receiving or having received anti-TNFα antibodies. Attached Figure Description

[0036] Figure 1 A flowchart of an example method according to this technology is shown.

[0037] Figure 2A and Figure 2B The method according to this technique is shown in the normality test of example screening and titration data. Figure 2A ) and normality test of example confirmatory data ( Figure 2B In the Shapiro-Wilk W test, the normality of a set of inhibition percentage values ​​is determined. Detailed Implementation

[0038] This technology includes a method for detecting anti-drug antibodies (ADAs) against a target antibody. The target antibody may include an anti-TNFα antibody. In some embodiments, the anti-TNFα antibody is a chimeric human-mouse monoclonal IgG1-κ antibody. In some embodiments, the target antibody comprises the heavy chain complementarity-determining region (CDR) of SEQ ID NO: 9 to SEQ ID NO: 11 and the light chain CDR of SEQ ID NO: 12 to SEQ ID NO: 14. In some embodiments, the target antibody comprises the heavy chain CDR of SEQ ID NO: 15 to SEQ ID NO: 17 and the light chain CDR of SEQ ID NO: 18 to SEQ ID NO: 20. In some embodiments, the target antibody comprises the heavy chain variable region of SEQ ID NO: 2 and the light chain variable region of SEQ ID NO: 5. In some embodiments, the target antibody comprises the heavy chain of SEQ ID NO: 1 and the light chain of SEQ ID NO: 4. This technology also includes a kit for detecting the target antibody in a sample according to the method of this technology.

[0039] While this technology can be embodied in many forms, the following description of several embodiments should be understood as illustrative of the technology and not intended to limit the technology to the specific embodiments shown. Headings are provided for convenience only and should not be construed as limiting the technology in any way. Embodiments shown under any heading may be combined with embodiments shown under any other heading.

[0040] Unless otherwise expressly stated, the use of numerical values ​​within the various quantitative ranges specified herein is as an approximation, as the minimum and maximum values ​​within the stated range are preceded by the word "about". It should be understood that, although not always explicitly stated, all numerical names are preceded by the term "about". It should be understood that this range format is used only for convenience and brevity and should be flexibly interpreted to include not only the numerical values ​​explicitly designated as the limits of the range, but also all individual numerical values ​​or subranges covering that range, as if each numerical value and subrange were explicitly specified. For example, ratios in the range of about 1 to about 200 should be understood to include the explicitly listed limits of about 1 and about 200, as well as individual ratios such as about 2, about 3, and about 4, and subranges such as about 10 to about 50, about 20 to about 100, etc. It should also be understood that, although not always explicitly stated, the reagents described herein are merely exemplary, and their equivalents are known in the art.

[0041] If any material incorporated herein by reference conflicts with this technology, this technology shall prevail.

[0042] definition

[0043] As used herein, the term “about” when referring to a measurable value such as amount or concentration means that it covers a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0044] As used herein, “affinity antibody” includes an anti-TNFα antibody. In some embodiments, an affinity antibody includes a target antibody. An affinity antibody may also contain an affinity marker having high affinity for a separate marker. For example, the affinity marker may be a first affinity marker (i.e., biotin) having high affinity for a second affinity marker (i.e., streptavidin). In some embodiments, an affinity antibody comprises an anti-TNFα antibody and an affinity marker. In some embodiments, an affinity antibody comprises a target antibody and an affinity marker.

[0045] As used herein, "detection antibody" includes a TNFα antibody. In some embodiments, a detection antibody comprises a target antibody. A detection antibody may also comprise a detection marker that can be detected directly or indirectly. In some embodiments, a detection antibody comprises an anti-TNFα antibody and a detection marker. In some embodiments, a detection antibody comprises a target antibody and a detection marker.

[0046] In some embodiments, a method for detecting ADA bound to the target antibody in a sample includes: (i) contacting the sample with one or more anti-TNFα conjugates to form a complex between the ADA and the anti-TNFα conjugate; and (ii) measuring the level of the anti-TNFα conjugate in the complex; wherein the method detects the ADA with a sensitivity of less than 5 ng / mL. In some embodiments, the one or more anti-TNFα conjugates comprise a detection antibody. In some embodiments, measuring the level of the anti-TNFα conjugate includes measuring the level of the detection marker. In some embodiments, the one or more anti-TNFα conjugates comprise an affinity antibody.

[0047] As used herein, “screening assay” generally refers to a method for detecting ADA bound to a target antibody. For example, a screening assay includes a method for detecting ADA bound to a target antibody, comprising: (i) incubating the sample with a composition comprising: (a) an anti-TNFα antibody (affinity antibody) conjugated to a first affinity label, and (b) an anti-TNFα antibody (detection antibody) conjugated to a detection label; (ii) isolating ADA that forms a complex with both the affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label bound to the first affinity label; and (iii) detecting the ADA by measuring the level of the detection label above a predetermined cutoff point. A screening assay may also include a method for detecting ADA, comprising: (i) contacting the sample with one or more anti-TNFα conjugates to form a complex between the ADA and the anti-TNFα conjugate; and (ii) measuring the level of the anti-TNFα conjugate in the complex; wherein the method detects the ADA with a sensitivity of less than 5 ng / mL.

[0048] In some embodiments, measuring the level of the detection marker includes contacting the solid phase with a marker configured to generate a chemiluminescence or electrochemiluminescence (ECL) signal when the marker is near the detection marker, and detecting the chemiluminescence or ECL signal. As used herein, the marker is considered "near" the detection marker when the distance between the marker and the detection marker is small enough to cause a reaction that generates a chemiluminescence or ECL signal. In some embodiments, the ECL is generated when the marker is near both the detection marker and the electrode.

[0049] The sample may be obtained from a mammal, including humans, rats, mice, rabbits, or other animals. In some embodiments, the sample is a blood, plasma, or serum sample. In some embodiments, the sample is plasma. In some embodiments, the sample is serum. In some embodiments, the sample is pretreated before detecting ADA according to the method of this technology. As used herein, "pretreatment" includes extracting tissue from the subject and identifying the sample (e.g., drawing a blood sample and identifying or separating a plasma or serum sample, or distinguishing a blood sample into a plasma sample and / or a serum sample).

[0050] Antidrug antibodies (ADA)

[0051] During or after administration of a drug (e.g., a therapeutic drug) to a subject, the subject's immune system may respond by producing anti-drug antibodies (ADAs). ADAs may bind to at least a portion of the drug, thereby isolating, neutralizing, and / or reducing the drug's efficacy.

[0052] ADA may be naturally produced as part of the subject's innate immune response. ADA may be a naturally occurring antibody produced by the subject, such as that produced by lymphocytes using variable (diversity) linkers (V(D)J). For example, ADA may be an immunoglobulin. In some embodiments, ADA is IgG, IgA, IgE, a monoclonal antibody, a polyclonal antibody, or any other type of antibody capable of binding to a target antibody.

[0053] Target antibody

[0054] During or after administration of the drug (i.e., the therapeutic drug), ADA (adjuvants to which at least a portion of the drug is bound) may be formed. In some embodiments, the drug contains an antibody, and when ADA is formed, the ADA may bind to the drug antibody. In some embodiments, the drug antibody includes an anti-TNFα antibody.

[0055] The presence of ADA and / or the binding of ADA to a drug antibody can be detected using antibodies that are identical to or mimic the drug antibody (i.e., "target antibodies"). In some embodiments, the target antibody comprises one or more anti-TNFα antibodies. An anti-TNFα antibody is an antibody that binds to at least a portion of the TNFα protein or peptide.

[0056] In some embodiments, the target antibody includes a monoclonal antibody.

[0057] In some embodiments, the target antibody of this technology is a chimeric antibody. Non-limiting examples of chimeric antibodies include human-mouse antibodies, human-rat antibodies, human-hamster antibodies, human-rabbit antibodies, human-bovine antibodies, and human-goat antibodies. In some embodiments, the chimeric antibody comprises a humanized antibody (e.g., a fully humanized antibody or a partially humanized antibody).

[0058] In some embodiments, the target antibody of this technology (e.g., an anti-TNFα antibody) may comprise an isotype that affects the functional properties and / or structural characteristics of the antibody. In some embodiments, the target antibody comprises an isotype selected from the group consisting of: IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. The light chain may comprise the isotype kappa (ĸ) or the isotype lambda (λ). In some embodiments, the target antibody comprises an IgG1-κ antibody.

[0059] Target antibody sequence

[0060] Heavy chain:

[0061] The target antibody of this technology comprises a heavy chain having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 1.

[0062] The target antibody of this technology comprises a heavy chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 1.

[0063] The target antibody of this technology comprises a heavy chain having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 1.

[0064] The heavy chain of the target antibody may contain a variable region domain (V) having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: H ): SEQ.ID NO: 2.

[0065] The heavy chain of the target antibody may contain a V with an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the following: H SEQ.ID NO: 2.

[0066] The heavy chain of the target antibody may contain a V with an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the following: H SEQ.ID NO: 2.

[0067] The heavy chain of the target antibody may contain a constant region having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 3.

[0068] The heavy chain of the target antibody may contain a constant region having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 3.

[0069] The heavy chain of the target antibody may contain a constant region having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the following: SEQ.ID NO: 3.

[0070] The target antibody of this technology comprises a heavy chain having at least one complementarity-determining region (CDR) sequence. This at least one CDR sequence confers binding to a specific antigen (e.g., TNFα peptide).

[0071] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 9 that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0072] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 9 that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0073] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 9 that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0074] In some embodiments, the target antibody comprises a heavy chain having a CDR of approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the following amino acid sequence: SEQ.ID NO: 10.

[0075] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 10 that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0076] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 10 that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0077] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 11 that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0078] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 11 that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0079] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 11 that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0080] In some embodiments, the target antibody comprises a heavy chain having a CDR of about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the following amino acid sequence: SEQ.ID NO: 15.

[0081] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 15 that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0082] In some embodiments, the target antibody comprises a heavy chain having a CDR of at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the following amino acid sequence: SEQ.ID NO: 15.

[0083] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 16 that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0084] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 16 that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0085] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 16 that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0086] In some embodiments, the target antibody comprises a heavy chain having a CDR of approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity with the following amino acid sequence: SEQ.ID NO: 17.

[0087] In some embodiments, the target antibody comprises a heavy chain having a CDR: SEQ.ID NO: 17 that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0088] In some embodiments, the target antibody comprises a heavy chain having a CDR of SEQ.ID NO: 17 that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following amino acid sequence.

[0089] In some embodiments, the target antibody comprises a heavy chain comprising (1) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 9, (2) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 10, and (3) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 11.

[0090] In some embodiments, the target antibody comprises a heavy chain comprising (1) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 9, (2) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 10, and (3) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 11.

[0091] In some embodiments, the target antibody comprises a heavy chain comprising (1) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 9, (2) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 10, and (3) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 11.

[0092] In some embodiments, the target antibody comprises a heavy chain comprising (1) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 15, (2) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 16, and (3) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 17.

[0093] In some embodiments, the target antibody comprises a heavy chain comprising (1) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 15, (2) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 16, and (3) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 17.

[0094] In some embodiments, the target antibody comprises a heavy chain comprising (1) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 15, (2) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 16, and (3) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 17.

[0095] In some embodiments, the heavy chain of the target antibody of this technology is encoded by a nucleotide sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 8.

[0096] In some embodiments, the heavy chain of the target antibody of this technology is encoded by a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 8.

[0097] In some embodiments, the heavy chain of the target antibody of this technology is encoded by a nucleotide sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 8.

[0098] Light chain:

[0099] The target antibody of this technology comprises a light chain having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 4.

[0100] The target antibody of this technology comprises a light chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 4.

[0101] The target antibody of this technology comprises a light chain having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 4.

[0102] The light chain of the target antibody may contain a variable region domain (V) having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: L ): SEQ.ID NO: 5.

[0103] The light chain of the target antibody may contain a V having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: L SEQ.ID NO: 5.

[0104] The light chain of the target antibody may contain a V having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following. L SEQ.ID NO: 5.

[0105] The light chain of the target antibody may contain a constant region having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 6.

[0106] The light chain of the target antibody may contain a constant region having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 6.

[0107] The light chain of the target antibody may contain a constant region having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 6.

[0108] The target antibody of this technology comprises a light chain having at least one CDR sequence. The at least one CDR sequence confers binding to a specific antigen (e.g., TNFα peptide).

[0109] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 12.

[0110] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 12.

[0111] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 12.

[0112] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 13.

[0113] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 13.

[0114] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 13.

[0115] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 14.

[0116] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 14.

[0117] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 14.

[0118] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 18.

[0119] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 18.

[0120] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 18.

[0121] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 19.

[0122] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 19.

[0123] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 19.

[0124] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 20.

[0125] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 20.

[0126] In some embodiments, the target antibody comprises a light chain having a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 20.

[0127] In some embodiments, the target antibody comprises a light chain comprising (1) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 12, (2) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 13, and (3) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 14.

[0128] In some embodiments, the target antibody comprises a light chain comprising (1) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 12, (2) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 13, and (3) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 14.

[0129] In some embodiments, the target antibody comprises a light chain comprising (1) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 12, (2) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 13, and (3) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 14.

[0130] In some embodiments, the target antibody comprises a light chain comprising (1) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 18, (2) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 19, and (3) a CDR having an amino acid sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 20.

[0131] In some embodiments, the target antibody comprises a light chain comprising (1) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 18, (2) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 19, and (3) a CDR having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 20.

[0132] In some embodiments, the target antibody comprises a light chain comprising (1) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 18, (2) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 19, and (3) a CDR having an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 20.

[0133] In some embodiments, the light chain of the target antibody of this technology is encoded by a nucleotide sequence that is approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 7.

[0134] In some embodiments, the light chain of the target antibody of this technology is encoded by a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 7.

[0135] In some embodiments, the light chain of the target antibody of this technology is encoded by a nucleotide sequence that is at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the following: SEQ.ID NO: 7.

[0136] Table 1: Sequences of exemplary anti-TNFα antibodies of this technology

[0137]

[0138] Antibody labeling

[0139] The target antibodies of this technology can be labeled, such as with affinity labeling or detection labeling. Non-limiting examples of antibody labeling include biotinylation, fluorescent labeling, enzyme labeling (e.g., horseradish peroxidase (HRP) and alkaline phosphatase (AP)), gold nanoparticles, radioisotopes, and drug molecules.

[0140] In some embodiments, the target antibody is conjugated to an additional molecule or compound to form an antibody-drug conjugate. Conjugation can be performed via covalent bonds.

[0141] In some embodiments, the target antibody of this technology is labeled directly (e.g., by conjugation) or indirectly with an affinity label or a detection label. The detection label can be any label that can be detected directly or indirectly. As used herein, “direct” detection of a detection label includes measuring the presence of the detection label or inducing the detection label to produce a detectable substance. For example, the detection label can be a luminescent compound. In some embodiments, detection is configured to generate a chemiluminescence or ECL signal in the presence of the marker. In some embodiments, the detection label is an enzyme configured to convert a chromogenic marker into a pigment. For example, the enzyme is HRP and the chromogenic marker can be 3,3',5,5'-tetramethylbenzidine (TMB). In some embodiments, the detection label comprises a sulfonate tag. The sulfonate tag (Ru-tag) can be contacted with a marker containing tripropylamine (TPA), and the level of the detection label can be measured by applying an electric current to the marker in the presence of the detection label to generate an ECL signal.

[0142] In some embodiments, the detection marker is determined by ultrasensitive electrochemiluminescence (MSD). In some embodiments, the detection marker is detected by ECL. In some embodiments, the detection marker is a ruthenium (Ru) metal ion. In some embodiments, the Ru ion can be detected when it is in close proximity to the electrode. As will be understood by those skilled in the art, “close proximity” between the Ru ion and the electrode, as used herein, means that the distance between the Ru ion and the electrode is small enough to cause a redox reaction detectable by ECL. In some embodiments, the solid phase includes the electrode. In some embodiments, when the Ru ion is in close proximity to the electrode, it can be detected by a charge-coupled device (CCD) camera.

[0143] In some embodiments, the detection marker is detected indirectly. “Indirect” detection of the detection antibody as used herein involves adding a separate component that interacts with the detection marker, wherein this separate component is detectable. For example, a secondary antibody may be added, wherein the secondary antibody is conjugated to a detectable luminescent probe or enzyme configured to convert a chromogenic substrate into a pigment (e.g., a detection antibody conjugated to the detection marker).

[0144] The affinity marker can be any molecular marker having affinity for the second affinity marker. In some embodiments, the first affinity marker is configured to form a covalent bond with the second affinity marker. For example, the first affinity marker can be biotin, and the second affinity marker can be streptavidin, or vice versa. The first affinity marker and the second affinity marker can be any pair of affinity markers known to those skilled in the art. In some embodiments, the first affinity marker can be glutathione, and the second affinity marker can be glutathione S-transferase, or vice versa. In some embodiments, the first affinity marker can be maltose, and the second affinity marker can be a maltose-binding protein, or vice versa. In some embodiments, the first affinity marker is chitin, and the second affinity marker is a chitin-binding protein, or vice versa.

[0145] In some embodiments, the detection tag may be a third affinity tag, and detecting the detection tag further includes contacting the third affinity tag with a fourth affinity tag coupled to a detectable compound, wherein the third affinity tag binds to the fourth affinity tag. For example, the fourth affinity tag may be coupled to a luminescent compound or enzyme configured to convert a chromogenic substrate into a pigment.

[0146] The third affinity marker can be any molecular marker that has an affinity for the fourth affinity marker. In some embodiments, the third affinity marker is configured to form a covalent bond with the fourth affinity marker. For example, the third affinity marker can be biotin, and the fourth affinity marker can be streptavidin, or vice versa. The third and fourth affinity markers can be any pair of affinity markers known to those skilled in the art. In some embodiments, the third affinity marker can be glutathione, and the fourth affinity marker can be glutathione S-transferase, or vice versa. In some embodiments, the third affinity marker can be maltose, and the fourth affinity marker can be a maltose-binding protein, or vice versa. In some embodiments, the third affinity marker is chitin, and the fourth affinity marker is a chitin-binding protein, or vice versa.

[0147] Composition

[0148] This technology includes compositions having antibodies for detecting ADA. In some embodiments, the composition comprises one or more target antibodies that are bound by ADA. The one or more target antibodies that are bound by ADA may include affinity antibodies and / or detection antibodies. Both affinity antibodies and detection antibodies may be anti-TNFα antibodies and / or may contain the heavy chain, light chain, or variable region (e.g., V) of this technology. H or V L One or more of the following: constant region (e.g., heavy chain constant region or light chain constant region), or CDR.

[0149] In some embodiments, the affinity antibody comprises an affinity label. The affinity antibody may be labeled directly or indirectly with an affinity label. Non-limiting examples of affinity labels include fluorescent labels, enzyme labels (e.g., horseradish peroxidase and alkaline phosphatase), biotin labels, streptavidin labels, and radioisotopes. In some embodiments, the detection antibody is conjugated to a detection label. Non-limiting examples of detection labels include biotin labels, fluorescent labels, enzyme labels (e.g., horseradish peroxidase and alkaline phosphatase), gold nanoparticles, and radioisotopes.

[0150] In some embodiments, the composition further comprises a second affinity antibody. The second affinity antibody may bind an affinity label, such as the affinity label conjugated to the first affinity antibody as described above. In some embodiments, the second affinity antibody binds to the affinity label when the first affinity antibody and / or detection antibody is bound by one or more ADAs. The second affinity antibody may be an unlabeled antibody. Non-limiting examples of unlabeled antibodies include antibodies not conjugated to an affinity label or detection label.

[0151] In some embodiments, the composition comprises a single composition having a first affinity antibody of the present technology, a detection antibody of the present technology, and a second affinity antibody of the present technology. In some embodiments, the composition comprises (1) a first composition having a first affinity antibody and a detection antibody, and (2) a second composition having a second affinity antibody. In some embodiments, the composition comprises (1) a first composition having a first affinity antibody, (2) a second composition having a detection antibody, and (3) a third composition having a second affinity antibody. In some embodiments, the composition comprises (1) a first composition having a first affinity antibody, and (2) a second composition having a detection antibody and a second affinity antibody. In some embodiments, the composition comprises (1) a first composition having a first affinity antibody and a second affinity antibody, and (2) a second composition having a detection antibody. In some embodiments, the first composition and the second composition are incubated simultaneously or sequentially with a sample. In some embodiments, the first composition and the third composition are incubated simultaneously or sequentially with a sample. In some embodiments, the second composition and the third composition are incubated simultaneously or sequentially with a sample. In some embodiments, the first composition, the second composition, and the third composition are each incubated simultaneously or sequentially with a sample.

[0152] Samples may include biological samples. Non-limiting examples of biological samples include serum, blood, synovial fluid, plasma, saliva, and respiratory secretions.

[0153] Methods for detecting ADA

[0154] This technology includes methods for detecting ADA in samples. Compositions based on this technology can be used to detect and / or separate ADA.

[0155] In some embodiments, detecting ADA involves incubating a sample with a composition of the present technology (e.g., a composition comprising one or more of an affinity antibody, a detection antibody, or an unlabeled antibody). In some embodiments, the affinity antibody is conjugated to a detection label.

[0156] In some embodiments, detecting ADA includes contacting a sample with a composition comprising a first affinity antibody of the present technology. In some embodiments, the affinity antibody is added at a concentration of about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 20 ng / mL, about 20 ng / mL to about 50 ng / mL, or about 50 ng / mL to about 100 ng / mL.

[0157] In some embodiments, affinity antibodies are added at concentrations of at least 1 ng / mL to at least 10 ng / mL, at least 10 ng / mL to at least 20 ng / mL, at least 20 ng / mL to at least 50 ng / mL, or at least 50 ng / mL to at least 100 ng / mL.

[0158] In some embodiments, affinity antibodies are added at concentrations of at least about 1 ng / mL to at least about 10 ng / mL, at least about 10 ng / mL to at least about 20 ng / mL, at least about 20 ng / mL to at least about 50 ng / mL, or at least about 50 ng / mL to at least about 100 ng / mL.

[0159] In some embodiments, affinity antibodies are added at a concentration of approximately 25 ng / mL.

[0160] In some embodiments, affinity antibodies are added at a concentration of at least 25 ng / mL.

[0161] In some embodiments, affinity antibodies are added at a concentration of at least about 25 ng / mL.

[0162] In some embodiments, detecting ADA includes incubating the sample together with a composition containing a detection antibody.

[0163] In some embodiments, the detection antibody is added at concentrations of about 0.001 mg / mL to about 0.01 mg / mL, about 0.01 mg / mL to about 0.1 mg / mL, about 0.1 mg / mL to about 1 mg / mL, and about 1 mg / mL to about 10 mg / mL. In some embodiments, an affinity antibody is added at a concentration of about 1.2 mg / mL. In some embodiments, the detection antibody comprises a ruthenium label (i.e., Ru-XTMAB-16) and is added at a concentration of about 1.2 mg / mL.

[0164] In some embodiments, the detection antibody is added at concentrations of at least 0.001 mg / mL to at least 0.01 mg / mL, at least 0.01 mg / mL to at least 0.1 mg / mL, at least 0.1 mg / mL to at least 1 mg / mL, and at least 1 mg / mL to at least 10 mg / mL. In some embodiments, an affinity antibody is added at a concentration of at least 1.2 mg / mL. In some embodiments, the detection antibody comprises a ruthenium label (i.e., Ru-XTMAB-16) and is added at a concentration of at least 1.2 mg / mL.

[0165] In some embodiments, the detection antibody is added at concentrations of at least about 0.001 mg / mL to at least about 0.01 mg / mL, at least about 0.01 mg / mL to at least about 0.1 mg / mL, at least about 0.1 mg / mL to at least about 1 mg / mL, and at least about 1 mg / mL to at least about 10 mg / mL. In some embodiments, an affinity antibody is added at a concentration of at least about 1.2 mg / mL. In some embodiments, the detection antibody comprises a ruthenium label (i.e., Ru-XTMAB-16) and is added at a concentration of at least about 1.2 mg / mL.

[0166] In some embodiments, the method further includes isolating ADA from the target antibody (e.g., an affinity antibody or a detection antibody) bound to the present technology. As used herein, “isolation” of ADA forming a complex with both the affinity antibody and the detection antibody includes recognizing and distinguishing the ADA that forms the complex from ADA that does not form a complex. Isolating ADA also includes recognizing and distinguishing ADA from other compounds that bind to the target antibody. ADA can be isolated using any technique known to those skilled in the art for isolating or purifying antibodies. For example, antibody pull-down or purification techniques can be used to isolate ADA. In some embodiments, ADA is isolated by contacting the ADA complex with a solid phase containing a second affinity label, such that a first affinity label of the ADA complex binds to the second affinity label on the solid phase.

[0167] As used herein, "solid phase" can be any medium that remains stationary or temporarily stationary while the mobile phase moves through or permeates it, allowing the components of the mobile phase to interact with the solid phase. The solid phase can include plates, chromatography columns, microbeads, magnetic microbeads, or any other solid phase known to those skilled in the art. In some embodiments, the solid phase is microbeads, a plate, or a column. In some embodiments, the solid phase is a plate. In some embodiments, the plate is coated with a second affinity label. In some embodiments, the solid phase is blocked with a blocking buffer before contacting the sample and the composition with it.

[0168] In some embodiments, separating the ADA complex includes: (a) providing a solid phase containing a second affinity label; (b) contacting the sample and the composition with the solid phase; and (c) washing the solid phase. In some embodiments, the solid phase is washed with a washing buffer that does not disrupt the interaction between ADA and the affinity antibody or the interaction between ADA and the detection antibody. In some embodiments, the washing buffer comprises phosphate-buffered saline (PBS). In some embodiments, the washing buffer comprises a phosphate-buffered saline solution containing a surfactant. In some embodiments, the washing buffer comprises Tween-20. In some embodiments, the washing buffer comprises 1x PBS containing 0.05% Tween-20. In some embodiments, the washing buffer comprises 50 mM Tris. In some embodiments, the washing buffer comprises 0.14 M NaCl. In some embodiments, the washing buffer has a pH of about 8.0. In some embodiments, the washing buffer comprises 50 mM Tris, 0.14 M NaCl, 0.05% Tween-20, and has a pH of about 8.0.

[0169] In some embodiments, the method further includes detecting a detection marker. In some embodiments, the detection marker is detected by measuring a luminescent compound. In some embodiments, the detection marker is detected by inducing an enzyme to convert a chromogenic substrate into a pigment, and by detecting and measuring the presence of the pigment. In some embodiments, detecting the detection marker includes converting a chromogenic substrate into a pigment and detecting the optical density of the sample. In some embodiments, detecting the detection marker includes contacting a sulfonated tag (Ru-tag) detection marker with a marker containing TPA, and the level of the detection marker can be detected by applying an electric current to the marker in the presence of the detection marker to generate a detectable ECL signal. Therefore, a higher level of the detection marker is associated with a higher amount of ADA in the sample.

[0170] In some embodiments, ADA is detected by determining whether a detection marker is present at a level higher than a predetermined level. In some embodiments, ADA is detected by determining that the ECL of the sample is higher than a predetermined cutoff point.

[0171] In some embodiments, the method further includes performing the method on a negative control sample. In some embodiments, the negative control sample does not contain detectable ADA. In some embodiments, the negative control sample is a drug-free sample from a healthy subject who has not received the target antibody. In some embodiments, the method is performed on the negative control sample in parallel with the sample. As used herein, performing the method in parallel includes performing the method on the negative control sample simultaneously with and / or on the same plate as the sample. Performing the method on the negative control sample includes: (i) incubating the control sample with a composition comprising an affinity antibody and a detection antibody; (ii) contacting the complex with a second affinity label that binds to a first affinity label; and (iii) measuring the level of the detection label.

[0172] In some embodiments, the method further includes performing the method on a positive control sample. In some embodiments, the positive control sample is a sample from a subject who has received the target antibody. In some embodiments, the positive control sample is a sample that showed the presence of ADA in a previous assay. The method can be performed on the positive control sample in parallel with the sample. The method can be performed on the sample, the negative control sample, and the positive control sample in parallel.

[0173] In some embodiments, the cutoff point is determined by performing the method on multiple negative control samples. In some embodiments, the negative control samples are untreated samples from healthy subjects who have never received the target antibody. In some embodiments, the cutoff point is determined by performing the method on at least 20, 30, 40, or 50 negative control samples. In some embodiments, the ECL signal from 50 negative control samples is normalized by the mean of all negative control samples. In some embodiments, outliers are identified and removed.

[0174] In some embodiments, a cutoff point is identified based on a false positive rate of approximately 0.01% to 0.05%, 0.05% to 0.1%, 0.1% to 0.5%, 0.5% to 1.0%, 1% to 2%, 2% to 5%, or 5% to 10%. In some embodiments, a cutoff point is identified based on a false positive rate of approximately 0.1%, 1%, or 5%. In some embodiments, a cutoff point is identified based on a false positive rate of approximately 5%. In some embodiments, a cutoff point is identified based on a false positive rate of approximately 1%. In some embodiments, a cutoff point is identified based on a false positive rate of approximately 0.1%.

[0175] In some embodiments, a cutoff point is identified based on a false positive rate of at least 0.01% to 0.05%, 0.05% to 0.1%, 0.1% to 0.5%, 0.5% to 1.0%, 1% to 2%, 2% to 5%, or 5% to 10%. In some embodiments, a cutoff point is identified based on a false positive rate of at least 0.1%, 1%, or 5%. In some embodiments, a cutoff point is identified based on a false positive rate of at least 5%. In some embodiments, a cutoff point is identified based on a false positive rate of at least 1%. In some embodiments, a cutoff point is identified based on a false positive rate of at least 0.1%.

[0176] In some embodiments, a cutoff point is identified based on a false positive rate of at least about 0.01% to 0.05%, 0.05% to 0.1%, 0.1% to 0.5%, 0.5% to 1.0%, 1% to 2%, 2% to 5%, or 5% to 10%. In some embodiments, a cutoff point is identified based on a false positive rate of at least about 0.1%, 1%, or 5%. In some embodiments, a cutoff point is identified based on a false positive rate of at least about 5%. In some embodiments, a cutoff point is identified based on a false positive rate of at least about 1%. In some embodiments, a cutoff point is identified based on a false positive rate of at least about 0.1%.

[0177] In some embodiments, the method further includes performing the method on quality control (QC) samples. One or more QC samples may be included to evaluate the performance of the method. In some embodiments, the QC samples are blank samples, matrix-adulterated samples (i.e., plasma or serum samples doped with ADA), field double-replicated samples, replicate samples, and / or reference material samples. The method may be performed on QC samples in parallel with the sample. The method may be performed on the sample, negative control sample, positive control sample, and QC samples in parallel.

[0178] In some embodiments, the predetermined cutoff point is an electrochemiluminescence (ECL) of about 0.1 to 1, 1 to 2, 2 to 5, or 5 to 10. In some embodiments, the predetermined cutoff point is about 1. In some embodiments, the predetermined cutoff point is about 1.1.

[0179] In some embodiments, the predetermined cutoff point is an ECL of at least 0.1 to 1, 1 to 2, 2 to 5, or 5 to 10. In some embodiments, the predetermined cutoff point is at least 1. In some embodiments, the predetermined cutoff point is at least 1.1.

[0180] In some embodiments, the predetermined cutoff point is an ECL of at least about 0.1 to 1, 1 to 2, 2 to 5, or 5 to 10. In some embodiments, the predetermined cutoff point is at least about 1. In some embodiments, the predetermined cutoff point is at least about 1.1.

[0181] In some embodiments, the sensitivity for detecting ADA is less than about 3 ng / mL, less than about 5 ng / mL, less than about 10 ng / mL, less than about 50 ng / mL, or less than about 100 ng / mL. In some embodiments, the sensitivity for detecting ADA is less than about 3 ng / mL. In some embodiments, the sensitivity for detecting ADA is less than about 2.77 ng / mL. In some embodiments, the sensitivity for detecting ADA is about 4 ng / mL. In some embodiments, the sensitivity for detecting ADA is about 6 ng / mL.

[0182] In some embodiments, the sensitivity for detecting ADA is less than 3 ng / mL, less than 5 ng / mL, less than 10 ng / mL, less than 50 ng / mL, or less than 100 ng / mL. In some embodiments, the sensitivity for detecting ADA is less than 3 ng / mL. In some embodiments, the sensitivity for detecting ADA is less than 2.77 ng / mL. In some embodiments, the sensitivity for detecting ADA is at least 4 ng / mL. In some embodiments, the sensitivity for detecting ADA is at least 6 ng / mL.

[0183] In some embodiments, the sensitivity for detecting ADA is less than about 3 ng / mL, less than about 5 ng / mL, less than about 10 ng / mL, less than about 50 ng / mL, or less than about 100 ng / mL. In some embodiments, the sensitivity for detecting ADA is less than about 3 ng / mL. In some embodiments, the sensitivity for detecting ADA is less than about 2.77 ng / mL. In some embodiments, the sensitivity for detecting ADA is at least about 4 ng / mL. In some embodiments, the sensitivity for detecting ADA is at least about 6 ng / mL.

[0184] In some embodiments, resistance to ADA is detected at concentrations of about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 50 ng / mL, about 50 ng / mL to about 100 ng / mL, about 100 ng / mL to about 1000 ng / mL, or about 1000 ng / mL to about 5000 ng / mL. In some embodiments, resistance is about 100 ng / mL.

[0185] In some embodiments, the detection of ADA resistance is performed at a concentration of at least 1 ng / mL to at least 10 ng / mL, at least 10 ng / mL to at least 50 ng / mL, at least 50 ng / mL to at least 100 ng / mL, at least 100 ng / mL to at least 1000 ng / mL, or at least 1000 ng / mL to at least 5000 ng / mL. In some embodiments, resistance is at least 100 ng / mL.

[0186] In some embodiments, the detection of ADA resistance is performed at a concentration of at least about 1 ng / mL to at least about 10 ng / mL, at least about 10 ng / mL to at least about 50 ng / mL, at least about 50 ng / mL to at least about 100 ng / mL, at least about 100 ng / mL to at least about 1000 ng / mL, or at least about 1000 ng / mL to at least about 5000 ng / mL. In some embodiments, resistance is at least about 100 ng / mL.

[0187] In some embodiments, the method includes dissociating ADA from the target by adding an acid wash solution to the sample. The acid wash solution can be any acid wash solution known to those skilled in the art for dissociating antibodies from their targets. In some embodiments, the acid wash solution may contain acetic acid. In some embodiments, the acid wash solution contains about 2 M acetic acid. In some embodiments, the acid wash solution is added to the sample prior to the addition of the anti-TNFα conjugate. In some embodiments, the acid wash solution contains about 300 mM acetic acid. In some embodiments, the acid wash solution contains about 0.1 M glycine-HCl. In some embodiments, the acid has a pH of about 2.5.

[0188] In some embodiments, the method is performed wholly or partially using an automated system. As used herein, “automated system” means a system that processes a sample according to steps performed by a robot or mechanical platform. The automated system can be fully automated or partially automated. In some embodiments, the sample and reagents of this technology are manually “loaded” into the automated system, which then performs the remaining steps. For example, the automated system could be a KingFisher™ Flex purification system. Other automated systems having at least the features described above and known to those skilled in the art are also within the scope of this technology.

[0189] In some embodiments, a method for detecting ADA binding to a target antibody in a sample includes: (i) incubating the sample together with a composition comprising: (a) an affinity antibody containing a first affinity label and (b) a detection antibody containing a detection label; (ii) providing a solid phase containing a second affinity label, wherein the second affinity label is configured to bind the first affinity label; (iii) contacting the sample and the composition with the solid phase; (iv) washing the solid phase; and (v) detecting ADA by measuring the level of the detection label above a predetermined cutoff point.

[0190] In some embodiments, a method for detecting ADA binding to a target antibody in a sample includes: (i) incubating the sample with a composition comprising an affinity antibody containing a first affinity label; (ii) incubating the sample with a detection antibody comprising a detection label; (iii) providing a solid phase comprising a second affinity label, wherein the second affinity label is configured to bind the first affinity label; (iv) contacting the sample and composition with the solid phase; (v) washing the solid phase; and (vi) detecting ADA by measuring the level of the detection label above a predetermined cutoff point.

[0191] In some embodiments, a method for detecting ADA binding to a target antibody in a sample includes: (i) contacting the sample with an affinity antibody containing a first affinity label and a detection antibody containing a detection label; and (ii) measuring the ADA that forms a complex with the affinity antibody and the detection antibody by detecting the level of the detection label in the complex; wherein the method detects the ADA with a sensitivity of less than 5 ng / mL.

[0192] This technology also includes a method for estimating the amount of ADA bound to a target antibody in a sample. The method includes: (i) incubating the sample with a composition comprising an affinity antibody and a detection antibody; (ii) isolating ADA that forms a complex with both the affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label bound to the first affinity label; (iii) detecting the ADA by measuring the level of the detection label above a predetermined cutoff point; (iv) performing steps (i) to (iii) on a known sample having a known concentration of the target antibody; and (v) comparing the level of the detection label in the sample with the level of the detection label in the known sample. As used herein, estimating the “amount” of ADA includes estimating the concentration of ADA. As used herein, “titration assay” generally refers to a method for estimating the amount of ADA bound to a target antibody.

[0193] As used herein, “known sample” includes a positive control where the concentration of ADA has been previously determined. In some embodiments, the known sample is serially diluted into multiple titer samples. As used herein, a “titer sample” is one of multiple titer samples diluted from the known sample. Thus, these multiple titer samples create a dilution profile.

[0194] In some embodiments, the titer sample has a concentration, for example, between about 0.49 ng / mL and about 4000 ng / mL. In some embodiments, the titer sample has concentrations, for example, about 4000 ng / mL, about 2000 ng / mL, about 1000 ng / mL, about 500 ng / mL, about 250 ng / mL, about 125 ng / mL, about 62.5 ng / mL, about 31.25 ng / mL, about 15.63 ng / mL, about 7.81 ng / mL, about 3.91 ng / mL, about 1.95 ng / mL, about 0.98 ng / mL, and about 0.49 ng / mL.

[0195] In some embodiments, the concentration of the titer sample is, for example, between at least 0.49 ng / mL and at least 4000 ng / mL. In some embodiments, the titer sample has concentrations of, for example, at least 4000 ng / mL, at least 2000 ng / mL, at least 1000 ng / mL, at least 500 ng / mL, at least 250 ng / mL, at least 125 ng / mL, at least 62.5 ng / mL, at least 31.25 ng / mL, at least 15.63 ng / mL, at least 7.81 ng / mL, at least 3.91 ng / mL, at least 1.95 ng / mL, at least 0.98 ng / mL, and at least 0.49 ng / mL.

[0196] In some embodiments, the titer sample has a concentration, for example, between at least about 0.49 ng / mL and at least about 4000 ng / mL. In some embodiments, the titer sample has concentrations, for example, at least about 4000 ng / mL, at least about 2000 ng / mL, at least about 1000 ng / mL, at least about 500 ng / mL, at least about 250 ng / mL, at least about 125 ng / mL, at least about 62.5 ng / mL, at least about 31.25 ng / mL, at least about 15.63 ng / mL, at least about 7.81 ng / mL, at least about 3.91 ng / mL, at least about 1.95 ng / mL, at least about 0.98 ng / mL, and at least about 0.49 ng / mL.

[0197] In some embodiments, the amount or concentration of a sample is estimated by comparing the level of a detection marker in the sample with the level of a detection marker in a known sample or a titer sample. In some embodiments, the method further includes generating a curve of the level of the detection marker in each titer sample and comparing the detection marker in the sample with the curve.

[0198] This technology also includes a method for estimating the amount of ADA binding to a target antibody in a sample. The method includes: (i) incubating the sample with a composition comprising an affinity antibody and a detection antibody, the affinity antibody comprising a first affinity label and the detection antibody comprising a detection label; (ii) providing a solid phase comprising a second affinity label, wherein the second affinity label is configured to bind the first affinity label; (iii) contacting the sample and composition with the solid phase; (iv) washing the solid phase; (v) detecting ADA by measuring the level of the detection label above a predetermined cutoff point; (vi) performing steps (i) through (v) on a known sample having a known concentration of the target antibody; and (vii) comparing the level of the detection label in the sample with the level of the detection label in the known sample. Step (vi) may be performed in parallel with steps (i) through (v).

[0199] This technology also includes a method for confirming the binding of an ADA compound to a target antibody in a sample by performing a competitive binding assay. This method includes any of the methods described herein for detecting ADA in a sample, wherein the composition further comprises an unlabeled anti-TNFα antibody (unlabeled antibody). As used herein, “confirmatory assay” or “confirmatory assay” generally refers to a method for confirming the binding of an ADA component to a target antibody.

[0200] In some embodiments, the method for confirming that an ADA compound binds to a target antibody includes: (i) incubating a sample with a composition comprising an affinity antibody, a detection antibody, and an unlabeled antibody; (ii) isolating ADA that forms a complex with both the affinity antibody and the detection antibody and / or the unlabeled antibody, wherein the ADA is isolated by contacting the complex with a second affinity label that binds to a first affinity label; and (iii) confirming ADA by measuring the level of the detection label, which is a predetermined factor lower than the level of the detection label in the absence of the unlabeled antibody.

[0201] In some embodiments, the unlabeled antibody is present in an excess concentration in the composition. For example, the excess concentration of the unlabeled antibody may be about 150 µg / mL or more, about 175 µg / mL or more, about 200 µg / mL or more, or about 250 µg / mL or more. In some embodiments, the excess concentration of the unlabeled antibody may be about 200 µg / mL or more.

[0202] In some embodiments, the excess concentration of unlabeled antibody may be at least 150 µg / mL or more, at least 175 µg / mL or more, at least 200 µg / mL or more, or at least 250 µg / mL or more. In some embodiments, the excess concentration of unlabeled antibody may be at least 200 µg / mL or more.

[0203] In some embodiments, the excess concentration of the unlabeled antibody may be at least about 150 µg / mL or more, at least about 175 µg / mL or more, at least about 200 µg / mL or more, or at least about 250 µg / mL or more. In some embodiments, the excess concentration of the unlabeled antibody may be at least about 200 µg / mL or more.

[0204] In some embodiments, the presence of ADA in a sample is confirmed by measuring the level of a detection marker that is lower than the level of the detection marker in the absence of an unlabeled antibody by a predetermined factor, which is about 10% or more, about 12% or more, about 14% or more, about 16% or more, about 18% or more, or about 20% or more. In some embodiments, the predetermined factor is about 16% or more.

[0205] In some embodiments, the presence of ADA in a sample is confirmed by measuring the level of a detection marker that is lower than the level of the detection marker in the absence of an unlabeled antibody by a predetermined factor, which is at least 10% or more, at least 12% or more, at least 14% or more, at least 16% or more, at least 18% or more, or at least 20% or more. In some embodiments, the predetermined factor is at least 16% or more.

[0206] In some embodiments, ADA in a sample is confirmed by measuring the level of a detection marker that is lower than the level of the detection marker in the absence of an unlabeled antibody by a predetermined factor, which is at least about 10% or more, at least about 12% or more, at least about 14% or more, at least about 16% or more, at least about 18% or more, or at least about 20% or more. In some embodiments, the predetermined factor is at least about 16% or more.

[0207] In some embodiments, the level of the labeled antibody is detected above a predetermined cutoff point when no labeled antibody is present.

[0208] In some embodiments, the method for confirming that the ADA compound binds to the target antibody includes: (i) incubating a sample with a composition comprising an affinity antibody, a detection antibody, and an unlabeled antibody; (ii) providing a solid phase comprising a second affinity label, wherein the second affinity label is configured to bind a first affinity label; (iii) contacting the sample and the composition with the solid phase; (iv) washing the solid phase; and (v) confirming ADA by measuring the level of the detection label, which is lower than the level of the detection label in the absence of the unlabeled antibody by a predetermined factor.

[0209] The method of this technology may further include steps for evaluating one or more of the following: hook effect, assay selectivity, drug resistance, intra-assay precision, inter-assay precision, and stability of the sample, reference standard, and / or antibody.

[0210] The hook effect (prozone effect) refers to the phenomenon where the measured signal changes from increasing to decreasing as the concentration of ADA increases. If all concentrations of positive antibodies produce positive results, the hook effect may not be important for ADA detection.

[0211] Stability assessment may include quantifying the method’s ability to detect ADA in the presence of other matrix components.

[0212] Drug resistance assessment may include evaluating the method's ability to correctly detect ADA. The presence of drug antibodies in the sample may interfere with the detection of ADA.

[0213] Stability assessment may include evaluating positive controls. For example, positive controls may be evaluated in biological samples such as human serum. The ADA of this technology remains stable through up to six freeze-thaw cycles from approximately -70°C (nominal) to ambient temperature, and remains stable at ambient temperature for up to approximately 24.5 hours.

[0214] Reagent test kit

[0215] This technology includes a kit for detecting ADA against a target antibody. In some embodiments, the technology includes a kit for estimating the amount of ADA in a sample. In some embodiments, the technology includes a kit for confirming ADA binding to a target antibody.

[0216] In some embodiments, the kit comprises an affinity antibody containing a first affinity label, a detection antibody, and a solid phase containing a second affinity label. In some embodiments, the kit comprises a composition containing an affinity antibody and a detection antibody.

[0217] In some embodiments, the solid phase is a plate. In some embodiments, the second affinity label is streptavidin. In some embodiments, the first affinity label is biotin. In some embodiments, the detection antibody comprises a sulfonyl-tagged anti-TNFα antibody. In some embodiments, the kit further comprises a biomarker configured to generate a chemiluminescent or ECL signal in the presence of the detection label. In some embodiments, the biomarker is TPA.

[0218] In some embodiments, the kit includes an undetected antibody. In some embodiments, the undetected antibody is at an excess concentration, for example, about 200 µg / mL or higher. The kit may also contain a composition comprising an affinity antibody, a detection antibody, and an unlabeled antibody.

[0219] In some embodiments, the affinity antibody comprises a first affinity label. In some embodiments, the first affinity label is biotin. In some embodiments, the detection antibody comprises a detection label. In some embodiments, the detection label is a luminescent compound, an enzyme configured to convert a chromogenic substrate into a pigment, or biotin. In some embodiments, the detection label is biotin.

[0220] In some embodiments, the kit includes a negative control. The negative control may be a sample from a subject who has not received drug treatment. In some embodiments, the negative control is a sample known to be free of ADA. In some embodiments, the kit includes a positive control. The positive control may be a sample from a subject who has received a target antibody. In some embodiments, the positive control is a sample known to contain ADA. In some embodiments, the kit may include ADA or an ADA solution and instructions for preparing the positive control. For example, the kit may include an ADA solution and instructions for “admixing” a plasma or serum sample with the ADA solution to prepare a positive control.

[0221] In some embodiments, the kit comprises a "high" positive control (HPC) and a "low" positive control (LPC), wherein the HPC has a higher concentration than the LPC. For example, the HPC may have the following concentrations: about 500 ng / mL to about 1000 ng / mL, about 1000 ng / mL to about 2000 ng / mL, about 2000 ng / mL to about 5000 ng / mL, or about 5000 ng / mL to about 10000 ng / mL. In some embodiments, the HPC has an ADA concentration of about 4000 ng / mL. The LPC concentration may be less than about 1 ng / mL, about 1 ng / mL to about 10 ng / mL, about 10 ng / mL to about 100 ng / mL, about 100 ng / mL to about 200 ng / mL, about 200 ng / mL to about 300 ng / mL, or about 300 ng / mL to about 500 ng / mL. In some embodiments, LPC has an ADA concentration of approximately 9.7 ng / mL.

[0222] In some embodiments, HPC may have the following concentrations: at least 500 ng / mL to at least 1000 ng / mL, at least 1000 ng / mL to at least 2000 ng / mL, at least 2000 ng / mL to at least 5000 ng / mL, or at least 5000 ng / mL to at least 10000 ng / mL. In some embodiments, HPC has an ADA concentration of at least 4000 ng / mL. LPC concentrations may be less than at least 1 ng / mL, at least 1 ng / mL to at least 10 ng / mL, at least 10 ng / mL to at least 100 ng / mL, at least 100 ng / mL to at least 200 ng / mL, at least 200 ng / mL to at least 300 ng / mL, or at least 300 ng / mL to at least 500 ng / mL. In some embodiments, LPC has an ADA concentration of at least 9.7 ng / mL.

[0223] In some embodiments, HPC may have the following concentrations: at least about 500 ng / mL to at least about 1000 ng / mL, at least about 1000 ng / mL to at least about 2000 ng / mL, at least about 2000 ng / mL to at least about 5000 ng / mL, or at least about 5000 ng / mL to at least about 10000 ng / mL. In some embodiments, HPC has an ADA concentration of at least about 4000 ng / mL. LPC concentrations may be less than at least about 1 ng / mL, at least about 1 ng / mL to at least about 10 ng / mL, at least about 10 ng / mL to at least about 100 ng / mL, at least about 100 ng / mL to at least about 200 ng / mL, at least about 200 ng / mL to at least about 300 ng / mL, or at least about 300 ng / mL to at least about 500 ng / mL. In some embodiments, LPC has an ADA concentration of at least about 9.7 ng / mL.

[0224] In some embodiments, the kit contains a known sample. In some embodiments, the known sample has the following ADA concentrations: about 4000 ng / mL or higher, about 40 µg / mL or higher, about 400 µg / mL or higher, or about 4000 µg / mL or higher. The known sample may have an ADA concentration of about 4000 ng / mL or higher. The kit may also contain one or more titer samples. For example, the kit may contain titer samples with the following ADA concentrations: about 4000 ng / mL, about 2000 ng / mL, about 1000 ng / mL, about 500 ng / mL, about 250 ng / mL, about 125 ng / mL, about 62.5 ng / mL, about 31.25 ng / mL, about 15.63 ng / mL, about 7.81 ng / mL, about 3.91 ng / mL, about 1.95 ng / mL, about 0.98 ng / mL, and about 0.49 ng / mL.

[0225] In some embodiments, the kit contains a known sample. In some embodiments, the known sample has the following ADA concentrations: at least 4000 ng / mL or higher, at least 40 µg / mL or higher, at least 400 µg / mL or higher, or at least 4000 µg / mL or higher. The known sample may have an ADA concentration of at least 4000 ng / mL or higher. The kit may also contain one or more titer samples. For example, the kit may contain titer samples with the following ADA concentrations: at least 4000 ng / mL, at least 2000 ng / mL, at least 1000 ng / mL, at least 500 ng / mL, at least 250 ng / mL, at least 125 ng / mL, at least 62.5 ng / mL, at least 31.25 ng / mL, at least 15.63 ng / mL, at least 7.81 ng / mL, at least 3.91 ng / mL, at least 1.95 ng / mL, at least 0.98 ng / mL, and at least 0.49 ng / mL.

[0226] In some embodiments, the kit contains a known sample. In some embodiments, the known sample has the following ADA concentrations: at least about 4000 ng / mL or higher, at least about 40 µg / mL or higher, at least about 400 µg / mL or higher, or at least about 4000 µg / mL or higher. The known sample may have an ADA concentration of at least about 4000 ng / mL or higher. The kit may also contain one or more titer samples. For example, the kit may contain samples with the following ADA concentrations: at least about 4000 ng / mL, at least about 2000 ng / mL, at least about 1000 ng / mL, at least about 500 ng / mL, at least about 250 ng / mL, at least about 125 ng / mL, at least about 62.5 ng / mL, at least about 31.25 ng / mL, at least about 15.63 ng / mL, at least about 7.81 ng / mL, at least about 3.91 ng / mL, at least about 1.95 ng / mL, at least about 0.98 ng / mL, and at least about 0.49 ng / mL.

[0227] Affinity antibodies and / or detection antibodies can be purified via affinity treatment. In some embodiments, affinity antibodies and / or detection antibodies can be stored in lyophilized powder form. In some embodiments, affinity antibodies and / or detection antibodies can be stored in reconstituted liquid antibody form.

[0228] Any component in the kit may be provided at a more concentrated concentration than it is intended for use. For example, components of the kit may be provided as “stock solutions” that must be diluted to the desired concentration of the “working solution.” In some embodiments, the stock solution may be concentrated to approximately 2, 5, 10, 50, 100, 500, 1000, 5000, or 10000 times the concentration of the working solution.

[0229] Affinity antibodies and / or detection antibodies can be lyophilized using methods known to those skilled in the art. One or more lyophilizing agents may be included. Examples of suitable lyophilizing agents include, for example, Tris-EDTA and sucrose. Any other lyophilizing agents commonly used in the art are also available. Determining suitable lyophilizing agents and the effective amounts of other lyophilizing agents is within the scope of conventional techniques in the art.

[0230] The kit may contain approximately 50 µg to approximately 100 µg, approximately 100 µg to approximately 500 µg, approximately 500 µg to approximately 1000 µg, approximately 1 mg to approximately 10 mg, approximately 10 mg to approximately 50 mg, approximately 50 mg to approximately 100 mg, approximately 100 mg to approximately 500 mg, or approximately 500 mg to approximately 1000 mg of affinity antibody and / or detection antibody. The kit may contain approximately 50 µg to approximately 200 µg of affinity antibody and / or detection antibody. The kit may contain approximately 100 mg of affinity antibody and / or detection antibody. The kit may contain approximately 1 mg of affinity antibody and / or detection antibody. The purity of the affinity antibody and / or the detection antibody may be approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99%.

[0231] The kit may include at least 50 µg to at least 100 µg, at least 100 µg to at least 500 µg, at least 500 µg to at least 1000 µg, at least 1 mg to at least 10 mg, at least 10 mg to at least 50 mg, at least 50 mg to at least 100 mg, at least 100 mg to at least 500 mg, or at least 500 mg to at least 1000 mg. The kit may include at least 50 µg to at least 200 µg of affinity antibody and / or detection antibody. The kit may include at least 100 mg of affinity antibody and / or detection antibody. The kit may include at least 1 mg of affinity antibody and / or detection antibody. The purity of the affinity antibody and / or the purity of the detection antibody may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99%.

[0232] The kit may include at least about 50 µg to at least about 100 µg, at least about 100 µg to at least about 500 µg, at least about 500 µg to at least about 1000 µg, at least about 1 mg to at least about 10 mg, at least about 10 mg to at least about 50 mg, at least about 50 mg to at least about 100 mg, at least about 100 mg to at least about 500 mg, or at least about 500 mg to at least about 1000 mg. The kit may include at least about 50 µg to at least about 200 µg of affinity antibody and / or detection antibody. The kit may include at least about 100 mg of affinity antibody and / or detection antibody. The kit may include at least about 1 mg of affinity antibody and / or detection antibody. The purity of the affinity antibody and / or the purity of the detection antibody may be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99%.

[0233] In some embodiments, the lyophilized affinity antibody and / or detection antibody may be reconstituted in a reconstitution buffer of about 50 µL to about 100 µL, about 100 µL to about 150 µL, about 150 µL to about 200 µL, about 200 µL to about 250 µL, about 250 µL to about 300 µL, about 300 µL to about 350 µL, about 350 µL to about 400 µL, about 400 µL to about 450 µL, about 350 µL to about 400 µL, about 400 µL to about 450 µL, or about 450 µL to about 500 µL.

[0234] In some embodiments, the lyophilized affinity antibody and / or detection antibody may be reconstituted in a reconstitution buffer of at least 50 µL to at least 100 µL, at least 100 µL to at least 150 µL, at least 150 µL to at least 200 µL, at least 200 µL to at least 250 µL, at least 250 µL to at least 300 µL, at least 300 µL to at least 350 µL, at least 350 µL to at least 400 µL, at least 400 µL to at least 450 µL, at least 350 µL to at least 400 µL, at least 400 µL to at least 450 µL, or at least 450 µL to at least 500 µL.

[0235] In some embodiments, the lyophilized affinity antibody and / or detection antibody may be reconstituted in at least about 50 μL to at least about 100 μL, at least about 100 μL to at least about 150 μL, at least about 150 μL to at least about 200 μL, at least about 200 μL to at least about 250 μL, at least about 250 μL to at least about 300 μL, at least about 300 μL to at least about 350 μL, at least about 350 μL to at least about 400 μL, at least about 400 μL to at least about 450 μL, at least about 350 μL to at least about 400 μL, at least about 400 μL to at least about 450 μL, or at least about 450 μL to at least about 500 μL of reconstitution buffer.

[0236] The reconstitution buffer may contain any component known to those skilled in the art. For example, the reconstitution buffer may contain water, PBS, DMSO, glycerol, or sodium azide. The reconstitution buffer may contain about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, or about 30% to about 35% glycerol. The reconstitution buffer may contain about 0.02% to about 0.04%, about 0.04% to about 0.06%, about 0.06% to about 0.08%, about 0.08% to about 0.1%, about 0.1% to about 0.12%, about 0.12% to about 0.14%, or about 0.14% to about 0.16% sodium azide. In some embodiments, the reconstitution buffer has a pH of about 6 to about 7 or about 7 to about 8. In some embodiments, the reconstitution buffer has a pH of about 7.2.

[0237] The reconstitution buffer may contain at least 10% to at least 15%, at least 15% to at least 20%, at least 20% to at least 25%, at least 25% to at least 30%, or at least 30% to at least 35% glycerol. The reconstitution buffer may contain at least 0.02% to at least 0.04%, at least 0.04% to at least 0.06%, at least 0.06% to at least 0.08%, at least 0.08% to at least 0.1%, at least 0.1% to at least 0.12%, at least 0.12% to at least 0.14%, or at least 0.14% to at least 0.16% sodium azide. In some embodiments, the reconstitution buffer has a pH of at least 6 to at least 7 or at least 7 to at least 8. In some embodiments, the reconstitution buffer has a pH of at least 7.2.

[0238] The reconstitution buffer may contain at least about 10% to at least about 15%, at least about 15% to at least about 20%, at least about 20% to at least about 25%, at least about 25% to at least about 30%, or at least about 30% to at least about 35% of glycerol. The reconstitution buffer may contain at least about 0.02% to at least about 0.04%, at least about 0.04% to at least about 0.06%, at least about 0.06% to at least about 0.08%, at least about 0.08% to at least about 0.1%, at least about 0.1% to at least about 0.12%, at least about 0.12% to at least about 0.14%, or at least about 0.14% to at least about 0.16% of sodium azide. In some embodiments, the reconstitution buffer has a pH of at least about 6 to at least about 7 or at least about 7 to at least about 8. In some embodiments, the reconstitution buffer has a pH of at least about 7.2.

[0239] In some embodiments, the kit includes a reconstitution buffer. In some embodiments, the kit includes reconstituted affinity antibodies and / or detection antibodies.

[0240] The kit may include detection antibodies and / or affinity antibodies in solution at concentrations of about 1 µg / mL to about 10 µg / mL, about 10 µg / mL to about 50 µg / mL, about 50 µg / mL to about 100 µg / mL, about 100 µg / mL to about 500 µg / mL, or about 500 µg / mL to about 1000 µg / mL, about 1 mg / mL to about 10 mg / mL, about 10 mg / mL to about 50 mg / mL, about 50 mg / mL to about 100 mg / mL, about 100 mg / mL to about 1000 mg / mL, or about 1000 mg / mL to about 10000 mg / mL.

[0241] The kit may include detection antibodies and / or affinity antibodies in solution at a concentration of at least 1 µg / mL to at least 10 µg / mL, at least 10 µg / mL to at least 50 µg / mL, at least 50 µg / mL to at least 100 µg / mL, at least 100 µg / mL to at least 500 µg / mL, or at least 500 µg / mL to at least 1000 µg / mL, at least 1 mg / mL to at least 10 mg / mL, at least 10 mg / mL to at least 50 mg / mL, at least 50 mg / mL to at least 100 mg / mL, at least 100 mg / mL to at least 1000 mg / mL, or at least 1000 mg / mL to at least 10000 mg / mL.

[0242] The kit may contain detection antibodies and / or affinity antibodies in solution at a concentration of at least about 1 µg / mL to at least about 10 µg / mL, at least about 10 µg / mL to at least about 50 µg / mL, at least about 50 µg / mL to at least about 100 µg / mL, at least about 100 µg / mL to at least about 500 µg / mL, or at least about 500 µg / mL to at least about 1000 µg / mL, at least about 1 mg / mL to at least about 10 mg / mL, at least about 10 mg / mL to at least about 50 mg / mL, at least about 50 mg / mL to at least about 100 mg / mL, at least about 100 mg / mL to at least about 1000 mg / mL, or at least about 1000 mg / mL to at least about 10000 mg / mL.

[0243] In some embodiments, the second affinity label is streptavidin. In some embodiments, the kit includes a solid phase, a second affinity label, and instructions for coating the solid phase with the second affinity label.

[0244] The solid phase can be any material known to those skilled in the art, which is image-capable and can bind a second affinity marker, including streptavidin. For example, the substrate can be a glass slide, plate, petri dish, or flask. In some embodiments, the kit comprises a solid phase coated with streptavidin. In some embodiments, the kit comprises a solid phase, streptavidin, and instructions for coating the solid phase with streptavidin.

[0245] The kit may contain one or more buffers. In some embodiments, the kit contains an acid wash buffer. In some embodiments, the kit further contains a cleaning buffer. In some embodiments, the kit contains a blocking buffer. The buffers of this technology can be formulated according to any formulation of this technology or formulations known to those skilled in the art.

[0246] According to some embodiments, the kit may contain one or more consumables for performing the methods of this technology. For example, the kit may include one or more plates containing multiple wells. In some embodiments, the plate may include 6, 9, 12, 16, 24, 36, 96, 384, or 1536 wells. In some embodiments, the kit or system includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than twenty assay plates.

[0247] The kit may also include instructions for performing all or part of the methods described in this technology. In some embodiments, the kit includes links (e.g., URLs or QR codes) guiding a user to obtain instructions for performing all or part of the methods of this technology. The kit may include software or other computer technology to facilitate performing the methods of this technology.

[0248] The components of the kit can be stored to provide stability for a particular component. For example, if the component contains a photosensitive compound, the kit component can be stored in an opaque container. In some embodiments, the kit component can be stored in a sealed container. In some embodiments, the kit component can be stored frozen, in liquid form, or in solid form. In some embodiments, the kit component is stored at about -195°C, -80°C, -20°C, 4°C, or 20°C.

[0249] In some embodiments, the kit comprises: (i) an affinity antibody containing biotin; (ii) a detection antibody containing a sulfonyl tag; (iii) a plate containing streptavidin; (iv) TPA; and (v) a washing buffer.

[0250] As used in this article, the term “about” immediately preceding a numerical value indicates a range of plus or minus 10%, for example, “about 50” means 45 to 55, “about 10” means “9 to 11”, and so on. Furthermore, the phrases “below about” or “greater than about” should be understood in conjunction with the definition of the term “about” provided in this article.

[0251] In another embodiment, the kit comprises one or more of the components, consumables, or reagents described in the following examples.

[0252] Example

[0253] Examples provided in this technology include an example method for detecting ADA in human samples using ECL assays, a method for validating the assay, and the validation results.

[0254] List of abbreviations and definitions: ADA: Anti-drug antibody; AT: Ambient temperature; Bio-XTMAB-16: Biotin-labeled XTMAB-16; CCP: Confirmatory cutoff; CP: Cutoff point; CV: Coefficient of variation; diH2O: Deionized water; ECL: Electrochemiluminescence; ECLIA: Electrochemiluminescence immunoassay; HPC: High positive control; iNHS: Individual unimmunized human serum; LPC: Low positive control; MRD: Minimum required dilution; MSD: Ultrasensitive electrochemiluminescence technology; NA: Not applicable; NC: Negative control; pNHS: Combined unimmunized human serum; PBS: Phosphate-buffered saline; QC: Quality control; rECL: Relative ECL; Ru-XTMAB-16: Ruthenium-labeled XTMAB-16; SCP: Screening cutoff point; S-CPF: Screening cutoff factor; SD: Standard deviation; TCP: Titration cutoff point; T-CPF: Titration cutoff factor.

[0255] Example 1: Method for detecting ADA in subjects using ECL and its validation method

[0256] Overview : Figure 1 An exemplary method (method 100) for detecting ADA bound to a target antibody in a sample is shown. Method 100 can be used for immunogenicity assessment of various antibodies, including but not limited to anti-TNFα antibodies. Method 100 includes: (i) incubating a sample with a composition comprising: (a) an anti-TNFα antibody (affinity antibody) conjugated to a first affinity label, and (b) an anti-TNFα antibody (detection antibody) 110 conjugated to a detection label; (ii) separating ADA that forms a complex with both the affinity antibody and the detection antibody, wherein ADA 120 is separated by contacting the complex with a second affinity label bound to the first affinity label; and (iii) detecting ADA 130 by measuring the level of the detection label above a predetermined cutoff point. Separation of ADA may include: (a) providing a solid phase containing the second affinity label; (b) contacting the sample and the composition with the solid phase; and (c) washing the solid phase.

[0257] Validation Summary: To support the assessment of the immunogenicity of XTMAB-16 in human samples, a method for detecting antibodies in human serum samples was evaluated.

[0258] MSD streptavidin plates were blocked to prevent nonspecific antibody binding. Control and experimental samples were incubated with Bio-XTMAB-16 and then transferred to the blocked MSD streptavidin plates. After incubation, the plates were washed and then incubated with Ru-XTMAB-16. After incubation with Ru-XTMAB-16, the MSD streptavidin plates were washed again and read using a microplate reader (MSDSector Imager S600). The validation method also included screening, confirmatory, and titration steps.

[0259] Screening methods are used to detect ADA in human serum samples that may be positive for XTMAB-16. Confirmatory methods are used to determine whether potentially ADA-positive samples are specific for XTMAB-16. Titration methods are used to estimate ADA levels in human serum samples that have been confirmed to be positive for XTMAB-16 antibodies.

[0260] Material The validation method used the reagents and controls listed in Table 2.

[0261] Table 2: Reagents and Controls Used in ECL Validation

[0262]

[0263] negative control : Combine 40 batches of iNHS (29 batches of males and 11 batches of females) of equal volume to prepare a combined batch of pNHS.

[0264] High positive control and low positive control: The ECL assay was validated for detecting anti-XTMAB-16 antibodies in human serum and for determining whether potentially ADA-positive samples are specific for anti-XTMAB-16. A titration method was incorporated to provide a quasi-quantitative estimate of ADA levels in human serum samples confirmed to be anti-XTMAB-16 positive. Preparation of positive controls is further detailed in Table 3.

[0265] Table 3: Preparation of positive control standards

[0266]

[0267] Reagent preparation PCs were prepared by adding anti-XTMAB-16 antibody to pNHS at high concentrations (4,000.0 ng / mL) for HPC and low concentrations (9.70 ng / mL) for LPC, respectively.

[0268] The reagent preparation included: MSD 2X read buffer (4x MSD read buffer diluted with diH2O), wash buffer (1x PBS containing 0.05% Tween-20), blocking buffer (protein-free), assay buffer (NGBI), and XTMAB-16 stock solution (XTMAB-16 dissolved in PBS to 10 mg / mL).

[0269] QC preparation included the preparation of NC (pNHS prepared from at least 40 individual batches) and positive controls for LPC and HPC. The HPC concentration was 4,000 ng / mL, and the final LPC concentration was prepared with a 99.9% confidence interval.

[0270] Establishment of LPC concentration In both screening and confirmatory assays, the LPC concentration is calculated using the mean of the corresponding sensitivity plus t. 0.99 , df X SD (sensitivity) calculation. The final LPC concentration is the higher LPC concentration between the screening LPC and the confirmatory LPC. Each sensitivity will be obtained from a separate sensitivity run. The screening sensitivity concentration will be interpolated at SCP based on the screening sensitivity curve. The confirmatory sensitivity concentration will be interpolated at CCP based on the confirmatory sensitivity curve. Once the LPC is finalized, each run will include at least two sets of LPC and HPC, and at least four sets of NC as plate controls.

[0271] System Applicability Standards System suitability will be assessed based on all accepted validation runs (excluding robustness runs and long-term stability runs, if any) to establish acceptable ranges for NC, LPC, and HPC assays. Control ranges will be set using response values ​​for NC, LPC, and HPC, as detailed below.

[0272] Example of calculation based on the reference range:

[0273] NC limit = Average NC response + 3xSD of NC response

[0274] LPC range = LPC response mean ± LPC response 3xSD

[0275] HPC lower limit = HPC response mean - HPC response 3xSD

[0276] In cutoff and sensitivity runs, at least 50% of each HPC level must have a %CV ≤ 20.0% for repeat samples.

[0277] Once the LPC concentration is finally determined, at least 50% of each level of the plate reference must have a %CV of ≤ 20.0%.

[0278] The accepted control sample relationships should be as follows:

[0279] For screening and titration assays, mean ECL: NC < SCP (if applicable) ≤ LPC (if applicable) < HPC

[0280] For confirmatory assays, inhibition percentage: NC < CCP (if applicable) ≤ LPC (if applicable) and HPC.

[0281] To be accepted for a run, at least 50% of each level of the control samples and at least 66.7% of the total control samples should meet the above criteria.

[0282] Acceptance criteria for screening test plate reference standards For screening assays, the CV for each plate control was ≤20.0%, and the plate controls were presented in double replicates. The plate NC was considered the mean of all accepted NCs, and %CV failures and outliers were excluded. The relationship between accepted control samples in screening assays was the mean response (ECL): NC < SCP ≤ LPC < HPC, and the ECL was within the range established during validation.

[0283] NC: ECL mean ≤ 158.8; LPC: 95.5 ≤ ECL mean ≤ 156.1; HPC: 4761.7 ≤ ECL mean.

[0284] The S-CPF is 1.10, and the plate-specific SCP is the value calculated by multiplying the NC by the S-CPF.

[0285] Acceptance criteria for confirmatory assay plate reference standards: For confirmatory assays, the CV for each plate control was 20.0% or less, and the plate controls were present in double replicates. The relationship between accepted control samples was inhibition %: NC < CCP ≤ LPC and HPC. Plate NC was the mean of all accepted NCs, excluding %CV failures and outliers. In the presence of XTMAB-16, the CCP was 16.45%, and the inhibition % was calculated using the following formula:

[0286] Inhibition % = 100 * (1 - [mean ECL of doped sample / mean ECL of undoped sample])

[0287] Acceptance criteria for titration test plate reference standards For titration assays, the CV for each plate reference is 20.0% or lower, and the plate references are presented in double replicates. The plate NC is considered the mean of all accepted NCs, and %CV failures and outliers are excluded. The relationship between accepted references in titration assays is the mean response (ECL): NC < SCP ≤ LPC < HPC, and the ECL is within the range established during validation.

[0288] NC: ECL mean ≤ 158.8; LPC: 95.5 ≤ ECL mean ≤ 156.1; HPC: 4761.7 ≤ ECL mean.

[0289] The titer reference standard is 25600 ≤ XTMAB-16 ADA PC at 4,000 mg / mL with a titer ≤ 102,400.

[0290] The T-CPF is 1.20, and the board-specific TCP is calculated by multiplying the T-CPF by the board NC.

[0291] Screening and testing sample acceptance criteria: The average ECL value of the screened samples had a %CV of 20.0% or lower. Samples with an average ECL value ≥ plate-specific SCP were classified as potentially positive. Samples with an average ECL value lower than plate-specific SCP were classified as negative.

[0292] Acceptance criteria for confirmatory testing samples: The mean ECL value of confirmatory samples has a %CV of 20.0% or less. A sample is classified as positive for anti-XTMAB-16 antibody if the following criteria are met: (1) the mean ECL of the sample in the screening assay is ≥ the plate-specific SCP and (2) the inhibition % in the confirmatory assay is ≥ the CCP. A sample is classified as negative for anti-XTMAB-16 antibody if any of the following criteria are met: (1) the mean ECL in the screening assay is < the plate-specific SCP and (2) the inhibition % in the confirmatory assay is < the CCP.

[0293] Acceptance criteria for titration determination samples: Undiluted samples with an ECL lower than the plate TCP are reported as MRD. Undiluted samples with an ECL higher than the TCP but an ECL lower than the plate TCP at the first dilution (minimum dilution) are reported as MRD. For samples with at least one dilution reaching an ECL lower than the TCP, the diluted sample must have at least two dilutions spanning the TCP, where %CV ≤ 20.0%.

[0294] The titer of a diluted sample with an ECL higher than that of a plate-specific TCP is considered to be the dilution factor of the last diluted sample with an ECL higher than that of a plate-specific TCP.

[0295] Determining the cutoff point: SCP: The ECL signal of each individual sample was normalized using plate NC (expressed as S / N). The data were then logarithmically transformed. After removing outliers, the data were tested for normality using JMP. Based on a 5% false positive rate, the S-CPF was calculated according to the data distribution using the following formula.

[0296] S-CPF = "mean + 1.645xSD" (if the data is normally distributed) or " 第95th percentile (if the data is not normally distributed)

[0297] Plate-specific SCP will be calculated using the following formula:

[0298] SCP = Average NC ECL value in the plate * S-CPF

[0299] CCP: CCP is defined as the inhibition rate of individual samples doped with and without the reference drug. Inhibition % and CCP (using a 1% false positive rate) are calculated based on the data distribution of JMP using the following equation.

[0300] Suppression % = 100 * ECL 无药物 -ECL 有药物 ) / ECL 无药物

[0301] CCP = "mean % suppression + 2.33 * SD % suppression" (if the data is normally distributed) or " 第 99th percentile (if the data is not normally distributed)

[0302] TCP: T-CPF will be calculated using S-CPF normalized data. T-CPF will be calculated based on a 0.1% false positive rate using the following equation according to the data distribution. T-CPF will be reported to two decimal places.

[0303] T-CPF = "mean + 3.09 x SD" (if the data is normally distributed) or "99.9 percentile" (if the data is not normally distributed)

[0304] Board TCP = Average NC ECL value on the board * T - CPF

[0305] The experimental design and target specifications for determining the cutoff point are summarized in Table 4.

[0306] Table 4: Experimental Design and Objective Specifications for Determining Cutoff Points

[0307]

[0308] Sensitivity: The sensitivity of the ADA assay can be defined by the lowest concentration of the positive control antibody preparation that consistently provides a positive signal in the assay. Assay sensitivity is determined in both drug-containing and drug-free conditions. Experimental designs for sensitivity assays for screening assays are summarized in Table 5, and experimental designs for sensitivity assays for confirmatory assays are summarized in Table 6.

[0309] Table 5: Screening Test Experiment Design and Target Specifications for Sensitivity Measurement

[0310]

[0311] Table 6: Confirmatory Test Design and Target Specifications for Sensitivity Measurement

[0312]

[0313] Hook effect: Positive control antibody samples for preband assessment were prepared at concentrations higher than HPC to demonstrate that higher concentrations may not lead to a decreased response. Prepared samples were frozen for at least 12 hours prior to serial dilution and analysis. On the day of assay, samples were diluted using negative serum as a diluent and then analyzed to yield concentrations higher than or equal to the assay sensitivity level. At least 80% of validation samples used for hook effect assessment had a %CV of ≥ 20.0%.

[0314] Intra- and inter-measurement precision: Based on %CV of NC, LPC, and HPC, a minimum of six independent formulations of the same sample were used in a single validation run, with screening and confirmatory assays employed to assess internal precision. The experimental design and target specifications for internal precision are summarized in Table 7.

[0315] Table 7: Intra-precision of screening and confirmatory assays

[0316]

[0317] In addition to robustness and long-term stability tests, inter-assay precision was evaluated based on the %CV of NC, LPC, and HPC results from all approved validation runs. The experimental design and target specifications for inter-assay precision are summarized in Table 8.

[0318] Table 8: Inter-assay precision of screening and confirmatory assays

[0319]

[0320] Selectivity and matrix interference: In both the screening and confirmatory tests, LPC-level ADA was incorporated into the normal matrix of at least 10 individuals to assess selectivity and matrix interference in the individual's normal matrix. In both the screening and confirmatory tests, LPC-level ADA was incorporated into the diseased matrix of at least 10 individuals to assess selectivity and matrix interference in the individual's diseased matrix. In the screening assay, matrix samples with and without ADA were analyzed and compared with SCP. In the confirmatory assay, the analyzed samples were compared with CCP. Samples may be frozen prior to analysis. Selectivity samples with pre-existing antibodies were not included in the selectivity assessment assays. The experimental design and target specifications for selectivity assessment are summarized in Table 9.

[0321] Table 9: Selective Evaluation Experimental Design and Objective Specifications

[0322]

[0323] Hemolysis and lipemia matrix interference Selective assessments included evaluation of hemolysis (2%) and lipemia (150 mg / dL) matrix. Serum samples with at least three control matrix pools were evaluated for both unadulterated and LPC-adulterated samples. Adulterated matrix samples were analyzed in comparison with plate SCP. In confirmatory assays, the analyzed samples were compared with CCP. Samples may be frozen prior to analysis. Matrix interference experimental design and target specifications are detailed in Table 10.

[0324] Table 10: Experimental Design and Target Specifications for Matrix Interference

[0325]

[0326] Drug resistance determined: Drug resistance is used to determine the highest level of drug that can be added to a sample containing a positive control antibody without interfering with the positive control antibody detection to produce a positive antibody response. Various concentrations of drug are incorporated into matrix samples containing various concentrations of positive control antibodies.

[0327] Drug tolerance limit can be defined as the highest drug concentration at which ADA remains detectable in the presence of the drug. ADA PC at 100 ng / mL and LPC levels will be tested. In the screening assay, the highest drug concentration that still maintains a positive response for each ADA level tested will be defined as the drug tolerance level for the screening assay. The experimental design and target specifications for testing drug tolerance are summarized in Table 11.

[0328] Table 11: Experimental Design and Target Specifications for Testing Drug Resistance

[0329]

[0330] Stability test: Samples were subjected to a combination of pressurized loading on the test bench (24 ± 6 hours or 8 ± 2 hours) and freeze-thaw cycles (from -70 °C ± 10 °C to room temperature, 6 or 3 cycles). Samples were prepared and stored overnight (at least 12 hours) at -70 °C ± 10 °C, then thawed at ambient temperature as the first cycle. For subsequent freeze-thaw cycles, samples were frozen under the same conditions for at least 12 hours, then thawed. Samples were left on the test bench for 2 ± 1 hours before being refrozen. The experimental design and target specifications are summarized in Table 12.

[0331] Table 12: Experimental Design and Objective Specifications for On-Site and Freeze-Thaw Stability Determination

[0332]

[0333] Data evaluation:Raw ECL count data were acquired using MSD Sector Imager S600 and Discovery Workbench software version 4.0. %CV, SD, and suppression percentage were calculated respectively. The cutoff factor was statistically evaluated using JMP software.

[0334] Performance characteristics to be measured: All samples and controls, including two LPC and HPC sets and at least four NC sets, were evaluated in double replicates. Text files imported from MSD Sector Imager S600 were used as source data for the analysis and verified against screenshots of the raw data. Source data were processed using Microsoft Excel to calculate the mean, %CV, and inhibition percentage of ECL values. S-CPF, T-CPF, and CCP were calculated using JMP 14 statistical software. The mean of all double or quadruple replicate ECL values ​​was evaluated in the analysis.

[0335] Failed test: The plate control test that caused the run to fail was repeated twice. If the repeated run fails again, and the failure is not due to experimental error, the method was investigated, and if necessary, the method was revised or optimized and the run was repeated.

[0336] Example 2: Validation of the method for detecting ADA in serum samples using electrochemiluminescence immunoassay

[0337] Cutoff point determined: To determine the cutoff factors for screening, confirmatory, and titration assays of anti-XTMAB-16 antibodies, two serum samples from 54 batches of unimmunized male and female human serum were tested over a five-day period. Each sample was tested six times in 18 individual runs.

[0338] The cutoff point statistical outliers were identified using the quantile range outlier method, and the identified outliers were removed. The normality of the cutoff point values ​​was tested using the Shapiro-Wilk W test with JMP software.

[0339] For each run, the mean ECL signal of each individual serum sample was normalized using the mean ECL of the plate NC. The normalized ECL (rECL) was then logarithmically transformed. Examples of data analyzed according to this technique are shown in Tables 12 through 14. Table 12 shows the raw data from one run of samples tested according to the method of this technique. Table 13 shows the raw data for S-CPF and T-CPF from the runs in Table 12. Table 14 shows an example of the summative normalized data calculated for S-CPF and T-CPF.

[0340] Table 12: Examples of raw data from a single run

[0341]

[0342] Table 13: Raw data for S-CPF and T-CPF

[0343]

[0344] *Each line represents a different batch

[0345] Table 14: Raw Data for S-CPF and T-CPF

[0346]

[0347] *Each line represents a different batch

[0348] Box plot analysis was used to identify analytical outliers on normalized and logarithmically transformed serum values ​​(Table 15). Biological outliers were identified and excluded from the analysis, and the normalized and logarithmically transformed data (if any) for each individual serum sample were averaged over six assays. Box plot analysis was then performed to identify biological outliers (Table 16). Examples of the analyzed outliers are shown in Tables 15 through 17. Tables 15, 16, 18, and 19 show examples of analytical and biological outlier identification from the batches evaluated in Tables 12 through 14. Table 17 shows an example of the summary data used to calculate the logarithmically transformed rECL for S-CPF and T-CPF after removing biological and analytical outliers (such as those shown in Tables 15, 16, 18, and 19).

[0349] Table 15: Analysis of Outliers and Identification of Biological Outliers (Example A)

[0350]

[0351] *Each line represents a different batch

[0352] Table 16: Analysis of Outliers and Identification of Biological Outliers (Example B)

[0353]

[0354] *Each line represents a different batch

[0355] Table 17: Logarithmic Transformation of S-CPF and T-CPF (rECL)

[0356]

[0357] *Each row represents a different batch. Table 18: Calculation of Outliers in SCP and TCP Analysis

[0358]

[0359] Table 19: Calculation of SCP and TCP Biotic Outlier Values

[0360]

[0361] Normality test of screening and titration data Using JMP software, the normality of 323 log-transformed normalized values ​​was tested using the Shapiro-Wilk W test. Figure 2A The normal distribution was confirmed, and the S-CPF and T-CPF were calculated using a parameterized method (Table 20).

[0362] Table 20: Calculated parameterized S-CPF and T-CPF

[0363]

[0364] CCP confirms: For each run, calculate the inhibition % for each sample using the following formula:

[0365] Inhibition % = 100 * (ECL) 不含药物 -ECL 含药物 ) / ECL 不含药物

[0366] Remove SCP and TCP from CCP analysis.

[0367] Box plot analysis was used to identify and exclude outliers from the CCP analysis based on the suppression % value (Table 21).

[0368] Table 21: Calculation of Outliers in CCP Analysis

[0369]

[0370] After identifying and excluding outliers, the inhibition percentage data for each individual serum sample were averaged across six assays, if available. Box plot analysis was performed on these values ​​to identify biological outliers (Table 22).

[0371] Table 22: Calculation of CCP Outlier Values

[0372]

[0373] Normality test for confirmatory data:The normality of the 323 inhibition % values ​​was tested using JMP software via the Shapiro-Wilk W test. Figure 2B The normal distribution was confirmed, and the S-CPF and T-CPF were calculated using a parameterized method (Table 23).

[0374] Table 23: Calculated parameterized CCP factors

[0375]

[0376] Cutoff values ​​for the application assay to detect ADA against XTMAB-16 in human serum were determined (Table 24). Examples of the assessed CCP values ​​are shown in Tables 25 through 28. Examples of the raw data values ​​assessed are shown in Table 25, and examples of these values ​​after removing outliers are shown in Tables 26 through 28, where no outliers were found.

[0377] Table 24: Determined detection and measurement cutoff values

[0378]

[0379] Table 25: Exemplary Raw Data

[0380]

[0381] * Each line represents a different batch.

[0382] Table 26: Values ​​after removing outliers (Example A)

[0383]

[0384] *Each line represents a different batch

[0385] Table 27: Values ​​after removing outliers (Example B)

[0386]

[0387] *Each line represents a different batch

[0388] Table 28: Values ​​after removing outliers (Example C)

[0389]

[0390] *Each line represents a different batch

[0391] Sensitivity:The sensitivity of the screening assay was evaluated six times using pooled human serum (triple) and three individual batches of human serum (once per batch). Each sample was doped with anti-XTMAB-16 antibody PC. The doped samples were serially diluted twofold (from 4,000 ng / mL to 0.49 ng / mL) in pNHS and individual mouse serum (iNMS). The screening sensitivity for each sensitivity group was determined by interpolation at SCP using Microsoft ExcelForecast (5% false positive). The final assay sensitivity in the screening assay was the mean of the concentrations calculated from all qualifying curves. The results showed that the screening sensitivity for the assay to detect anti-XTMAB-16 antibody in human serum was 2.77 ng / mL. Examples of such results are shown in Table 29.

[0392] Table 29: Screening Sensitivity

[0393]

[0394] The confirmatory assay sensitivity was determined by incorporating XTMAB-16 into diluted samples prepared for sensitivity screening. The confirmatory sensitivity (1% false positive) for each sensitivity group was determined by interpolation at the CCP using Microsoft Excel Forecast. The final assay sensitivity for confirmatory assays was the mean concentration calculated from all qualifying curves. The results showed that the confirmatory sensitivity for the assay of anti-XTMAB-16 antibody was 4.25 ng / mL. Examples of such results are shown in Table 30.

[0395] Table 30: Confirmatory Sensitivity

[0396]

[0397] Sensitivity data were also used to determine the LPC to be measured. The LPC was based on a sensitivity with a 1% false positive rate, calculated as the mean of sensitivity + t0.99, df × SD (sensitivity) for both the screening and confirmatory assays. The LPC concentrations for the screening and confirmatory assays were 6.80 ng / mL and 9.70 ng / mL, respectively. The results are detailed in Table 31. The calculated LPC concentration, 9.70 ng / mL, represents the higher LPC between the screening and confirmatory assays.

[0398] Table 31: LPC Determination for Screening and Confirmatory Tests

[0399]

[0400] Hook effect determination:The hook (prozone) effect is the phenomenon where the detection signal changes from enhancement to deterioration as ADA concentration increases. If all concentrations of positive antibody produce positive results, the hook effect may not be significant for ADA detection. The hook effect was assessed using samples containing extremely high concentrations of anti-XTMAB-16 antibody (32,000 ng / mL) in pNHS, prepared by incorporating ADA PC into pooled serum and freezing for at least 12 hours. The samples were then serially diluted twofold to concentrations below the plate-specific TCP. All samples with concentrations of anti-XTMAB-16 antibody of 7.81 ng / mL or higher showed positive ECL values ​​compared to the plate-specific TCP, indicating no prozone effect was observed. Examples of data demonstrating this result are shown in Table 32.

[0401] Table 32: Determination of Hook Effect

[0402]

[0403] Selectivity: Selectivity is the ability of this assay to detect anti-XTMAB-16 antibodies in the presence of other matrix components. Samples with LPC concentrations (9.70 ng / mL) were prepared from 10 batches of normal human serum, 10 batches of patient serum, 3 batches of hemolyzed human serum, and 3 batches of lipemic human serum, both unadulterated and ADA-adulterated. These sera were initially tested four times in screening and confirmatory assays. For individual batches of normal human serum, 9 out of 10 unadulterated samples (90%) were confirmably negative, while 9 out of 10 LPC-adulterated samples (90%) were confirmably positive. For individual batches of patient serum, 10 out of 10 unadulterated samples (100%) were confirmably negative, while 10 out of 10 LPC-adulterated samples (100%) were confirmably positive. For individual batches of hemolyzed human serum, 3 out of 3 unadulterated samples (100%) were negative, while 3 out of 3 samples with LPC admixture were positive. For individual batches of lipemic human serum, 3 out of 3 unadulterated samples (100%) were negative, while 3 out of 3 samples with LPC admixture were positive, meeting the acceptance criteria. This indicates that there is no matrix interference for detecting anti-XTMAB-16 antibodies in human serum. Examples of data showing this result are presented in Table 33.

[0404] Table 33: Evaluation of Selectivity and Matrix Interference

[0405]

[0406] Drug resistance:The presence of the drug XTMAB-16 in the sample may interfere with the detection of anti-XTMAB-16 antibodies; therefore, the ability of the assay system to correctly detect ADA was evaluated. Drug tolerance limit was defined as the highest concentration of the drug at which ADA can still be detected in the presence of the drug. Positive controls prepared at concentrations of 100 ng / mL and 9.7 ng / mL were used to assess potential drug interference. These controls were doped with XTMAB-16 at concentrations ranging from 5.0 µg / mL to 400 µg / mL. In the sample doped with 100 ng / mL, the addition of up to 100.0 µg / mL of XTMAB-16 had no effect on ADA detection; in the sample doped with 9.7 ng / mL, the addition of up to 5.0 µg / mL of XTMAB-16 did affect ADA detection, leading to false negative results. Examples of data demonstrating this result are shown in Table 34.

[0407] Table 34: Assessment of Drug Resistance

[0408]

[0409] Determine internal precision: Intra-run precision (intra-run performance evaluation of the reference standard) was assessed using six independent formulations of HPC (4,000 ng / mL), LPC (9.70 ng / mL), and NC in a single run. Results showed that the reference standard met the intra-run precision criteria of ≤20.0% ECL signal for both screening and confirmatory assays. The reference standard also met the criteria for ECL in screening assays: NC < plate-specific SCP ≤ LPC < HPC, and for inhibition percentage in confirmatory assays: NC < CCP ≤ LPC and HPC. The intra-run precision for screening assays of HPC and LPC was 18.2% and 4.3%, respectively. The intra-run precision for confirmatory assays of ECL signal of HPC and LPC was 6.3% and 4.1%, respectively. The intra-run precision for percentage of HPC and LPC was 0.2% and 9.1%, respectively. Examples of data showing these results are presented in Table 35.

[0410] Table 35: Evaluation of Internal Precision

[0411]

[0412] Inter-measurement precision:Inter-assay precision (an assessment of the inter-assay performance of the reference standard) was evaluated based on HPC (4,000 ng / mL), LPC (9.7 ng / mL), and NC results from all accepted validation runs other than the robustness run PC. The screening inter-assay precision of this method was 14.1% (HPC) and 7.9% (LPC). The reference standard met the inter-assay precision criteria for ≤20.0% HPC and LPC. The confirmatory inter-assay precision for inhibition % of HPC and LPC was 0.2% and 13.3%, respectively. The reference standard also met the criteria for ECL: NC < plate-specific SCP ≤ LPC < HPC for screening assays, and the criteria for inhibition % of inhibitory concentration for confirmatory assays: NC < CCP ≤ LPC and HPC. Examples of data demonstrating these results are shown in Table 36.

[0413] Table 36: Evaluation of inter-assay precision

[0414]

[0415] stability: The stability of the samples was assessed using the stability of positive controls in human serum. Freeze-thaw (-70℃ ± 10℃ to ambient temperature, 6 cycles) and on-site (ambient temperature, 24 hours) stability tests were performed. The acceptance criteria for stable samples were as follows: (1) for screening assays, NC < plate-specific SCP ≤ LPC < HPC; (2) for confirmatory assays, inhibition %: NC < CCP ≤ LPC and HPC; (3) for NC, LPC and HPC, %CV ≤ 20%; (4) at least 66.7% (two-thirds) of the stable samples must meet the above acceptance criteria.

[0416] The tested samples were stored at -70°C (nominal) for at least 24 hours before the first thaw and for 12 hours before each subsequent thaw. Before refrozen, the samples were placed at ambient temperature for 1 to 2 hours. After six freeze / thaw cycles, three aliquots of each control (HPC, LPC, and NC) were tested on a plate using screening and confirmatory assays, with frozen PC and NC used for plate acceptance. The results met the acceptance criteria; therefore, anti-XTMAB-16 antibodies in human serum remained stable during up to six freeze-thaw cycles from -70°C (nominal) to ambient temperature. Examples of these results are shown in Table 37.

[0417] Table 37: Stability Assessment (Example A)

[0418]

[0419] The tested samples were stored at -70°C (nominal) for at least 24 hours prior to initial thawing. Triples of each pre-existing frozen control (HPC, LPC, and NC) were thawed on a benchtop for 24.5 hours and then analyzed using both screening and confirmatory assays for the frozen PC and NC to be accepted for plate testing. All HPC, LPC, and NC stability samples met the acceptance criteria, thus anti-XTMAB-16 antibodies in human serum were stable for up to 24.5 hours under ambient conditions. Examples of these results are shown in Table 38.

[0420] Table 38: Stability Assessment (Example B)

[0421]

[0422] Acceptance criteria for bioanalytical studies in the reference standard research phase The system suitability criteria were established using data from all valid runs, excluding robustness runs, during validation. These criteria will be used as acceptance criteria for accepting or rejecting runs in future sample analyses. The %CV criterion for repeated internal measurements of the reference standard and samples is ≤ 20.0%.

[0423] Acceptance criteria for NC included screening and titration assays, and ECL:NC < plate-specific SCP and ECL ≤ 142.2 for the mean NC. These results are further summarized in Table 39.

[0424] Table 39: Acceptable Range of Standard Reference Materials

[0425]

[0426] The acceptance criteria for NC also include a confirmatory assay, in which inhibition percentage: NC < CCP (16.45%).

[0427] The acceptance criteria for LPCs include screening and titration assays, and ECL: LPC ≥ plate-specific SCP, wherein the mean ECL of LPCs is ≥ 96.8 or ≤ 156.8.

[0428] The acceptance criteria for LPC also include confirmatory assays and inhibition %: LPC ≥ CCP (16.45%).

[0429] Acceptance criteria for HPC include screening and titration determination, where the ECL of HPC is greater than that of LPC, and the mean ECL of HPC is 4695.6 ≤.

[0430] Acceptance criteria for HPC also include a confirmatory assay, in which inhibition of % HPC ≥ CCP (16.45%).

[0431] Acceptance criteria for titer reference standards include a titer reference standard value equal to ±2 × the total average titer of sensitivity operation (inclusive), and a titer of XTMAB-16 ADA PC at a concentration of 4000 ng / mL in pNHS ranging from 25600 to 102400.

[0432] Determining robustness To determine the robustness of the assays, the parameters were tested. In the screening and confirmatory assays, HPC, LPC, and NC were tested in two runs with different incubation times. The results of both runs met the method acceptance criteria, and the reference standards were within the established range, indicating the robustness of the assay. Examples of these results are shown in Tables 40 and 41.

[0433] Table 40: Assessment of robustness (Example A)

[0434]

[0435] Table 41: Assessment of robustness (Example B)

[0436]

[0437] Other embodiments

[0438] This technology includes, but is not limited to, the specific embodiments described in the following paragraphs

[0334] -

[0410] :

[0439] 1. A method for detecting anti-drug antibodies (ADAs) in a sample that bind to a target antibody including an anti-TNFα antibody, the method comprising:

[0440] (i) Incubate the sample together with a composition comprising:

[0441] (a) A first-affinity antibody comprising an anti-TNFα antibody conjugated to a first-affinity label, and

[0442] (b) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker;

[0443] (ii) Isolating the ADA that forms a complex with both the first affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label bound to the first affinity label; and

[0444] (iii) The ADA is detected by measuring the level of the detection mark above a predetermined cutoff point.

[0445] 2. The method according to Example 1, wherein separating the ADA includes:

[0446] (a) Providing a solid phase containing the second affinity marker;

[0447] (b) bringing the sample and the composition into contact with the solid phase; and

[0448] (c) Wash the solid phase.

[0449] 3. The method according to Example 1 or 2, wherein the target antibody, the affinity antibody and / or the detection antibody comprises a chimeric human-mouse monoclonal antibody IgG1-κ antibody.

[0450] 4. The method according to any one of Examples 1 to 3, wherein the target antibody, the affinity antibody and / or the detection antibody comprises SEQ ID NO: 1 and SEQ ID NO: 4.

[0451] 5. The method according to any one of Examples 2 to 4, wherein the solid phase is a plate.

[0452] 6. The method according to any one of Examples 1 to 5, wherein the first affinity label is biotin.

[0453] 7. The method according to any one of Examples 1 to 6, wherein the second affinity label is streptavidin.

[0454] 8. The method according to any one of Examples 1 to 7, wherein the detection antibody comprises a sulfonyl-tagged anti-TNFα antibody.

[0455] 9. The method according to any one of Examples 2 to 8, wherein measuring the level of the detection marker comprises:

[0456] (1) The solid phase is brought into contact with a marker, the marker being configured to generate a chemiluminescent or electrochemiluminescent signal when the marker is near the detection marker; and

[0457] (2) Detect the chemiluminescence signal or electrochemiluminescence signal.

[0458] 10. The method according to Example 9, wherein the marker is tripropylamine (TPA), and measuring the level of the detection marker further includes applying an electric current to the marker to generate a chemiluminescent or electrochemiluminescent signal.

[0459] 11. The method according to any one of Examples 2 to 10, wherein the solid phase is blocked with a blocking buffer before the sample and the composition come into contact with the solid phase.

[0460] 12. The method according to any one of Examples 1 to 11, further comprising performing steps (i) to (iii) on a control sample.

[0461] 13. The method according to Example 12, wherein the control sample is incubated together with the composition and contacted with the second affinity marker.

[0462] 14. The method according to any one of Examples 1 to 13, wherein the predetermined cutoff point corresponds to a level with a false positive rate of 5%.

[0463] 15. The method according to Example 14, wherein the 5% false positive rate is determined by measuring the detection marker in multiple control samples and calculating the 5% false positive rate.

[0464] 16. The method according to Example 9, wherein the chemiluminescence or electrochemiluminescence signal is measured as electrochemiluminescence (ECL).

[0465] 17. The method according to Example 16, wherein the predetermined cutoff point is an ECL value of at least 1.

[0466] 18. The method according to embodiment 16 or 17, wherein the predetermined cutoff point is an ECL value of approximately 1.10.

[0467] 19. The method according to any one of Examples 1 to 18, wherein the method has a sensitivity of less than 6 ng / mL.

[0468] 20. The method according to Example 19, wherein the sensitivity is less than 4 ng / mL.

[0469] 21. The method according to any one of Examples 1 to 20, wherein the method has a drug resistance of about 100 ng / mL.

[0470] 22. A method for detecting anti-drug antibodies (ADAs) in a sample that bind to a target antibody including an anti-TNFα antibody, the method comprising:

[0471] (i) Contacting the sample with one or more anti-TNFα conjugates to form a complex between the ADA and the anti-TNFα conjugate; and

[0472] (ii) Measure the level of the anti-TNFα conjugate in the complex;

[0473] This method detects ADA with a sensitivity of less than 5 ng / mL.

[0474] 23. The method according to Example 22, wherein the one or more anti-TNFα conjugates comprise an affinity antibody comprising an anti-TNFα antibody conjugated to an affinity label.

[0475] 24. The method according to Example 22 or 23, wherein the one or more anti-TNFα conjugates comprise a detection antibody comprising an anti-TNFα antibody conjugated to a detection marker.

[0476] 25. The method according to Example 23, wherein the affinity label comprises biotin.

[0477] 26. The method according to Example 24, wherein the detection marker comprises a sulfonate label.

[0478] 27. The method according to any one of Examples 22 to 26, wherein the method detects the ADA with a sensitivity of less than 3 ng / mL.

[0479] 28. The method according to any one of Examples 22 to 27, wherein the method detects the ADA with a sensitivity of 2.77 ng / mL or lower.

[0480] 29. A method for estimating the amount of anti-drug antibody (ADA) binding to a target antibody including an anti-TNFα antibody in a sample, the method comprising:

[0481] (i) Incubate the sample together with a composition comprising:

[0482] (a) An affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity marker;

[0483] (b) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker;

[0484] (ii) Isolating the ADA that forms a complex with both the affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label bound to the first affinity label;

[0485] (iii) The ADA is detected by measuring the level of the detection marker above a predetermined cutoff point;

[0486] (iv) Perform steps (i) to (iii) on a known sample with a known ADA concentration; and

[0487] (v) Compare the level of the detection marker in the sample with the level of the detection marker in the known sample.

[0488] 30. The method according to Example 29, wherein the known sample is continuously diluted into a plurality of titer samples, wherein comparing the level of the detection marker in the sample with the level of the detection marker in the known sample comprises: generating a curve of the level of the detection marker for each titer sample in the titer samples, and comparing the detection marker in the sample with the curve.

[0489] 31. A method for confirming that an anti-drug antibody (ADA) in a sample binds to a target antibody including an anti-TNFα antibody by performing a competitive binding assay, the method comprising performing the sample according to the method of Example 1, wherein the composition further comprises an unlabeled anti-TNFα antibody.

[0490] 32. The method according to Example 31, wherein the presence of the ADA in the sample is confirmed by measuring the level of the detection marker, the level of the detection marker being lower by a predetermined factor than the level of the detection marker in the absence of the unlabeled anti-TNFα antibody.

[0491] 33. A kit for detecting anti-drug antibodies (ADAs) in a sample that bind to a target antibody including an anti-TNFα antibody, the kit comprising:

[0492] (i) An affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label;

[0493] (ii) a detection antibody comprising an anti-TNFα antibody conjugated to a detection marker; and

[0494] (iii) A solid phase comprising a second affinity tag, wherein the second affinity tag is bound to the first affinity tag.

[0495] 34. The kit according to Example 33, wherein the solid phase is a plate.

[0496] 35. The kit according to Example 33 or 34, wherein the second affinity label is streptavidin.

[0497] 36. The kit according to any one of Examples 33 to 35, wherein the first affinity label is biotin.

[0498] 37. The kit according to any one of Examples 33 to 36, wherein the detection antibody comprises a sulfonyl-tagged anti-TNFα antibody.

[0499] 38. The kit according to any one of Examples 33 to 37 further comprises a marker configured to generate a chemiluminescent or electrochemiluminescent signal when the marker is in proximity to the detection marker.

[0500] 39. The kit according to Example 38, wherein the marker is tripropylamine (TPA).

[0501] 40. The kit according to any one of Examples 33 to 39, further comprising a blocking buffer.

[0502] 41. The kit according to any one of Examples 33 to 40, further comprising a washing buffer.

[0503] 42. A kit for estimating the amount of anti-drug antibody (ADA) binding to a target antibody including an anti-TNFα antibody in a sample, the kit comprising:

[0504] (i) An affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label;

[0505] (ii) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker;

[0506] (iii) a solid phase comprising a second affinity tag, wherein the second affinity tag is bound to the first affinity tag; and

[0507] (iv) Positive control.

[0508] 43. The kit according to Example 42, wherein the positive control is a plurality of titer samples.

[0509] 44. A kit for identifying anti-drug antibodies (ADAs) that bind to target antibodies including anti-TNFα antibodies, the kit comprising:

[0510] (i) An affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label;

[0511] (ii) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker;

[0512] (iii) Unlabeled anti-TNFα antibody; and

[0513] (iv) A solid phase comprising a second affinity marker, wherein the second affinity marker is bound to the first affinity marker.

[0514] 45. The use of target antibodies, including anti-TNFα antibodies, for the detection of ADA bound to the target antibody in a sample, the use including:

[0515] (i) Incubate the sample together with a composition comprising:

[0516] (a) A first-affinity antibody comprising an anti-TNFα antibody conjugated to a first-affinity label, and

[0517] (b) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker;

[0518] (ii) Isolating the ADA that forms a complex with both the first affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label bound to the first affinity label; and

[0519] (iii) The ADA is detected by measuring the level of the detection mark above a predetermined cutoff point.

[0520] 46. ​​The use according to Example 45, wherein separating the ADA comprises:

[0521] (a) Providing a solid phase containing the second affinity marker;

[0522] (b) bringing the sample and the composition into contact with the solid phase; and

[0523] (c) Wash the solid phase.

[0524] 47. The use according to Example 45 or 46, wherein the target antibody, the affinity antibody and / or the detection antibody comprises a chimeric human-mouse monoclonal IgG1-κ antibody.

[0525] 48. The use according to any one of Examples 45 to 47, wherein the target antibody, the affinity antibody, and / or the detection antibody comprises SEQ ID NO: 1 and SEQ ID NO: 4.

[0526] 49. The use according to any one of Examples 46 to 48, wherein the solid phase is a plate.

[0527] 50. The use according to any one of Examples 45 to 49, wherein the first affinity label is biotin.

[0528] 51. The use according to any one of Examples 45 to 50, wherein the second affinity label is streptavidin.

[0529] 52. The use according to any one of Examples 45 to 51, wherein the detection antibody comprises a sulfonyl-tagged anti-TNFα antibody.

[0530] 53. The use according to any one of embodiments 46 to 52, wherein measuring the level of the detection marker comprises:

[0531] (1) The solid phase is brought into contact with a marker, the marker being configured to generate a chemiluminescent or electrochemiluminescent signal when the marker is near the detection marker; and

[0532] (2) Detect the chemiluminescence signal or electrochemiluminescence signal.

[0533] 54. The use according to Example 53, wherein the marker is tripropylamine (TPA), and measuring the level of the detection marker further comprises applying an electric current to the marker to generate the chemiluminescence or electrochemiluminescence signal.

[0534] 55. The use according to any one of Examples 46 to 54, wherein the solid phase is blocked with a blocking buffer before the sample and the composition come into contact with the solid phase.

[0535] 56. The use according to any one of Examples 45 to 55 further includes performing steps (i) to (iii) on a control sample.

[0536] 57. The use as described in Example 56, wherein the control sample is incubated together with the composition and contacted with the second affinity marker.

[0537] 58. The use according to any one of Examples 45 to 57, wherein the predetermined cutoff point corresponds to a level with a false positive rate of 5%.

[0538] 59. The use according to Example 58, wherein the 5% false positive rate is determined by measuring the detection marker in multiple control samples and calculating the 5% false positive rate.

[0539] 60. The use according to Example 53, wherein the chemiluminescence or electrochemiluminescence signal is measured as electrochemiluminescence (ECL).

[0540] 61. The use according to embodiment 60, wherein the predetermined cutoff point is an ECL value of at least 1.

[0541] 62. The use according to embodiment 60 or 61, wherein the predetermined cutoff point is an ECL value of about 1.10.

[0542] 63. The use according to any one of Examples 45 to 62, wherein the method has a sensitivity of less than 6 ng / mL.

[0543] 64. The use according to Example 63, wherein the sensitivity is less than 4 ng / mL.

[0544] 65. The use according to any one of Examples 45 to 64, wherein the method has a resistance of about 100 ng / mL.

[0545] 66. The use of one or more anti-TNFα conjugates for detecting anti-drug antibodies (ADAs) in a sample that bind to a target antibody including an anti-TNFα antibody, the use including:

[0546] (i) Contacting the sample with one or more anti-TNFα conjugates to form a complex between the ADA and the anti-TNFα conjugate; and

[0547] (ii) Measure the level of the anti-TNFα conjugate in the complex;

[0548] This method detects ADA with a sensitivity of less than 5 ng / mL.

[0549] 67. The use according to Example 66, wherein the one or more anti-TNFα conjugates comprise an affinity antibody comprising an anti-TNFα antibody conjugated to an affinity label.

[0550] 68. The use according to Example 66 or 67, wherein the one or more anti-TNFα conjugates comprise a detection antibody comprising an anti-TNFα antibody conjugated to a detection marker.

[0551] 69. The use according to Example 67, wherein the affinity label comprises biotin.

[0552] 70. The use according to Example 68, wherein the detection mark comprises a sulfonate label.

[0553] 71. The use according to any one of Examples 66 to 70, wherein the method detects ADA with a sensitivity of less than 3 ng / mL.

[0554] 72. The use according to any one of Examples 66 to 71, wherein the method detects ADA with a sensitivity of 2.77 ng / mL or lower.

[0555] 73. The use of a target antibody, including an anti-TNFα antibody, to estimate the amount of an anti-drug antibody (ADA) binding to the target antibody, including the anti-TNFα antibody, in a sample, the use including:

[0556] (i) Incubate the sample together with a composition comprising:

[0557] (a) An affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity marker;

[0558] (b) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker;

[0559] (ii) Isolating the ADA that forms a complex with both the affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label bound to the first affinity label;

[0560] (iii) The ADA is detected by measuring the level of the detection marker above a predetermined cutoff point;

[0561] (iv) Perform steps (i) to (iii) on a known sample with a known ADA concentration; and

[0562] (v) Compare the level of the detection marker in the sample with the level of the detection marker in the known sample.

[0563] 74. The use according to Example 73, wherein the known sample is continuously diluted into a plurality of titer samples, wherein comparing the level of the detection marker in the sample with the level of the detection marker in the known sample includes generating a curve of the level of the detection marker for each titer sample in the titer samples, and comparing the detection marker in the sample with the curve.

[0564] 75. The use of a target antibody including an anti-TNFα antibody for confirming, by performing a competitive binding assay, an anti-drug antibody (ADA) in a sample that binds to the target antibody including the anti-TNFα antibody, comprising performing the method according to Example 1 on the sample, wherein the composition further comprises an unlabeled anti-TNFα antibody.

[0565] 76. The use according to Example 75, wherein the ADA in the sample is confirmed by measuring the level of the detection marker, the level of the detection marker being lower by a predetermined factor than the level of the detection marker in the absence of the unlabeled anti-TNFα antibody.

[0566] In summary, it should be understood that specific embodiments of the present technology have been described herein for illustrative purposes, but various modifications can be made without departing from the scope of the present technology. Therefore, the present technology is limited only by the appended claims.

Claims

1. A method for detecting anti-drug antibodies (ADAs) in a sample that bind to a target antibody including an anti-TNFα antibody, the method comprising: (i) Incubate the sample together with a composition comprising: (a) A first-affinity antibody comprising an anti-TNFα antibody conjugated to a first-affinity label, and (b) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker; (ii) Isolating the ADA that forms a complex with both the first affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label bound to the first affinity label; as well as (iii) The ADA is detected by measuring the level of the detection marker above a predetermined cutoff point.

2. The method of claim 1, wherein separating the ADA comprises: (a) Providing a solid phase containing the second affinity tag; (b) Contact the sample and the composition with the solid phase; as well as (c) Wash the solid phase.

3. The method according to claim 1 or 2, wherein the target antibody, the affinity antibody and / or the detection antibody comprises a chimeric human-mouse monoclonal IgG1-κ antibody.

4. The method according to any one of claims 1 to 3, wherein the target antibody, the affinity antibody and / or the detection antibody comprises SEQ ID NO: 1 and SEQ ID NO:

4.

5. The method according to any one of claims 2 to 4, wherein the solid phase is a plate.

6. The method according to any one of claims 1 to 5, wherein the first affinity label is biotin.

7. The method according to any one of claims 1 to 6, wherein the second affinity marker is streptavidin.

8. The method according to any one of claims 1 to 7, wherein the detection antibody comprises a sulfonyl-tagged anti-TNFα antibody.

9. The method according to any one of claims 2 to 8, wherein measuring the level of the detection marker comprises: (1) The solid phase is brought into contact with a marker, the marker being configured to generate a chemiluminescence signal or an electrochemiluminescence signal when the marker is near the detection mark; as well as (2) Detect the chemiluminescence signal or electrochemiluminescence signal.

10. The method of claim 9, wherein the marker is tripropylamine (TPA), and measuring the level of the detection marker further comprises applying an electric current to the marker to generate the chemiluminescent signal or electrochemiluminescent signal.

11. The method according to any one of claims 2 to 10, wherein the solid phase is blocked with a blocking buffer before the sample and the composition are contacted with the solid phase.

12. The method according to any one of claims 1 to 11, further comprising performing steps (i) to (iii) on a control sample.

13. The method of claim 12, wherein the control sample is incubated together with the composition and contacted with the second affinity label.

14. The method according to any one of claims 1 to 13, wherein the predetermined cutoff point corresponds to a level with a false positive rate of 5%.

15. The method of claim 14, wherein the 5% false positive rate is determined by measuring the detection marker in a plurality of control samples and calculating the 5% false positive rate.

16. The method of claim 9, wherein the chemiluminescence signal or electrochemiluminescence signal is measured as electrochemiluminescence (ECL).

17. The method of claim 16, wherein the predetermined cutoff point is an ECL value of at least 1.

18. The method of claim 16 or 17, wherein the predetermined cutoff point is an ECL value of about 1.

10.

19. The method according to any one of claims 1 to 18, wherein the method has a sensitivity of less than 6 ng / mL.

20. The method of claim 19, wherein the sensitivity is less than 4 ng / mL.

21. The method according to any one of claims 1 to 20, wherein the method has a drug resistance of about 100 ng / mL.

22. A method for detecting an anti-drug antibody (ADA) in a sample that binds to a target antibody including an anti-TNFα antibody, the method comprising: (i) Contact the sample with one or more anti-TNFα conjugates to form a complex between the ADA and the anti-TNFα conjugate; as well as (ii) Measure the level of the anti-TNFα conjugate in the complex; The method described herein detects the ADA with a sensitivity of less than 5 ng / mL.

23. The method of claim 22, wherein the one or more anti-TNFα conjugates comprise an affinity antibody, the affinity antibody comprising an anti-TNFα antibody conjugated to an affinity label.

24. The method of claim 22 or 23, wherein the one or more anti-TNFα conjugates comprise a detection antibody, the detection antibody comprising an anti-TNFα antibody conjugated to a detection marker.

25. The method of claim 23, wherein the affinity marker comprises biotin.

26. The method of claim 24, wherein the detection marker comprises a sulfonate label.

27. The method according to any one of claims 22 to 26, wherein the method detects the ADA with a sensitivity of less than 3 ng / mL.

28. The method according to any one of claims 22 to 27, wherein the method detects the ADA with a sensitivity of 2.77 ng / mL or lower.

29. A method for estimating the amount of anti-drug antibody (ADA) binding to a target antibody including an anti-TNFα antibody in a sample, the method comprising: (i) Incubate the sample together with a composition comprising: (a) An affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity marker; (b) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker; (ii) Isolating the ADA that forms a complex with both the affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label bound to the first affinity label; (iii) The ADA is detected by measuring the level of the detection marker above a predetermined cutoff point; (iv) Perform steps (i) to (iii) on known samples with known ADA concentrations; as well as (v) Compare the level of the detection marker in the sample with the level of the detection marker in the known sample.

30. The method of claim 29, wherein the known sample is serially diluted into a plurality of titer samples, wherein comparing the level of the detection marker in the sample with the level of the detection marker in the known sample comprises generating a curve of the level of the detection marker for each titer sample and comparing the detection marker in the sample with the curve.

31. A method for confirming that an anti-drug antibody (ADA) in a sample binds to a target antibody including an anti-TNFα antibody by performing a competitive binding assay, the method comprising performing the sample according to the method of claim 1, wherein the composition further comprises an unlabeled anti-TNFα antibody.

32. The method of claim 31, wherein the ADA in the sample is confirmed by measuring the level of the detection marker, the level of the detection marker being lower by a predetermined factor than the level of the detection marker in the absence of the unlabeled anti-TNFα antibody.

33. A kit for detecting anti-drug antibodies (ADAs) in a sample that bind to a target antibody including an anti-TNFα antibody, said kit comprising: (i) An affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label; (ii) a detection antibody comprising an anti-TNFα antibody conjugated to a detection marker; and (iii) A solid phase comprising a second affinity marker, wherein the second affinity marker is bound to the first affinity marker.

34. The kit according to claim 33, wherein the solid phase is a plate.

35. The kit according to claim 33 or 34, wherein the second affinity label is streptavidin.

36. The kit according to any one of claims 33 to 35, wherein the first affinity label is biotin.

37. The kit according to any one of claims 33 to 36, wherein the detection antibody comprises a sulfonyl-tagged anti-TNFα antibody.

38. The kit according to any one of claims 33 to 37, further comprising a marker configured to generate a chemiluminescent or electrochemiluminescent signal when the marker is near the detection marker.

39. The kit according to claim 38, wherein the marker is tripropylamine (TPA).

40. The kit according to any one of claims 33 to 39, further comprising a blocking buffer.

41. The kit according to any one of claims 33 to 40, further comprising a washing buffer.

42. A kit for estimating the amount of anti-drug antibody (ADA) binding to a target antibody including an anti-TNFα antibody in a sample, said kit comprising: (i) An affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label; (ii) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker; (iii) a solid phase comprising a second affinity tag, wherein the second affinity tag is bound to the first affinity tag; and (iv) Positive control.

43. The kit according to claim 42, wherein the positive control is a plurality of titer samples.

44. A kit for identifying anti-drug antibodies (ADAs) that bind to a target antibody including an anti-TNFα antibody, said kit comprising: (i) An affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label; (ii) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker; (iii) Unlabeled anti-TNFα antibody; and (iv) A solid phase comprising a second affinity marker, wherein the second affinity marker is bound to the first affinity marker.

45. The use of a target antibody, including an anti-TNFα antibody, for detecting ADA bound to the target antibody in a sample, the use comprising: (i) Incubate the sample together with a composition comprising: (a) A first-affinity antibody comprising an anti-TNFα antibody conjugated to a first-affinity label, and (b) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker; (ii) Isolating the ADA that forms a complex with both the first affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label bound to the first affinity label; as well as (iii) The ADA is detected by measuring the level of the detection marker above a predetermined cutoff point.

46. ​​The use according to claim 45, wherein separating the ADA comprises: (a) Providing a solid phase containing the second affinity tag; (b) Contact the sample and the composition with the solid phase; as well as (c) Wash the solid phase.

47. The use according to claim 45 or 46, wherein the target antibody, the affinity antibody, and / or the detection antibody comprises a chimeric human-mouse monoclonal IgG1-κ antibody.

48. The use according to any one of claims 45 to 47, wherein the target antibody, the affinity antibody and / or the detection antibody comprises SEQ ID NO: 1 and SEQ ID NO:

4.

49. The use according to any one of claims 46 to 48, wherein the solid phase is a plate.

50. The use according to any one of claims 45 to 49, wherein the first affinity label is biotin.

51. The use according to any one of claims 45 to 50, wherein the second affinity marker is streptavidin.

52. The use according to any one of claims 45 to 51, wherein the detection antibody comprises a sulfonyl-tagged anti-TNFα antibody.

53. The use according to any one of claims 46 to 52, wherein measuring the level of the detection marker comprises: (1) The solid phase is brought into contact with a marker, the marker being configured to generate a chemiluminescence signal or an electrochemiluminescence signal when the marker is near the detection mark; as well as (2) Detect the chemiluminescence signal or electrochemiluminescence signal.

54. The use according to claim 53, wherein the marker is tripropylamine (TPA), and measuring the level of the detection marker further comprises applying an electric current to the marker to generate the chemiluminescent signal or electrochemiluminescent signal.

55. The use according to any one of claims 46 to 54, wherein the solid phase is blocked with a blocking buffer before the sample and the composition are contacted with the solid phase.

56. The use according to any one of claims 45 to 55, further comprising performing steps (i) to (iii) on a control sample.

57. The use according to claim 56, wherein the control sample is incubated together with the composition and contacted with the second affinity marker.

58. The use according to any one of claims 45 to 57, wherein the predetermined cutoff point corresponds to a level having a false positive rate of 5%.

59. The use according to claim 58, wherein the 5% false positive rate is determined by measuring the detection marker in a plurality of control samples and calculating the 5% false positive rate.

60. The use according to claim 53, wherein the chemiluminescence signal or electrochemiluminescence signal is measured as electrochemiluminescence (ECL).

61. The use according to claim 60, wherein the predetermined cutoff point is an ECL value of at least 1.

62. The use according to claim 60 or 61, wherein the predetermined cutoff point is an ECL value of about 1.

10.

63. The use according to any one of claims 45 to 62, wherein the method has a sensitivity of less than 6 ng / mL.

64. The use according to claim 63, wherein the sensitivity is less than 4 ng / mL.

65. The use according to any one of claims 45 to 64, wherein the method has a resistance of about 100 ng / mL.

66. The use of one or more anti-TNFα conjugates for detecting anti-drug antibodies (ADAs) in a sample that bind to a target antibody including an anti-TNFα antibody, said use comprising: (i) Contact the sample with one or more anti-TNFα conjugates to form a complex between the ADA and the anti-TNFα conjugate; as well as (ii) Measure the level of the anti-TNFα conjugate in the complex; The method described herein detects the ADA with a sensitivity of less than 5 ng / mL.

67. The use according to claim 66, wherein the one or more anti-TNFα conjugates comprise an affinity antibody, the affinity antibody comprising an anti-TNFα antibody conjugated to an affinity label.

68. The use according to claim 66 or 67, wherein the one or more anti-TNFα conjugates comprise a detection antibody, the detection antibody comprising an anti-TNFα antibody conjugated to a detection marker.

69. The use according to claim 67, wherein the affinity marker comprises biotin.

70. The use according to claim 68, wherein the detection mark comprises a sulfonate label.

71. The use according to any one of claims 66 to 70, wherein the method detects the ADA with a sensitivity of less than 3 ng / mL.

72. The use according to any one of claims 66 to 71, wherein the method detects the ADA with a sensitivity of 2.77 ng / mL or lower.

73. The use of a target antibody including an anti-TNFα antibody for estimating the amount of an anti-drug antibody (ADA) binding to the target antibody including the anti-TNFα antibody in a sample, the use including: (i) Incubate the sample together with a composition comprising: (a) An affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity marker; (b) Detection antibody, which contains an anti-TNFα antibody conjugated to a detection marker; (ii) Isolating the ADA that forms a complex with both the affinity antibody and the detection antibody, wherein the ADA is isolated by contacting the complex with a second affinity label bound to the first affinity label; (iii) The ADA is detected by measuring the level of the detection marker above a predetermined cutoff point; (iv) Perform steps (i) to (iii) on known samples with known ADA concentrations; as well as (v) Compare the level of the detection marker in the sample with the level of the detection marker in the known sample.

74. The use according to claim 73, wherein the known sample is continuously diluted into a plurality of titer samples, wherein comparing the level of the detection marker in the sample with the level of the detection marker in the known sample comprises generating a curve of the level of the detection marker for each titer sample in the titer samples and comparing the detection marker in the sample with the curve.

75. The use of a target antibody comprising an anti-TNFα antibody for confirming, by performing a competitive binding assay, an anti-drug antibody (ADA) comprising the target antibody comprising the anti-TNFα antibody in a sample, the use comprising performing the method of claim 1 on the sample, wherein the composition further comprises an unlabeled anti-TNFα antibody.

76. The use according to claim 75, wherein the ADA in the sample is confirmed by measuring the level of the detection marker, the level of the detection marker being lower by a predetermined factor than the level of the detection marker in the absence of the unlabeled anti-TNFα antibody.

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