Method for detecting neutralizing antibody (NAb) against anti-TNF alpha antibody

Abstract

The present technology includes a method for detecting a neutralizing antibody (NAb) against a target antibody. In some embodiments, the target antibody comprises an anti-tumor necrosis factor-alpha (TNF alpha) antibody. In some embodiments, the target antibody comprises SEQ ID NO: 9 to 11 or 15 to 17 and SEQ ID NO: 12 to 14 or 18 to 20. The technology of the invention also includes kits for performing the methods of the technology of the invention.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 506,911, filed June 8, 2023. The contents of that provisional application are incorporated herein by reference in their entirety.

[0002] sequence list This application contains a sequence list conforming to ST.26, which was submitted in XML format via the Patent Centre and incorporated herein by reference in its entirety. A copy of the .xml file created on May 17, 2024, is named 140505-8006WO00_SL.xml and has a size of 20,939 bytes. Technical Field

[0003] The present invention includes an in vitro assay for identifying neutralizing antibodies (NAb). Background Technology

[0004] Anti-tumor necrosis factor-α (TNFα) antibodies are novel biological agents with the potential to treat a variety of diseases. One obstacle to the development and implementation of anti-TNFα antibodies as a treatment is that administration of such antibodies to subjects may trigger an immune response against them. The immunogenicity of anti-TNFα antibodies in subjects, particularly the production of neutralizing antibodies, will therefore reduce the efficacy of anti-TNFα antibodies in treating the subjects' diseases. To advance the development of treatments using anti-TNFα antibodies, reliable methods are needed to determine the presence and quantity of anti-TNFα neutralizing antibodies in subjects who have received or been administered them. Summary of the Invention

[0005] In some embodiments, the present invention includes a method for detecting a neutralizing antibody (NAb) against a target antibody in a sample, comprising: (i) dissociating the NAb from the target antibody by adding an acid wash to the sample; (ii) purifying the NAb in the sample; (iii) contacting the purified NAb with a detection antibody comprising an anti-TNFα antibody conjugated to a detection label; (iv) contacting the purified NAb and the detection antibody with a TNFα-coated substrate; and (v) detecting the NAb when the detection antibody binds to the NAb but not to the TNFα-coated substrate, wherein the target antibody comprises an anti-TNFα chimeric human-mouse monoclonal IgG1-κ antibody containing SEQ ID NO: 9-11 or 15-17 and SEQ ID NO: 12-14 or 18-20.

[0006] In some implementations, the method also includes detecting the detection marker before detecting NAb.

[0007] In some implementations, the method further includes washing the TNFα-coated substrate before detecting NAb.

[0008] In some implementations, the pickling solution contains acetic acid.

[0009] In some embodiments, the purification step (ii) of NAb includes (a) contacting the sample with an affinity antibody containing an anti-TNFα antibody conjugated to a first affinity label; (b) contacting the sample with a first solid phase coated with a second affinity label, wherein the first affinity label binds to the second affinity label; (c) washing the first solid phase; and (d) eluting the NAb from the first solid phase.

[0010] In some implementations, the first affinity marker is biotin.

[0011] In some embodiments, the first solid phase is microspheres. In some embodiments, the first solid phase is magnetic microspheres.

[0012] In some implementations, the second affinity marker is streptavidin.

[0013] In some implementations, NAb is purified using an automated purification system.

[0014] In some embodiments, the detection tag comprises a luminescent compound. In some embodiments, the present invention includes a method in which the detection tag comprises an enzyme configured to convert a chromogenic substrate into a pigment. In some embodiments, the present invention includes a method in which the detection tag is a third affinity tag.

[0015] In some embodiments, detecting the detection marker further includes contacting a third affinity marker with a fourth affinity marker, the fourth affinity marker being conjugated to a luminescent compound or an enzyme configured to convert a chromogenic substrate into a pigment, wherein the third affinity marker binds to the fourth affinity marker.

[0016] In some implementations, the third affinity marker is biotin, and the fourth affinity marker is streptavidin.

[0017] In some embodiments, detecting the detection marker includes detecting a luminescent compound. In some embodiments, detecting the detection marker includes converting a chromogenic substrate into a pigment and detecting 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.

[0018] In some implementations, the enzyme is horseradish peroxidase (HRP).

[0019] In some implementations, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB).

[0020] In some implementations, steps (i) to (v) are performed on negative control samples that do not have detectable NAb.

[0021] In some embodiments, the detection antibody binds to the substrate coated with NAb instead of TNFα when the detection marker is present at a level below a predetermined level. In some embodiments, the detection marker is present at a level below a predetermined level when the optical density of the sample is above a predetermined cutoff point.

[0022] In some implementations, the predetermined cutoff point corresponds to a false positive rate of 3% or lower. In some implementations, the predetermined cutoff point corresponds to a false positive rate of 1%. In some implementations, the predetermined cutoff point is 0.5-2. In some implementations, the predetermined cutoff point is 0.91.

[0023] In some implementations, the sensitivity for detecting NAb is less than 500 ng / mL. In some implementations, the sensitivity for detecting NAb is less than 250 ng / mL. In some implementations, the sensitivity for detecting NAb is less than 150 ng / mL.

[0024] In some implementations, the target tolerance is approximately 1000 ng / mL.

[0025] In some embodiments, the target antibody comprises SEQ ID NO: 2 and 5. In some embodiments, the target antibody comprises SEQ ID NO: 1 and 4.

[0026] In some embodiments, the present invention includes a method for detecting NAb (neutralizing antibody) against a target antibody in a sample, comprising: (i) purifying the NAb in the sample; (ii) determining the presence of the NAb in the sample by performing a competitive binding assay, wherein the NAb is present if the NAb binds to an anti-TNFα antibody with a higher affinity for TNFα than the anti-TNFα antibody binds to TNFα; and (iii) detecting the NAb; wherein the method has a sensitivity of 250 ng / mL or less, wherein the target antibody comprises an anti-TNFα chimeric human-mouse monoclonal IgG1-κ antibody containing SEQ ID NO: 9-11 or 15-17 and SEQ ID NO: 12-14 or 18-20.

[0027] In some implementations, the method further includes dissociating the NAb from the target antibody by adding an acid washing solution to the sample prior to purification.

[0028] In some embodiments, the competitive binding assay includes: (a) contacting purified NAb with a detection antibody containing an anti-TNFα antibody conjugated to a detection tag; (b) contacting the purified NAb and the detection antibody with a TNFα-coated substrate; (c) washing the TNFα-coated substrate; and (d) detecting the detection tag.

[0029] In some implementations, detecting NAb includes determining whether the detection marker is present at a level below a predetermined level.

[0030] In some implementations, when the optical density is above a predetermined cutoff point, the detection marker is present at a level below a predetermined level.

[0031] In some implementations, the pickling solution contains acetic acid.

[0032] In some embodiments, purifying NAb includes: (a) contacting a sample with an affinity antibody containing an anti-TNFα antibody conjugated to a first affinity label; (b) contacting the sample with a first solid phase coated with a second affinity label, wherein the first affinity label binds to the second affinity label; (c) washing the first solid phase; and (d) eluting the NAb from the first solid phase.

[0033] In some implementations, the first affinity marker is biotin.

[0034] In some embodiments, the first solid phase is microspheres. In some embodiments, the first solid phase is magnetic microspheres.

[0035] In some implementations, the second affinity marker is streptavidin.

[0036] In some implementations, NAb is purified using an automated purification system.

[0037] In some implementations, the detection marker contains a luminescent compound.

[0038] In some embodiments, the detection tag comprises an enzyme configured to convert a chromogenic substrate into a pigment. In some embodiments, the detection tag is a third affinity tag.

[0039] In some embodiments, detecting the detection marker further includes contacting a third affinity marker with a fourth affinity marker, the fourth affinity marker being conjugated to a luminescent compound or an enzyme configured to convert a chromogenic substrate into a pigment, wherein the third affinity marker binds to the fourth affinity marker.

[0040] In some implementations, the third affinity marker is biotin, and the fourth affinity marker is streptavidin.

[0041] In some embodiments, detecting the detection marker includes detecting a luminescent compound. In some embodiments, detecting the detection marker includes converting a chromogenic substrate into a pigment and detecting 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.

[0042] In some implementations, the enzyme is horseradish peroxidase (HRP).

[0043] In some implementations, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB).

[0044] In some implementations, steps (i) to (iii) are performed on negative control samples that do not contain detectable NAb.

[0045] In some implementations, the predetermined cutoff point corresponds to a 1% false positive rate. In some implementations, the predetermined cutoff point is 0.91.

[0046] In some implementations, the sensitivity for detecting NAb is less than 500 ng / mL. In some implementations, the sensitivity for detecting NAb is less than 250 ng / mL. In some implementations, the sensitivity for detecting NAb is less than 150 ng / mL.

[0047] In some implementations, the target tolerance is approximately 1000 ng / mL.

[0048] In some embodiments, the target antibody comprises SEQ ID NO: 2 and 5. In some embodiments, the target antibody comprises SEQ ID NO: 1 and 4.

[0049] In some embodiments, the present invention includes a kit for detecting a neutralizing antibody (NAb) against a target antibody in a sample, the target antibody comprising an anti-TNFα chimeric human-mouse monoclonal IgG1-κ antibody containing SEQ ID NO: 9-11 or 15-17 and SEQ ID NO: 12-14 or 18-20, the kit comprising: (i) an affinity antibody containing the target antibody conjugated to a first affinity label; (ii) a first solid phase coated with a second affinity label; (iii) a detection antibody containing the target antibody conjugated to a detection label; (iv) a substrate; and (v) TNFα, wherein the target antibody comprises an anti-TNFα chimeric human-mouse monoclonal IgG1-κ antibody containing SEQ ID NO: 9-11 or 15-17 and SEQ ID NO: 12-14 or 18-20.

[0050] In some implementations, the first affinity marker is biotin.

[0051] In some implementations, the second affinity marker is streptavidin.

[0052] In some implementations, the detection marker is a luminescent compound, an enzyme configured to convert a chromogenic substrate into a pigment, or biotin.

[0053] In some embodiments, the first solid phase is microspheres. In some embodiments, the first solid phase is magnetic microspheres.

[0054] In some implementations, the detection marker is biotin.

[0055] In some implementations, the kit also includes an acid washing buffer.

[0056] In some implementations, the kit also includes a washing buffer.

[0057] In some implementations, the kit also includes an elution buffer. Attached Figure Description

[0058] Figure 1 A flowchart illustrating an example method of the present invention is shown.

[0059] Figure 2 A flowchart illustrating another example method of the present invention is shown.

[0060] Figure 3 An example normal distribution of 295 log-transformed normalized values ​​from a control sample prepared by a method according to the present invention is shown. Detailed Implementation

[0061] This invention includes a method for detecting neutralizing antibodies (NAbs) against a target antibody. The target antibody may comprise 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: 9-11 and the light chain CDR of SEQ ID NO: 12-14. In some embodiments, the target antibody comprises the heavy chain CDR of SEQ ID NO: 15-17 and the light chain CDR of SEQ ID NO: 18-20. 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, in a sample obtained from a subject who has received or been administered the target antibody, the target antibody comprises the heavy chain of SEQ ID NO: 1 and the light chain of SEQ ID NO: 4. This invention also includes a kit for detecting NAbs in a sample using a method according to the invention.

[0062] definition The term "about" means a quantity, level, value, number, frequency, percentage, size, volume, weight, or length that varies according to levels acceptable in the art. Typically, such variation can be up to 10% above or below a reference quantity, level, value, number, frequency, percentage, size, volume, weight, or length, and such variation can be influenced by standard applicable measurement practices. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated value.

[0063] As used herein, an "affinity antibody" includes an anti-TNFα antibody. In some embodiments, the affinity antibody includes a target antibody. An affinity antibody may also include an affinity marker having high affinity for a separate label. 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, the affinity antibody includes an anti-TNFα antibody and an affinity marker. In some embodiments, the affinity antibody includes a target antibody and an affinity marker.

[0064] As used herein, "detection antibody" includes a TNFα antibody. In some embodiments, the detection antibody includes a target antibody. The detection antibody may also include a detection marker that can be detected directly or indirectly. In some embodiments, the detection antibody includes an anti-TNFα antibody and a detection marker. In some embodiments, the detection antibody includes a target antibody and a detection marker.

[0065] In some embodiments, a method for detecting NAbs against a target antibody in a sample includes (i) dissociating the NAbs from the target antibody by adding an acid wash solution to the sample; (ii) purifying the NAbs from the sample; (iii) contacting the purified NAbs with an anti-TNFα antibody conjugate (i.e., "detection antibody") containing an anti-TNFα antibody conjugated to a detection tag; (iv) contacting the purified NAbs and the detection antibody with a TNFα-coated substrate; and (v) detecting the NAbs by determining whether the detection tag is present at a level above a predetermined cutoff point.

[0066] In some implementations, a method for detecting NAbs against a target antibody includes (i) purifying the NAb in a sample; (ii) determining the presence of the NAb in the sample by performing a competitive binding assay, wherein the NAb is present if the NAb binds to the detection antibody with a higher affinity than the detection antibody binds to TNFα; and (iii) detecting the NAb; wherein the method has a sensitivity of about 250 ng / mL or less.

[0067] In some embodiments, the competitive binding assay includes (a) contacting the purified NAb with a detection antibody; (b) contacting the purified NAb and the detection antibody with a TNFα-coated substrate; (c) washing the TNFα-coated substrate; and (d) detecting the detection label.

[0068] 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 the method according to the invention is used to detect NAb. 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).

[0069] Antibody Neutralizing antibodies (NAb) During or after administration of a drug (e.g., a therapeutic drug) to a subject, the subject's immune system may respond by producing NAbs. NAbs can bind at least a portion of the drug, thereby isolating, neutralizing, and / or reducing the drug's efficacy. In some embodiments, the drug is a drug antibody. When NAbs are present, they can bind at least a portion of the drug antibody, including but not limited to binding the heavy chain, light chain, or variable region (e.g., the heavy chain variable region domain (V)) of the drug antibody. H ) or light chain variable region structural domain (V L One or more amino acids of the constant region, Fc domain, Fab domain, hinge region, disulfide bond, or complementarity-determining region (CDR).

[0070] NAbs can be naturally produced as part of a subject's innate immune response. NAbs can be subject-generated, such as naturally occurring antibodies produced by lymphocytes using variable (diversity) linker (V(D)J) recombination. For example, NAbs can be immunoglobulins. In some embodiments, NAbs are IgG, IgA, IgE, monoclonal antibodies, polyclonal antibodies, or any other type of antibody capable of binding to a target antibody.

[0071] Target antibody The presence of NAbs and / or the binding of NAbs to drug antibodies 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. Anti-TNFα antibodies are antibodies that bind to at least a portion of the TNFα protein or peptide.

[0072] In some implementations, the target antibody comprises a monoclonal antibody.

[0073] In some embodiments, the target antibody of the present invention 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).

[0074] In some embodiments, the target antibody of the present invention 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 IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, and IgE. The light chain may comprise an isotype kappa (κ) or an isotype lambda (λ). In some embodiments, the target antibody comprises an IgG1-κ antibody.

[0075] Target antibody sequence The target antibody of this invention may comprise an anti-TNFα antibody or at least a portion thereof. For example, the target antibody may comprise a heavy chain, a light chain, or a variable region (e.g., V) derived from an anti-TNFα antibody. H or V L The NAb of this invention can bind to one or more amino acid residues of the amino acid sequence, including the following: structural domain, constant region, Fc domain, Fab domain, hinge region, disulfide bond, or CDR.

[0076] In some implementations, the portion of the anti-TNFα antibody contains approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as those in Table 1.

[0077] In some implementations, the portion of the anti-TNFα antibody contains at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as those in Table 1.

[0078] In some implementations, the portion of the anti-TNFα antibody contains at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as those in Table 1.

[0079] Heavy chain ： The target antibody of the present invention comprises a heavy chain having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 1.

[0080] The target antibody of the present invention comprises a heavy chain having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 1.

[0081] The target antibody of the present invention comprises a heavy chain having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 1.

[0082] The heavy chain of the target antibody may contain a variable region domain (V) having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 2. H ).

[0083] The heavy chain of the target antibody may contain a V having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 2. H .

[0084] The heavy chain of the target antibody may contain a V having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 2. H .

[0085] The heavy chain of the target antibody may contain a constant region having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 3.

[0086] The heavy chain of the target antibody may contain a constant region having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 3.

[0087] The heavy chain of the target antibody may contain a constant region having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 3.

[0088] The target antibody of this invention comprises a heavy chain having at least one complementarity-determining region (CDR) sequence. At least one CDR sequence confers binding to a specific antigen (e.g., TNFα peptide).

[0089] In some embodiments, the target antibody comprises a heavy chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 9.

[0090] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 9.

[0091] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 9.

[0092] In some embodiments, the target antibody comprises a heavy chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 10.

[0093] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 10.

[0094] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 10.

[0095] In some embodiments, the target antibody comprises a heavy chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 11.

[0096] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 11.

[0097] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 11.

[0098] In some embodiments, the target antibody comprises a heavy chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 15.

[0099] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 15.

[0100] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 15.

[0101] In some embodiments, the target antibody comprises a heavy chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 16.

[0102] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 16.

[0103] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 16.

[0104] In some embodiments, the target antibody comprises a heavy chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 17.

[0105] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 17.

[0106] In some embodiments, the target antibody comprises a heavy chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 17.

[0107] In some embodiments, the target antibody comprises a heavy chain comprising: (1) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 9; (2) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 10; and (3) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 11.

[0108] In some embodiments, the target antibody comprises a heavy chain comprising: (1) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 9; (2) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 10; and (3) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 11.

[0109] In some embodiments, the target antibody comprises a heavy chain comprising: (1) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 9; (2) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 10; and (3) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 11.

[0110] In some embodiments, the target antibody comprises a heavy chain comprising: (1) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 15; (2) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 16; and (3) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 17.

[0111] In some embodiments, the target antibody comprises a heavy chain comprising: (1) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 15; (2) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 16; and (3) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 17.

[0112] In some embodiments, the target antibody comprises a heavy chain comprising: (1) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 15; (2) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 16; and (3) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 17.

[0113] In some embodiments, the heavy chain of the target antibody of the present invention is encoded by about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same nucleotide sequence as SEQ. ID NO: 8.

[0114] In some embodiments, the heavy chain of the target antibody of the present invention is encoded by at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same nucleotide sequence as SEQ. ID NO: 8.

[0115] In some embodiments, the heavy chain of the target antibody of the present invention is encoded by at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same nucleotide sequence as SEQ. ID NO: 8.

[0116] Light chain: The target antibody of the present invention comprises a light chain having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 4.

[0117] The target antibody of the present invention comprises a light chain having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 4.

[0118] The target antibody of the present invention comprises a light chain having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 4.

[0119] The light chain of the target antibody may contain a variable region domain (V) having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 5. L ).

[0120] The light chain of the target antibody may contain a V having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 5. L .

[0121] The light chain of the target antibody may contain a V having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 5. L .

[0122] The light chain of the target antibody may contain a constant region having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 6.

[0123] The light chain of the target antibody may contain a constant region having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 6.

[0124] The light chain of the target antibody may contain a constant region having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 6.

[0125] The target antibody of this invention 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).

[0126] In some embodiments, the target antibody comprises a light chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 12.

[0127] In some embodiments, the target antibody comprises a light chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 12.

[0128] In some embodiments, the target antibody comprises a light chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 12.

[0129] In some embodiments, the target antibody comprises a light chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 13.

[0130] In some embodiments, the target antibody comprises a light chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 13.

[0131] In some embodiments, the target antibody comprises a light chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 13.

[0132] In some embodiments, the target antibody comprises a light chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 14.

[0133] In some embodiments, the target antibody comprises a light chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 14.

[0134] In some embodiments, the target antibody comprises a light chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 14.

[0135] In some embodiments, the target antibody comprises a light chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 18.

[0136] In some embodiments, the target antibody comprises a light chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 18.

[0137] In some embodiments, the target antibody comprises a light chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 18.

[0138] In some embodiments, the target antibody comprises a light chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 19.

[0139] In some embodiments, the target antibody comprises a light chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 19.

[0140] In some embodiments, the target antibody comprises a light chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 19.

[0141] In some embodiments, the target antibody comprises a light chain having a CDR having approximately 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 20.

[0142] In some embodiments, the target antibody comprises a light chain having a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 20.

[0143] In some embodiments, the target antibody comprises a light chain having a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 20.

[0144] In some embodiments, the target antibody comprises a light chain comprising: (1) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 12; (2) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 13; and (3) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 14.

[0145] In some embodiments, the target antibody comprises a light chain comprising: (1) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 12; (2) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 13; and (3) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 14.

[0146] In some embodiments, the target antibody comprises a light chain comprising: (1) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 12; (2) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 13; and (3) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 14.

[0147] In some embodiments, the target antibody comprises a light chain comprising: (1) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 18; (2) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 19; and (3) a CDR having about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 20.

[0148] In some embodiments, the target antibody comprises a light chain comprising: (1) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 18; (2) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 19; and (3) a CDR having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 20.

[0149] In some embodiments, the target antibody comprises a light chain comprising: (1) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 18; (2) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 19; and (3) a CDR having at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same amino acid sequence as SEQ. ID NO: 20.

[0150] In some embodiments, the light chain of the target antibody of the present invention is encoded by about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same nucleotide sequence as SEQ. ID NO: 7.

[0151] In some embodiments, the light chain of the target antibody of the present invention is encoded by a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to that of SEQ. ID NO: 7.

[0152] In some embodiments, the light chain of the target antibody of the present invention is encoded by at least about 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the same nucleotide sequence as SEQ. ID NO: 7.

[0153] Table 1: Sequences of example anti-TNFα antibodies of the present invention

[0154] Dissociation of NAb Pickling solution In some embodiments, the method includes dissociating the NAb 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 the antibody from its target. In some embodiments, the acid wash solution may contain acetic acid.

[0155] In some embodiments, the pickling solution contains about 2M acetic acid. In some embodiments, the pickling solution contains about 300mM acetic acid. In some embodiments, the pickling solution contains 0.1M glycine-HCl.

[0156] In some embodiments, the pickling solution contains about 10 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1 M, about 1.5 M, about 2 M, about 2.5 M, about 3 M, about 3.5 M, about 4 M, about 4.5 M, or about 5 M acetic acid.

[0157] In some embodiments, the pickling solution contains at least 10 mM, at least 50 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 500 mM, at least 600 mM, at least 700 mM, at least 800 mM, at least 900 mM, at least 1 M, at least 1.5 M, at least 2 M, at least 2.5 M, at least 3 M, at least 3.5 M, at least 4 M, at least 4.5 M, or at least 5 M acetic acid.

[0158] In some embodiments, the pickling solution contains at least about 10 mM, at least about 50 mM, at least about 100 mM, at least about 150 mM, at least about 200 mM, at least about 250 mM, at least about 300 mM, at least about 350 mM, at least about 400 mM, at least about 500 mM, at least about 600 mM, at least about 700 mM, at least about 800 mM, at least about 900 mM, at least about 1 M, at least about 1.5 M, at least about 2 M, at least about 2.5 M, at least about 3 M, at least about 3.5 M, at least about 4 M, at least about 4.5 M, or at least about 5 M acetic acid.

[0159] In some embodiments, the pickling solution contains about 0.01M, about 0.02M, about 0.03M, about 0.04M, about 0.05M, about 0.06M, about 0.07M, about 0.08M, about 0.09M, about 0.1M, about 0.2M, about 0.3M, about 0.4M, about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, or about 1.0M glycine-HCl.

[0160] In some embodiments, the pickling solution contains at least 0.01M, at least 0.02M, at least 0.03M, at least 0.04M, at least 0.05M, at least 0.06M, at least 0.07M, at least 0.08M, at least 0.09M, at least 0.1M, at least 0.2M, at least 0.3M, at least 0.4M, at least 0.5M, at least 0.6M, at least 0.7M, at least 0.8M, at least 0.9M, or at least 1.0M glycine-HCl.

[0161] In some embodiments, the pickling solution contains at least about 0.01M, at least about 0.02M, at least about 0.03M, at least about 0.04M, at least about 0.05M, at least about 0.06M, at least about 0.07M, at least about 0.08M, at least about 0.09M, at least about 0.1M, at least about 0.2M, at least about 0.3M, at least about 0.4M, at least about 0.5M, at least about 0.6M, at least about 0.7M, at least about 0.8M, at least about 0.9M, or at least about 1.0M glycine-HCl.

[0162] In some implementations, the pH of the pickling solution is about 0.5, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, or about 4.0.

[0163] In some implementations, the pH of the pickling solution is at least 0.5, at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, or at least 4.0.

[0164] In some embodiments, the pH of the pickling solution is at least about 0.5, at least about 1.0, at least about 1.5, at least about 2.0, at least about 2.5, at least about 3.0, at least about 3.5, or at least about 4.0.

[0165] Purification of NAb In some embodiments, the method includes purifying NAb. In some embodiments, NAb is purified using affinity chromatography. In some embodiments, NAb is purified using a microbead-based purification technique. For example, NAb can be purified using a magnetic bead purification system. In some embodiments, antibody purification includes adding ionizing liquid ions.

[0166] Affinity antibodies The antibodies of this invention can be labeled, such as with affinity labels or detection labels. 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.

[0167] In some implementations, the antibody is conjugated with another molecule or compound to form an antibody conjugate. Conjugation can occur via covalent bonds.

[0168] In some embodiments, the antibodies of the present invention are directly labeled (e.g., via conjugation labels) or indirectly labeled with affinity labels or detection labels. 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.

[0169] For example, the detection label may be a luminescent compound. In some embodiments, the detection is configured to generate a chemiluminescent signal or ECL signal in the presence of the label. 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 may be 3,3',5,5'-tetramethylbenzidine (TMB). In some embodiments, the detection label comprises a sulfonyl tag. The sulfonyl tag (Ru-tag) may contact a marker containing tripropylamine (TPA), and the level of the detection label may be measured by applying an electric current to the marker in the presence of the detection label to generate an ECL signal.

[0170] In some embodiments, the detection marker is detected by mesoscale detection (MSD). In some embodiments, the detection marker is detected by ECL. In some embodiments, the detection marker is ruthenium (Ru) metal ions. In some embodiments, the ions are detected when Ru ions are in close proximity to the electrode. As used herein, "close proximity" between Ru ions and the electrode means that the distance between the Ru ions and the electrode is small enough to induce a detectable redox reaction by ECL, as understood by those skilled in the art. In some embodiments, the electrode is contained in a solid phase. In some embodiments, Ru ions are detected using a charge-coupled device (CCD) camera when they are in close proximity to the electrode.

[0171] In some implementations, the detection label is detected indirectly. As used herein, “indirect” detection of the detection antibody involves adding a separate component that interacts with the detection label, wherein the separate component is detectable. For example, a secondary antibody may be added, wherein the secondary antibody is conjugated to a detectable luminescent probe or is configured to convert a chromogenic substrate into a pigment (e.g., a detection antibody conjugated to the detection label).

[0172] 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.

[0173] 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 conjugated to the detectable compound, wherein the third affinity tag binds to the fourth affinity tag. For example, the fourth affinity tag may be conjugated to a luminescent compound or be an enzyme configured to convert a chromogenic substrate into a pigment.

[0174] The third affinity marker can be any molecular marker that has 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 maltose-binding protein or vice versa. In some embodiments, the third affinity marker is chitin and the fourth affinity marker is chitin-binding protein or vice versa.

[0175] Purification methods In some embodiments, purifying NAb includes contacting the sample with an anti-TNFα antibody conjugated to a first affinity label (i.e., "affinity antibody"), and then contacting the sample containing NAb and the affinity antibody with a first solid phase coated with a second affinity label. In some embodiments, the affinity antibody is added at concentrations of about 10,000 ng / mL to 20,000 ng / mL, about 20,000 ng / mL to 30,000 ng / mL, about 30,000 ng / mL to 40,000 ng / mL, or about 40,000 ng / mL to 50,000 ng / mL. In some embodiments, the affinity antibody is added at a concentration of about 30,000 ng / mL.

[0176] In some embodiments, the affinity antibody is added at a concentration of at least 10,000 ng / mL to 20,000 ng / mL, at least 20,000 ng / mL to 30,000 ng / mL, at least 30,000 ng / mL to 40,000 ng / mL, or at least 40,000 ng / mL to 50,000 ng / mL. In some embodiments, the affinity antibody is added at a concentration of at least 30,000 ng / mL.

[0177] In some embodiments, the affinity antibody is added at a concentration of at least about 10,000 ng / mL to 20,000 ng / mL, at least about 20,000 ng / mL to 30,000 ng / mL, at least about 30,000 ng / mL to 40,000 ng / mL, or at least about 40,000 ng / mL to 50,000 ng / mL. In some embodiments, the affinity antibody is added at a concentration of at least about 30,000 ng / mL.

[0178] As used herein, "solid phase" can be any medium that remains stationary or temporarily stationary when the mobile phase moves across or through it, allowing components of the mobile phase to interact with the solid phase. The solid phase can include plates, columns, microspheres, magnetic microspheres, or any other solid phase known to those skilled in the art. In some embodiments, the first solid phase is microspheres, a plate, or a column. In some embodiments, the first solid phase is microspheres. In some embodiments, the first solid phase is magnetic microspheres.

[0179] In some embodiments, purifying NAbs includes contacting the sample with an affinity antibody; contacting the sample containing the NAb and the affinity antibody with a first solid phase coated with a second affinity label; and washing the first solid phase. In some embodiments, the solid phase is washed with a wash buffer that does not disrupt the interaction between the NAb and the affinity antibody. In some embodiments, the wash buffer comprises phosphate-buffered saline (PBS). In some embodiments, the wash buffer comprises a phosphate-buffered saline solution containing a surfactant. In some embodiments, the wash buffer contains Tween-20. In some embodiments, the wash buffer comprises 1x PBS containing about 0.05% Tween-20. In some embodiments, the wash buffer contains about 50 mM Tris. In some embodiments, the wash buffer contains about 0.14 M NaCl. In some embodiments, the pH of the wash buffer is about 8.0. In some embodiments, the wash buffer contains about 50 mM Tris, about 0.14 M NaCl, about 0.05% Tween-20, and has a pH of about 8.0.

[0180] In some embodiments, purifying NAb includes contacting the sample with an affinity antibody; contacting the sample containing NAb and the affinity antibody with a first solid phase coated with a second affinity label; washing the first solid phase; and eluting the NAb from the first solid phase. In some embodiments, the NAb is eluted using an elution buffer that disrupts the interaction between the NAb and the affinity antibody. In some embodiments, the elution buffer contains glycine and sodium chloride (NaCl). In some embodiments, the elution buffer contains about 0.1 M glycine and about 0.15 M NaCl. In some embodiments, the elution buffer is acidic. In some embodiments, the pH of the elution buffer is about 2 to about 3. In some embodiments, the pH of the elution buffer is about 2.2. In some embodiments, the pH of the elution buffer is about 2.8.

[0181] In some embodiments, an automated purification system is used for NAb purification. As used herein, "automated purification system" refers to a system that purifies antibodies according to steps performed partially by a robotic or mechanical platform. The automated purification system can be fully automated or partially automated. In some embodiments, the sample and reagents of the present invention are "loaded" into the automated purification system, and the automated purification system performs the remaining steps. For example, the automated purification system may be the KingFisher™ Flex purification system or another automated purification system using magnetic particle separation technology. Other automated purification systems that have at least the foregoing features and are known to those skilled in the art are within the scope of the present invention.

[0182] The method also includes contacting purified NAb with a detection antibody, which is an anti-TNFα antibody conjugated to a detection tag. The detection tag can be any tag that can be detected directly or indirectly. As used herein, “direct” detection of a detection tag includes measuring the presence of the detection tag or inducing the detection tag to produce a detectable substance. For example, the detection tag can be a luminescent compound. In some embodiments, the detection tag comprises an enzyme configured to convert a chromogenic substrate into a pigment. For example, the enzyme is horseradish peroxidase (HRP) and the chromogenic substrate can be 3,3',5,5'-tetramethylbenzidine (TMB). In some embodiments, the detection tag is detected by mesoscale detection (MSD) assay. In some embodiments, the detection tag is detected by electrochemiluminescence. In some embodiments, the detection tag is a ruthenium (Ru) metal ion. In some embodiments, the Ru ion is detected when it is in close proximity to the electrode. As used herein, “close proximity” between the Ru ion and the electrode means that the distance between the Ru ion and the electrode is small enough to induce a detectable redox reaction by electrochemiluminescence, as understood by those skilled in the art. In some embodiments, the TNFα-coated substrate contains electrodes. In some embodiments, Ru ions are detected using a CCD camera when they are in close proximity to the electrodes.

[0183] In some embodiments, the detection antibody is added at concentrations of approximately 10,000 ng / mL to 20,000 ng / mL, approximately 20,000 ng / mL to 30,000 ng / mL, approximately 30,000 ng / mL to 40,000 ng / mL, or approximately 40,000 ng / mL to 50,000 ng / mL. In some embodiments, the detection antibody is added at a concentration of approximately 30,000 ng / mL.

[0184] In some embodiments, the detection antibody is added at a concentration of at least 10,000 ng / mL to 20,000 ng / mL, at least 20,000 ng / mL to 30,000 ng / mL, at least 30,000 ng / mL to 40,000 ng / mL, or at least 40,000 ng / mL to 50,000 ng / mL. In some embodiments, the detection antibody is added at a concentration of at least 30,000 ng / mL.

[0185] In some embodiments, the detection antibody is added at a concentration of at least about 10,000 ng / mL to 20,000 ng / mL, at least about 20,000 ng / mL to 30,000 ng / mL, at least about 30,000 ng / mL to 40,000 ng / mL, or at least about 40,000 ng / mL to 50,000 ng / mL. The method of this invention involves contacting purified NAb and an anti-TNFα antibody conjugate with a TNFα-coated substrate. The TNFα-coated substrate can be any substrate coated with TNFα by methods known to those skilled in the art. In some embodiments, human TNFα is used to coat the substrate.

[0186] The substrate can be coated with TNFα at concentrations of approximately 50 µg / mL to 200 µg / mL, approximately 75 µg / mL to 150 µg / mL, or approximately 90 µg / mL to 110 µg / mL. In some embodiments, the substrate is coated with approximately 100 µg / mL TNFα.

[0187] The substrate can be coated with at least 50 µg / mL-200 µg / mL, at least 75 µg / mL-150 µg / mL, or at least 90 µg / mL-110 µg / mL TNFα. In some embodiments, the substrate is coated with at least 100 µg / mL.

[0188] The substrate can be coated with at least about 50 µg / mL to 200 µg / mL, at least about 75 µg / mL to 150 µg / mL, or at least about 90 µg / mL to 110 µg / mL. In some embodiments, the substrate is coated with at least about 100 µg / mL.

[0189] In some embodiments, the TNFα substrate is blocked to prevent non-specific binding to the substrate. Any blocking buffer or reagent known to those skilled in the art can be used to block the TNFα substrate. In some embodiments, the substrate is a plate.

[0190] In some embodiments, the substrate is washed after the purified NAb and detection antibody have been contacted with the TNFα-coated substrate. In some embodiments, the TNFα-coated substrate is washed with a wash buffer that does not disrupt the interaction between the NAb and the TNFα-coated substrate or the interaction between the NAb and the detection antibody. The wash buffer may be configured to remove NAbs bound to the detection antibody from the substrate, but not NAbs bound to the TNFα-coated substrate. In some embodiments, the substrate is a plate.

[0191] In some embodiments, the method further includes detecting the 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, then detecting the pigment and measuring its presence. In some embodiments, detecting the detection marker includes converting the chromogenic substrate into a pigment and detecting the optical density (OD) of the sample. In some embodiments, the detection marker is a Ru metal ion, and detecting the detection marker includes using a camera to detect light emitted by Ru metal ions in close proximity to a TNFα-coated substrate containing an electrode.

[0192] The method also includes detecting NAb when the detection antibody binds to the NAb instead of the TNFα-coated substrate. When the detection antibody binds to the NAb instead of the TNFα-coated substrate, less detection antibody will bind to the substrate, thus resulting in a lower detection marker when NAb is present. In some embodiments, the substrate is a plate. In embodiments where the detection marker is detected with a luminescent compound, the presence of NAb can be measured by a lower luminescence intensity. In embodiments where the detection marker is detected with a pigment, the presence of NAb can be measured by a lower pigment density. In embodiments where the detection marker is detected with a pigment, the presence of NAb can be measured by a higher OD (i.e., more light passes through the sample because less pigment is present). In embodiments where the detection marker is a Ru metal ion, the presence of NAb can be measured by the higher intensity of light emitted when the Ru metal ion is in close proximity to the TNFα-coated substrate.

[0193] In some implementations, NAb is detected by determining whether a detection marker is present at a level below a predetermined level. In other implementations, NAb is detected by determining that the OD of the sample is above a predetermined cutoff point.

[0194] control sample In some embodiments, the method further includes performing a method on a negative control sample. In some embodiments, the negative control sample does not contain detectable NAbs. 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, "parallel" method execution includes performing the method on the negative control sample at the same time and / or on the same plate as the sample. Performing a method on the negative control sample includes (i) treating the negative control sample with an acid wash; (ii) performing a purification step on the negative control sample; (iii) contacting the negative control sample with a detection antibody; (iv) contacting the negative control sample containing the detection antibody with a TNFα-coated substrate; and (v) detecting the absence of NAbs by measuring the anti-TNFα antibody conjugate in contact with the TNFα-coated substrate.

[0195] 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 has shown the presence of NAb 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.

[0196] In some embodiments, the cutoff point is determined by methodologically processing 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 methodologically processing at least 50 negative control samples. In some embodiments, the OD signals from the 50 negative control samples are normalized by the mean of all negative control samples. In some embodiments, outliers are identified and removed. In some embodiments, the cutoff point is identified based on a false positive rate of 0.5%-1%, 1%-1.5%, 1.5%-2%, 2%-2.5%, or 2.5%-3%. In some embodiments, the cutoff point is identified based on a 3% false positive rate. In some embodiments, the cutoff point is identified based on a 1% false positive rate.

[0197] 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, QC samples are blank samples, matrix-doped samples (i.e., plasma or serum samples doped with NAb), field duplicate samples, duplicate samples, and / or reference material samples. The method may be performed on QC samples in parallel with the sample samples. The method may be performed on the sample samples, negative control samples, positive control samples, and QC samples in parallel.

[0198] In some embodiments, the predetermined cutoff point is an OD of 0.5–2, 0.5–1, 1–1.5, or 1.5–2. In some embodiments, the predetermined cutoff point is about 1. In some embodiments, the predetermined cutoff point is about 0.91.

[0199] Sensitivity and target tolerance In some embodiments, the sensitivity for detecting NAb is less than about 150 ng / mL, less than about 200 ng / mL, less than about 250 ng / mL, less than about 300 ng / mL, less than about 350 ng / mL, less than about 400 ng / mL, less than about 450 ng / mL, less than about 500 ng / mL, less than about 550 ng / mL, or less than about 600 ng / mL. In some embodiments, the sensitivity for detecting NAb is less than about 500 ng / mL. In some embodiments, the sensitivity for detecting NAb is less than about 250 ng / mL.

[0200] In some embodiments, the sensitivity for detecting NAb is less than 150 ng / mL, less than 200 ng / mL, less than 250 ng / mL, less than 300 ng / mL, less than 350 ng / mL, less than 400 ng / mL, less than 450 ng / mL, less than 500 ng / mL, less than 550 ng / mL, or less than 600 ng / mL. In some embodiments, the sensitivity for detecting NAb is less than 500 ng / mL. In some embodiments, the sensitivity for detecting NAb is less than 250 ng / mL.

[0201] In some embodiments, the sensitivity for detecting NAb is less than at least about 150 ng / mL, less than at least about 200 ng / mL, less than at least about 250 ng / mL, less than at least about 300 ng / mL, less than at least about 350 ng / mL, less than at least about 400 ng / mL, less than at least about 450 ng / mL, less than at least about 500 ng / mL, less than at least about 550 ng / mL, or less than at least about 600 ng / mL. In some embodiments, the sensitivity for detecting NAb is less than at least about 500 ng / mL. In some embodiments, the sensitivity for detecting NAb is less than at least about 250 ng / mL.

[0202] In some implementations, the target tolerance for NAb detection is approximately 500 ng / mL–750 ng / mL, approximately 750 ng / mL–1000 ng / mL, approximately 1000 ng / mL–1250 ng / mL, or approximately 1250 ng / mL–1500 ng / mL. In some implementations, the target tolerance for NAb detection is approximately 1000 ng / mL.

[0203] In some implementations, the target tolerance for NAb detection is at least 500 ng / mL–750 ng / mL, at least 750 ng / mL–1000 ng / mL, at least 1000 ng / mL–1250 ng / mL, or at least 1250 ng / mL–1500 ng / mL. In some implementations, the target tolerance for NAb detection is at least 1000 ng / mL.

[0204] In some implementations, the target tolerance for NAb detection is at least about 500 ng / mL to 750 ng / mL, at least about 750 ng / mL to 1000 ng / mL, at least about 1000 ng / mL to 1250 ng / mL, or at least about 1250 ng / mL to 1500 ng / mL. In some implementations, the target tolerance for NAb detection is at least about 1000 ng / mL.

[0205] Reagent test kit The present invention also includes a kit for detecting NAbs against a target antibody, comprising: an affinity antibody; a first solid phase coated with a second affinity label; a detection antibody; a substrate; and TNFα. The concentrations of the reagents in the kit are formulated to enable those skilled in the art to perform the present invention.

[0206] In some embodiments, the affinity antibody comprises a first affinity marker. In some embodiments, the first affinity marker is biotin. In some embodiments, the detection antibody comprises a detection marker. In some embodiments, the detection marker is a luminescent compound, an enzyme configured to convert a chromogenic substrate into a pigment, or biotin. In some embodiments, the detection marker is biotin.

[0207] 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 NAb. 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 NAb. In some embodiments, the kit may include NAb or a NAb solution and instructions for preparing the positive control. For example, the kit may include a NAb solution and instructions for "doping" a plasma or serum sample with the NAb solution to prepare the positive control.

[0208] In some implementations, the kit includes 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 concentrations of approximately 300 ng / mL–400 ng / mL, approximately 400 ng / mL–500 ng / mL, approximately 500 ng / mL–600 ng / mL, approximately 600 ng / mL–700 ng / mL, approximately 700 ng / mL–800 ng / mL, approximately 800 ng / mL–900 ng / mL, approximately 900 ng / mL–1000 ng / mL, or greater than approximately 1000 ng / mL. The LPC concentration may be less than approximately 1 ng / mL, approximately 1 ng / mL–10 ng / mL, approximately 10 ng / mL–100 ng / mL, approximately 100 ng / mL–200 ng / mL, approximately 200 ng / mL–300 ng / mL, or approximately 300 ng / mL–500 ng / mL.

[0209] In some implementations, HPC has a concentration of at least 300 ng / mL-400 ng / mL, at least 400 ng / mL-500 ng / mL, at least 500 ng / mL-600 ng / mL, at least 600 ng / mL-700 ng / mL, at least 700 ng / mL-800 ng / mL, at least 800 ng / mL-900 ng / mL, at least 900 ng / mL-1000 ng / mL, or greater than at least 1000 ng / mL. LPC concentration can be less than at least 1 ng / mL, at least 1 ng / mL-10 ng / mL, at least 10 ng / mL-100 ng / mL, at least 100 ng / mL-200 ng / mL, at least 200 ng / mL-300 ng / mL, or at least 300 ng / mL-500 ng / mL.

[0210] In some embodiments, HPC has a concentration of at least about 300 ng / mL to 400 ng / mL, at least about 400 ng / mL to 500 ng / mL, at least about 500 ng / mL to 600 ng / mL, at least about 600 ng / mL to 700 ng / mL, at least about 700 ng / mL to 800 ng / mL, at least about 800 ng / mL to 900 ng / mL, at least about 900 ng / mL to 1000 ng / mL, or greater than at least about 1000 ng / mL. LPC concentration can be less than at least about 1 ng / mL, at least about 1 ng / mL to 10 ng / mL, at least about 10 ng / mL to 100 ng / mL, at least about 100 ng / mL to 200 ng / mL, at least about 200 ng / mL to 300 ng / mL, or at least about 300 ng / mL to 500 ng / mL.

[0211] Affinity antibodies and / or detection antibodies can be affinity purified. In some embodiments, affinity antibodies and / or detection antibodies can be stored as lyophilized powders. In some embodiments, affinity antibodies and / or detection antibodies can be stored as reconstituted liquid antibodies.

[0212] Any component of 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 solutions may be approximately 2X, 5X, 10X, 50X, 100X, 500X, 1000X, 5000X, or 10000X more concentrated than the working solution.

[0213] 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 the appropriate lyophilizing agent, as well as the effective amount of other lyophilizing agents, is within the scope of conventional techniques in the art.

[0214] The kit may include approximately 50µg-100µg, approximately 100µg-500µg, approximately 500µg-1000µg, approximately 1µg-10mg, approximately 10µg-50mg, approximately 50µg-100mg, approximately 100µg-500mg, or approximately 500µg-1000mg of affinity antibodies and / or detection antibodies. The kit may include approximately 50µg-200µg of affinity antibodies and / or detection antibodies. The kit may include approximately 100mg of affinity antibodies and / or detection antibodies. The kit may include approximately 1mg of affinity antibodies and / or detection antibodies. The purity of the affinity antibodies and / or detection antibodies may be approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher than 99%.

[0215] The kit may include at least 50µg-100µg, at least 100µg-500µg, at least 500µg-1000µg, at least 1µg-10mg, at least 10µg-50mg, at least 50µg-100mg, at least 100µg-500mg, or at least 500µg-1000mg of affinity antibody and / or detection antibody. The kit may include at least 50µg-200µg of affinity antibody and / or detection antibody. The kit may include at least 100mg of affinity antibody and / or detection antibody. The kit may include at least 1mg of affinity antibody and / or detection antibody. The purity of the affinity antibody and / or detection antibody may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher than 99%.

[0216] The kit may include at least about 50µg-100µg, at least about 100µg-500µg, at least about 500µg-1000µg, at least about 1µg-10mg, at least about 10µg-50mg, at least about 50µg-100mg, at least about 100µg-500mg, or at least about 500µg-1000mg of affinity antibody and / or detection antibody. The kit may include at least about 50µg-200µg of affinity antibody and / or detection antibody. The kit may include at least about 100mg of affinity antibody and / or detection antibody. The kit may include at least about 1mg of affinity antibody and / or detection antibody. The purity of the affinity antibody and / or detection antibody may be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher than 99%.

[0217] In some implementations, the lyophilized affinity antibodies and / or detection antibodies can be reconstituted in reconstitution buffers of about 50 µg-100 µL, about 100 µg-150 µL, about 150 µg-200 µL, about 200 µg-250 µL, about 250 µg-300 µL, about 300 µg-350 µL, about 350 µg-400 µL, about 400 µg-450 µL, or about 450 µg-500 µL.

[0218] In some implementations, the lyophilized affinity antibodies and / or detection antibodies can be reconstituted in reconstitution buffers of at least 50 µg-100 µL, at least 100 µg-150 µL, at least 150 µg-200 µL, at least 200 µg-250 µL, at least 250 µg-300 µL, at least 300 µg-350 µL, at least 350 µg-400 µL, at least 400 µg-450 µL, at least 350 µg-400 µL, at least 400 µg-450 µL, or at least 450 µg-500 µL.

[0219] In some implementations, the lyophilized affinity antibodies and / or detection antibodies can be reconstituted in at least about 50 µg-100 µL, at least about 100 µg-150 µL, at least about 150 µg-200 µL, at least about 200 µg-250 µL, at least about 250 µg-300 µL, at least about 300 µg-350 µL, at least about 350 µg-400 µL, at least about 400 µg-450 µL, at least about 350 µg-400 µL, at least about 400 µg-450 µL, or at least about 450 µg-500 µL of reconstitution buffer.

[0220] The reconstitution buffer may contain any components known to those skilled in the art. For example, the reconstitution buffer may contain water, PBS, DMSO, glycerol, or sodium azide.

[0221] The reconstitution buffer may contain about 10%-15%, about 15%-20%, about 20%-25%, about 25%-30%, or about 30%-35% glycerol. The reconstitution buffer may contain about 0.02%-0.04%, about 0.04%-0.06%, about 0.06%-0.08%, about 0.08%-0.1%, about 0.1%-0.12%, about 0.12%-0.14%, or about 0.14%-0.16% sodium azide. In some embodiments, the pH of the reconstitution buffer is about 6 to about 7 or about 7 to about 8. In some embodiments, the pH of the reconstitution buffer is about 7.2.

[0222] The reconstitution buffer may contain at least 10%-15%, at least 15%-20%, at least 20%-25%, at least 25%-30%, or at least 30%-35% glycerol. The reconstitution buffer may contain at least 0.02%-0.04%, at least 0.04%-0.06%, at least 0.06%-0.08%, at least 0.08%-0.1%, at least 0.1%-0.12%, at least 0.12%-0.14%, or at least 0.14%-0.16% sodium azide. In some embodiments, the pH of the reconstitution buffer is at least 6 to at least 7 or at least 7 to at least 8. In some embodiments, the pH of the reconstitution buffer is at least 7.2.

[0223] The reconstitution buffer may contain at least about 10%-15%, at least about 15%-20%, at least about 20%-25%, at least about 25%-30%, or at least about 30%-35% glycerol. The reconstitution buffer may contain at least about 0.02%-0.04%, at least about 0.04%-0.06%, at least about 0.06%-0.08%, at least about 0.08%-0.1%, at least about 0.1%-0.12%, at least about 0.12%-0.14%, or at least about 0.14%-0.16% sodium azide. In some embodiments, the pH of the reconstitution buffer is at least about 6 to at least about 7 or at least about 7 to at least about 8. In some embodiments, the pH of the reconstitution buffer is at least about 7.2.

[0224] In some embodiments, the kit includes a reconstitution buffer. In some embodiments, the kit includes reconstituted affinity antibodies and / or detection antibodies.

[0225] The kit may include detection antibodies and / or affinity antibodies in solution at concentrations of approximately 1 µg / mL to 10 µg / mL, approximately 10 µg / mL to 50 µg / mL, approximately 50 µg / mL to 100 µg / mL, approximately 100 µg / mL to 500 µg / mL, approximately 500 µg / mL to 1000 µg / mL, approximately 1 mg / mL to 10 mg / mL, approximately 10 mg / mL to 50 mg / mL, approximately 50 mg / mL to 100 mg / mL, approximately 100 mg / mL to 1000 mg / mL, or approximately 1000 mg / mL to 10000 mg / mL.

[0226] The kit may include detection antibodies and / or affinity antibodies in solution at concentrations of at least 1 µg / mL to 10 µg / mL, at least 10 µg / mL to 50 µg / mL, at least 50 µg / mL to 100 µg / mL, at least 100 µg / mL to 500 µg / mL, at least 500 µg / mL to 1000 µg / mL, at least 1 mg / mL to 10 mg / mL, at least 10 mg / mL to 50 mg / mL, at least 50 mg / mL to 100 mg / mL, at least 100 mg / mL to 1000 mg / mL, or at least 1000 mg / mL to 10000 mg / mL.

[0227] The kit may include detection antibodies and / or affinity antibodies in solution at a concentration of at least about 1 µg / mL to 10 µg / mL, at least about 10 µg / mL to 50 µg / mL, at least about 50 µg / mL to 100 µg / mL, at least about 100 µg / mL to 500 µg / mL, at least about 500 µg / mL to 1000 µg / mL, at least about 1 mg / mL to 10 mg / mL, at least about 10 mg / mL to 50 mg / mL, at least about 50 mg / mL to 100 mg / mL, at least about 100 mg / mL to 1000 mg / mL, or at least about 1000 mg / mL to 10000 mg / mL.

[0228] The first solid phase may include any solid phase known to those skilled in the art for antibody affinity purification. In some embodiments, the first solid phase is microbeads. In some embodiments, the first solid phase is magnetic microbeads. The kit may include microbeads in solution at concentrations of 1 µg / mL-10 µg / mL, 10 µg / mL-50 µg / mL, 50 µg / mL-100 µg / mL, 100 µg / mL-500 µg / mL, or 500 µg / mL-1000 µg / mL, 1 mg / mL-10 mg / mL, 10 mg / mL-50 mg / mL, 50 mg / mL-100 mg / mL, 100 mg / mL-1000 mg / mL, or 1000 mg / mL-10000 mg / mL. The kit may include microbeads in solutions of 0.5 mL-1 mL, 1 mL-1.5 mL, 1.5 mL-2 mL, 2 mL-2.5 mL, 2.5 mL-3 mL, 3 mL-3.5 mL, 3.5 mL-4 mL, 4 mL-4.5 mL, or 4.5 mL-5 mL.

[0229] 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.

[0230] The substrate can be any imageable material known to those skilled in the art capable of binding TNFα. For example, the substrate can be a glass slide, plate, dish, or flask. In some embodiments, the kit includes a substrate coated with TNFα. In some embodiments, the kit includes a substrate, TNFα, and instructions for coating the substrate with TNFα. For example, the kit may include about 1µg-5µg, 5µg-10µg, 10µg-20µg, or 20µg-50µg of human TNFα. In some embodiments, TNFα is a human TNFα solution of 1ng / mL-10ng / mL, 10ng / mL-100ng / mL, 100ng / mL-1000ng / mL, or 1000ng / mL-10000ng / mL. The TNFα solution may contain any medium known to those skilled in the art, such as phosphate-buffered saline (PBS).

[0231] The kit may include one or more buffers. In some embodiments, the kit includes an acid wash buffer. In some embodiments, the kit further includes a wash buffer. In some embodiments, the kit further includes an elution buffer. The buffers of the present invention can be formulated according to any formulation of the present technology or formulations known to those skilled in the art.

[0232] According to some embodiments, the kit may contain one or more consumables for performing the methods of the present invention. For example, the kit may include one or more plates containing multiple wells. In some embodiments, the plates 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 assay plates.

[0233] The kit may also include instructions for performing all or part of the methods described in this invention. In some embodiments, the kit includes a link (e.g., a URL or QR code) directing the user to the instructions for performing all or part of the methods of this invention. The kit may include software or other computer technology to facilitate the methods of performing this invention.

[0234] The kit components can be stored to provide stability for specific components. For example, if the kit components contain a photosensitive compound, the components can be stored in an opaque container. In some embodiments, the kit components can be stored in an airtight container. In some embodiments, the kit components are stored frozen in liquid or solid form. In some embodiments, the kit components are stored at approximately -195°C, -80°C, -20°C, 4°C, or 20°C.

[0235] In another embodiment, the kit includes one or more of the components, consumables, or reagents described in the following examples.

[0236] While the present invention can be embodied in various forms, the following description of several embodiments is made under the understanding that this disclosure is intended to be illustrative of the invention and not to limit the invention to the specific embodiments illustrated. The headings are provided for convenience only and should not be construed as limiting the invention in any way. Embodiments illustrated under any heading may be combined with embodiments illustrated under any other heading.

[0237] Based on the foregoing, it should be understood that specific embodiments of the present invention have been described herein for illustrative purposes, but various modifications can be made without departing from the scope of the present invention. Therefore, the present invention is not limited except by the appended claims.

[0238] Example abbreviation ADA: Anti-drug antibody; AT: Ambient temperature; Bio-MAB-16: Biotin-labeled MAB-16; CP: Cutoff point; Conc.: Concentration; %CV: Percentage of coefficient of variation; diH2O: Deionized water; ECL: Electrochemiluminescence; ECLIA: Electrochemiluminescence immunoassay; FT: Freeze / thaw; HPC: High positive control; LPC: Low positive control; MSD: Mesoscale detection; NA: Not applicable; NC: Negative control; pNHS: Combined primary human serum; PBS: Phosphate-buffered saline; QC: Quality control; rECL: Relative ECL; Ru-MAB-16: Ruthenium-labeled MAB-16; SCP: Screening cutoff point; S-CPF: Screening cutoff coefficient; SD: Standard deviation; TCP: Titration cutoff point; T-CPF: Titration cutoff coefficient.

[0239] Overview: Figure 1 An example of a method for detecting NAbs against a target antibody in a sample is illustrated, comprising (i) dissociating the NAb from the target antibody by adding an acid wash solution to the sample 110; (ii) purifying the NAb from the sample 120; (iii) contacting the purified NAb with an antiTNFα antibody conjugate (i.e., "detection antibody") containing an antiTNFα antibody conjugated to a detection label 130; (iv) contacting the purified NAb and the detection antibody with a TNFα-coated substrate 140; and (v) detecting the NAb by determining whether the detection label is present at a level above a predetermined cutoff point 150.

[0240] Figure 2 An example of a method for detecting NAbs against a target antibody is provided, comprising (i) purifying NAb210 from a sample; (ii) determining the presence of NAb in the sample by performing a competitive binding assay, wherein NAb220 is present if the affinity of the NAb for the detection antibody is higher than the affinity of the detection antibody for TNFα; and (iii) detecting NAb230; wherein the sensitivity of the method is 250 ng / mL or lower.

[0241] Example 1: A method for detecting NAb The presence of NAbs against XTMAB-16 in human serum was evaluated using a competitive ligand binding (CLB) assay. XTMAB-16 is an anti-TNFα monoclonal antibody used to treat patients with sarcoidosis. XTMAB-16 is an example of an anti-TNFα chimeric human-mouse monoclonal IgG1-κ antibody (i.e., a “target antibody”) comprising SEQ ID NO: 1 and 4 of the present invention.

[0242] Buffer and solution Assay buffer: Combine 33.3 mL or 30% BSA with 5 mL of 10% Tween-20 and approximately 800 mL of DPBS. Adjust the QS (appropriate amount) solution to 1.0 L with DPBS. Store at 2°C–8°C for up to 6 months from the date of preparation.

[0243] Diluent / Wash Buffer: Combine 16.7 mL of 30% BSA, 100 mL of 10x DPBS (pH 7.4 ± 0.1), 10 mL of 10% Tween-20, 0.5 mL of ProClin 300, and approximately 800 mL of deionized water until dissolved. Adjust the volume of the solution to 1.0 L with deionized water. Store at 2°C–8°C for up to 6 months from the date of preparation.

[0244] Biotin-XTMAB-16 working solution in 2M Tris-HCl (30,000 ng / mL): Dilute the biotin-XTMAB-16 stock solution to 30,000 ng / mL in 2M Tris-HCl (pH 9.6).

[0245] Coating solution (100 ng / mL human TNFα in DPBS): Dilute human TNFα to 100 ng / mL in DPBS.

[0246] Neutralization buffer: Add 1 mL of 1 M HEPES (pH 9.0) to 23 mL of dilution / wash buffer.

[0247] Termination solution (1M phosphoric acid solution): Dilute 68.0 mL of 85% phosphoric acid to 1.0 L with diH2O.

[0248] Preparation plate control Preparation of negative control (NC): NC is pNHS.

[0249] Preparation of high positive control (HPC): Anti-XTMAB-16 antibody (91F9D4, 0.781 mg / mL) was used as NAbPC. The concentration of HPC was 600 ng / mL. After calculating SCP, the final concentration of LPC (220.6 ng / mL) was prepared with a 99% confidence interval (1% failure rate).

[0250] Table 2: NAb concentration in the control group

[0251] Analysis program On day 1, coat the wells with TNFα (100 µg / mL) and incubate at 2°C–8°C for 16–48 hours. On day 2, add controls and samples to their respective wells. Add dilution / wash buffer and acid wash buffer (2 M acetic acid) to the wells. Shake the plate at AT for 30 minutes.

[0252] Add biotin-XTMAB-16 working solution (30,000 ng / mL) to each well and allow to shake at AT for 60 minutes.

[0253] Add streptavidin magnetic bead solution to the wells and shake the plate at AT for 60 minutes. Add dilution / wash buffer to the wells of the other four wells (further designated as Wash 1, 2, 3, and 4 plates).

[0254] Add 0.1M glycine, 0.15M NaCl, pH 2.2 (elution solution) to all wells of the well plate (further referred to as the acid elution plate). Wash the beads and perform acid elution.

[0255] Add neutralization buffer to the wells and mix. Add 100 µL / well of elution control / sample to a new unbound plate and add 120 µL / well of biotin-XTMAB-16 working solution. Incubate the plate at 450 rpm for 60 ± 10 minutes at AT.

[0256] Wash the wells with washing buffer. Add strong blocking solution III to each well and shake the plate at 450 rpm for 1–2 hours at AT. Wash the coated plate with washing buffer.

[0257] Transfer 100 µL / well of elution control / sample to a TNFα-coated plate. Incubate the plate at 450 rpm for 60 ± 10 min in AT and wash with wash buffer. Add 100 µL / well of streptavidin-HRP working solution (1:50000) to each well and incubate the plate at 450 rpm for 60 ± 10 min in AT.

[0258] The plate was then washed with washing buffer, and TMB solution was added to the wells.

[0259] Add stop solution to each well and read the plate at 450 nm using a 630 nm reference filter on a microplate reader.

[0260] Data analysis: Raw data (OD) were captured using a SpectraMax M2e microplate reader. JMP software was used for statistical evaluation of the cutoff coefficient.

[0261] Boards are subject to acceptance standards. Average Response Value (OD): NC < Plate Measurement Cutoff Point ≤ LPC <HPC。

[0262] The %CV of the average response value for NC, HPC, and LPC must be ≤25%. The board NC is the average of all accepted NCs. Values ​​exceeding the average will be identified as outliers. The NC range is excluded from the board NC calculation.

[0263] Table 3: Range of Measured Response Values

[0264] The cutoff coefficient was determined to be 0.91. The plate-specific cutoff was calculated using the following formula: Plate-specific measurement cutoff point = Average OD value of all NCs in the plate x Measurement cutoff point coefficient Two-thirds of the QC samples on each board (at least half (50%) of each level) are required to meet the acceptance criteria.

[0265] Sample Acceptance Standards The mean OD value of the samples is ≤25% CV. Samples with a mean OD value ≤ the plate-specific assay cutoff are classified as positive. Samples with a mean OD value > the plate-specific assay cutoff and %CVS 25% are classified as negative.

[0266] verify A CLB assay for the detection of NAbs against XTMAB-16 in human serum was developed and validated. Validation parameters included cutoff point determination, sensitivity, drug tolerance, intra-assay and inter-assay precision, selectivity, matrix interference, specificity, stability, and robustness.

[0267] Each run includes NC, HPC, and LPC. Cutoff and sensitivity runs do not include LPC. Each NAb assay plate includes two sets of HPC and LPC, and four sets of NC.

[0268] Assay validation includes the evaluation of the following parameters: assay cutoff point, determination of LPC concentration, assay sensitivity and drug tolerance, intra-assay precision and inter-assay precision, system suitability (control acceptance criteria), selectivity and matrix interference, specificity, stability and robustness.

[0269] Determining the cutoff point Assay cutoff data were generated from multiple runs of analysis of samples that did not receive drug treatment. Individual human serum samples from at least 50 batches of healthy volunteers were screened across a total of six distinct groups. The experimental design is outlined in Table 4.

[0270] The OD signal was normalized using the plate NC mean. After identifying and removing outliers, the data were tested for normality using JMP software. The determination cutoff factor (ACPF) was calculated based on a 1% false positive rate using the following equation based on the data distribution.

[0271] ACPF = "mean - 2.33 x SD" (if the data is normally distributed) or "first percentile" (if the data is normally distributed) (Not normally distributed) Calculate the plate-specific screening cutoff point using the following formula: Plate intercept (ACP) = Average NC OD value of the plate Table 4: Experimental Design and Objective Description for Intercept Point Determination

[0272] Sensitivity The sensitivity of the competitive ligand binding assay is limited by the lowest concentration of the positive control antibody formulation that consistently provides a positive signal during the assay. The experimental design is summarized in Table 5.

[0273] Table 5: Experimental Design and Objectives for Sensitivity Measurement

[0274] The LPC concentration was limited based on the sensitivity values ​​obtained during the validation period.

[0275] Determination of drug tolerance XTMAB-16 was prepared in pNHS at the desired 2X concentration of the sample. Antibody samples were prepared in pNHS at 2X levels. Each 2X sample was combined with each 2X drug sample at a 1:1 ratio. The samples were incubated at room temperature with stirring for at least 1 hour prior to analysis. The samples were frozen prior to analysis. The mean response of the PC samples pre-incubated with the drug was compared with the response of the matched PC samples. The experimental design is summarized in Table 6.

[0276] Table 6: Experimental Design and Objectives of Drug Tolerance Determination

[0277] Intra-measurement accuracy and inter-measurement accuracy Internal precision (IP) was evaluated by analyzing multiple samples at each run acceptance control level in a single validation run. IIP was calculated using NC, LPC, and HPC responses. Positive control formulations were prepared from six LPC and six HPC aliquots, and each was analyzed in duplicate. NC was analyzed from six aliquots, with each analysis performed in duplicate. The experimental design is summarized in Table 7.

[0278] Table 7: Experimental Design and Objectives for Determining Internal Accuracy

[0279] Inter-assay accuracy was evaluated by analyzing samples at each run acceptance control level across all applicable acceptable validation runs. Inter-assay accuracy was calculated using the LPC / NC ratio. The experimental design is summarized in Table 8.

[0280] Table 8: Experimental Design and Objectives for Inter-measurement Accuracy

[0281] Selectivity and matrix interference Selectivity was evaluated by doping NAb LPC into at least 20 individual serum samples from sarcoidosis patients. NAb-doped and undoped matrix samples were analyzed and compared with ACP. Once prepared, samples were preheated at room temperature with stirring for at least 1 hour, and then frozen at -70°C (-80°C to -60°C) prior to analysis. The experimental design is summarized in Table 9.

[0282] Table 9: Experimental Design and Objectives for Matrix Selectivity Determination

[0283] Selective assessments included hemolysis and lipemia matrix evaluation. At least six plate controls, both undoped and doped with interfering substances (hemolyzed whole blood and lipids), were evaluated in NC and LPC. The experimental design is summarized in Table 10.

[0284] Table 10: Experimental Design and Objective Description for Interference Measurement

[0285] Specificity refers to the ability to detect only NAbs without detecting other components in the matrix. In this assay, the tolerance for target (TNFα) interference was determined. Various target concentrations covering the expected target level in the sample were doped into the matrix sample at HPC, LPC, and NC levels. The experimental design is summarized in Table 11.

[0286] Table 11: Experimental Design and Objective Description for Specificity Assay

[0287] Stability test The combined stress samples were tested on a workbench (24 ± 6 h or 8 ± 2 h) and subjected to freeze / thaw cycles (-70 °C ± 10 °C to 6 or 3 AT cycles). For the first cycle, the sample was prepared and stored overnight (at least 12 h) at -70 °C ± 10 °C, then thawed at AT. For subsequent freeze / thaw cycles, the sample was frozen under the same conditions for at least 12 h, then thawed. Before refrozen, the sample was placed on the workbench for 2 ± 1 h. The experimental design is summarized in Table 12.

[0288] Table 12: Experimental Design and Objectives for Determining Freeze / Thaw Stability of the Workbench

[0289] Evaluation of robustness Robustness testing included an incubation time range. Incubation times are shown in Table 13. Robustness determination used three sets of HPC, LPC, and NC. Only qualified robustness parameters were used in sample analysis. Acceptance criteria are as follows: NC, LPC, and HPC: %CV ≤ 25% for each replicate sample. The average LPC signal and average HPC signal must be less than or equal to the measurement cutoff point calculated for the corresponding board, and the average NC signal must be greater than the board's value. Determine the cutoff point.

[0290] All robustness samples at each level must exceed the acceptance criteria.

[0291] Table 13: Incubation time for each incubation step

[0292] All the minimum and maximum incubation times listed above are target times; the actual minimum and maximum times for evaluation can be adjusted.

[0293] The results of at least 12 acceptable validation runs are used to determine the acceptance criteria for the assay control used in the sample analysis.

[0294] HPC lower limit = Overall average response value - 3xSD LPC acceptance range = overall average response value ± 3xSD NC limit = Overall average response value + 3xSD The acceptance ranges for the LPC / NC response value ratio are determined post-validation and are not applied to validation runs. These acceptance ranges are set at a failure rate of 1%.

[0295] The LPC / NC ratio is based on the average response value, where LPC / NC ratio acceptance range = average ratio ± 2.33 x SD All acceptable validation run measurement control tables and LPC / NC ratio acceptance range tables will be included in the validation report.

[0296] Determination of the measurement intercept point according to the present invention Fifty individual serum samples were screened six times by two analysts to calculate the assay cutoff coefficient. For each run, the mean OD signal of each individual serum sample was normalized by dividing the mean OD of the sample by the mean OD of the negative control. The normalized OD was then logarithmically transformed.

[0297] No sample had a %CV higher than 25.0%, and all 300 individual serum values ​​were used for screening cutoff analysis.

[0298] Box plot analysis was performed on all 300 normalized values. The formulas are shown in Table 14. Five (5) outliers were identified.

[0299] Table 14: Formulas for identifying outliers

[0300] Identification of biological outliers After identifying and excluding outliers from the analysis, the normalized and logarithmically transformed data (295 values) for each individual serum sample across 6 defects were averaged (if available). Box plot analysis was performed on the 295 normalized average values. Formulas for identifying biological outliers are provided in Table 15. No biological outliers were identified.

[0301] Table 15: Formulas for identifying biological outliers

[0302] Normality test for screening data The normality of the normalized values ​​of 295 logarithmic transformations was tested using JMP 14.0 software. Figure 3 The data shows a non-normal distribution as determined by the Shapiro-Wilk test. Therefore, a non-parametric method is used to calculate the cutoff point.

[0303] Table 16: Calculation of Intercept Coefficient for Nonparametric Measurements

[0304] formula: Log(ACPF, 1% false positive) = 1% percentile of rOD value Plate intercept (ACP) = Average NC OD value of the plate Summarize No %CV failure was identified, and all 300 individual serum values ​​were used for the assay cutoff analysis. During the determination of the assay cutoff, five (5) analytical outliers were identified, and no biological outliers were identified. The final assay ACPF was determined to be 0.91 using a nonparametric method based on 295 log-transformed normalized data values.

[0305] For example, Tables 17-20 show examples of data analyzed according to the technology of the present invention. Table 17 shows the raw data from one run of samples tested by the method according to the technology of the present invention. Table 18 shows the logarithmically transformed OD data from six screenings of samples used to identify analytical outliers. Table 19 shows the OD data used to identify biological outliers after removing analytical outliers (no biological outliers were identified). Table 20 shows the data used to identify cutoff points after removing all outliers.

[0306] Table 17: Raw Data

[0307] Each row represents a single sample. Table 18: OD data after logarithmic transformation

[0308] Each row represents a single sample. Table 19: Log-transformed OD data after outlier removal

[0309] Each row represents a single sample. Table 20: Data used to identify cutoff points

[0310] Each row represents a single sample. Verification Summary Determine performance characteristics All samples and controls, two LPC and HPC assays, and at least four NC assays were evaluated in duplicate. Text files imported from the SpectraMax M2e reader were used as source data for the analysis. Assay cutoffs were calculated using JMP 14.0 statistical software. The study-phase bioanalytical acceptance criteria for the OD ranges of HPC, LPC, and NC were calculated by a Reliable Scientist (RS). All mean OD values ​​from duplicate (or quadruplicate, if applicable) were evaluated in the analysis.

[0311] Determine the cutoff point To determine the assay cutoff point for detecting NAbs against XTMAB-16, two analysts tested 50 individual virgin human serum samples. Each sample was tested six times in 12 individual runs. The assay cutoff factor (0.91) was calculated as per the present invention technique.

[0312] Using this cutoff factor, the plate-specific cutoff point for each measurement run is calculated as follows: Plate-specific cutoff point The average OD value of the NC on each plate. When the OD value of a sample is less than or equal to the cutoff point for plate specificity assay, the sample is classified as presumed positive.

[0313] Sensitivity Sensitivity was evaluated 12 times. Serial 1.5-fold dilutions (600.00 ng / mL to 52.67 ng / mL) of doped samples in pNHS were analyzed. Sensitivity assessment in human serum was based on the sample with the lowest ADA concentration that consistently produced results above the plate-specific assay cutoff. The assay sensitivity for each sensitivity group was determined by interpolation at the assay cutoff using point-to-point linear regression. The final assay sensitivity was calculated using the concentrations from all qualifying curves. Results showed that the assay sensitivity for detecting anti-XTMAB-16 antibody in human serum was 102.7 ng / mL.

[0314] Sensitivity data were also used to determine the LPC for this assay. The LPC used for the assay was calculated based on the corresponding sensitivity with a 1% false positive rate, which was calculated as mean (sensitivity) + 99%,df x SD (sensitivity), where 99%,df = 2.821 (df = 9). The final LPC concentration used for the assay was calculated to be 220.6 ng / mL (Table 21).

[0315] Table 21: Sensitivity Data Selective Selectivity is the ability of this assay to detect anti-XTMAB-16 NAb in the presence of other matrix components. NAb at LPC levels (220.6 ng / mL) was doped into the serum of 20 individual patients. One of the 20 undoped samples tested positive. All 20 NAb-doped samples at LPC levels were confirmed positive. These results met the selectivity acceptance criteria (Table 22).

[0316] Table 22: Exemplary operational selectivity

[0317] Each row represents a single undoped sample. Matrix interference was evaluated in lipemic samples at 300 mg / dL or in 2% hemolyzed samples. Six pooled serum samples containing 300 mg / dL triglycerides were evaluated, both undoped and doped at LPC levels (220.6 ng / mL) and HPC levels (600 ng / mL). As shown in Figure 7, all undoped samples were confirmed negative, and all LPC and HPC-doped samples were confirmed positive. These results met the acceptance criteria, indicating that the 300 mg / dL lipemic matrix did not interfere with the assay results.

[0318] Six pooled serum samples containing 2% hemolyzed serum were evaluated, both undoped and doped with LPC (220.6 ng / mL) and HPC (600 ng / mL) levels. As shown in Table 23, all undoped samples were confirmed negative, and all LPC and HPC doped samples were confirmed positive. These results meet the acceptance criteria, indicating that the 2% hemolyzed matrix does not interfere with the assay results.

[0319] Table 23: Matrix Interference Data

[0320] Drug tolerance The presence of the drug (XTMAB-16) in the sample may interfere with the detection of anti-XTMAB-16 NAb; therefore, the ability of the assay system to correctly detect NAbs must be evaluated. Drug tolerance is defined as the highest level of drug that will not cause a sample / control to change from positive to negative or from negative to positive.

[0321] Potential interference from the drug was evaluated using serum samples prepared at NC (0.0 ng / mL), LPC (220.6 ng / mL), and HPC (600.0 ng / mL) levels. XTMAB-16 was doped into controls at all three levels at concentrations of 0.0 µg / mL, 12.5 µg / mL, 25.0 µg / mL, 50.0 µg / mL, 100.0 µg / mL, and 200.0 µg / mL.

[0322] As shown in Table 24, no interference was found when up to 200 µg / mL XTMAB-16 was added to the NC, LPC, and HPC levels.

[0323] Table 24: Interference Data Target tolerance The presence of the target (TNFα) in the sample may interfere with the detection of anti-XTMAB-16 NAb. Target tolerance is defined as the highest level of the target that will not cause a sample / control to change from positive to negative or from negative to positive.

[0324] Potential interference from the target TNFα was assessed using serum samples prepared at NC (0.0 ng / mL), LPC (220.6 ng / mL), and HPC (600.0 ng / mL) levels. Four samples were prepared for each control level and doped with 0.0 µg / mL, 0.25 µg / mL, 0.50 µg / mL, or 1.0 µg / mL TNFα. The addition of 0.96 µg / mL TNFα interfered with NAb detection in the NC-level doped samples, but no target interference was observed in the HPC and LPC-level samples doped with up to 1 µg / mL TNFα (Table 25).

[0325] Table 25: Interference data from the target

[0326] Accuracy In a single run, six sets of HPC (600.0 ng / mL), LPC (220.6 ng / mL), and NC (0.0 ng / mL) were used to evaluate the intra-analytical precision, i.e., the evaluation of the intra-analytical performance of the reference standards. The reference standards should meet the OD criteria of NC > plate-specific cutoff point ≥ LPC > HPC. The CV of the OD for HPC and LPC should also be ≤25.0%. All plate controls met the above criteria. The results shown in Table 26 demonstrate that the intra-analytical precisions of HPC, LPC, and NC were 21.6%, 5.6%, and 3.0%, respectively.

[0327] Table 26: Measurement Internal Accuracy

[0328] Inter-analysis accuracy was evaluated based on all accepted validation runs of LPC / NC and HPC / NC listed in Tables 27-29, i.e., the inter-analysis performance of the control. After removing outliers, the inter-analysis accuracies of LPC / NC and HPC / NC were 8.5% and 24.6%, respectively. The results shown in Tables 27-29 demonstrate that the control meets the inter-analysis accuracy criteria.

[0329] Table 27: Inter-measurement Accuracy (Part 1)

[0330] Table 28: Inter-measurement Accuracy (Part 2)

[0331] Table 29: Inter-measurement Accuracy (Part 3)

[0332] stability Sample stability was assessed using HPC, LPC, and NC controls from combined human serum. Freeze / thaw cycles (6 cycles from -70℃ ± 10℃ to room temperature) and benchtop (23 hours) stability were tested. Acceptance criteria for stable samples are as follows: %CV ≤ 25% for LPC and HPC.

[0333] Average OD: NC > ACP 2: LPC > HPC At least 66.7% of the stability samples must meet the above acceptance criteria.

[0334] Test samples were stored at -70°C (nominal) for at least 12 hours prior to the first thaw and for at least 12 hours prior to each additional thaw. Before refreezing, samples were placed at AT for 2 ± 1 hours. Three aliquots of each control (HPC, LPC, and NC) were tested after undergoing six freeze / thaw cycles on plates with frozen PCs and NCs for plate acceptance. All stability samples met the acceptance criteria (Table 30); therefore, anti-XTMAB-16 NAbs in human serum were stable for up to six freeze / thaw cycles from -70°C (nominal) to AT.

[0335] Table 30: Freeze / Thaw Stability

[0336] Prior to stability testing, test samples were stored overnight at -70°C (nominal). Before analysis with the frozen PC and NC used for plate acceptance, three aliquots of each pre-existing frozen control (HPC, LPC, and NC) were thawed on the bench for 23 hours. All stability samples met the acceptance criteria (Table 30). Therefore, anti-XTMAB-16NAb in human serum was stable at AT for up to 23 hours (bench).

[0337] Acceptance criteria for bioanalytical studies in the control phase System suitability criteria are based on data from all valid runs during the validation operation. The %CV standard for repeated internal measurements of controls and samples is ≤25%.

[0338] NC acceptance criteria: NC limit = Overall average response value + 3xSD and > ACP The upper limit of the OD value for NC is 1.92, including the end value.

[0339] LPC acceptance criteria: The LPC acceptance range is: overall average response value ± 3xSD and ≤ ACP.

[0340] The OD values ​​of LPC (220.6 ng / mL XTMAB-16 NAb in pNHS) range from 0.56 to 1.27, including the end values.

[0341] HPC acceptance criteria: HPC lower limit = overall average response value - 3xSD and <LPC。

[0342] The lower limit of the OD value for HPC (600.0 ng / mL XTMAB-16 NAb in pNHS) is 0.03, including the end value.

[0343] LPC / INC's acceptance criteria: LPC / NC ratio acceptance range = average ratio ± 2.33 x SD The OD value range for LPC / NC is 0.57 to 0.85, including the end values.

[0344] Determining robustness To determine the robustness of the assay, the parameters of the assay were tested. HPC, LPC, and NC were tested in two runs with different incubation times in the assay and confirmatory assays, according to the conditions shown in Table 13.

[0345] The operation met the method acceptance criteria, and the control was within the established range. As shown in Table 31, the assay demonstrated robustness at both maximum and minimum incubation times.

[0346] Table 31: Robustness under minimum and maximum incubation times

[0347] Table 32: Summary of Results

[0348] Additional Implementation Plan Clause 1. A method for detecting a neutralizing antibody (NAb) against a target antibody in a sample, comprising: (i) dissociating the NAb from the target antibody by adding an acid wash to the sample; (ii) purifying the NAb in the sample; (iii) contacting the purified NAb with a detection antibody comprising an anti-TNFα antibody conjugated to a detection label; (iv) contacting the purified NAb and the detection antibody with a TNFα-coated substrate; and (v) detecting the NAb when the detection antibody binds to the NAb but not to the TNFα-coated substrate, wherein the target antibody comprises an anti-TNFα chimeric human-mouse monoclonal IgG1-κ antibody containing SEQ ID NO: 9-11 or 15-17 and SEQ ID NO: 12-14 or 18-20.

[0349] Clause 2. The method as described in Clause 1 further includes detecting the detection marker prior to detecting the NAb.

[0350] Clause 3. The method as described in Clause 1 further includes washing the TNFα-coated substrate prior to detecting the NAb.

[0351] Clause 4. The method as described in Clause 1, wherein the pickling solution contains acetic acid.

[0352] Clause 5. The method as described in Clause 1 or 4, wherein the purification step (ii) of the NAb comprises: (a) contacting the sample with an affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label; (b) contacting the sample with a first solid phase coated with a second affinity label, wherein the first affinity label binds to the second affinity label; (c) washing the first solid phase; and (d) eluting the NAb from the first solid phase.

[0353] Clause 6. The method as described in Clause 5, wherein the first affinity marker is biotin.

[0354] Clause 7. The method as described in Clause 5 or 6, wherein the first solid phase is microbeads.

[0355] Clause 8. The method of any one of Clauses 5-7, wherein the first solid phase is magnetic microspheres.

[0356] Clause 9. The method as described in any one of Clauses 5-8, wherein the second affinity marker is streptavidin.

[0357] Clause 10. The method of any one of Clauses 1-9, wherein the NAb is purified using an automated purification system.

[0358] Clause 11. The method of any one of Clauses 1-9, wherein the detection marker comprises a luminescent compound.

[0359] Clause 12. The method of any one of Clauses 1-9, wherein the detection marker comprises an enzyme configured to convert a chromogenic substrate into a pigment.

[0360] Clause 13. The method of any one of Clauses 1-9, wherein the detection marker is a third affinity marker.

[0361] Clause 14. The method of Clause 13, wherein detecting the detection marker further comprises contacting the third affinity marker with a fourth affinity marker, the fourth affinity marker being conjugated to a luminescent compound or configured to convert a chromogenic substrate into a pigment, wherein the third affinity marker is bound to the fourth affinity marker.

[0362] Clause 15. The method as described in Clause 14, wherein the third affinity marker is biotin and the fourth affinity marker is streptavidin.

[0363] Clause 16. The method as described in Clause 11 or 14, wherein detecting the detection mark includes detecting the luminescent compound.

[0364] Clause 17. The method as described in Clause 12 or 14, wherein detecting the detection mark comprises converting the chromogenic substrate into the pigment and detecting the pigment.

[0365] Clause 18. The method as described in Clause 12 or 14, wherein detecting the detection marker comprises converting the chromogenic substrate into the pigment and detecting the optical density of the sample.

[0366] Clause 19. The method as described in any one of Clauses 12 or 14-18, wherein the enzyme is horseradish peroxidase (HRP).

[0367] Clause 20. The method as described in Clause 19, wherein the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB).

[0368] Clause 21. The method as described in any one of Clauses 1-20, wherein steps (i) to (v) are performed on a negative control sample that does not contain detectable NAb.

[0369] Clause 22. The method of any one of Clauses 1-21, wherein the detection antibody is detected when the detection marker is present at a level below a predetermined level, and the detection antibody binds to the NAb rather than the TNFα-coated substrate.

[0370] Clause 23. The method as described in Clause 22, wherein when the optical density of the sample is above a predetermined cutoff point, the detection marker is present at a level below the predetermined level.

[0371] Clause 24. The method as described in Clause 23, wherein the predetermined cutoff point corresponds to a level of 3% or lower false positive rate.

[0372] Clause 25. The method as described in any one of Clauses 23-24, wherein the predetermined cutoff point is a level corresponding to a 1% false positive rate.

[0373] Clause 26. The method as described in any one of Clauses 23-25, wherein the predetermined cutoff point is 0.5-2.

[0374] Clause 27. The method as described in Clause 26, wherein the predetermined cutoff point is 0.91.

[0375] Clause 28. The method of any one of Clauses 1-27, wherein the sensitivity for detecting the NAb is less than 500 ng / mL.

[0376] Clause 29. The method as described in Clause 28, wherein the sensitivity for detecting the NAb is less than 250 ng / mL.

[0377] Clause 30. The method as described in any one of Clauses 28 or 29, wherein the sensitivity for detecting the NAb is less than 150 ng / mL.

[0378] Clause 31. The method as described in any one of Clauses 1-30, wherein the target tolerance is about 1000 ng / mL.

[0379] Clause 32. The method of any one of Clauses 1-31, wherein the target antibody comprises SEQ ID NO: 2 and 5.

[0380] Clause 33. The method of any one of Clauses 1-32, wherein the target antibody comprises SEQ ID NO: 1 and 4.

[0381] Clause 34. A method for detecting a NAb against a target antibody in a sample, comprising: (i) purifying the NAb in the sample; (ii) determining the presence of the NAb in the sample by performing a competitive binding assay, wherein the NAb is present if the NAb binds to the anti-TNFα antibody with a higher affinity than the anti-TNFα antibody for TNFα; and (iii) detecting the NAb; wherein the method has a sensitivity of 250 ng / mL or less, wherein the target antibody comprises an anti-TNFα chimeric human-mouse monoclonal IgG1-κ antibody containing SEQ ID NO: 9-11 or 15-17 and SEQ ID NO: 12-14 or 18-20.

[0382] Clause 35. The method of Clause 34, wherein prior to purifying the NAb, the method further comprises dissociating the NAb from the target antibody by adding an acid washing solution to the sample.

[0383] Clause 36. The method of Clause 34 or 35, wherein the competitive binding assay comprises: (a) contacting the purified NAb with a detection antibody comprising an antiTNFα antibody conjugated to a detection tag; (b) contacting the purified NAb and the detection antibody with a TNFα-coated substrate; (c) washing the TNFα-coated substrate; and (d) detecting the detection tag.

[0384] Clause 37. The method as described in Clause 36, wherein detecting the NAb includes determining whether the detection marker is present at a level below a predetermined level.

[0385] Clause 38. The method as described in Clause 37, wherein when the optical density is above a predetermined cutoff point, the detection marker exists at a level below the predetermined level.

[0386] Clause 39. The method of any one of Clauses 35-38, wherein the pickling solution comprises acetic acid.

[0387] Clause 40. The method of any one of Clauses 34-39, wherein purifying the NAb comprises: (a) contacting the sample with an affinity antibody comprising an anti-TNFα antibody conjugated to a first affinity label; (b) contacting the sample with a first solid phase coated with a second affinity label, wherein the first affinity label binds to the second affinity label; (c) washing the first solid phase; and (d) eluting the NAb from the first solid phase.

[0388] Clause 41. The method as described in Clause 40, wherein the first affinity marker is biotin.

[0389] Clause 42. The method as described in Clause 40 or 41, wherein the first solid phase is microbeads.

[0390] Clause 43. The method of any one of Clauses 40-42, wherein the first solid phase is magnetic microbeads.

[0391] Clause 44. The method as described in any one of Clauses 40-43, wherein the second affinity marker is streptavidin.

[0392] Clause 45. The method of any one of Clauses 34-44, wherein the NAb is purified using an automated purification system.

[0393] Clause 46. The method of any one of Clauses 36-45, wherein the detection marker comprises a luminescent compound.

[0394] Clause 47. The method of any one of Clauses 36-45, wherein the detection marker comprises an enzyme configured to convert a chromogenic substrate into a pigment.

[0395] Clause 48. The method of any one of Clauses 36-45, wherein the detection marker is a third affinity marker.

[0396] Clause 49. The method of Clause 48, wherein detecting the detection marker further comprises contacting the third affinity marker with a fourth affinity marker, the fourth affinity marker being conjugated to a luminescent compound or configured to convert a chromogenic substrate into a pigment, wherein the third affinity marker is bound to the fourth affinity marker.

[0397] Clause 50. The method as described in Clause 49, wherein the third affinity marker is biotin and the fourth affinity marker is streptavidin.

[0398] Clause 51. The method as described in Clause 46 or 49, wherein detecting the detection mark includes detecting the luminescent compound.

[0399] Clause 52. The method as described in Clause 47 or 49, wherein detecting the detection mark comprises converting the chromogenic substrate into the pigment and detecting the pigment.

[0400] Clause 53. The method as described in Clause 47 or 49, wherein detecting the detection marker comprises converting the chromogenic substrate into the pigment and detecting the optical density of the sample.

[0401] Clause 54. The method as described in any one of Clauses 47 or 49-52, wherein the enzyme is horseradish peroxidase (HRP).

[0402] Clause 55. The method as described in Clause 54, wherein the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB).

[0403] Clause 56. The method as described in any one of Clauses 34-55, wherein steps (i) to (iii) are performed on a negative control sample in which no detectable NAb is available.

[0404] Clause 57. The method as described in Clause 38, wherein the predetermined cutoff point corresponds to a level of 1% false positive rate.

[0405] Clause 58. The method described in Clause 38 or 57, wherein the predetermined cutoff point is 0.91.

[0406] Clause 59. The method of any one of Clauses 34-58, wherein the sensitivity for detecting the NAb is less than 500 ng / mL.

[0407] Clause 60. The method of any one of Clauses 34-59, wherein the sensitivity for detecting the NAb is less than 250 ng / mL.

[0408] Clause 61. The method of any one of Clauses 34-60, wherein the sensitivity for detecting the NAb is less than 150 ng / mL.

[0409] Clause 62. The method as described in any one of Clauses 34-61, wherein the target tolerance is about 1000 ng / mL.

[0410] Clause 63. The method of any one of Clauses 34-62, wherein the target antibody comprises SEQ ID NO: 2 and 5.

[0411] Clause 64. The method of any one of Clauses 34-63, wherein the target antibody comprises SEQ ID NO: 1 and 4.

[0412] Clause 65. A kit for detecting NAbs against a target antibody in a sample, comprising: (i) an affinity antibody containing the target antibody conjugated to a first affinity label; (ii) a first solid phase coated with a second affinity label; (iii) a detection antibody containing the target antibody conjugated to a detection label; (iv) a substrate; and (v) TNFα, wherein the target antibody comprises an anti-TNFα chimeric human-mouse monoclonal IgG1-κ antibody containing SEQ ID NO: 9-11 or 15-17 and SEQ ID NO: 12-14 or 18-20.

[0413] Clause 66. The kit as described in Clause 65, wherein the first affinity marker is biotin.

[0414] Clause 67. The kit as described in Clause 65 or 66, wherein the second affinity marker is streptavidin.

[0415] Clause 68. The kit as described in any one of Clauses 65-67, wherein the detection marker is a luminescent compound, an enzyme configured to convert a chromogenic substrate into a pigment, or biotin.

[0416] Clause 69. The kit as described in any one of Clauses 65-68, wherein the first solid phase is microbeads.

[0417] Clause 70. The kit as described in any one of Clauses 65-69, wherein the first solid phase is magnetic microbeads.

[0418] Clause 71. The kit as described in any one of Clauses 65-70, wherein the detection marker is biotin.

[0419] Clause 72. The kit as described in any one of Clauses 65-71 further includes an acid wash buffer.

[0420] Clause 73. The kit as described in any one of Clauses 65-72 also includes a washing buffer.

[0421] Clause 74. The kit as described in any one of Clauses 65-73 further includes elution buffer. (Claims set)

Claims

1. A method for detecting neutralizing antibodies (NAb) against a target antibody in a sample, comprising: (i) dissociating the NAb from the target antibody by adding an acid wash to the sample; (ii) purifying the NAb in the sample; (iii) contacting the purified NAb with a detection antibody comprising an anti-TNFa antibody conjugated to a detection label; (iv) contacting the purified NAb and the detection antibody with a TNFa-coated substrate; and (v) detecting the NAb when the detection antibody binds to the NAb and not the TNFa-coated substrate, wherein the target antibody is an anti-TNFa chimeric human-mouse monoclonal IgGl- kappa antibody comprising SEQ ID NOs: 9-11 or 15-17 and SEQ ID NOs: 12-14 or 18-20.

2. The method of claim 1, further comprising detecting the detection label prior to detecting the NAb.

3. The method of claim 1, further comprising washing the TNFa-coated substrate prior to detecting the NAb.

4. The method of claim 1, wherein the acid wash comprises acetic acid.

5. The method of claim 1 or 4, wherein purifying the NAb of step (ii) comprises: (a) contacting the sample with an affinity antibody comprising an anti-TNFa antibody conjugated to a first affinity label; (b) contacting the sample with a first solid phase coated with a second affinity label, wherein the first affinity label binds to the second affinity label; (c) washing the first solid phase; and (d) eluting the NAb from the first solid phase.

6. The method of claim 5, wherein the first affinity label is biotin.

7. The method of claim 5 or 6, wherein the first solid phase is a microbead.

8. The method of any one of claims 5-7, wherein the first solid phase is a magnetic microbead.

9. The method of any one of claims 5-8, wherein the second affinity label is streptavidin.

10. The method of any one of claims 1-9, wherein the NAb is purified using an automated purification system.

11. The method of any one of claims 1-9, wherein the detection label comprises a luminescent compound.

12. The method of any one of claims 1-9, wherein the detection label comprises an enzyme configured to convert a chromogenic substrate to a pigment.

13. The method of any one of claims 1-9, wherein the detection label is a third affinity label.

14. The method of claim 13, wherein detecting the detection label further comprises contacting the third affinity label with a fourth affinity label conjugated to a luminescent compound or an enzyme configured to convert a chromogenic substrate to a pigment, wherein the third affinity label binds to the fourth affinity label.

15. The method of claim 14, wherein the third affinity label is biotin and the fourth affinity label is streptavidin. ​ 16. The method of claim 11 or 14, wherein detecting the detection marker comprises detecting the luminescent compound.

17. The method of claim 12 or 14, wherein detecting the detection marker comprises converting the chromogenic substrate to the chromogen and detecting the chromogen.

18. The method of claim 12 or 14, wherein detecting the detection marker comprises converting the chromogenic substrate to the chromogen and detecting the optical density of the sample.

19. The method of any one of claims 12 or 14-18, wherein the enzyme is horseradish peroxidase (HRP).

20. The method of claim 19, wherein the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB).

21. The method of any one of claims 1-20, wherein steps (i) to (v) are performed on a negative control sample that has no detectable NAb.

22. The method of any one of claims 1-21, wherein the detection antibody that binds the NAb but not the TNFα-coated substrate is detected when the detection marker is present at a level below a predetermined level.

23. The method of claim 22, wherein the detection marker is present at a level below the predetermined level when the optical density of the sample is above a predetermined cut-off point.

24. The method of claim 23, wherein the predetermined cut-off point is a level corresponding to a false positive rate of 3% or less.

25. The method of any one of claims 23-24, wherein the predetermined cut-off point is a level corresponding to a 1% false positive rate.

26. The method of any one of claims 23-25, wherein the predetermined cut-off point is 0.5-2.

27. The method of claim 26, wherein the predetermined cut-off point is 0.

91.

28. The method of any one of claims 1-27, wherein the sensitivity of detecting the NAb is less than 500 ng / mL.

29. The method of claim 28, wherein the sensitivity of detecting the NAb is less than 250 ng / mL.

30. The method of any one of claims 28 or 29, wherein the sensitivity of detecting the NAb is less than 150 ng / mL.

31. The method of any one of claims 1-30, wherein the target margin is about 1000 ng / mL.

32. The method of any one of claims 1-31, wherein the target antibody comprises SEQ ID NOs: 2 and 5.

33. The method of any one of claims 1-32, wherein the target antibody comprises SEQ ID NOs: 1 and 4.

34. A method for detecting a NAb against a target antibody in a sample, comprising: (i) purifying the NAb in the sample; (ii) determining whether the NAb is present in the sample by performing a competitive binding assay, wherein the NAb is present if it binds the anti-TNFa antibody with a higher affinity than the anti-TNFa antibody binds TNFa; and (iii) detecting the NAb. (iii) detecting the NAb; wherein the target antibody comprises an anti-TNFa chimeric human-mouse monoclonal IgGl-kappa antibody comprising SEQ ID NOs: 9-11 or 15-17 and SEQ ID NOs: 12-14 or 18-20, and wherein the method has a sensitivity of 250 ng / mL or less.

35. The method of claim 34, wherein prior to purifying the NAb, the method further comprises dissociating the NAb from the target antibody by adding an acid wash to the sample.

36. The method of claim 34 or 35, wherein the competitive binding assay comprises: (a) contacting the purified NAb with a detection antibody comprising an anti-TNFa antibody conjugated to a detection label; (b) contacting the purified NAb and the detection antibody with a TNFa-coated substrate; (c) washing the TNFa-coated substrate; and (d) detecting the detection label.

37. The method of claim 36, wherein detecting the NAb comprises determining whether the detection label is present at a level below a predetermined level.

38. The method of claim 37, wherein the detection label is present at a level below the predetermined level when the optical density is above a predetermined cutoff.

39. The method of any one of claims 35-38, wherein the acid wash comprises acetic acid.

40. The method of any one of claims 34-39, wherein purifying the NAb comprises: (a) contacting the sample with an affinity antibody comprising an anti-TNFa antibody conjugated to a first affinity label; (b) contacting the sample with a first solid phase coated with a second affinity label, wherein the first affinity label binds to the second affinity label; (c) washing the first solid phase; and (d) eluting the NAb from the first solid phase.

41. The method of claim 40, wherein the first affinity label is biotin.

42. The method of claim 40 or 41, wherein the first solid phase is a microbead.

43. The method of any one of claims 40-42, wherein the first solid phase is a magnetic microbead.

44. The method of any one of claims 40-43, wherein the second affinity label is streptavidin.

45. The method of any one of claims 34-44, wherein the NAb is purified using an automated purification system.

46. The method of any one of claims 36-45, wherein the detection label comprises a luminescent compound.

47. The method of any one of claims 36-45, wherein the detection label comprises an enzyme configured to convert a chromogenic substrate to a pigment.

48. The method of any one of claims 36-45, wherein the detection label is a third affinity label. ​ 49. The method of claim 48, wherein detecting the detection label further comprises contacting the third affinity label with a fourth affinity label conjugated to a luminescent compound or an enzyme configured to convert a chromogenic substrate to a chromogen, wherein the third affinity label binds to the fourth affinity label.

50. The method of claim 49, wherein the third affinity label is biotin and the fourth affinity label is streptavidin.

51. The method of claim 46 or 49, wherein detecting the detection label comprises detecting the luminescent compound.

52. The method of claim 47 or 49, wherein detecting the detection label comprises converting the chromogenic substrate to the chromogen and detecting the chromogen.

53. The method of claim 47 or 49, wherein detecting the detection label comprises converting the chromogenic substrate to the chromogen and detecting the optical density of the sample.

54. The method of any one of claims 47 or 49-52, wherein the enzyme is horseradish peroxidase (HRP).

55. The method of claim 54, wherein the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB).

56. The method of any one of claims 34-55, wherein steps (i) to (iii) are performed on a negative control sample that has no detectable NAb.

57. The method of claim 38, wherein the predetermined cut-off point is a level corresponding to a 1% false positive rate.

58. The method of claim 38 or 57, wherein the predetermined cut-off point is 0.

91.

59. The method of any one of claims 34-58, wherein the sensitivity of detecting the NAb is less than 500 ng / mL.

60. The method of any one of claims 34-59, wherein the sensitivity of detecting the NAb is less than 250 ng / mL.

61. The method of any one of claims 34-60, wherein the sensitivity of detecting the NAb is less than 150 ng / mL.

62. The method of any one of claims 34-61, wherein the target margin is about 1000 ng / mL.

63. The method of any one of claims 34-62, wherein the target antibody comprises SEQ ID NOs: 2 and 5.

64. The method of any one of claims 34-63, wherein the target antibody comprises SEQ ID NOs: 1 and 4.

65. A kit for detecting a NAb against a target antibody in a sample, comprising: (i) an affinity antibody comprising the target antibody conjugated to a first affinity label; (ii) a first solid phase coated with a second affinity label; (iii) a detection antibody comprising the target antibody conjugated to a detection label; (iv) a substrate; and (v) TNFα, wherein the target antibody is a chimeric human-murine monoclonal IgGl-kappa antibody to TNFa comprising SEQ ID NOs: 9-11 or 15-17 and SEQ ID NOs: 12-14 or 18-20.

66. The kit of claim 65, wherein the first affinity label is biotin.

67. The kit of claim 65 or 66, wherein the second affinity label is streptavidin.

68. The kit of any one of claims 65-67, wherein the detection label is a luminescent compound, an enzyme configured to convert a chromogenic substrate to a pigment, or biotin.

69. The kit of any one of claims 65-68, wherein the first solid phase is a microbead.

70. The kit of any one of claims 65-69, wherein the first solid phase is a magnetic microbead.

71. The kit of any one of claims 65-70, wherein the detection label is biotin.

72. The kit of any one of claims 65-71, further comprising an acid wash buffer.

73. The kit of any one of claims 65-72, further comprising a wash buffer.

74. The kit of any one of claims 65-73, further comprising an elution buffer.

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