IMPROVED METHOD AND TEST SYSTEM FOR IN-VITRO DETERMINATION OF DRUG ANTIBODIES IN BLOOD
20230305021 · 2023-09-28
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Cpc classification
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Abstract
Method of determining therapeutic drug antibodies in a sample of bodily fluid of a subject receiving a medication containing a therapeutic drug antibodies against tumor necrosis factor alpha. The method is used in an lateral flow immunochromatographic test wherein the immunochromatographic bridging and binding in the test line comprises the use of an anti-idiotypic scFv fragment or Fab fragment fused to a carrier protein which is not involved in nor plays a role in the inherent or developed immune system. The fusion protein may contain human serum albumin, chicken ovalbumin, human haptoglobin or human alpha-1- antitrypsin. The immunological reaction is therefore not impaired, augmented or interfered by members of the complement system or by autoantibodies such as the rheumatoid factor. This is of particular importance and favorable when determining the concentration of tumor necrosis alpha blockers such as adalimumab or in-fliximab in serum or blood of patients.
Claims
1-11. (canceled)
12. A method of determining the concentration of a therapeutic drug antibody in a blood sample of a human subject receiving the therapeutic drug antibody by a lateral flow immunoassay, comprising the steps of: a) providing at least one chemically modified, labeled, or tagged receptor that binds the therapeutic drug antibody; and b) providing a solid phase comprising an immobilized target protein that binds to or is bound by the therapeutic drug antibody, wherein the receptor or the target protein or both have been previously prepared from a complement determining region (CDR) or an antigen-binding fragment (Fab) or a single-chain variable fragment (scFv) of an anti-idiotypic single-chain antibody and fused to an amino acid chain of a large protein selected so as not to be bound by molecules of the human immune system or by other molecules present in the body fluid, and c) sequentially contacting a defined sample of body fluid with said chemically modified, labeled, or tagged receptor and said target protein immobilized on said solid phase; and d) determining the amount of chemically modified, labeled, or tagged receptors in the body fluid sample to quantify the concentration of said therapeutic drug antibody in the body fluid sample without any interference from the inert or acquired immune system or molecules in the body fluid sample that can bind to the Fc-fragment of an antibody.
13. A method of determining the concentration of a therapeutic drug antibody in a blood sample of a human subject receiving the therapeutic drug antibody by lateral flow immunoassay, comprising the steps of: a) providing at least one chemically modified, labeled, or tagged target protein that binds to or is bound by the therapeutic drug antibody; b) providing a solid phase having an immobilized receptor that binds to the therapeutic antibody; wherein the receptor or the target protein or both have been previously prepared from a complement determining region (CDR) or an antigen-binding fragment (Fab) or a single-chain variable fragment (scFv) of an anti-idiotypic single-chain antibody and fused to an amino acid chain of a large protein selected so as not to be bound by molecules of the human immune system or by other molecules present in the body fluid, and c) sequentially contacting a defined sample of the body fluid with the chemically modified, labeled, or tagged peptide and the receptor immobilized on the solid phase; and d) determining the amount of chemically modified, labeled, or tagged receptors in the body fluid sample to quantify the concentration of said therapeutic drug antibody in the body fluid sample without any interference from the inert or acquired immune system or molecules in the body fluid sample that can bind to the Fc-fragment of an antibody.
14. The method of claim 12, wherein the immobilized target peptide or the receptor is fused with an amino acid chain from a protein selected from human serum albumin, human haptoglobin, and human hemoglobin.
15. The method of claim 12, wherein the immobilized target peptide is an anti-idiotypic single-chain variable fragment (scFv) fused with an amino acid chain encoding for human serum albumin or chicken ovalbumin or human haptoglobin or human alpha-1-antitrypsin.
16. The method of claim 12, wherein the receptor is His6-tagged anti-idiotypic Fab fragment (Fab) labeled by any one or more of gold nanoparticles, dyed latex particles, cellulose nanobeads, carbon nanobeads, magnetic nanobeads or a detection enzyme.
17. The method of claim 12, wherein the receptor binds a human, humanized, or chimeric monoclonal antibody selected from the group comprising antibodies binding a member of the tumor necrosis factor ligand family and tumor necrosis factor alpha (tumor necrosis factor ligand superfamily member 2) blocker.
18. The method of claim 12, wherein the receptor binds any one of Adalimumab (Humira®), Infliximab (Remicade ®), Golimumab (CNTO 148 or Simponi®), certolizumab (Cimzia®), Vedolizumab (Entyvio®, Tanaka), Etanercept antibody (Enbrel®, Pfizer), Ustekinumab (Stelara ®, Janssen-Cilag), Rituximab, Brentuximab (Adcetrix).
19. The method of claim 12, wherein the fluid sample is whole blood, plasma, or serum.
20. The method of claim 12, comprising the use of a test system that includes a test device adapted to house a lateral flow test which test device displays one or more reference images and a region of interest of said lateral flow test, which bears one or more visible response zones indicating the presence and content of said therapeutic antibody (T) in a said test sample and a control zone (C), and a portable processor device such as a smartphone comprising a digital camera, a source of light and a processor, wherein the processor is configured to process digital images captured by the said camera and to represent an analytical result based on the determination of the optical intensities of said visual zones.
21. The method of claim 12, comprising the use of a test system wherein the application pad of said lateral flow assay contains a fleece that can withhold the particulate components of a blood sample.
22. The method of claim 12, comprising the use of a test kit comprising a measuring capillary for taking a defined amount of blood; a vessel with a known amount of buffer for dilution of the blood sample and for being a running buffer in a lateral flow immunochromatography; and a lateral flow immunochromatographic test device comprising a tagged or labeled receptor which binds the therapeutic drug antibody, and a target protein as described in claim 12 immobilized in a zone on the membrane of the immunochromatographic strip.
23. The method of claim 12, comprising the use of a lateral flow immunochromatographic assay for monitoring the level of therapeutic antibody in the blood or serum of a patient receiving a medication containing said therapeutic antibody, wherein the target protein in the test line contains an antigen-binding fragment (Fab) or a single-chain variable fragment (scFv) of said therapeutic antibody fused with an amino acid chain from a protein or peptide that is not bound by molecules related to the immune system, including autoantibodies, rheumatoid factors, and CCP antibodies.
24. The method of claim 13, wherein the immobilized target peptide or the receptor is fused with an amino acid chain from a protein selected from human serum albumin, human haptoglobin, and human hemoglobin.
25. The method of claim 13, wherein the immobilized target peptide is an anti-idiotypic single-chain variable fragment (scFv) fused with an amino acid chain encoding for human serum albumin or chicken ovalbumin or human haptoglobin or human alpha-1-antitrypsin.
26. The method of claim 13, wherein the receptor is His6-tagged anti-idiotypic Fab fragment (Fab) labeled by any one or more of gold nanoparticles, dyed latex particles, cellulose nanobeads, carbon nanobeads, magnetic nanobeads or a detection enzyme.
27. The method of claim 13, wherein the receptor binds a human, humanized, or chimeric monoclonal antibody selected from the group comprising antibodies binding a member of the tumor necrosis factor ligand family and tumor necrosis factor alpha (tumor necrosis factor ligand superfamily member 2) blocker.
28. The method of claim 13, wherein the receptor binds any one of Adalimumab (Humira®), Infliximab (Remicade ®), Golimumab (CNTO 148 or Simponi®), certolizumab (Cimzia®), Vedolizumab (Entyvio®, Tanaka), Etanercept antibody (Enbrel®, Pfizer), Ustekinumab (Stelara ®, Janssen-Cilag), Rituximab, Brentuximab (Adcetrix).
29. The method of claim 13, wherein the fluid sample is whole blood, plasma, or serum.
30. The method of claim 13, comprising the use of a test system that includes a test device adapted to house a lateral flow test which test device displays one or more reference images and a region of interest of said lateral flow test, which bears one or more visible response zones indicating the presence and content of said therapeutic antibody (T) in a said test sample and a control zone (C), and a portable processor device such as a smartphone comprising a digital camera, a source of light and a processor, wherein the processor is configured to process digital images captured by the said camera and to represent an analytical result based on the determination of the optical intensities of said visual zones.
31. The method of claim 13, comprising the use of a test system wherein the application pad of said lateral flow assay contains a fleece that can withhold the particulate components of a blood sample.
32. The method of claim 13, comprising the use of a test kit comprising a measuring capillary for taking a defined amount of blood; a vessel with a known amount of buffer for dilution of the blood sample and for being a running buffer in a lateral flow immunochromatography; and a lateral flow immunochromatographic test device comprising a tagged or labeled receptor which binds the therapeutic drug antibody, and a target protein as described in claim 13 immobilized in a zone on the membrane of the immunochromatographic strip.
33. The method of claim 13, comprising the use of a lateral flow immunochromatographic assay for monitoring the level of therapeutic antibody in the blood or serum of a patient receiving a medication containing said therapeutic antibody, wherein the target protein in the test line contains an antigen-binding fragment (Fab) or a single-chain variable fragment (scFv) of said therapeutic antibody fused with an amino acid chain from a protein or peptide that is not bound by molecules related to the immune system, including autoantibodies, rheumatoid factors, and CCP antibodies.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] In the drawings:-
[0036]
[0037]
[0038]
[0039]
[0040]
DETAILED DESCRIPTION OF THE INVENTION
[0041] The disclosure relates to in vitro methods and compositions for determination of drug antibody (a tumor necrosis factor alpha (TNF-α)-blocker such as adalimumab (ADL) or infliximab, etc.) in a serum sample from a patient receiving or who shall receive therapeutic drug antibodies. The method is preferably based on a gold-labeled anti-idiotypic antibody as shown in
[0042] The instant disclosure relates to an anti-idiotypic receptor which recognizes the therapeutic drug antibody. The anti-idiotypic receptor may be bound to a solid phase such as a gold particle or to the membrane of a lateral flow immunochromatography. This binding may be covalently or by Van-der-Waal forces which however excludes the use of a pure Fab fragment of scFv fragment. The coupling to a solid phase of such a small molecule seems to grossly interfere with the immunological binding reaction. The anti-idiotypic receptor could be covalently coupled to another antibody fragments, avidin, streptavidin, but as the oral administration of supplementary biotin has become accepted, allegedly or presumed to influence the fertility rate and having other beneficial activity, the use of such highly binding tags is prohibited as circulating biotin may interfere with test results.
[0043] The conventional anti-idiotypic immunoglobulin antibodies are large heterodimeric molecules composed of heavy and light polypeptide chains. Light chains are divided into kappa (k) and lambda (λ) types. By enzymatic cleavage, the fragment-antigen binding (Fab) portion of the molecule can be separated from the fragment constant (Fc). The Fab fragments contain the variable domains, which consist of three antibody hypervariable amino acid domains responsible for the antibody specificity embedded into constant regions. The autoantibodies however bind primarily the non-variable regions of such antibodies.
[0044] Therapeutic monoclonal antibodies are a group of biological drugs designed for diseases that are difficult to treat by conventional small molecule drugs. These diseases include autoimmune disorders such as inflammatory bowel disease (IBD) and rheumatoid arthritis. The therapeutic principle in both disorders is the blockade of endogenous tumor necrosis factor α (TNFα). By inhibiting TNFα, the autoimmune inflammation cascade is interrupted, and the symptoms of the disease are suppressed. On the other hand, such patients have typically developed “uncontrolled” auto-immunoreactions which interfere with a conventional determination of the concentration of the therapeutic drug antibody.
[0045] Adalimumab is a TNF-inhibiting, anti-inflammatory humanized antibody-drug that binds selectively to tumor necrosis factor alpha (TNFα). Adalimumab is used to treat a wide range of diseases such as rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn’s disease, ulcerative colitis, chronic psoriasis, hidradenitis suppurativa, and juvenile idiopathic arthritis. Treatment with adalimumab may however increase the risk of infections. The monoclonal antibody therapy may further be associated with adverse reactions such as hypersensitivity or reduced efficacy of biological drugs. These adverse reactions are often induced by anti-drug antibodies (ADA) of different isotypes. Foreign murine epitopes in the variable region of chimeric antibodies may be responsible for the formation of ADA. However, immediate-type reactions with severe allergic symptoms and even fatal anaphylaxis are reported for the drug cetuximab. ADA may also be involved during therapy in the reduced efficacy of therapeutic antibodies, resulting in clinical non-responsiveness. It has also been shown that pre-existing antibodies against biological drugs may be present in some individuals.
[0046] Bodily fluids are liquids originating from animal and human subjects, including fluids that are excreted or secreted from the body. Samples of bodily fluids for use in the present method may be blood, serum, plasma, urine, saliva.
[0047] It has been discovered that anti-drug antibodies are not only developed against chimeric antibodies, such as infliximab (IFX), but also during treatment with so-called humanized antibodies, such as adalimumab (ADL) (Harding et al., MAbs. 2:256-65, 2010). These anti-drug antibodies are directed against the antigen-binding region and, thus, prevent binding to TNF-α resulting in treatment failure (Frederiksen et al., Inflamm Bowel Di. 20:1714-21, 2014). The specific immunogenic peptide sequences of IFX and their localization in the TNF-α binding site have been discovered (Homann et al., J Transl Med 13:339, 2015). However, the relevant immunogenic peptide sequences of adalimumab involved in the binding by anti-drug antibodies have so far not been described. Epitopes recognized by serum IgG from infliximab-treated patients are often located in the variable part of the Fab region but usually not in the complement determining region. Thus, as may be expected from a chimeric antibody, non-human peptide sequences evoke an anti-idiotype immune response in the human host. However, anti-drug antibodies may also develop during treatment with humanized therapeutic antibodies like adalimumab.
[0048] The present disclosure overcome these problems created by the binding of anti-drug antibodies to the drug infliximab or adalimumab. The present application further discloses a method for detecting the presence of anti-adalimumab antibodies, which allow for enhanced detection sensitivity and facilitate formation of immunological complexes.
EXAMPLES
Example 1
[0049] Rapid test for Infliximab using an Fd-ovalbumin fusion protein. The Fd chain of Fab in the Fab-ds format was fused with chicken ovalbumin (UniProtKB – P01012 (OVAL_CHICK) – Gallus gallus) so that the fusion product can be coupled to the membrane of a rapid immuno lateral flow test without causing any interference of the Fab binding to the drug antibody infliximab. The chicken ovalbumin was chosen for not introducing any antigenic determinant which is recognized or bound by human antibodies or serum proteins. The native ovalbumin conformer can be transformed into a thermostabilized conformer under alkaline conditions as Ser-165, Ser-237 and Ser-321 may take on a D-configuration. Cloning of the ovalbumin fragment via Eco RI and Hindlll in a licensed standard HuCAL expression vector pMx11 (containing the Fab-Fragment of an antibody against infliximab). With respect to the structure of the HUCL expression vector pMx11, see
TABLE-US-00001 SEQ ID NO: 1 KSC EFAAA GSIGAASMEF CFDVFKELKV HHANENIFYC PIAIMSALAM VYLGAKDSTR TQINKWRFD KLPGFGDSIE AQCGTSVNVH SSLRDILNQI TKPNDVYSFS LASRLYAEER YPILPEYLQC VKELYRGGLE PINFQTAADQ ARELINSWVE SQTNGIIRNV LQPSSVDSQT AMVLVNAIVF KGLWEKAFKD EDTQAMPFRV TEQESKPVQM MYQIGLFRVA SMASEKMKIL ELPFASGTMS MLVLLPDEVS GLEQLESIIN FEKLTEWTSS NVMEERKIKV YLPRMKMEEK YNLTSVLMAM GITDVFSSSA NLSGISSAES LKISQAVHAA HAEINEAGRE WGSAEAGVD AASVSEEFRA DHPFLFCIKH IATNAVLFFG RCVSPGSHHHHHH
[0050] The expressed fusion product of the Fd chain and chicken S-ovalbumin bound infliximab in a serum sample when bound to a membrane. Human serum contained no antibodies nor molecules of the complement system which cross-reacted with the Fd chain or ovalbumin. The immunological reaction was not affected (augmented, interfered or inhibited) by members of the complement system or by autoantibodies such as the rheumatoid factor. This will prove favorable when determining the concentration of tumor necrosis alpha blocker infliximab in serum or blood of patients. Thus, it this construct is useful for building a lateral flow rapid test for a quantitative determination of infliximab in blood or serum.
Example 2
[0051] Rapid test for Infliximab using an Fd-human albumin fusion protein. The following isoform of human albumin (UniProtKB – P02768 (ALBU_HUMAN) was used for a fusion protein with the Fd chain is given below, wherein KSC represents again the C-terminus of the Fd chain, EFAA an introduced EcoR1 cloning site, GSHHHHHH a GS-linker plus His6-tag for facilitating purification of the expressed Fd-human albumin fusion protein.
TABLE-US-00002 SEQ ID NO: 2 KSC EFAAAD AHKSEVAHRF KDLGEENFKAL VLIAFAQYLQ QCPFEDHVKL VNEVTEFAKT CVADESAENC DKSLHTLFGD KLCTVATLRET YGEMADCCAK QEPERNECFL QHKDDNPNLP RLVRPEDVMC TFHDNEETFL KKYLYEIARRH PYFYAPELLF FAKRYKAAFT ECCQAADKAA CLLPKLDELR DEGKASSAKQ RLKCASLQKFG ERAFKAWAVA RLSQRFPKAE FAEVSKLVTD LTKVHTECCH DLLECADRAD LAKYICENQDS ISSKLKECCE KPLLEKSHCI AEVENDEMPA DLPSLAADFV ESKDVCKNYA EAKDVFLGMF LYEYARRHPD YSVVLLLRLA KTYETTLEKC CAAADPHECY AKVFDFKPLV EEQNLIKQNC ELFEQLGEYK FQNALLVRYT KKVPQVSTPT LVEVSRNLGK VGSKCCKHPE AKRMPCAEDY LSVVLNQLCV LHEKTPVSDR VTKCCTESLV NRRPCFSALE VDETYVPKEF NETFTFHADC TLSEKERQIK KQTALVELVK HKPKATKEQL KAVMDDFAAF VEKCCKADDK ETCFAEEGKK LVAASQAALG LGSHHHHHH
[0052] The purified fusion product of the Fd chain and human albumin should not cross-bind any antibodies nor molecules of the complement system or rheumatoid factor when bound to a membrane. Thus, this construct can be used in a lateral flow rapid test for rapid quantitative determination of infliximab in serum.
Example 3
[0053] Rapid test for Infliximab using a Fd-human alpha-1-antitrypsin fusion protein. The following isoform of human alpha-1-anti-trypsin (UniProtKB – P01009 (A1AT_HUMAN)) was used for a fusion protein with the Fd chain, since it has only moderate affinity for plasmin and thrombin. In the sequence given below KSC represents the C-terminus of the Fd chain, EFAA an introduced EcoR1 cloning site, GSHHHHHH a GS-linker plus His6-tag for facilitating purification of the expressed Fd-human albumin fusion protein.
TABLE-US-00003 SEQ ID NO: 3 KSC EF AAA EDPQGDAAQK TDTSHHDQDH PTFNKITPNL AEFAFSLYRQ LAHQSNSTNI FFSPVSIATA FAMLSLGTKA DTHDEILEGL NFNLTEIPEA QIHEGFQELL RTLNQPDSQL QLTTGNGLFL SEGLKLVDKF LEDVKKLYHS EAFTVNFGDT EEAKKQINDY VEKGTQGKIV DLVKELDRDT VFALVNYIFF KGKWERPFEV KDTEEEDFHV DQVTTVKVPM MKRLGMFNIQ HCKKLSSWVL LMKYLGNATA IFFLPDEGKL QHLENELTHD IITKFLENED RRSASLHLPK LSITGTYDLK SVLGQLGITK VFSNGADLSG VTEEAPLKLS KAVHKAVLTI DEKGTEAAGA MFLEAIPMSI PPEVKFNKPF VFLMIEQNTK SPLFMGKWN PTQKGSHHHHHH
[0054] The purified fusion product of the Fd chain and alpha-1-antitrypsin should bind the other not augment, inhibit, or interfered by molecules of the complement or the rheumatoid factor (RF). The rheumatoid factor is an auto-antibody against the Fc portion of IgG, which has been deleted. Consequently, RF present in serum cannot form an immune complex in a lateral flow assay with the stationary receptor, a fusion product as described. Thus, this Fd-fusion construct can be used in a lateral flow rapid test for rapid quantitative determination of infliximab in serum.
Example 4
[0055] LFA-test for infliximab using a fusion of HAS with a single-chain Fv fragment. A lateral flow immunochromatographic assay test to detect infliximab® (human anti-human-TNFα-mouseCDR) was prepared using anti-IFX single-chain Fv fused with human serum albumin (conc. 1.5 mg/mL) immobilized on a nitrocellulose membrane (240-280 .Math.m nitrocellulose with a backing of 100 .Math.m polyester (clear) and 100 .Math.m polyester (white)). The membrane had a capillary speed down web of 90 to 180 seconds per 40 mm. The conjugate pad contained gold-labeled anti-IFX-Fab-.sub.K-ds-H (FLAG-His6) at 200 .Math.L/30 which could bind laterally the CDR of IFX. The sample was 10 .Math.l whole blood in 500 microliters MOPS running buffer (100 mmol 3-(N-morpholino) propanesulfonic acid, 200 mmol NaCl, pH 7.5, 0.095 (w/v) sodium azide). The sample was applied on a blood particle separation pad which also acted as a sponge for the running sample fluid. Once soaked, the sample fluid flew to the conjugate pad which contained in a salt-sugar matrix gold-labeled anti-IFX-Fab-.sub.K-ds-H (His6). The conjugate pad also contained the reagents for an optimized chemical binding between the infliximab analyte and the gold labeled anti-IFX-Fab as they pass 9through the pad and continue across to the test and control lines. The control line (C ) contained rabbit anti-chicken IgY at 0.6 mg/mL and labeled chicken IgY was used as control.
[0056]
[0057] This example was repeated using whole EDTA blood samples (5) and serum samples (5) from patients having rheumatoid arthritis or another rheumatic disease who have not yet received a therapeutic treatment with anti-TNFα-drug antibodies (Infliximab®, Remicade®, Remsima®, Inflectra®, Adalimumab®, or any other biosimilar).These blood or serum samples contained rheumatoid factors (RF) from 29.5 IU/mL to 420 IU/mL (healthy reference < 25 IU/mL) and/or CCP (cyclic citrullinated peptide) antibodies from 23.9 to 1876 IU/mL (healthy reference < 7 IU/mL) as determined by an external clinical laboratory.
[0058] In brief, CCP autoantibodies attack healthy tissue in joints, and they are found in more than 75 percent of people who have rheumatoid arthritis but almost never in people without that disease. Rheumatoid arthritis is a progressive, autoimmune disease that causes pain, swelling, and stiffness in the joints. Despite the name, the level of rheumatoid factors (RF) in blood is not specific to rheumatoid arthritis. The normal range of RF is from 0-20 IU/ml and there are other reasons the RF level may be elevated. Some conditions and medical procedures that can raise RF levels include other autoimmune diseases, certain chronic infections, diabetes, bacterial endocarditis, cancer, normal aging, vaccinations, and transfusions. Once the RF level is elevated, it will often remain so even if the disease goes into remission. Thus, increased RF levels may be found even in healthy people.
[0059] The patient’s blood and serum samples containing RF and CCP antibodies were spiked with infliximab® drug antibodies in same concentrations as described above and their levels in blood or serum determined by an LFA-test and using a smart phone as line readerwhich contained a proprietary OuantOn® IFX (ImmundiagnostikAG, Bensheim, DE) application as described in WO 2019/215199 and PCT/EP2021/054106. The QuantOnO IFX reader had been calibrated against said Optrilyzer® line reader.
[0060] The LFA-test lower limit of detection was again 0.5 .Math.g/mL and the determination not impacted by the presence of RF or CCP autoantibodies. The re-finding of IFX spikes in said samples was equivalent to that of IFX spikes in RF- or CCP negative samples.
SYNOPSIS
[0061] The examples therefore provide a LFA test and a method of determining the level of therapeutic drug antibodies in a sample of blood or serum of a subject receiving a medication containing a therapeutic drug antibodies, such as TNFα blockers. The method is comprising in a lateral flow immunochromatographic test wherein the bridging and binding in the test line is obtained by an anti-idiotypic scFv fragment or Fab fragment fused to a carrier protein which is not involved in nor plays a role in the inherent or developed immune system. The fusion protein may be with human serum albumin, chicken ovalbumin, human haptoglobin or human alpha-1-antitrypsin. The immunological reaction is therefore not impaired, augmented or interfered by components of the complement system or autoantibodies such as the rheumatoid factor or CCP antibodies. This is of particular importance and favorable when determining the level TNFα blockers in patients treated for rheumatoid arthritis or a rheumatic disease such as systemic lupus erythematosus (SLE), Sjögren syndrome, dermatomyositis/polymyositis, Scleroderma/systemic sclerosis, ankylosing spondylitis, psoriatic arthritis, enteropathic arthritis, inflammatory bowel diseases (Crohn’s disease and ulcerative colitis), granulomatous polyangiitis (Wegener’s granulomatosis), polyangiitis, fibromyalgia and many more. More than 200 rheumatic diseases have been identified so far.