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WO2017137613A1 - Dosage immunologique pour la détection et la surveillance des réponses inflammatoires - Google Patents

Dosage immunologique pour la détection et la surveillance des réponses inflammatoires Download PDF

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Publication number
WO2017137613A1
WO2017137613A1 PCT/EP2017/053086 EP2017053086W WO2017137613A1 WO 2017137613 A1 WO2017137613 A1 WO 2017137613A1 EP 2017053086 W EP2017053086 W EP 2017053086W WO 2017137613 A1 WO2017137613 A1 WO 2017137613A1
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Prior art keywords
antibody
tenascin
immunoassay
immunoassay according
human
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Kim Suzanne Midwood
David Pritchard
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NASCIENT Ltd
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NASCIENT Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

  • the present disclosure relates an immunoassay suitable for the detection and monitoring of inflammatory and/or autoimmune diseases, such as rheumatoid arthritis and kits for use in the said assay.
  • RA Rheumatoid Arthritis
  • RA is diagnosed using a defined set of clinical features, including a selection of biomarkers: such as auto-antibodies against citrullinated peptides (anti-CCP) and rheumatoid factor (RF).
  • anti-CCP citrullinated peptides
  • RF rheumatoid factor
  • CRP C-reactive protein
  • ESR erythrocyte sedimentation rate
  • RA The pathology of RA is complex. It is characterised by a chronic synovial inflammation and destruction of joint cartilage and bone, which is mediated by the recruitment and invasion of immune cells into the acellular synovium (the joint lining). This cellular invasion is thought to be a result of the production of a variety of cytokines, matrix-degrading enzymes and small molecule mediators such as prostaglandins and nitric oxide.
  • cytokines cytokines
  • matrix-degrading enzymes small molecule mediators
  • a selection of immune cells including CD4 + T cells, B cells and macrophages, are activated by certain synoviocyte cells which exhibit macrophage and fibroblast-like properties.
  • the RA synoviocytes also recruit surrounding cells.
  • synovium Whilst the healthy synovium protects the joints and facilitates joint movement; in RA, the synoviocytes show increased proliferation, the ability to proliferate in anchorage-independent conditions and defective contact inhibition. This results in a hyperplastic intimal lining and the formation of aggressive fronts of tissue called pannus, which lead the destruction of the articular structures within the joint.
  • synoviocytes in RA have are thought to be oligoclonality, particularly the synoviocytes involved in the pannus regions.
  • synoviocytes taken from osteoarthritis patients (OA) show normal behaviour. This has resulted in the hypothesis that the expanding synovium is a locally invasive tumour.
  • an immunoassay for the detection of a Tenascin in a biological sample, comprising at least a capture antibody and a conjugate antibody, wherein at least one of said antibodies has a human VH specific to the Tenascin.
  • the sample is an ex vivo patient sample.
  • the immunoassay is a method, for the detection of a Tenascin in a biological sample, comprising at least a capture antibody and a conjugate antibody, said method comprising the step of contacting an ex vivo patient derived sample with said antibodies, wherein at least one of said antibodies has a human VH specific to the Tenascin.
  • the immunoassay of the present disclosure is suitable for identifying levels of Tenascin (or specific forms thereof) indicative of an autoimmune disease and/or an inflammatory disease, such as rheumatoid arthritis.
  • the immunoassay is for identifying the presence or levels of Tenascin (or specific forms thereof) indicative chronic inflammatory responses.
  • the capture antibody is specific to the Tenascin, for example is specific to a region of Tenascin selected from the group comprising domain B, EGF-like repeats, FBG domain (in particular a conserved portion, section, fragment thereof), FNIII 1-3 region (in particular a conserved section thereof), such as the FBG domain and FNIII 1-3 region, in particular the FBG domain.
  • conjugate antibody is specific to the capture antibody.
  • the conjugate antibody is specific to Tenascin, for example is specific to the FBG domain of Tenascin.
  • the capture antibody is specific to an FBG region in the Tenascin.
  • the Tenascin is Tenascin C.
  • the at least one human VH is specific to the FBG region.
  • the at least one human VH is specific to the FBG region of Tenascin-C.
  • the human VH is selected from a sequence shown in SEQ ID NO: 4, 12, 19,
  • the capture antibody is from a non-human mammal, for example a mouse, a rat, a rabbit or a camelid (others non-human mammals include sheep, cows, llamas, camels, goats, donkey and pigs), for example one or more, such as a six CDRs from the murine monoclonal antibody 9F8, in particular the VH domain and VL domain from said antibody.
  • a non-human mammal for example a mouse, a rat, a rabbit or a camelid
  • non-human mammals include sheep, cows, llamas, camels, goats, donkey and pigs
  • one or more such as a six CDRs from the murine monoclonal antibody 9F8, in particular the VH domain and VL domain from said antibody.
  • the conjugate antibody comprises a human variable domain, for example the antibody or binding domain (VH and VL) thereof is fully human.
  • the conjugate antibody is from a non-human mammal, for example mouse, rat, rabbit or camelid (others non-human mammals include sheep, cows, llamas camels, goats, donkey and pigs), for example one or more, such as a six CDRs from the murine monoclonal antibody 9F8, in particular the VH domain and VL domain from said antibody.
  • a non-human mammal for example mouse, rat, rabbit or camelid
  • non-human mammals include sheep, cows, llamas camels, goats, donkey and pigs
  • one or more such as a six CDRs from the murine monoclonal antibody 9F8, in particular the VH domain and VL domain from said antibody.
  • the capture and/or conjugate antibody is humanized.
  • both the capture antibody and the conjugate antibody comprise a human VH, for example a VH of SEQ ID NO: 12.
  • both the capture antibody and the conjugate antibody comprise a VH with the same amino acid sequence, for example a VH disclosed herein such as SEQ ID NO: 12.
  • the immunoassay of the present disclosure is capable of detecting and/or differentiating oligomeric Tenascin molecules.
  • the assay of the present disclosure is capable of detecting and/or differentiating a Tenascin form associated with pathogenesis of rheumatoid arthritis.
  • the immunoassay is an ELISA assay.
  • the immunoassay is a self-ELISA.
  • kits for use in an immunoassay defined herein comprising a capture antibody and a conjugate antibody wherein at least one of said antibodies has a human VH specific to the Tenascin, in particular as described herein.
  • the immunoassay according to the present disclosure is not specific for citrullinated Tenascin, for example an antibody employed in the present immunoassay is not specific to citrullinated Tenascin-C.
  • the immunoassay according to the present disclosure does not identify the presence or levels of patient antibodies specific to citrullinated Tenascin (such as citrullinated Tenascin C).
  • an antibody or binding fragment as specifically disclosed herein or a composition comprising the same for example for use in diagnosis (including in vivo analysis) and/or treatment.
  • Figure 1 shows a graph of serum concentration in a sample against the percentage of maximum absorbance generated by each assay, demonstrating the linearity of each reaction.
  • IBL is a commercially available assay available from IBL (Tenascin-C large (FNIII-B) Assay Kit Code No: 27767).
  • 9F8 is a murine antibody specific to the FNIII
  • B12-VH refers to a human antibody C3 disclosed herein.
  • Figure 3 shows a graph of absorbance response generated by incubation of sera samples
  • Figure 5 shows a graph of absorbance response generated by incubation of sera samples
  • Figure 7 shows a graph of absorbance response generated by incubation of sera samples
  • Figure 9 shows complete sera testing with self-sandwich assay employing a B12 antibody or
  • Figure 10 shows a ROC curve comparison of 9F8 (capture antibody): B12-VH (conjugate antibody) and IBL (FNIII-C) assay. After about 0.5 on the X-axis the IBL assay is incapable of discriminating normal samples from RA samples. In contrast the 9F8:B12-VH assay is capable of discriminating sample at all points bar one (at about 0.9).
  • Figure 11 shows a ROC curve of serum testing including single antibody sandwich assays
  • a fully human VH region employed in an immunoassay of the present disclosure provides better results than employing commercially available murine antibodies.
  • the analysis employing a human VH provides at least comparable results to currently available commercial kits for the analysis of Tenascin (such as the kit available from IBL). What is more employing a human VH in the analysis of Tenascin, may allow identification of nuances in the data, which are not detected by currently available commercial kits.
  • the immunoassays of the present disclosure may provide more linear correlation than one or more currently available kits.
  • the detectable ranges vary widely between currently available assay kits, as does the particular splice being detected.
  • the immunoassay of the present disclosure may be able to assess multiple forms of tenascin.
  • the information generated from the immunoassays of the present disclosure may have significant implications for the diagnosis and analysis of the prognosis of inflammatory diseases, such as RA.
  • an ELISA assay comprising the same human VH in the capture antibody and conjugate antibody (referred to herein as a self- sandwich/self-ELISA/Single antibody sandwich) gave good discrimination between healthy samples and RA samples. This may allow, for example for oligomeric forms of Tenascin to be analysed.
  • Immunoassay as employed herein refers to is a biochemical test that measures the presence or concentration of a macromolecule, in this instance Tenascin, in a sample using an antibody.
  • the immunoassay of the present disclosure is a sandwich assay.
  • an immunoassay of the present disclosure is an ELISA assay.
  • ELISA assay as employed herein refers to an enzyme-linked immunosorbent assay.
  • ELISA assays are well known in the art.
  • Capture antibody as employed herein refers to an antibody which binds the Tenascin.
  • the capture antibody is labelled, for example wherein binding to the antigen (in this case Tenascin) can be detected as the colour or change in colour of the label.
  • Conjugate antibody as employed herein refers to an antibody which is conjugated to a label, wherein the antibody in the conjugate binds the antigen or binds to and epitope on the capture antibody. Binding of the conjugate antibody is generally detected as the colour or change in colour of the label.
  • one or more photochromic molecules or compounds are employed as labels, for example a fluorescent label is employed.
  • labels such radiolabels can be employed.
  • radiolabels are generally less convenient to use than fluorescent labels.
  • Fluorescent labels can be detected by measuring absorbance or a change in absorbance.
  • Tenascin as employed herein refers to the family of extracellular matrix glycoproteins, which includes Tenascin C, Tenascin R, Tenascin X and Tenascin W, in particular a human form of said protein or proteins.
  • Tenascin C is a large hexameric protein of 1.5 million Da. Each chain comprises different domains, including an assembly domain (TA), EGF-like repeats (EGF-L), fibronectin type Ill-like repeats (TNIII) and the fibrinogen-like globe (FBG).
  • TA assembly domain
  • EGF-like repeats EGF-like repeats
  • TNIII fibronectin type Ill-like repeats
  • FBG fibrinogen-like globe
  • Antibody as employed herein refers to an immunoglobulin, a molecule comprising an immunoglobulin, such as a multispecific antibody molecule, or a binding fragment thereof.
  • binding fragment refers to a fragment capable of binding a target peptide or antigen with sufficient affinity to characterise the fragment as specific for the peptide or antigen.
  • Binding fragments include but are not limited to Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single domain antibodies (VH or VLs), scFv, Fv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217).
  • Linkers suitable for use in recombinant antibodies or binding fragments include but are not limited to hinge linkers as shown in SEQ ID NOs: 160 to 168, flexible linkers as shown in SEQ ID NOs: 169 to 208 and rigid linkers PPP and as shown in SEQ ID NOs: 209 210.
  • Multispecific antibody molecule refers to a molecule with the ability to specifically bind at least two distinct antigens, for example different antigens.
  • the multispecific molecule is a bispecific, trispecific or tetraspecific molecule, in particular a bispecific or trispecific molecule.
  • VH refers to a variable heavy domain sequence, i.e. a heavy framework in combination with CDRHl, CDRH2 and CDRH3, for example B12-VH refers to the variable heavy domain of the antibody B12, as shown in SEQ ID NO: 12.
  • VL as employed herein refers to a variable light domain sequence, i.e. a light framework in combination with CDRL1, CDRL2 and CDRL3.
  • a variant in this context is intended to refer to where one, two, three, four or five amino acids in a naturally occurring sequence have been replaced or deleted, for example to optimize the properties of the domain such as by eliminating undesirable properties but wherein the characterizing feature(s) of the domain is/are retained.
  • modifications are those to remove glycosylation sites, GPI anchors, or solvent exposed lysines. These modifications can be achieved by replacing the relevant amino acid residues with a conservative amino acid substitution.
  • Highly similar as employed herein is intended to refer to an amino acid sequence which over its full length is 95% similar or more, such as 96, 97, 98 or 99% similar.
  • the present disclosure extends to antibodies having at least 95% sequence identity, such as 96, 96, 98 or 99% identity to the sequences disclosed herein.
  • Antibodies generated against the antigen polypeptide may be obtained, where immunisation of an animal is necessary, by administering the polypeptides to an animal, preferably a non-human animal, using well-known and routine protocols, see for example Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep, cows, camels or pigs may be immunized. However, mice, rabbits, pigs and rats are generally most suitable.
  • Monoclonal antibodies may be prepared by any method known in the art such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72) and the EBV- hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).
  • Antibodies may also be generated using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNAs generated from single lymphocytes selected for the production of specific antibodies by, for example, the methods described by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92/02551; WO2004/051268 and WO2004/106377.
  • the antibodies for use in the present disclosure can also be generated using various phage display methods known in the art and include those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough
  • Humanised which include CDR-grafted antibodies
  • CDR-grafted antibodies refers to molecules having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule (see, e.g. US 5,585,089; WO91/09967). It will be appreciated that it may only be necessary to transfer the specificity determining residues of the CDRs rather than the entire CDR (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanised antibodies may optionally further comprise one or more framework residues derived from the non-human species from which the CDRs were derived.
  • the term 'humanised antibody molecule' refers to an antibody molecule wherein the heavy and/or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g. a murine monoclonal antibody) grafted into a heavy and/or light chain variable region framework of an acceptor antibody (e.g. a human antibody).
  • a donor antibody e.g. a murine monoclonal antibody
  • acceptor antibody e.g. a human antibody
  • only one or more of the specificity determining residues from any one of the CDRs described herein above are transferred to the human antibody framework (see for example, Kashmiri et al., 2005, Methods, 36, 25-34).
  • only the specificity determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework.
  • only the specificity determining residues from each of the CDRs described herein above are transferred to the human antibody framework.
  • any appropriate acceptor variable region framework sequence may be used having regard to the class/type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions.
  • the humanised antibody according to the present disclosure has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs provided herein.
  • human frameworks which can be used in the present disclosure are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Rabat et al., supra).
  • KOL and NEWM can be used for the heavy chain
  • REI can be used for the light chain and EU
  • LAY and POM can be used for both the heavy chain and the light chain.
  • human germline sequences may be used; these are available at: http://vbase.rnrc-cpe.carn.ac.uk/
  • the acceptor heavy and light chains do not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains.
  • the framework regions need not have exactly the same sequence as those of the acceptor antibody. For instance, unusual residues may be changed to more frequently-occurring residues for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions may be changed so that they correspond to the residue found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to recover the affinity of the donor antibody.
  • a protocol for selecting residues in the acceptor framework regions which may need to be changed is set forth in WO91/09967.
  • Fully human antibody as used herein is intended to describe antibodies in which the variable regions and the constant regions (where present) of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, not necessarily from the same antibody.
  • Fully human antibodies may include antibodies produced, for example by the phage display methods described above and antibodies produced by mice in which the murine immunoglobulin variable and optionally the constant region genes have been replaced by their human counterparts eg. as described in general terms in EP0546073 Bl, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, US5,770,429, EP 0438474 and EP0463151.
  • Antibody 163 02 Dll (derived from 2 A5)
  • B12-B12 refers to full-length IgG B12 as the capture antibody and as the conjugate antibody.
  • the plate coating procedure was based on using pH 9.6 bicarbonate coating buffer and a coating concentration of 10 ⁇ g/ml. 100 ⁇ of coating solution was dispensed into the wells of a high-binding microtitre plate (Biomat, Sir), and incubated for 24hrs at room temperature. The buffer was decanted, the plate was blotted dry and 100 ⁇ blocking buffer (lOmM PBS, 1% BSA) was dispensed into the well before another 24hr incubation time at room temperature. The blocking buffer was decanted, the wells were thoroughly washed x3 with overcoat buffer (lOmM PBS, 0.1% BSA, 5% sucrose) and the plate was placed in a 37°C incubator for at least 2 hrs until completely dry. The plate was then stored in a sealed bag with desiccant at 2-8°C until use.
  • pH 9.6 bicarbonate coating buffer 100 ⁇ of coating solution was dispensed into the wells of a high-binding microtitre plate (Biomat, Sir
  • Lightning- Link® Horseradish Peroxidase kits (LL-HRP) (Innova Biosciences Ltd.) were chosen for use due to the simplicity, time efficiency and reported reproducibility of the procedure.
  • the solution was disturbed to allow full resuspension of the freeze-dried conjugation mix, and allowed to incubate at room temperature overnight, before the appropriate amount of LL-quencher reagent was added to the glass vial.
  • the resulting conjugate stock solution was then stored at 2-8°C to be diluted to working strength as desired for later use.
  • Standard solutions were made up to concentration using Human Tenascin-C Purified Protein (Merck Millipore Corporation, Cat No: CC065) in a lOmM PBS 1%, BSA buffer. This was the only full protein extraction available on market at initiation of project work. The protein had been extracted from Human glioma cell line (U251) after induced expression, and is quoted to migrate at 280-300kDa by SDS-PAGE analysis.
  • TMB substrate solution FMHBP130
  • FMHBP140 stop solution
  • Standard deviation (S.D.) and coefficient of variance (C.V.) was calculated each time. The C.V. values were monitored, and if they crept above 15% then the test was repeated.
  • the wells of a plate were coated with either Human TNC Purified Protein (Merck Millipore) or BSA.
  • ⁇ of each conjugate antibody solution (at [ ⁇ g/ml], in lOmM PBS, 1% BSA) was placed in each well and incubated at room temperature for 1 hr. The plate was washed.
  • ⁇ of substrate solution (FMHBP130) was placed in each well, and the plate was incubated at room temperature until sufficient colour had developed, with the incubation time noted.
  • ⁇ of stop solution (FMHBP140) was added to each well, and the plate was read with a plate reader.
  • Conjugate antibody was coated on a plate (at [ ⁇ /ml]), before being incubated with ⁇ of substrate solution (FMHBP130) at room temperature for 1 hr. ⁇ of stop solution (FMHBP140) was added to each well and the plate was read with a plate reader.
  • TMB substrate solution FMHBP130
  • conjugate solution [ ⁇ g/ml] in lOmM PBS, 1% BSA
  • the reaction mix was allowed to incubate for 2 minutes at room temperature before ⁇ of stop solution (FMHBP140) was added to the mix.
  • the solution was then made up to 1ml by the addition of 800 ⁇ 1 of RO water, mixed by inversion, and read at A450nm on a spectrophotometer.
  • Each antibody was coated on a plate, and tested against increasing concentrations of TNC solutions (0, 10, 50 and 100ng/ml), with each different antibody as conjugate (at [ ⁇ g/ml] in lOmM PBS, 1% BSA). Substrate incubation times varied, to allow sufficient colour to develop. Results were graphed, and ratings were generated based on the absorbance response of each antibody pair before analysis with respect to dose response and strength of reaction.
  • Serum sample N2 (ProMedDx, Lie), selected after preliminary testing using the IBL (FNIII-C) assay was diluted using a double-down serial dilution with buffer (lOmM PBS, 1% BSA, 0.5% Tween 20) to 3.125% and tested against the 9F8:B12-VH and IBL (FNIII-C) assays, with the addition of a 1/50 dilution point on the IBL (FNIII-C) assay. Simultaneously, the IBL (FNIII-C) kit conjugate and plate was tested against the 9F8 coated plate and B12-VH conjugate, respectively.
  • buffer lOmM PBS, 1% BSA, 0.5% Tween 20
  • lOmM PBS 400 ⁇ of lOmM PBS, 1% BSA buffer and sera samples were made up to 500 ⁇ by the addition of 100 ⁇ of increasing concentrations of TNC spike solution.
  • Spike solutions were made up using TNC purified protein and sample diluent (lOmM PBS, 1% BSA, 0.5% Tween20) at concentrations of 0, 25, 50, 75, 100, 200ng/ml. The spiked samples were then simultaneously tested against the 9F8:B12-VH and IBL (FNIII-C) assays.
  • lOmM PBS 400 ⁇ 1 of lOmM PBS, 1% BSA buffer and sera samples were made up to 500 ⁇ l by the addition of ⁇ of increasing concentrations of TNC spike solution.
  • Spike solutions were made up using TNC purified protein and sample diluent (lOmM PBS, 1% BSA, 0.5% Tween20) at concentrations of 0, 25, 50, 75, 100, 200ng/ml. The spiked samples were then simultaneously tested against the 9F8:B12-VH and IBL (FNIII-C) assays.
  • Serum was tested on IBL (FNIII-C) and (FNIII-B) kits (protocols available online,), purchased from Takara Bio, Inc. (Cat. No: 27751A and 27767A, respectively), as well as against a selection of antibody pairs.
  • the results of the 9F8:B12-VH and IBL (FNIII-C) assays were used as comparison when performing any analysis to somewhat compensate for the lack of suitable standard solutions.
  • ROC Curve analysis was generated using Analyse-it software (Analyse-it Software, Ltd).
  • Plates were coated with TNC purified protein and incubated with sera samples for 1 hr, before washing and blotting dry. The plates were then incubated with ⁇ of w/s anti-Human IgG-HRP conjugate (FCCP620) from the ASD anti-CCP EIA (FCCP600) for 1 hr, before washing and blotting. ⁇ ofTMB substrate (FMHBP130) was placed in each well and incubated at room temperature for 105 mins to allow colour to develop. ⁇ of stop solution (FMHBP140) was added and the plate was read at A450nm on the plate reader.
  • FCCP620 anti-Human IgG-HRP conjugate
  • FCCP600 ASD anti-CCP EIA
  • the LL-HRP kits were chosen for a variety of reasons, including the economy and ease of use, and due to the elegance of HRP as a small conjugate enzyme with a variety of available substrates, which are both fast-acting and affordable.
  • Error bars are included, with the "error" of each being the S.D. of each sample performance in assay. Where the mean results of two different assay runs are represented, the error bars are the mean S.D. produced by the sample, in place of representing the full spread of each sample alongside the mean each time.
  • Standard solutions were found to be non-commutable with TNC in serum, with significantly weaker reactions than those seen in serum. For example, in the case of the 9F8:B12-VH assay, where sample N2 gives an absorbance value of 0.74 units, and a standard solution made up to be[24 ng/ml] TNC gives an absorbance value of 0.34 units. This would make the calculated concentration of TNC in sample N2 to be 402.60 ng/ml (concentrations calculated using the IBL assay protocol). The mean TNC concentration in normal serum is quoted as being 20.34 ng/ml 2 , suggesting that this result is extremely exaggerated.
  • Normalised graphs present each absorbance value as a percentage of the maximum absorbance achieved by each assay, to adjust for variance in sensitivity and N.S.B between assays. RESULTS
  • Figure 1 suggests that the an assay based on murine capture antibody 9F8 and the conjugate antibody B12 (or the VH form thereof) provides a more linear correlation to the levels of Tenascin in samples than commercially available assays.
  • FIG. 2 shows that the data generated with the capture antibody 9F8 and the conjugate antibody B12-VH is reproducible.
  • FIGS 3 and 4 show that normalised data for B12 (capture antibody) and 9F8 (conjugate antibody) shows similar trends to the commercially available assay IBL (FNIII-C) for many samples.
  • FIGs 5 and 6 show that normalised data for B12-VH (capture antibody) and 9F8 (conjugate antibody) shows similar trends to the commercially available assay IBL (FNIII-C) for many samples, especially normal samples.
  • Figures 9 shows that the B12-B12 self-sandwich assay shows a significant difference in between the normal and RA samples.
  • the data generated with the self -sandwich assays may suggest that polymeric forms of Tenascin-C exist in the RA serum samples.
  • Figure 10 associated data.

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Abstract

La présente invention concerne un dosage immunologique pour la détection et la surveillance de maladies inflammatoires et/ou auto-immunes. Le dosage immunologique détecte la présence d'une ténascine dans un échantillon et comprend un anticorps de capture et de conjugué, au moins un des anticorps ayant un VH humain spécifique de la ténascine. L'invention concerne également un kit comprenant ledit dosage immunologique.
PCT/EP2017/053086 2016-02-10 2017-02-10 Dosage immunologique pour la détection et la surveillance des réponses inflammatoires Ceased WO2017137613A1 (fr)

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