DIAGNOSTIC FOR SJORGREN'S SYNDROME BASED ON A BIOMARKER

Abstract

A method, assay, peptide and antibody that forms an antibody-antigen conjugate with the peptide comprising the amino acid sequence: GPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPAQGGSKSQSARAPPGKPQGPPQQEGNNPQGPPPPAGGNPQQPQAPP or an amino acid sequence that is at least 75% identical to said sequence, and the formation of an antigen-antibody conjugate is a positive indicator for Sjgren's Syndrome.

Claims

1. A method for detecting a biomarker for Sjgren's Syndrome comprising: obtaining a body fluid sample from a patient suspected of having Sjgren's Syndrome, incubating proteins or peptides in the sample with an antibody specifically binding a peptide comprising the amino acid sequence SEQ ID NO: 5 or an amino acid sequence that is at least 75% identical, and is a biomarker for Sjgren's Syndrome, and detecting the presence of binding between the antibody and the peptide.

2. The method of claim 1 further comprising: adding a second antibody specifically binding the peptide, and wherein binding between the peptide to both the antibody and the second antibody is detected.

3. The method of claim 1 wherein the antibody is tagged.

4. The method of claim 3 further comprising adding at least one reagent that interacts with the tag to produce a readily detectable signal.

5. The method of claim 4 wherein the readily detectable signal is measured quantitatively.

6. The method of claim 3 wherein the tag is a radioactive atom, a fluorescent molecule, an enzyme, or an insoluble solid phase.

7. The method of claim 1 further comprising adding a tagged peptide comprising the amino acid sequence SEQ ID NO: 5 or an amino acid sequence that is at least 75% identical.

8. The method of claim 7 wherein the tagged peptide is incubated with and binds to the antibody before the sample contacts the antibody.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0026] FIG. 1. MALDI-TOF mass spectra (A) heat map representation (B) doubly-charged peaks at m/z=3803.4 and (C) protonated molecular ion at m/z 7606.8 for saliva of confirmed Sjgren's Syndrome patient.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Development of a diagnostic test for Sjgren's Syndrome (SjS) starts with collection of saliva to isolate and capture an active form of the peptide or protein fragment responsible for the SjS specific peptide sequence with MW of 7606 Da

TABLE-US-00002 GPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPAQGGSKSQSARAPPGK PQGPPQQEGNNPQGPPPPAGGNPQQPQAPP
SEQ ID NO:5 followed by the generation of antibodies against this peptide/protein fragment. Once the antibodies have been generated, the next step is testing the antibody with saliva from patients with confirmed SjS relative to normal control saliva samples. Any conventional immunoassay format may be used. From this point on, the form of a test could be as simple as a test strip impregnated with the antibody to a solution containing the antibody that would react with the peptide in patient saliva samples or other biological fluids being detected by a color change or a colorimetric device that could yield quantitative results about the disease. An active form of the peptide or protein fragment is then synthesized and tested.

[0028] Antibodies are pirated to the active form of the peptide/protein fragment responsible for the sequence with MW of 7606 Da. Typically, this is done by exposing rabbits to the peptide/protein fragment and harvesting serum antibodies once the rabbit develops an immune response.

[0029] Since this peptide/protein fragment is present in the full protein of healthy patients, a discriminating antibody must specifically bind the fragment but not react with the full protein. Antibodies that bind to the full protein would produce false positives for the disease. Once the antibody has been generated, the next step is testing the antibody with saliva from patients with confirmed SjS relative to normal control saliva samples.

[0030] In one embodiment, the antibodies bind specifically to the marker peptide/protein fragment responsible for the sequence with MW of 7606 Da and do not react to the full protein are placed on a solid phase such as a test strip along with other optional components either in the test strip or, mixed with the sample or separately added so that a detectable change (e.g. a change color) upon combination of the antibody with the peptide.

[0031] In another embodiment, other immunoassay formats may be used with different components bound to different solid phases or in solution and different detection schemes used. However, in each format, the critical step is the specific binding of the antibody of the present invention to the peptide of the invention occurs.

[0032] A saliva sample or other body fluid (e.g. tears, nasal secretions, etc.) is collected from a patient. This sample serves as the peptide containing liquid. The sample may be fractionated to at least partially purify the sample or to remove any potentially interfering substance(s). The antibody specific to the SJS peptide is exposed to the sample. Binding is typically detected by a positive color change that indicates whether or not the saliva sample contains a peptide as a marker specific for SJS.

[0033] In another embodiment, the antibody-peptide binding is quantified. In such a situation, typically, the signal from a labeled antibody or a labeled peptide is quantitatively measured. For example, the amount of a color change may be measured by a colorimetric device. Spectrophotometers serve well for this purpose. Some other common diagnostic tests that can be developed include a multi-strip test that can be used to test multiple samples simultaneously. This strip could be a device with six or twelve wells coated with antibodies and an indicator that changes color when reacted with an antigen. This type of a device, similar to the diagram shown below, could be used to perform replicate analyses of a single sample or different biological samples or different concentrations of either sample or reagents to either quantify the results and/or to perform a confirmation.

Types of Antibody-Antigen Reactions that May be Used in this Invention and to Build a Test.

[0034] Precipitation reaction. When an antigen-antibody reaction takes place, the antigens and antibodies cross-link to form a lattice-like structure that precipitates out of the solution, settling onto the bottom of a vial. Observation of this precipitate can confirm the presence of a specific peptide indicative of SjS. To enhance effectiveness, plural antibodies binding to different portions of the peptide may be used. Also, a secondary antibody or other agent such as Protein A may be added that binds to the antibody to enhance precipitation. In a competitive type assay, the lack of a precipitate can confirm the presence of the SjS specific peptide in the sample.

[0035] Agglutination reaction. Agglutination refers to a clumping that occurs when an antigen comes into contact with its corresponding antibody. Observation of an agglutination reaction (or lack of agglutination) aids in the detection of a specific peptide, such as those produced in SjS.

[0036] Complement fixation refers to a reaction in which an antigen binds with an antibody, forming a combination that causes complement to become fixed at the same site. Detection of this complement fixation reaction leads to identification of the peptide produced by a SjS patient.

[0037] Immunofluorescent assay is a technique in which specific antibodies are tagged with a fluorescent dye. When these antibodies bind to the SjS indicator peptide, they appear as fluorescent, glowing particles under a fluorescent microscope, or are measured in a fluorescent spectrometer, thereby revealing their location, particularly when bound to the peptide as an indicator of the peptide being present in the sample. Likewise, the peptide may be tagged with a fluorescent dye for the opposite effect. The tagged peptide may be either the sample peptide or a previously synthesized peptide.

[0038] Enzyme immunoassays uses tagging with an enzyme. An enzyme substrate or product is a readily detectable substance which measurement of the formation or reduction of the readily detectable substances determines the presence of the antibody or peptide tagged. Typically, a developing solution is added that the enzyme catalyzes to form a color change. Thus a simple viewing of the color determine whether or not the sample comes from a patient with SJS. Of particular interest is an Enzyme-linked immunosorbent assay (ELISA). In this format the antibody is first bound to a solid phase such as the inside of a container or on a small particle. The sample containing a peptide is added and allowed to bind to the antibody. A tagged second specific antibody is then added which binds to the peptide so that the peptide is sandwiched between the antibodies. Optionally, a developing solution is added for the tag to react with and the result is measured or observed. Presence of the readily detectable signal indicates presence of the peptide in the sample. Alternatively, the binding of antibody to solid phase may be performed later in the assay and the antibody-peptide binding may be performed before adding it to a container.

[0039] Radioimmunoassay (RIA) is a similar technique to an immunofluorescent assay that tags antibody or peptide with radioactive material. Detection is typically performed by a scintillation counter.

[0040] Some of these tests can be incorporated into a diagnostic test.

[0041] In another embodiment of the present invention, the SjS specific peptide of the present invention may be isolated from patient fluid samples but is preferably chemically synthesized to form a pure chemical compound. Because of natural polymorphisms and mutations, variations in the amino acid sequence may be present. Furthermore, as the peptide is presumed to be a degradation product, a slightly truncated or lengthened peptide is possible In as much as only small portions of the entire sequence serves as antibody binding site(s), such changes may be made to the sequence as still be encompassed by the present invention, provided that SjS specific reactivity remains. Up to about 25% variation in the amino acid sequence is acceptable provided that at least one SjS specific antibody can be made. Of particular interest is a truncated peptide containing the 57 amino acids at the carboxy-terminal of SEQ ID NO: 1.

[0042] The SjS specific peptide of the present invention may be chemically coupled to itself or even polymerized to form a molecule with multiple identical antibody binding sites. This has certain advantages with some immunoassay formats. Such techniques are known per se.

[0043] The SjS specific peptide of the present invention may be chemically coupled to a diverse protein or peptide that is strongly antigenic (such as KLH, albumin, etc.) so as to elicit a strong immune response when used to immune an animal for the purposes of producing antibodies.

[0044] The SjS specific peptide defined by SEQ ID NO: 1 and antibody that specifically binds to it may separately be chemically coupled to a tag for the purposes of producing a reagent for an immunoassay to detect SjS. A large number of tags, known per se, have been used in various different immunoassays such as fluorescent, radioactive or enzyme molecules or solid phase particles.

[0045] The term antibody is used to broadly define a specific binding partner. While conventional polyclonal antibodies are typically used, monospecific antibodies, monoclonal antibodies, antibody fragments, recombinant molecules such as single-chain antibodies (scFv) and other specific binding molecules such as those determined by panning a library of molecules, or the natural biological receptor (or portions thereof) that are naturally bound by the SjS specific peptide of the present invention and the like. In all situations, the antibody should bind with high affinity and with sufficient specificity to distinguish SjS samples from non-SjS samples.

[0046] The assay of the present invention may be in the form of a kit. Such a kit contains at least a container containing an antibody specific for the SjS specific peptide of the present invention and instruction on using it in an immunoassay. Tagged antibody or tagged peptide is preferably included. Developing reagents such as enzyme substrates and apparatus such as a multi-welled plate may also be present.

[0047] The assay of the present invention may be used in conjunction with other assays for SjS to make a combination method and combination kit containing reagents for plural SjS assays based on different peptides/proteins. For example anti-nuclear antigen and rheumatoid factor are known to be associated with SjS. Others have also been proposed such as Maliska et al. Reumatologia. 2017; 55(3): 113-119, Tzartos et al, Rheumatology (Oxford) 2017 Aug. 31, Nezos et al, Clin Immunol. 2017 Sep. 14 and Zoukhri et al, Journal of Oral Science Vol. 54 (2012) No. 1 March P 61-70. Any of these and/or other assays may be included in the combination kit along with the reagents used for detecting the SjS specific peptide of the invention in a sample.

Example 1

[0048] Saliva samples from known SjS patients and control humans were taken by suction. Two l of salvia were processed for mass spectrometry analyses on a prOTOF 2000 matrix-assisted laser desorption/ionization orthogonal time of flight (MALDI O-TOF) mass spectrometer. Spectral data obtained for 750-12000 daltons is shown in FIG. 1. The results were highly reproducible and contained many peptides and peptide fragments in the 750-7,500 Da range.

[0049] Bioinformatic data analysis using spectral data were analyzed using PROFILE, a proprietary biomarker discovery platform optimized for signatures in high-resolution MALDI-TOF mass spectra. Die bioinformatics have been previously used for other biomarker discovery; for example, Lopez et al, (2005) Clin Chem 51, 1946-1954, Avasarala et al (2005) J Mol Neurosci 25, 119-125 and Brouwers et al (2005) Endocr Relat Cancer 12, 263-272. The saliva samples were fractionated by HPLC and then peptides sequenced by Edman degradation. By comparison of SjS and normal samples, the SjS specific peptide sequence with MW of 7606 Da having the SEQ ID NO: 5 was determined and found only in SjS samples and not in normal samples.

Example 2

[0050] An antibody is made by injecting the peptide from Example 1 that indicates SjS into an animal. After 6 weeks, blood is withdrawn and serum collected. This polyclonal antibody containing antisera is used for detecting the SjS specific peptide. The antibody is absorbed onto the inside of wells in a 96 well plate. Albumin is later adsorbed on the inside of the same wells. The plate is washed in phosphate buffered saline (PBS). Saliva samples from both normal and SJS patients are added to each well in duplicate and at 10 fold and 100 fold dilutions. After allowing to incubate for 15 minutes at 37 degrees C., the plate is emptied and rinsed with additional PBS. Antibody previously tagged with enzyme is added and again incubated for 15 minutes at 37 degrees C. and rinsed in PBS. Enzyme substrate is added and the color change (clear to blue) observed visually and quantitative measured in a standard Microtiter plate reader. Samples from SjS patients are noticeably different.

Example 3

[0051] Another test similar to Example 1 is made using tiny polystyrene beads as the solid phase instead of the 96 well plate. Albumin-blocked 96 well plates are used as vessels for the binding and developing reactions. Filter containing pipette tips are used for aspirating liquid and beads in lieu of emptying. The tagged antibody component is tagged with fluorescein isothiocyanate. The conditions of the method of Example 1 is repeated except for a final pipetting beads on a glass slide and visualizing under ultraviolet light instead of adding enzyme substrate. The results are seen visually (and optionally may be measured quantitatively

[0052] It will be understood by a person skilled in the art that a number of antibodies and similar molecules are within the scope and spirit of the invention; that many known assays can be adapted to the diagnosis of SjS and that any diagnostic kit that is not specifically recited herein are within the spirit and intent of this invention. All references cited herein are incorporated by reference.

REFERENCES

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