Methods, uses and kits for diagnosing gingivitis

Abstract

To disclose an in vitro method for assessing whether a human patient has gingivitis.SOLUTION: The concentration of a specific combination of proteins is measured in a sample of saliva from a patient having gingivitis. One such combination is α-1-acid glycoprotein (A1AGP) and S100 calcium-binding protein A8 (S100A8), and at least one of Hemoglobin subunit β (Hb-β), Keratin 4 (K-4) and Pyruvate Kinase (PK). The other combination is α-1-acid glycoprotein (A1AGP), Hemoglobin subunit β (Hb-β) and Keratin 4 (K-4). Based on the measured concentration, a testing value is compared with a threshold reflecting in the same manner the joint concentrations associated with gingivitis, so as to assess whether the testing value is indicative of gingivitis in the patient.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to saliva-based diagnosis of periodontal diseases in the field of oral care. In particular, the present invention relates to kits, uses and methods for diagnosing gingivitis.

Background Art

[0002] Gingival inflammation, i.e., gingivitis, is a non-destructive periodontal disease mainly caused by the attachment of dental bacterial biofilms, i.e., dental plaque, to the tooth surface. If detected and untreated, reversible gingivitis usually causes inflammation of the tissues around the teeth (i.e., periodontal tissues), which is a condition defined as irreversible periodontal disease, where tissue destruction and alveolar bone loss occur, ultimately leading to tooth loss. During the progression of gum disease, related clinical signs and symptoms such as gum swelling, color change from light red to dark red, gum bleeding, shortness of breath, gum tenderness or pain appear.

[0003] Periodontal disease is a chronic multifactorial inflammatory disease caused by oral microorganisms, characterized by progressive destruction of hard (bone) and soft (periodontal ligament) tissues, ultimately leading to tooth mobility and loss. This should be distinguished from gingivitis, which is a reversible infection and inflammation of the gum tissue. Inflammatory periodontal disease is one of the most common chronic human diseases and the main cause of tooth loss in adults. In addition to the substantial adverse effects of periodontal disease on oral health, there is also increasing evidence that periodontal disease has systemic consequences and is a risk factor for several systemic diseases including heart disease (e.g., atherosclerosis, stroke), diabetes, pregnancy complications, rheumatoid arthritis and respiratory infections.

[0004] Therefore, early and accurate diagnosis of periodontal diseases is important from the perspectives of oral and general health.

[0005] In general dental practice, the diagnosis of periodontal diseases remains inadequate, resulting in a relatively low rate of therapeutic intervention and a significant number of untreated cases. Current diagnosis relies on inaccurate and subjective clinical examinations of oral tissues by dentists (color, swelling, degree of bleeding on probing, probing pocket depth, and bone loss by oral X-rays). These conventional methods are time-consuming, and some of the techniques used (pocket depth, X-rays) reflect historical events such as past disease activity rather than current disease activity or further disease susceptibility. Therefore, a more objective, rapid, accurate, and convenient diagnostic method (ideally with predictive values) is desirable, and one that can be implemented by non-experts is preferred. Thereby, it is desirable to determine the current disease activity and the subject's further susceptibility to periodontal diseases.

[0006] Saliva or oral fluid has long been proposed as a diagnostic fluid for oral and general diseases. Also, with the emergence of miniaturized biosensors such as lab-on-a-chip, rapid point-of-care test diagnostic methods have gathered greater scientific and clinical interest. Particularly in the detection of periodontal diseases, inflammatory biomarkers associated with tissue inflammation and destruction are likely to reach saliva easily due to their proximity, suggesting that saliva has great potential for the detection of periodontal diseases. In fact, this field has attracted great interest and promising results have been obtained. For example, Non-Patent Document 1 identified host- and bacterium-derived biomarkers correlated with periodontal diseases. However, no clear test has been established yet.

[0007] A biomarker is a biological indicator that supports clinical symptoms and an objective measure for diagnosing the clinical outcome of periodontal diseases. Ultimately, with proven biomarkers, it is possible to assess the risk of future diseases, identify diseases at a very early stage, identify the response to initial treatment, and implement preventive strategies.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] Regarding the development of a clinical trial for biomarkers in saliva, there have been limitations so far, such as the lack of a technology applicable to in-clinic use and the impossibility of analyzing multiple biomarkers in individual samples. In addition, the selection of multiple biomarkers to be included in the trial has not been sufficiently addressed in the literature and has not been implemented in practical trials.

[0010] It is desired to provide a simpler process, especially a process for collecting a small amount of saliva sample from a patient, preferably by the patient himself / herself. It is desirable that the sample can be input into an in vitro diagnostic device capable of classifying saliva samples and, based on the measurement, return an index that can be classified as an indication of the patient's gingivitis.

Means for Solving the Problems

[0011] To better address the above desires, in one aspect, the present invention provides an in vitro method for evaluating whether a human patient has gingivitis, comprising the following: (i) at least one of α-1-acid glycoprotein (A1AGP), S100 calcium-binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK); or, (ii) α-1-acid glycoprotein (A1AGP), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); a step of detecting the protein concentration of; - a step of determining a test value reflecting the binding concentration determined for the protein; - a step of comparing the test value with a threshold value that reflects the binding concentration related to gingivitis in the same manner to evaluate whether the test value becomes an indicator of the patient's gingivitis; including.

[0012] In another aspect, the present invention provides the use of a protein of the first aspect in a human patient's saliva sample as a biomarker for assessing whether the patient has gingivitis.

[0013] In some cases, the patient's age may also be used as a biomarker.

[0014] In a further aspect, a system for assessing whether a human patient has gingivitis, comprising the following: - in a saliva sample of a human patient: (i) at least one of alpha-1-acid glycoprotein (A1AGP) and S100 calcium-binding protein A8 (S100A8), and hemoglobin subunit beta (Hb-beta), keratin 4 (K-4) and pyruvate kinase (PK); or, (ii) alpha-1-acid glycoprotein (A1AGP), hemoglobin subunit beta (Hb-beta), and keratin 4 (K-4); detection means capable of detecting such proteins and adapted accordingly; - a processor adapted to determine an indicator of a patient with gingivitis from the determined concentration of said protein; A system is provided that includes.

[0015] The system may optionally be capable of presenting information, preferably including information such as the age of the subject and optionally other information such as gender and / or BMI (body mass index), and includes a data connection to an interface, particularly a graphical user interface, which may be part of the system or a remote interface.

[0016] In some cases, one or more of said items, particularly the processor, can function by means of an Internet-based application, i.e., "in the cloud" rather than on a fixed machine.

[0017] In a further aspect, the present invention is a kit for detecting at least three biomarkers for gingivitis in a saliva sample of a human patient, comprising the following: (i) alpha-1-acid glycoprotein (A1AGP) and S100 calcium-binding protein A8 (S100A8), and at least one of hemoglobin subunit beta (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK); or, (ii) alpha-1-acid glycoprotein (A1AGP), hemoglobin subunit beta (Hb-beta), and keratin 4 (K-4); The kit is provided, comprising one or more detection reagents for detecting the same.

[0018] Typically, three or more detection reagents are used, each reagent binding to a different biomarker. In one embodiment, the first detection reagent can detect A1AGP, the second detection reagent can detect S100A8, and the third detection reagent can detect one of Hb-beta, K-4, and PK. In a further embodiment, the first detection reagent can detect A1AGP, the second detection reagent can detect Hb-beta, and the third detection reagent can detect K-4. In another embodiment, the first detection reagent can detect A1AGP, the second detection reagent can detect S100A8, the third detection reagent can detect one of Hb-beta, K-4, and PK, and the fourth detection reagent can detect a different one of Hb-beta, K-4, and PK.

[0019] In yet another aspect, the present invention is an in vitro method for determining a change in the state of gingivitis over a period of time from a first time point t1 to a second time point t2 in a human patient suffering from gingivitis, in at least one sample of saliva obtained from the patient, at t1 and in at least one sample of saliva obtained from the patient, the following: (i) At least one of α-1-acid glycoprotein (A1AGP), S100 calcium-binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK); or, (ii) α-1-acid glycoprotein (A1AGP), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); a step of detecting the concentration of the protein; and, a step of comparing the concentration such that at least one, two, or more differences in the concentration reflect a change in state; A method comprising the above is provided.

[0020] In a further aspect, the present invention is a method for diagnosing whether a human patient has gingivitis, comprising the following in a saliva sample of the human patient: (i) At least one of α-1-acid glycoprotein (A1AGP), S100 calcium-binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK); or, (ii) α-1-acid glycoprotein (A1AGP), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); a step of detecting the protein; and, a step of evaluating the presence of gingivitis in the patient based on the concentration of the protein in the sample; The method of this aspect may include a further step of treating the patient's gingivitis in some cases.

[0021] In still a further aspect, the present invention is a method for detecting the following proteins in a human patient: (i) At least one of α-1-acid glycoprotein (A1AGP), S100 calcium-binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK); or, (ii) α-1-acid glycoprotein (A1AGP), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); The method is as follows: (a) Obtaining a saliva sample from a human patient; and, (b) Contacting the sample with one or more detection reagents that bind to the protein to detect whether the protein is present in the sample, and detecting the binding between each protein and the one or more detection reagents; Provided is a method comprising. Usually, as described elsewhere herein, at least first and second, and optionally third and fourth detection reagents are present.

Brief Description of the Drawings

[0022]

Figure 1

Modes for Carrying Out the Invention

[0023] In a general sense, the present invention is based on the insightful observation that gingivitis can be distinguished from a healthy oral cavity with sufficient accuracy based on the measurement of a few protein biomarkers. In particular, it has been found that just three proteins in a saliva sample of a human patient function as biomarkers for identifying the presence or absence of gingivitis.

[0024] The biomarker proteins are α-1-acid glycoprotein (A1AGP), S100 calcium-binding protein A8 (S100A8), hemoglobin subunit beta (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK). The following combinations of these proteins: α-1-acid glycoprotein (A1AGP) and S100 calcium-binding protein A8 (S100A8), and at least one of hemoglobin subunit β (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK); or, α-1-acid glycoprotein (A1AGP), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4) are used for the diagnosis of gingivitis according to the present invention.

[0025] The age of the subject may, in some cases, be included as an additional marker.

[0026] Alpha-1-acid glycoprotein (A1AGP) is a plasma alpha-globulin glycoprotein mainly synthesized in the liver. It functions as a transport protein in the blood, also known as orosomucoid, and acts as a carrier for basic and neutrally charged lipophilic compounds. It is also thought to regulate the interaction between blood cells and endothelial cells.

[0027] S100 calcium-binding protein A8 (S100A8) is a calcium- and zinc-binding protein that plays an important role in the regulation of inflammatory processes and immune responses. It can induce neutrophil chemotaxis and adhesion.

[0028] S100 calcium-binding protein A9 (S100A9), also known as calgranulin B, is a calcium- and zinc-binding protein that plays an important role in the regulation of inflammatory processes and immune responses. It can induce neutrophil chemotaxis and adhesion, promote phagocytosis through the activation of SYK, PI3K / AKT, and ERK1 / 2, enhance the bactericidal activity of neutrophils, and induce neutrophil degranulation through a MAPK-dependent mechanism.

[0029] Hemoglobin (Hb) is an iron-containing oxygen-transport metalloprotein in the red blood cells of tissues of almost all vertebrates and some invertebrates. Hemoglobin beta (also known as beta-globin, HBB, beta globin, and hemoglobin subunit beta) is a globin protein and, together with alpha-globin (HBA), constitutes HbA, the most common form of adult hemoglobin. Hb-beta is usually 146 amino acids in length and has a molecular weight of 15,867 Da. Normal adult HbA is a heterotetramer consisting of two alpha chains and two beta chains. Hb-beta is encoded by the HBB gene on human chromosome 11.

[0030] The hemoglobin subunit delta (also known as delta globin, HBD, δ-globin, hemoglobin delta) is a globin protein that, together with alpha globin (HBA), constitutes a less common form of adult hemoglobin HbA-2. Hb delta usually consists of 147 amino acids and has a molecular weight of 16,055 Da. Adult human HbA-2 is a heterotetramer consisting of two alpha chains and two delta chains. Hb delta is encoded by the HBD gene on human chromosome 11.

[0031] Keratin-4 (K4), also known as cytokeratin-4 (CYK4) or cytoskeletal keratin-4 (CK-4), is a protein encoded by the KRT4 gene in humans. It belongs to the keratin gene family. Type II cytokeratins consist of basic or neutral proteins that are arranged in a pair of heterotypic keratin chains that are co-expressed during the differentiation of simple and stratified epithelial tissues. Type II cytokeratin CK4 is specifically expressed in the differentiated layers of the mucosal and esophageal epithelia of the KRT13 family. Mutations in this gene are associated with white sponge nevus, which is characterized by leukoplakia of the oral cavity, esophagus, and anus. Type II cytokeratins form a cluster in the region of chromosome 12q12-q13.

[0032] Pyruvate kinase catalyzes the final step of glycolysis. There are four tissue-specific isozymes of pyruvate kinase, each with specific kinetic properties required for different tissues.

[0033] Profilin is an actin-binding protein involved in the dynamic turnover and remodeling of the actin cytoskeleton and is found in most cells. It is important for the spatially and temporally controlled growth of actin microfilaments, which are essential processes for cell movement and cell morphological changes. Human profilin-1 is usually 140 amino acids in length when expressed but is often further processed into a mature form.

[0034] The above proteins are known in the art. Those skilled in the art know their structures and methods for detecting them in aqueous samples such as saliva samples. The following: (i) at least one of α-1-acid glycoprotein (A1AGP) and S100 calcium-binding protein A8 (S100A8), and hemoglobin subunit β (Hb-beta), keratin 4 (K-4) and pyruvate kinase (PK); or, (ii) α-1-acid glycoprotein (A1AGP), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); the protein biomarkers of are collectively referred to as the "biomarker panel of the present invention".

[0035] Table 1 of the examples provides 21 particularly preferred combinations of protein biomarkers according to the present invention.

[0036] In one embodiment, the biomarker panel of the present invention can consist of the following identified protein biomarkers. Preferably, the biomarker panel of the present invention consists of no more than 4 of the protein biomarkers identified in the present invention, for example, 3 or 4 protein biomarkers of the present invention. In addition to the biomarker panel of the present invention, other biomarkers and / or data such as demographic data (e.g., age, gender) can be included in the set of data applied to the determination of gingivitis. Examples of other protein biomarkers include hemoglobin subunit delta (Hb-delta), profilin or S100 calcium-binding protein A9 (S100A9). One of these additional proteins is included in some of the panels exemplified in Table 1 below.

[0037] When other biomarkers are included in some cases, the total number of biomarkers (i.e., the biomarker panel of the present invention + other biomarkers) is usually 3, 4, 5 or 6.

[0038] However, a desirable advantage of the present invention is that the classification of a patient's gingivitis can preferably be determined by measuring no more than four biomarkers, such as three or four protein biomarkers. In particular, for this determination, advantageously, no other data providing a simple and convenient diagnostic test is required.

[0039] This method may, if necessary, simply collect a small amount of saliva sample, such as a droplet, from the subject. The size of the sample is typically in the range of 0.1 μl to 2 ml, such as 1 to 2 ml, whereby a smaller amount, such as 0.1 to 100 μl, can be used for in vitro device processing, and it is also possible to collect a larger sample, such as up to 20 ml, such as 7.5 to 17 ml.

[0040] This sample is input into an in vitro diagnostic device that measures the concentration of the protein involved and returns a diagnostic result, and classifies the subject based on the likelihood of gingivitis.

[0041] Due to the simplicity of use of the present invention, most dental patients who have gingivitis or are at high risk of developing gingivitis can be tested regularly (e.g., as part of regular dental examinations or even at home). Thereby, in particular, since the presence of gingivitis can be detected immediately after the onset of gingivitis, more timely oral care can be performed, the progression to periodontal disease can be prevented, and the progression of gingivitis can be reversed. Alternatively, for example, if it is known that a patient is at high risk of gingivitis, the onset of gingivitis can be identified by this method in the patient's first test. In particular, this method is also suitable for self-diagnosis, and the steps of collecting the sample and putting it into the device can be performed by the patient himself / herself.

[0042] When the present invention is carried out for the confirmation of the presence of gingivitis, the patient is usually known or suspected to have gingivitis. Thus, in certain embodiments, the method assesses whether a human patient known or suspected to have gingivitis actually has gingivitis. When performing a "healthy or gingivitis classification" on a subject, it is already known or assumed that the subject does not suffer from periodontitis. This can be known, for example, from previously performed periodontitis detection / classification procedures, or can be assumed, for example, from the health records of the subject's oral cavity.

[0043] The method of the present invention generally involves using one or more detection reagents to detect the proteins that make up the biomarker panel of the present invention, and optionally, additional biomarker proteins.

[0044] The "saliva" tested according to the present invention may be undiluted saliva that can be obtained by spitting or swabbing, or diluted saliva that can be obtained by rinsing the mouth with a liquid. Diluted saliva can be obtained by having the patient rinse or gargle with sterile water (e.g., 5 ml or 10 ml) or other suitable fluid for a few seconds and then spit into a container. Such diluted saliva is sometimes referred to as oral rinse fluid.

[0045] "Detecting" means measuring, quantifying, scoring, or assaying the concentration of a biomarker protein. Methods for evaluating biological compounds containing biomarker proteins are known in the art. It is recognized that methods for detecting protein biomarkers include both direct and indirect measurements. One of ordinary skill in the art will be able to select an appropriate method for assaying a particular biomarker protein.

[0046] The term "concentration" with respect to protein biomarkers is given its ordinary meaning, i.e., the amount of protein present in a volume. Protein concentration is measured as mass per volume. Most commonly, it is mg / ml or μg / ml, but sometimes it is measured as low as pg / ml. Another measure is molarity (or molar concentration), moles / L or "M". Concentration can be measured by detecting and measuring the amount of protein in a known, determined, or predetermined volume of sample.

[0047] Instead of measuring concentration, one may measure the absolute amount of a protein biomarker in a sample, or measure the mass-fraction of the biomarker in the sample, e.g., measure the amount of the biomarker relative to the total of all other proteins in the sample.

[0048] A "detection reagent" is a substance or compound that specifically (or selectively) binds to, interacts with, or detects a target protein biomarker. The detection reagent can include, but is not limited to, an antibody, a polyclonal antibody, or a monoclonal antibody that preferentially binds to the protein biomarker.

[0049] The terms "binds specifically (or selectively)" or "has specific (or selective) immunoreactivity" when referring to a detection reagent, refers to a binding reaction that measures the presence of a protein biomarker in a heterogeneous population of proteins and other biological agents. Thus, under specified immunological measurement conditions, a particular detection reagent (e.g., an antibody) binds to a particular protein at least 2-fold over background and does not substantially bind in significant amounts to other proteins present in the sample. Specific binding under such conditions requires an antibody selected for specificity to a particular protein. Antibodies that are specifically immunoreactive with a particular protein can be selected using a variety of immunological measurement formats. For example, the solid-phase ELISA immunoassay (enzyme immunoassay) is routinely used for the selection of antibodies that are specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective reaction is at least 2-fold over background signal or noise and more typically will exceed 10- to 100-fold over background.

[0050] "Antibody" refers to a polypeptide ligand that specifically binds to and recognizes an epitope (e.g., an antigen) and is substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof. The immunoglobulin genes recognized include the κ and λ light chain constant region genes, the α, γ, δ, ε, and μ heavy chain constant region genes, and numerous immunoglobulin variable region genes. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. These include, for example, Fab’ and F(ab)’2 fragments. As used herein, the term "antibody" also encompasses antibody fragments produced by modification of intact antibodies or newly synthesized antibody fragments using recombinant DNA methods, and also encompasses polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single-chain antibodies. The "Fc" portion of an antibody means the portion of an immunoglobulin heavy chain that contains one or more heavy chain constant region domains CH1, CH2, and CH3 but does not contain the heavy chain variable region. An antibody can be a bispecific antibody, for example, an antibody having a first variable region that specifically binds to a first antigen and a second variable region that specifically binds to a second, different antigen. At least one bispecific antibody can be used to reduce the number of detection reagents required.

[0051] Diagnostic methods vary in sensitivity and specificity. The "sensitivity" of a diagnostic assay is the proportion of patients in whom the test was positive (the proportion of "true positives"), and patients with the disease who were not detected by the assay are "false negatives". Subjects who are not diseased but test negative are called "true negatives", and the "specificity" of a diagnostic test is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of non-diseased subjects who test positive.

[0052] The biomarker protein of the present invention can be detected in a sample by any means. Preferred methods for biomarker detection are antibody-based assays, protein array assays, mass spectrometry (MS)-based assays, and (near-infrared spectroscopy-based assays. For example, immunological assays include competitive and non-competitive assay systems using techniques such as Western blot, radioimmunoassay, ELISA "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, fluorescence immunoassay, etc., but are not limited thereto. Such assays are conventional means and are well known in the art. Exemplary immunological assays are briefly described below (but are not intended to be limiting).

[0053] The immunoprecipitation protocol generally involves lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) to which a protein phosphatase and / or protease inhibitor (e.g., EDTA, PMSF, aprotinin, sodium vanadate) has been added, adding the antibody of interest to the cell lysate, incubating at 4°C for a certain period (e.g., 1 to 4 hours), adding protein A and / or protein G sepharose beads to the cell lysate, incubating at 4°C for about 1 hour or more, washing the beads in the lysis buffer, and resuspending the beads in SDS / sample buffer. The immunoprecipitation ability of the antibody for a specific antigen can be evaluated, for example, by Western blot analysis. Those skilled in the art will have knowledge of parameters that can be modified to enhance the binding of the antibody to the antigen and reduce the background (e.g., pre-clearing the cell lysate with sepharose beads).

[0054] Western blot analysis generally involves the preparation of protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8-20% SDS-PAGE depending on the molecular weight of the antigen), transfer of the protein samples from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking of the membrane in a blocking solution (e.g., PBS containing 3% BSA or non-fat milk), washing of the membrane in a washing buffer (e.g., PBS-Tween20), blocking of the membrane with a primary antibody (antibody of interest) diluted in the blocking buffer, washing of the membrane in the washing buffer, blocking of the membrane with a primary antibody conjugated to an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (e.g., 32P or 125I) that recognizes the primary antibody (e.g., anti-human antibody), washing of the membrane in the blocking buffer, and detection of the presence of the antigen. Those skilled in the art will have knowledge of parameters that can be modified to enhance the detected signal and reduce background noise.

[0055] ELISA typically involves the preparation of an antigen (i.e., the biomarker protein of interest or a fragment thereof), coating of the wells of a 96-well microtiter plate with the antigen, addition of the antibody of interest conjugated to a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells, incubation for a period of time, and detection of the presence of the antigen. In ELISA, it is not necessary to conjugate the antibody of interest to a detectable compound; instead, a secondary antibody (that recognizes the antibody of interest) conjugated to a detectable compound can be added to the wells. Further, instead of coating the wells with the antigen, the wells can be coated with an antibody. In this case, the secondary antibody conjugated to a detectable compound can be added after adding the antigen-coated wells. Those skilled in the art will have knowledge of parameters that can be modified to enhance the detected signal and other variations of ELISA known in the art.

[0056] Since multiple markers are used, a threshold value can be determined based on the binding concentration of the biomarker. The threshold value determines whether a patient is classified as having gingivitis. The present invention reflects the insight that gingivitis can be detected with sufficient accuracy based on the measurement of the combination of the above biomarkers.

[0057] This insight is in other aspects, namely, in a saliva sample of a human patient, the following as biomarkers for assessing whether a patient has gingivitis: (i) α-1-acid glycoprotein (A1AGP) and S100 calcium-binding protein A8 (S100A8), and at least one of hemoglobin subunit beta (Hb-beta), keratin 4 (K-4) and pyruvate kinase (PK); or, (ii) α-1-acid glycoprotein (A1AGP), hemoglobin subunit beta (Hb-beta), and keratin 4 (K-4); supports the use of the protein.

[0058] This use can be carried out by the methods described herein and substantially described herein.

[0059] The method of the present invention includes measuring at least one test value that reflects the binding concentration measured for the protein. The binding concentration value can be any value obtained by the input of the measured concentration and the arithmetic operation of these values. This can be, for example, a simple addition of concentrations. It can also include multiplying each concentration by a coefficient that reflects the desired weight of these concentrations and then summing the results. It can also include multiplying the concentrations by each other, or including any combination of multiplication, division, subtraction, exponentiation, and addition. Furthermore, it can include increasing the concentration to a certain extent.

[0060] In some cases, the test value reflects the binding concentration measured for the protein in combination with the age of the subject.

[0061] The obtained binding concentration value can be compared with a threshold value that reflects the binding concentration related to the presence of gingivitis in the same manner. By this comparison, it can be evaluated whether the test value can be an indicator of the presence of gingivitis in the patient whose saliva was tested.

[0062] The threshold value can be based on, for example, the binding concentration values obtained in the same manner based on the concentration determined for the same protein in a reference sample related to the presence of gingivitis, that is, in a patient diagnosed with gingivitis. Usually, a value that reflects that the binding concentration is the same or higher indicates that the patient being tested has gingivitis. Similarly, a value that reflects a low binding concentration in the saliva of a tested gingivitis patient indicates that the patient does not have gingivitis. However, it will be understood that the threshold value can also be calculated (for example, by using a negative multiplier) such that the test value indicating gingivitis is below the threshold value and the test value indicating no gingivitis is above the threshold value.

[0063] The threshold value can also be determined based on measuring the concentration of this biomarker protein in a series of samples, including patients known to be diagnosed with gingivitis and "non-" gingivitis. Thereby, the measured concentration values can be subjected to statistical analysis, probably including machine learning methods, to identify patients classified as having gingivitis and patients classified as not having gingivitis with a desired sensitivity and specificity. Thereby, the desired threshold value can be obtained. Based on this threshold value, the test sample can be subjected to the same concentration measurement, and then, in the same manner as when the threshold value was obtained, the concentration value can be processed to determine a binding concentration value that can be compared with the threshold value, and thus, the tested sample can be classified as having gingivitis or not.

[0064] In an interesting embodiment, the binding concentration value is obtained in the form of a score as follows. A numerical value (for example, a protein concentration value in ng / ml) is assigned to each measurement, and these values are used in a linear or non-linear combination to calculate a score between zero and one. As described above, when the threshold value is determined based on a set of subjects, usually, the score from 0 to 1 is calculated using a sigmoid function with the binding concentration as the input (as further shown above).

[0065] When the score exceeds a certain threshold, it indicates that the patient is suffering from gingivitis. The threshold can be selected based on the desired sensitivity and specificity.

[0066] According to the present invention, when performing "gingivitis classification" on a subject, it will be understood that the subject may be a subject without knowledge or recognition of the state of gingivitis, or a subject at risk of gingivitis or considered to have gingivitis. This prior knowledge is usually, for example, known from a previously performed diagnosis of gingivitis, but either has no such discriminatory ability or is assumed from, for example, the oral health record of the subject.

[0067] The clinical definitions recognized in the art are based on the following: Gingival Index (GI) In the following cases, the Löe modified gingival index (MGI) is evaluated on a scale of 0 to 4, and the overall oral gingival index is recorded based on this: - 0 = No inflammation - 1 = Mild inflammation; not all of the marginal or papillary gingival units, but some parts have a slight change in color texture - 2 = Mild inflammation; however, covering the entire margin or papilla - 3 = Moderate inflammation; opening, redness, swelling and / or hypertrophy of the margin or papilla - 4 = Severe inflammation; marked redness, swelling and / or thickening of the marginal or papillary gingival units, spontaneous bleeding, congestion, ulceration.

[0068] Probing Depth (PD) Using UNC-15 manual periodontal probing, record the probing depth to the nearest mm. The probing depth refers to the distance from the tip of the probe (assumed to be the base of the pocket) to the gingival margin.

[0069] Gingival Recession (REC) ​​​​​Gingival recession is recorded to the nearest mm using UNC-15 manual periodontal probing. Gingival recession is the distance from the free gingival margin to the cementoenamel junction. Gingival recession is indicated by a positive number, and gingival overgrowth is indicated by a negative number.

[0070] Clinical Attachment Loss (CAL) Clinical attachment loss is calculated as the sum of the probing depth + recession at each site.

[0071] Bleeding on Probing (BOP) After probing, bleeding at each site during probing is evaluated. If bleeding occurs within 30 seconds after probing, a score of 1 is assigned to that site; otherwise, a score of 0 is assigned.

[0072] The resulting definition of the subject group (patient group) is as follows: - Healthy group (H): PD ≤ 3 mm at all sites (however, up to 4 mm pocket is possible at the distal of the final standing molar), sites without proximal attachment loss, GI ≥ 2.0, ≤ 10%, %BOP score ≤ 10%; - Gingivitis group (G): ≥ 3.0 at sites where GI exceeds 30%, no sites with proximal attachment loss, no sites with PD exceeding 4 mm, %BOP score exceeding 10%; - Mild to moderate periodontal disease group (MP): Interproximal PD of 5 - 7 mm (equivalent to approximately 2 - 4 mm CAL) in 8 or more teeth, %BOP score > 30%; - Advanced periodontal disease group (AP): Interproximal PD of 7 mm or more in 12 or more teeth (equivalent to CAL of approximately 5 mm or more), %BOP score > 30%.

[0073] ​​​​In one embodiment, the method of the present invention uses the system schematically shown in FIG. 1. The system may be a single device having various device components (units) integrated therein. Also, the system may have its various components, or a part of these components, as separate devices. The components shown in FIG. 1 are a measuring device (A), a graphical user interface (B), and a computer processing unit (C).

[0074] As described above, the system of the present invention includes a data connection to the interface, whereby the interface itself may be part of the system or a remote interface. The latter may use another device, preferably a handheld device such as a smartphone or a tablet computer, to provide the actual interface. The data connection in such a case preferably includes wireless data transfer by Wi-Fi or Bluetooth or other technologies or standards.

[0075] The measuring device (A) is configured to receive a saliva sample by dropping saliva droplets onto a cartridge (A1) insertable into the device (A). The device may be an existing device capable of determining the concentration of proteins from the same saliva sample.

[0076] The processing unit (C) receives the numerical value of the protein concentration from part (A). Unit (C) is attached with software (usually embedded software) capable of calculating a score (S) from 0 to 1. The software further includes a numerical value of a threshold (T). When the calculated value (S) exceeds (T), unit (C) outputs an instruction (I) of "gingivitis" to the GUI (B), and outputs "not gingivitis" otherwise. Further embodiments can indicate the certainty with which the instruction (I) is made using a specific value of (S). This is a probability score, 0.5 is a possible threshold, for example, a score S = 0.8 indicates the probability of gingivitis. Interesting options are as follows: Based on the score S, S = 0.8 can directly indicate an 80% certainty of gingivitis; or, When R1 < S < R2, define and display the range of R1 to R2 such that I becomes "uncertain".

[0077] The specific calculation of the score can be performed, for example, by logistic regression using the sigmoid function:

[0078]

Equation

[0079] When the score S is high in gingivitis patients and low in non - gingivitis / healthy controls, existing regression analysis methods and machine learning methods (such as linear regression, neural networks, support vector machines, etc.) can be used.

[0080] In particular, use clinical trials on subjects who are either in the state of gingivitis or healthy oral cavity (identified by clinical evaluation through current standards by dental experts such as those of the American Academy of Periodontology) to apply the treatment (in the examples). Evaluate the performance of various biomarker combinations by leave - one - out cross - validation to obtain the preferred biomarker combination of the present invention.

[0081] Regarding the above system, the present invention also provides, in a further aspect, a system for assessing whether a human patient has gingivitis, comprising the following: - in a saliva sample of a human patient: (i) at least one of alpha-1-acid glycoprotein (A1AGP), S100 calcium-binding protein A8 (S100A8), and hemoglobin subunit beta (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK); or, (ii) alpha-1-acid glycoprotein (A1AGP), hemoglobin subunit beta (Hb-beta), and keratin 4 (K-4); and detection means adapted to detect such proteins, and as described above, such means are known and readily accessible to those skilled in the art. Usually, a container for receiving an oral sample of a subject is provided, and the container is equipped with the detection means; - a processor adapted to determine an indicator of a patient with gingivitis from the determined concentration of the protein; comprising.

[0082] Optionally, the system may include a user interface (or a data connection to a remote interface), in particular a graphical user interface (GUI) capable of presenting information. The GUI is a type of user interface that enables a user to interact with an electronic device via visual indicators such as graphical icons and secondary notations instead of a text-based user interface, typed command labels, or text navigation (not excluded in the present invention). The GUI is generally known and is typically used in handheld mobile devices such as MP3 players, portable media players, gaming devices, smartphones, and smaller home, office, and industrial controls. As described above, the interface can optionally be selected to allow entry of information such as the subject's age, gender, BMI (Body Mass Index), etc.

[0083] The present invention also provides a kit for detecting at least three biomarkers for gingivitis in a saliva sample of a human patient, either separately or as part of the above system, comprising the following: (i) α-1-acid glycoprotein (A1AGP) and S100 calcium-binding protein A8 (S100A8), and at least one of hemoglobin subunit beta (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK); or, (ii) α-1-acid glycoprotein (A1AGP), hemoglobin subunit beta (Hb-beta), and keratin 4 (K-4); The kit is provided, comprising one or more detection reagents for detecting the same.

[0084] Typically, the kit comprises three or more detection reagents, each directed to a different biomarker. In one embodiment, the first detection reagent detects A1AGP, the second detection reagent detects S100A8, and the third detection reagent detects Hb-beta, K-4, or PK. In other embodiments, the first detection reagent detects A1AGP, the second detection reagent detects Hb-beta, and the third detection reagent detects K-4. In a further aspect, the first detection reagent detects A1AGP, the second detection reagent detects S100A8, the third detection reagent detects Hb-beta, K-4, or PK, and the fourth detection reagent detects a different one of Hb-beta, K-4, or PK.

[0085] As described above with respect to the methods of the present invention, the kit may comprise additional detection reagents for other proteins and the like. In a preferred aspect, the detection reagents available in the kit consist of detection reagents for detecting three or four proteins, including the biomarker panel of the present invention, as described above. In a further aspect, separate detection reagents are provided for each of the biomarker proteins present in the combinations exemplified in Table 1 in the following examples.

[0086] Preferably, the kit includes a chip, a microtiter plate, or a solid support such as beads or resin containing the detection reagent. In some embodiments, the kit includes mass spectrometry probing such as ProteinChip™.

[0087] The kit can also provide a non-binding detection reagent or a washing solution and / or a detection reagent specific to either of the biomarkers (sandwich assay).

[0088] In an interesting embodiment, the recognition of the biomarker panel of the present invention is applied to the longitudinal monitoring of the state of gingivitis in a human patient. Thus, the present invention also provides an in vitro method for determining changes in the state of gingivitis over time from a first time point t1 to a second time point t2 in a human patient suffering from gingivitis, comprising, in at least one sample of saliva obtained from said patient, at t1 and in at least one sample of saliva obtained from said patient, the following: (i) at least one of alpha-1-acid glycoprotein (A1AGP), S100 calcium-binding protein A8 (S100A8), and hemoglobin subunit beta (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK); or, (ii) alpha-1-acid glycoprotein (A1AGP), hemoglobin subunit beta (Hb-beta), and keratin 4 (K-4); a step of detecting the concentration of the protein; and, a step of comparing the concentrations such that at least one, two, or more differences in said concentrations reflect the change in state; The present invention provides a method comprising. This difference can be reexamined as a difference in concentration and thus can be directly compared without first generating a number between 0 and 1 or any other classification. It will also be understood that the measured values received at both time points can be processed in the same manner as is done when determining the state of the patient as described above.

[0089] The present invention also provides a method for diagnosing whether a human patient has gingivitis, comprising, in a saliva sample of said human patient, the following: (i) At least one of α-1-acid glycoprotein (A1AGP), S100 calcium-binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK); or, (ii) α-1-acid glycoprotein (A1AGP), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); a step of detecting the protein; and, a step of evaluating the presence of gingivitis in a patient based on the concentration of the protein in the sample; A method is provided that includes.

[0090] The presence of gingivitis in a patient is usually evaluated based on the concentration of the protein in the sample. In some cases, the method of this aspect includes a further step of treating the patient's gingivitis. This optional treatment step can include the administration of known therapeutic agents or dental procedures, or a combination of therapeutic agents and dental procedures. Known therapeutic agents include the administration of antibacterial agent-containing agents, such as mouthwashes, chips, gels, or microspheres. A typical antibacterial agent used in the treatment of gingivitis is chlorhexidine. Other therapeutic agents include antibiotics, antibiotics usually administered orally, and enzyme inhibitors such as doxycycline. Known non-surgical treatment techniques include scaling and root planing (SRP). Known surgical procedures include surgical pocket restoration, flap surgery, gingival grafts, or bone grafts, but these are usually only used for advanced periodontitis and are not usually used for the treatment of gingivitis.

[0091] The present invention further provides, in a human patient, the following: (i) At least one of α-1-acid glycoprotein (A1AGP), S100 calcium-binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), keratin 4 (K-4), and pyruvate kinase (PK); or, (ii) α-1-acid glycoprotein (A1AGP), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); A method for detecting a protein, comprising the following: (a) Obtaining a saliva sample from a human patient; and, (b) Contacting the sample with one or more detection reagents that bind to the protein to detect whether the protein is present in the sample, and detecting the binding between each protein and the one or more detection reagents; A method is provided that includes the above.

[0092] The present invention is further illustrated with reference to the following non-limiting examples.

Example

[0093] A clinical trial was conducted on 74 subjects, 35 diagnosed with gingivitis and 39 with healthy gums. Using a panel containing up to four protein biomarkers as shown below, an area under the Receiver-Operator-Characteristic curve value > 0.75 was obtained. It can be seen that when at least three biomarkers are combined, an AUC score close to 0.8 is obtained.

[0094] The Receiver Operator Characteristic (ROC) area-under-curve (AUC) value was obtained. The performance of various biomarker combinations was evaluated by logistic regression using leave-one-out cross validation (LOOCV), and a preferred biomarker combination as described herein was obtained.

[0095] In statistics, the Receiver-Operator-Characteristic curve, i.e., the ROC curve, is a graphical plot that shows the performance of a binary classifier system as the discrimination threshold changes. This curve is created by plotting the true positive rate (TPR) against the false positive rate (FPR) at various threshold settings. The true positive rate is also known as sensitivity, recall, or the probability of detection in machine learning. The false positive rate is also known as fallout or false alarm probability and can be calculated as (1 - specificity). Thus, the ROC curve is the sensitivity as a function of fallout. Generally, if the probability distributions of both detection and false alarm are known, the ROC curve can be generated by plotting the value of the cumulative distribution function of the detection probability (the area under the probability distribution from -∞ to the discrimination threshold) on the y-axis and the value of the cumulative distribution function of the false alarm probability on the x-axis. The accuracy of a test depends on how well the test can separate the test population into groups with and without the disease in question. The accuracy is measured by the area under the ROC curve. An area of 1 represents a perfect test, and an area of 0.5 represents a worthless test. The guide for classifying the accuracy of a diagnostic test is the traditional academic point system: -0.90 - 1 = Excellent (A) -0.80 - 0.90 = Good (B) -0.70 - 0.80 = Fair (C) -0.60 - 0.70 = Poor (D) -0.50 - 0.60 = Fail (F) From the above, in the results of the above clinical trial, when the ROC AUC value exceeds 0.75, it is considered to represent a desirable accuracy for conducting the test related to the present invention.

[0096] The following 21-panel protein biomarkers are up to four and have been found to provide an AUC LOOCV > 0.75 for classifying gingivitis versus oral hygiene:

[0097]

Table 1

[0098] In this table, following the marker age, the following numerous 8 protein markers (previously not associated with oral health) are considered: -A1AGP -Hb-beta -Hb-delta -Keratin 4 -Profilin -Pyruvate kinase -S100A8 -S100A9 To consider the 21 panels identified with AUC > 0.75: · With 8 protein markers and, optionally, age as a marker, 324 panels with a maximum of 4 protein markers are possible (panels with only age are not considered). It is unexpected that a limited set of 21 reliable biomarker panels available in saliva has been identified from a total of 324 possibilities.

[0099] · Without restricting the number of protein markers within the panel, a large number of 510 possible panels can be obtained from these 8 markers (panels with only age are not considered).

[0100] Although the present invention has been described and illustrated in detail in the drawings and the foregoing description, such description and illustration should be considered as exemplary and typical, and not restrictive, and the present invention is not limited to the disclosed embodiments.

[0101] For example, detection reagents can be presented in different units for different biomarkers. Alternatively, conveniently, the kits of the present invention can include a fixed set of detection reagents for flexible modules including detection reagents for all embodiments, for example, A1AGP, MMP8 or HGF, and optionally other biomarkers such as S100A8 and / or K-4, etc.

[0102] Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the term "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" (original text) does not exclude a plurality. The mere fact that certain features of the present invention are described in mutually different dependent claims does not indicate that a combination of these features cannot be advantageously used. The reference signs in the claims should not be construed as limiting their scope.

[0103] If desired, the inventors disclose an in vitro method for assessing whether a human patient is suffering from gingivitis. This method is based on insights into determining biomarker proteins. Accordingly, in a saliva sample from the patient, the concentration of the proteins described herein is measured. Based on the measured concentration, a value reflecting the binding concentration of the protein is determined. This value is compared with a threshold value that similarly reflects the gingivitis-related binding concentration. By this comparison, it can be evaluated whether the test value is an indicator of the presence of gingivitis in the patient. Thereby, usually, a test value reflecting a binding concentration less than the binding concentration reflected by the threshold value is an indicator that the patient does not have gingivitis, and a test value reflecting a binding concentration at or above the binding concentration reflected by the threshold value is an indicator that the patient has gingivitis.

Claims

1. 1. An in vitro method for assessing whether a human patient has gingivitis, comprising: in a saliva sample from said human patient: (i) alpha-1-acid glycoprotein (A1AGP), S100 calcium binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); or (ii) A1AG, S100A8 and Hb-beta: detecting the concentration of the protein; - determining a test value reflecting the binding concentration of said detected protein; - comparing said test value with a threshold value which in the same way reflects a binding concentration associated with gingivitis to assess whether said test value is indicative of the presence of gingivitis in said human patient; The in vitro method includes:

2. The method of claim 1 , wherein the human patient is suspected of having gingivitis.

3. 3. The method of claim 1 or 2, wherein the age of the human patient is determined and the test value reflects the binding concentration of the detected protein in combination with the age of the human patient.

4. The method according to any one of claims 1 to 3, wherein said threshold value is based on a concentration determined for said detected protein in one or more reference samples each associated with the presence of gingivitis or the absence of gingivitis.

5. The method of any one of claims 1 to 3, wherein the threshold value is based on the concentration of a protein in a set of samples comprising samples from subjects with gingivitis and samples from subjects without gingivitis.

6. The method according to any one of claims 1 to 5, wherein the determined concentration values ​​are arithmetically processed into numbers between 0 and 1.

7. In a saliva sample of a human patient, the following as biomarkers for assessing whether said human patient has gingivitis: (i) alpha-1-acid glycoprotein (A1AGP), S100 calcium binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); or (ii) A1AG, S100A8 and Hb-beta: Use of protein.

8. The use according to claim 7, wherein the age of the human patient is also used as a biomarker.

9. 1. A system for assessing whether a human patient has gingivitis, comprising: - in saliva samples from human patients: (i) alpha-1-acid glycoprotein (A1AGP), S100 calcium binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); or (ii) A1AG, S100A8 and Hb-beta: a detection means adapted to receive the concentration of each of the proteins; - a processor adapted to determine an indication of the presence of gingivitis in said human patient from the determined concentration of said protein; Including, the system.

10. 10. The system of claim 9, further comprising a container for receiving an oral fluid sample, said container comprising said detection means.

11. In addition, the following: - a user interface for presenting said indicators to a user; - a data connection between said processor and said user interface for transferring instructions from said processor to said user interface; The system according to claim 9 or 10, comprising:

12. The system of any one of claims 9 to 11, wherein the processor is operable by an internet-based application.

13. The system of claim 11, wherein the user interface is capable of inputting information regarding the age of the human patient, and the processor is capable of and adapted to determine an indication of the presence of gingivitis in the human patient from the determined concentration.

14. 1. A kit for detecting at least three biomarkers for gingivitis in a saliva sample from a human patient, comprising: (i) alpha-1-acid glycoprotein (A1AGP), S100 calcium binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); or (ii) A1AG, S100A8 and Hb-beta: A kit comprising a detection reagent for detecting

15. The kit of claim 14, wherein the one or more detection reagents comprise: (i) a first detection reagent that detects A1AGP, a second detection reagent that detects S100A8, a third detection reagent that detects Hb-beta, and a fourth detection reagent that detects K-4; or (ii) a first detection reagent that detects A1AGP, a second detection reagent that detects S100A8, and a third detection reagent that detects Hb-beta.

16. 16. The kit of claim 14 or 15, wherein the one or more detection reagents are contained on a solid support.

17. 1. A method of operating a processor in a system for determining a change in a state of gingivitis in a human patient suffering from gingivitis over time from a first time point t1 to a second time point t2, comprising: determining in at least one sample of saliva obtained from the human patient at t1 and in at least one sample of saliva obtained from the human patient at t2: (i) alpha-1-acid glycoprotein (A1AGP), S100 calcium binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); or (ii) A1AG, S100A8 and Hb-beta: and comparing the concentrations of each of said proteins such that a difference in concentration of each reflects a change in condition; The method includes:

18. 1. A method of operation of a processor in a system for diagnosing whether a human patient has gingivitis, the method comprising detecting in a saliva sample of the human patient: (i) alpha-1-acid glycoprotein (A1AGP), S100 calcium binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); or (ii) A1AG, S100A8 and Hb-beta: and assessing the presence of gingivitis in a human patient based on the concentration of said protein in said saliva sample.

19. In a human patient, a biomarker for assessing whether the human patient has gingivitis is one of the following: (i) alpha-1-acid glycoprotein (A1AGP), S100 calcium binding protein A8 (S100A8), hemoglobin subunit β (Hb-beta), and keratin 4 (K-4); or (ii) A1AG, S100A8 and Hb-beta:

1. A method for detecting a protein comprising: (a) obtaining a saliva sample from the human patient; and (b) detecting whether said protein is present in the sample by contacting the sample with one or more detection reagents that bind to said protein, and detecting binding between each of said proteins and said one or more detection reagents; A method comprising:

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