Methods, uses and kits for monitoring or predicting response to periodontal disease treatment

Abstract

An in vitro method for assessing or predicting the response of a human patient to periodontal disease treatment is disclosed. The method is based on the insight that biomarker proteins are determined. Thus, in a saliva sample of a patient, the concentration of a specific combination of proteins is measured. One such combination is hemoglobin subunit delta (Hb-delta) and pyruvate kinase (PK). Another combination is at least two of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metallopeptidase-8 (MMP-8), and keratin-4 (K-4). A third combination is alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8). Based on the measured concentrations, at least one value is determined that reflects the joint concentration of this proteins. The at least one value can be indicative of the likelihood that the human patient has or will successfully treat periodontitis. The at least one value can be compared to at least one threshold value that reflects the joint concentration in the same way that is associated with successful treatment of periodontitis. This comparison allows to assess whether the test value is an indication of the periodontal treatment status of the patient.

Description

Technical Field

[0001] The present invention belongs to the field of oral care and relates to saliva-based assessment of response to periodontal disease treatment. In particular, the present invention relates to a kit, use and method for assessing or predicting the response to treatment of a patient suffering from periodontal disease. Background Art

[0002] Gum inflammation, or gingivitis, is a non-destructive periodontal disease caused primarily by the adhesion of dental bacterial biofilm, or plaque, to the tooth surface. If not detected and treated, reversible gingivitis typically leads to inflammation of the tissues surrounding the teeth (i.e., the periodontium), a condition defined as periodontitis, which is irreversible and results in tissue destruction and alveolar bone loss, ultimately leading to tooth loss. As gum disease progresses, there are often associated clinical signs and symptoms, such as swollen gums, a change in color from pink to dark red, bleeding gums, bad breath, and gums that become tender or sore to the touch.

[0003] Periodontitis is a chronic, multifactorial inflammatory disease caused by oral microorganisms and is characterized by the progressive destruction of hard (bone) and soft (periodontal ligament) tissues, ultimately leading to tooth movement and loss. This is to be distinguished from gingivitis, which is a reversible infection and inflammation of the gum tissue. Inflammatory periodontitis is one of the most common chronic human diseases and is the leading cause of tooth loss in adults. In addition to its substantial negative impact on oral health, there is increasing evidence that periodontitis 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 disease is important from both the perspective of oral and overall health. Furthermore, it is desirable to be able to accurately determine whether a treatment for periodontal disease is or is likely to be effective for a patient. In particular, it is desirable to be able to predict whether a treatment for periodontal disease is likely to be effective before it is offered to a patient or at an early stage of treatment.

[0005] Periodontal disease remains poorly diagnosed in general dental practice, resulting in relatively low rates of therapeutic intervention and a significant number of untreated cases. Current diagnosis relies on inaccurate, subjective clinical examination by dental professionals of the condition of oral tissues (color, swelling, degree of bleeding on probing, depth of probing pockets, and bone loss as detected by oral X-rays). These traditional 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 susceptibility to further disease.

[0006] Similarly, for patients undergoing treatment for periodontal disease, response to treatment must currently be assessed clinically after treatment via these traditional methods, resulting in high costs and potentially inaccurate monitoring of the patient's condition. Furthermore, predicting the likelihood of treatment response can be of great value, as treatment strategies can be tailored accordingly. Therefore, it would be desirable to measure current disease activity, a subject's susceptibility to further periodontal disease, and what treatments are likely to be successful for that patient. Therefore, a more objective, faster, more accurate, easier to use diagnostic—ideally with predictive value—that can preferably also be performed by non-specialists is desirable.

[0007] Saliva or oral fluid has long been advocated as a diagnostic fluid for oral and general diseases, and with the advent of miniaturized biosensors (also known as lab-on-a-chip), point-of-care diagnostics for rapid chairside testing have gained greater scientific and clinical attention. In particular, for periodontal disease detection, inflammatory biomarkers associated with tissue inflammation and decomposition can be readily terminated in saliva due to proximity, suggesting that saliva has a strong potential for periodontal disease detection. In fact, this field has therefore received significant attention and has presented encouraging results. For example, Ramsier et al. (J Periodontol. 2009 Mar; 80(3): 436-46) identified host and bacteria-derived biomarkers associated with periodontal disease. However, no clear test has yet emerged.

[0008] Biomarkers represent biological indicators that support clinical manifestations and are therefore objective indicators of clinical outcome in diagnosing periodontal disease. Ultimately, validated biomarkers can be utilized to assess risk for future disease, identify disease at a very early stage, identify response to initial therapy, and allow the implementation of preventive strategies.

[0009] Factors that have previously limited the development of point-of-care tests for salivary biomarkers include the lack of technology suitable for chairside use and the inability to analyze multiple biomarkers in individual samples. Furthermore, the choice of which multiple biomarkers to include in such tests has not been adequately addressed in the literature or implemented in actual testing.

[0010] Furthermore, periodontitis can manifest itself across a spectrum of severity, from mild to advanced disease. To easily assess the severity of the condition, dentists often categorize patients with periodontitis into two groups: those with mild periodontitis and those with advanced periodontitis. However, available methods for performing this assessment involve labor-intensive processes that dentists cannot routinely perform for every patient and / or every visit, and are impossible for users to perform (self-diagnosis).

[0011] It would be desirable to provide a simpler method, and in particular a method that only requires the collection of a small saliva sample from the patient, which the patient can collect themselves. It would be desirable to input such a sample into an in vitro diagnostic device that allows the saliva sample to be classified based on the measurements so that the device can return an indication as to the likelihood that the patient's periodontal disease is being or could be effectively treated. Summary of the Invention

[0012] To better meet the above expectations, in one aspect, the present invention relates to an in vitro method for assessing or predicting a human patient's response to a periodontal disease treatment, the method comprising detecting the concentration of at least the following proteins in a saliva sample from a human patient suffering from periodontal disease:

[0013] (i) hemoglobin subunit delta (Hb-δ) and pyruvate kinase (PK); or

[0014] (ii) at least two of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8), and keratin-4 (K-4); or

[0015] (iii) α-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8);

[0016] At least one of the test values ​​is determined that reflects the combined concentrations determined for the proteins, and the test value is compared to a threshold value that in the same manner reflects the combined concentrations associated with successful treatment of periodontal disease to assess whether the test value is indicative of successful treatment of the periodontal disease for the patient.

[0017] In another aspect, the present invention provides for the use of the above-identified proteins in saliva samples of human patients as biomarkers for assessing whether the patient will respond to or has responded to periodontal disease treatment.

[0018] Optionally, the patient's age is also used as a biomarker.

[0019] In another aspect, the present invention is a system for assessing or predicting a human patient's response to a periodontal disease treatment, the system comprising:

[0020] a detection device capable of and adapted to detect the protein identified in the first aspect in a saliva sample of a human patient; and

[0021] A processor is capable and adapted to determine an indication of whether the patient's periodontal disease has been or will be successfully treated based on the determined concentration of the protein.

[0022] The system optionally comprises a data connection to an interface, in particular a graphical user interface, which enables presentation of information and preferably also input of information, said interface being part of the system or being a remote interface.

[0023] Optionally, one or more of the aforementioned items, in particular the processor, is enabled to function "in the cloud", ie not on a fixed machine, but by means of an Internet-based application.

[0024] In yet another aspect, the present invention provides a kit for detecting at least two biomarkers for periodontal disease in a saliva sample of a human patient, the kit comprising a detection agent for detecting:

[0025] Hemoglobin subunit delta (Hb-delta) and pyruvate kinase (PK); or

[0026] at least two of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8), and keratin-4 (K-4); or

[0027] α-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8).

[0028] Typically, two or more, three or four, detection agents are used, each of which binds to a different biomarker. In one embodiment, the first detection agent is used to detect Hb-δ, and the second detection agent is used to detect PK. In another embodiment, the first detection agent is used to detect K-4, the second detection agent is used to detect A1AGP, and the third detection agent is used to detect PK. In another embodiment, the first detection agent is used to detect K-4, the second detection agent is used to detect A1AGP, and the third detection agent is used to detect MMP-8. In another embodiment, the first detection agent is used to detect K-4, the second detection agent is used to detect MMP-8, and the third detection agent is used to detect PK. In another embodiment, the first detection agent is used to detect A1AGP, the second detection agent is used to detect PK, and the third detection agent is used to detect S100A8. In a further embodiment, the first detection agent is used to detect keratin-4 (K-4), the second detection agent is used to detect alpha-1-acid glycoprotein (A1AGP), the third detection agent is used to detect pyruvate kinase (PK), and the fourth detection agent is used to detect matrix metalloproteinase-8 (MMP-8).

[0029] In yet another aspect, the present invention provides an in vitro method for determining a change in the state of a periodontal disease in a human patient due to treatment of the disease over a time interval from a first time point t1 to a second time point t2, the method comprising detecting the concentration of the following proteins in at least one saliva sample obtained from the patient at t1 and at least one saliva sample obtained from the patient at t2:

[0030] Hemoglobin subunit delta (Hb-delta) and pyruvate kinase (PK); or

[0031] at least two of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8), and keratin-4 (K-4); or

[0032] alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8);

[0033] and comparing concentrations, whereby a difference in any one, two, three, four or more of the concentrations reflects a change in state.

[0034] In another aspect, the present invention provides a method for determining whether a human patient has been successfully treated, is currently being successfully treated, or will be successfully treated for periodontal disease, comprising detecting a protein identified in the first aspect above in a saliva sample of the human patient, and assessing whether the human patient has been successfully treated or will be successfully treated for periodontal disease based on the concentration of the protein in the sample. Optionally, the method comprises the further step of treating periodontitis in the patient.

[0035] In yet another aspect, the present invention provides a method for detecting a protein identified in the first aspect in a human patient, comprising:

[0036] (a) obtaining a saliva sample from a human patient; and

[0037] (b) detecting the presence of a protein in the sample by contacting the sample with two or more detection agents that bind to the protein and detecting binding of each protein to the two or more detection agents. As described elsewhere herein, typically, at least a first detection agent and a second detection agent are present, and sometimes a third detection agent and a fourth detection agent are present. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A system for use in the method of the present disclosure is schematically shown. DETAILED DESCRIPTION

[0039] In a general sense, the present invention is based on the insight that certain combinations of protein biomarkers in saliva samples of human patients can be used to assess or predict a patient's response to a treatment for periodontal disease. The biomarker combinations in saliva are able to distinguish a successful response to a periodontitis treatment from an unsuccessful response to a periodontitis treatment. This insight is based, at least in part, on the discovery that a biomarker's usefulness for diagnosing periodontal disease (e.g., prior to treatment) does not necessarily mean it is also useful for monitoring treatment of that periodontal disease. The present disclosure proposes clinical definitions of various levels of treatment response and identifies salivary protein marker combinations that enable assessment or prediction of treatment response from measurements of post-treatment or pre-treatment (for prognostic) concentrations of the salivary protein marker combinations.

[0040] The biomarker proteins are hemoglobin subunit delta (Hb-δ), pyruvate kinase (PK), keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), matrix metalloproteinase-8 (MMP-8), and S100 calcium binding protein A8 (S100A8). According to the present invention, the following combination of these proteins is used to monitor or predict the treatment of periodontal disease:

[0041] i. Hemoglobin subunit delta (Hb-delta) and pyruvate kinase (PK); or

[0042] ii. at least two of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8), and keratin-4 (K-4); or

[0043] iii. α-1-acid glycoprotein (A1AGP), pyruvate kinase (PK) and S100 calcium binding protein A8 (S100A8).

[0044] The age of the subject may optionally be included as an additional marker.

[0045] In one embodiment, the method assesses the response of a human patient previously diagnosed with periodontitis and who has received treatment for the periodontitis.Concentrations of hemoglobin subunit delta (Hb-delta) and pyruvate kinase (PK) protein may typically be measured in this embodiment.

[0046] In another embodiment, the method predicts the response of a human patient to the treatment of periodontitis. This can be done when the patient has not yet received treatment. Alternatively, treatment may have been recently applied to the patient, and it is desirable to know whether the treatment is likely to be effective in the early stages. Usually before using the method of the present invention to assess a patient, treatment is first applied for at least about one week, about two weeks, or about three weeks, for example, at least about 7 days, at least about 14 days, or at least about 21 days. Treatment can begin between about one week and about one month before the assessment. Alternatively, the patient can be assessed at intervals of two weeks, three weeks, or four weeks after the first treatment, for example, at the 3rd week, 6th week, and 9th week after the treatment, or at the 4th week, 8th week, and 12th week after the treatment. In these predictive embodiments, the concentration of at least two proteins of keratin-4 (K-4), and alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8) can be detected. Alternatively, the concentrations of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium binding protein A8 (S100A8) proteins can be measured.

[0047] Hemoglobin (Hb) is an iron- and oxygen-transporting metalloprotein found in the red blood cells of nearly all vertebrates and in the tissues of some invertebrates. Hemoglobin subunit δ (also known as δ-globin, HBD, δ-globin, and hemoglobin δ) is a globin that, along with α-globin (HBA), makes up the less common form of adult hemoglobin, HbA-2. Hb-δ is typically 147 amino acids long and has a molecular weight of 16,055 Da. Adult HbA-2 is a heterotetramer composed of two α chains and two δ chains. Hb-δ is encoded by the HBD gene on human chromosome 11.

[0048] Pyruvate kinase catalyzes the final step of glycolysis. There are four tissue-specific isoenzymes of pyruvate kinase, each with specific kinetic properties required for use in different tissues.

[0049] Keratin-4 (K4), also known as cytoskeletal keratin-4 (CYK4) or cytokeratin-4 (CK-4), is a protein encoded in humans by the KRT4 gene, a member of the keratin gene family. Type II cytokeratins are composed of basic or neutral proteins that are paired in heterotypic keratin chain pairs that are co-expressed during differentiation of simple and stratified epithelial tissues. Type II cytokeratin CK4 is specifically expressed in the differentiated layers of mucosal and esophageal epithelia, along with family member KRT13. Mutations in these genes are associated with white sponge nevus, characterized by white patches of the mouth, esophagus, and anus. Type II cytokeratins are clustered in the region of chromosome 12q12-q13.

[0050] Alpha-1-acid glycoprotein (A1AGP) is a plasma alpha-globulin glycoprotein, sometimes also called serum mucoid, synthesized primarily by the liver. It functions as a transport protein in the blood that acts as a carrier for basic and neutrally charged lipophilic compounds. Alpha-1-acid glycoprotein is also believed to regulate interactions between blood cells and endothelial cells.

[0051] MMPs are a family of enzymes responsible for the degradation of extracellular matrix components such as collagen, proteoglycans, laminin, elastin, and fibronectin. MMPs play a crucial role in periodontal ligament (PDL) remodeling under both physiological and pathological conditions. MMP-8, also known as neutrophil collagenase or PMNL collagenase (MNL-CL), is a collagenase present in the connective tissue of most mammals.

[0052] S100 calcium-binding protein A8 is a calcium-binding and zinc-binding protein that plays a significant role in the regulation of inflammatory processes and immune responses. S100 calcium-binding protein A8 can induce neutrophil chemotaxis and adhesion.

[0053] The above proteins are known in the art. Those skilled in the art are aware of their structures and methods for detecting them in aqueous samples (such as saliva samples). The following protein biomarker combinations are collectively referred to as the "biomarker panel of the present invention":

[0054] i. Hemoglobin subunit delta (Hb-delta) and pyruvate kinase (PK); or

[0055] ii. at least two of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK) and matrix metalloproteinase-8 (MMP-8), and keratin-4 (K-4); or

[0056] iii. α-1-acid glycoprotein (A1AGP), pyruvate kinase (PK) and S100 calcium binding protein A8 (S100A8).

[0057] In one embodiment, the biomarker panel of the present invention can be composed of identified protein biomarkers. Preferably, the biomarker panel of the present invention is composed of no more than four protein biomarkers identified by 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 data set for determining the type of periodontitis. Additional protein biomarkers include inhibitory protein, hemoglobin-δ, matrix metalloproteinase-9, and hepatocyte growth factor, in this example, each of which is present in the effective biomarker panel.

[0058] When additional biomarkers are optionally included, the total number of biomarkers (ie, the biomarker panel of the invention plus the additional biomarkers) is typically four, five, or six.

[0059] However, one desired advantage of the present invention is that the classification of a patient's periodontal disease can be determined by measuring preferably no more than four biomarkers, and more preferably only three biomarkers, or even in some embodiments, only two biomarkers. A particularly preferred biomarker panel consists of the following biomarkers:

[0060] (i) hemoglobin subunit delta (Hb-δ) and pyruvate kinase (PK); or

[0061] (ii) alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium binding protein A8 (S100A8); or

[0062] (iii) keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), and pyruvate kinase (PK); or

[0063] (iv) keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), and matrix metalloproteinase-8 (MMP-8); or

[0064] (v) keratin-4 (K-4), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8); or

[0065] (vi) keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8).

[0066] In particular, such a determination need not involve the use of other data, which advantageously provides a simple and direct diagnostic test.The biomarker panels identified herein allow for the detection of successful outcomes of periodontitis treatments.

[0067] As needed, the method only requires obtaining a small amount (e.g., a droplet-sized) saliva sample from the subject. The sample size is typically 0.1 μl-2 ml, such as 1 ml-2 ml, so that smaller amounts (e.g., 0.1 μl-100 μl) can be used for in vitro device processing, and thus, extracting larger samples, such as up to 20 ml, such as 7.5 ml-17 ml, is also possible.

[0068] This sample is input into an in vitro diagnostic device, which measures the concentration of the proteins involved and returns results that categorize the subject based on the likelihood of successful periodontal disease treatment.

[0069] The ease of use of the present invention allows for regular testing of most dental patients with periodontal disease (e.g., as part of a regular dental checkup or even at home). This allows, in particular, for detection of whether treatment for periodontitis is successful or about to be successful, thereby enabling more informed periodontitis treatment approaches, whereby successful treatments can be continued and unsuccessful treatments discontinued or not initiated. The ability to assess whether a therapy is successful is beneficial in confirming, for example, that a patient's current treatment is satisfactory. In particular, the method is also suitable for self-diagnosis, whereby the steps of obtaining a sample and inserting the sample into the device can be performed by the patient themselves.

[0070] When practicing the present invention, it is generally known that a patient has periodontal disease. When practicing the present invention, it is generally known that a patient has periodontitis. Periodontitis can be mild or advanced. Therefore, in certain embodiments, the present methods are used to assess whether a human patient known to have periodontitis is being (or will be) successfully treated for the condition.

[0071] The treatment that is assessed as successful or unsuccessful can be any treatment for periodontal disease. Therapeutic agents and dental surgery, or a combination of therapeutic agents and dental surgery, are known and can be used. Known therapeutic agents include the administration of antimicrobial-containing agents, such as mouthwashes, chips, gels or microspheres. A typical antimicrobial agent for treating gingivitis and periodontitis is chlorhexidine. Other therapeutic agents include antibiotics, typically oral antibiotics, and enzyme inhibitors, such as doxycycline. Known non-surgical treatment procedures include: root instruments that destroy subgingival plaque biofilms; scaling and root planing (SRP); and interdental cleaning. Known surgical treatments include surgical pocket reduction, flap surgery, gum grafting or bone grafting. Treatment is preferably a therapeutic agent, such as an antimicrobial, typically an antimicrobial mouthwash.

[0072] The need and number of optional visits to the dentist or hygienist for treatment can vary according to clinical needs and patient preferences. The initial stage of clinical treatment is usually completed in a fewer but longer number of visits. In this embodiment, the patient can go to the dental clinic for two visits, each visit lasting about 1 hour to 2 hours for the initial treatment. Subsequently, frequent return visits in the later stages of early treatment can check the cure, motivate the patient, strengthen oral hygiene, and provide prevention for the removal of re-formed dental plaque. This is usually achieved by short visits (e.g., 15-30 minutes) at intervals of 2-4 weeks within 1-2 months after the initial treatment period.

[0073] Thus, in certain embodiments, a treatment phase may include one to two clinic visits for root instrumentation. Thereafter, the patient may return to the dentist or hygienist approximately three, six, or nine weeks later for follow-up visits (preventive, motivational, and reinforcement oral hygiene). Within this timeline, the treatment plan may be implemented based on individual clinical needs.

[0074] In certain embodiments, the assessment or prediction methods of the present invention can be performed at any clinic visit following initial therapy.

[0075] The methods of the present invention generally comprise detecting at least two of the above proteins comprising the biomarker panel of the present invention and optionally other biomarker proteins using one or more detection agents.

[0076] Typically, treatment is considered successful when inflammation levels are significantly reduced while pocket depths remain relatively high (i.e., compared to healthy individuals or patients with gingivitis). Therefore, for patients after treatment, disease classification based on salivary protein markers of the disease itself (e.g., distinguishing between healthy, gingivitis, mild periodontitis, and severe periodontitis) may not be suitable for assessing treatment response. The biomarker proteins of the present invention overcome this problem and are able to specifically determine response to treatment.

[0077] According to the present invention, the "saliva" tested can be undiluted saliva that can be obtained by spitting or wiping, or diluted saliva that can be obtained by rinsing the mouth with a fluid. Diluted saliva can be obtained by the patient rinsing the mouth or gargling for a few seconds with sterile water (e.g., 5 ml or 10 ml) or other appropriate fluid and spitting into a container. Diluted saliva can sometimes be referred to as an oral rinse.

[0078] "Detecting" refers to measuring, quantifying, scoring, or determining the concentration of a biomarker protein. Methods for evaluating biological compounds, including biomarker proteins, are known in the art. It is generally recognized that methods for detecting protein biomarkers include direct and indirect measurements. One skilled in the art will be able to select an appropriate method for determining a particular biomarker protein.

[0079] The term "concentration" of a protein biomarker is given its usual meaning, i.e., the abundance of the protein in a volume. Protein concentration is typically measured in mass / volume, most commonly in mg / ml or μg / ml, but sometimes as low as pg / ml. Another alternative measurement is molarity, mol / L or "M." Concentration can be determined by measuring the amount of protein in a known, determined, or predetermined volume of a sample.

[0080] An alternative to determining concentration is to determine the absolute amount of the protein biomarker in the sample, or to determine the mass fraction of the biomarker in the sample, such as the amount of the biomarker relative to the total amount of all other proteins in the sample.

[0081] A "detection agent" is an agent or compound that specifically (or selectively) binds to, interacts with, or detects a protein biomarker of interest. Such detection agents may include, but are not limited to, antibodies, polyclonal antibodies, or monoclonal antibodies that preferentially bind to a protein biomarker.

[0082] The term "periodontal disease" refers to both gingivitis and periodontitis (mild and severe). Patients without periodontal disease are considered healthy.

[0083] When referring to a detection agent, the phrase "specifically (or selectively) binds" or "particularly (or selectively) immunoreacts with..." refers to a binding reaction that determines the presence of a protein biomarker in a heterogeneous population of proteins and other biological agents. Thus, under specified immunoassay conditions, a particular detection agent (e.g., an antibody) binds to a particular protein at least twice the background level and does not substantially bind to other proteins present in the sample in significant amounts. Specific binding under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid-phase ELISA immunoassays (enzyme-linked immunosorbent assays) are routinely used to select antibodies that specifically immunoreact with a protein (for a description of immunoassay formats and conditions that can be used to determine specific immunoreactions, see, for example, Harlow & Lane, Antibodies, A Laboratory Manual (1988)). Typically, a specific or selective reaction will be at least twice the background signal or noise, and more typically 10 to 100 times or more the background.

[0084] "Antibody" refers to a polypeptide ligand substantially encoded by one or more immunoglobulin genes or fragments of immunoglobulin genes, which specifically binds to and recognizes an epitope (e.g., an antigen). Recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon, and mu heavy chain constant region genes, and a large number of immunoglobulin variable region genes. Antibodies exist, for example, as intact immunoglobulins or as several well-characterized fragments produced by digestion with various peptidases. This includes, for example, Fab' and F(ab)'2 fragments. The term "antibody" as used herein also includes modifications of antibody fragments produced from intact antibodies or modified antibody fragments synthesized de novo using recombinant DNA methods. Antibodies also include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single-chain antibodies. The "Fc" portion of an antibody refers to a portion of an immunoglobulin heavy chain, including one or more heavy chain constant region domains, CH1, CH2, and CH3, but excluding 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 different, second antigen. The use of at least one bispecific antibody can reduce the number of detection agents required.

[0085] Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay refers to the percentage of sick individuals that test positive (the percentage of "true positives"). Sick individuals that are not detected by the assay are "false negatives." Subjects who are not sick and test negative in the assay are called "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, where the "false positive" rate is defined as the proportion of individuals who are not sick but test positive. Specificity can also be referred to as the true negative rate.

[0086] The (one or more) biomarker proteins 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, immunoassays include, but are not limited to, competitive and non-competitive assay systems using techniques such as protein immunoblotting (western blots), radioimmunoassays, ELISAs, "sandwich" immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, and fluorescent immunoassays. Such assays are conventional and well known in the art. Exemplary immunoassays will be briefly described below (but are not intended to be limiting).

[0087] Immunoprecipitation protocols generally include lysing a cell population in a lysis buffer supplemented with protein phosphatase and / or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), 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% tricalcium alcohol), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and / or protein G agarose beads to the cell lysate, incubating for about an hour or longer at 4° C., washing the beads in lysis buffer, and resuspending the beads in SDS / sample buffer. The ability of the antibody to immunoprecipitate a specific antigen can be assessed by, for example, Western blot analysis. Those skilled in the art are familiar with parameters that can be modified to increase antibody binding to the antigen and reduce background (e.g., pre-clearing the cell lysate with agarose beads).

[0088] Western blot analysis generally includes preparing a protein sample in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), electrophoresing the protein sample, transferring the protein sample from the polyacrylamide gel to a membrane, such as nitrocellulose, PVDF, or nylon, blocking the membrane in a blocking solution (e.g., PBS or non-fat milk with 3% BSA), washing the membrane in a wash buffer (e.g., PBS-Tween 20), blocking the membrane with a primary antibody (antibody of interest) diluted in a blocking buffer, washing the membrane in a wash buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody), conjugated to an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (e.g., 32P or 125I) diluted in a blocking buffer, washing the membrane in a wash buffer, and detecting the presence of the antigen. Those skilled in the art are familiar with parameters that can be modified to increase the detection signal and reduce the background.

[0089] ELISA generally includes preparing an antigen (i.e., a biomarker protein of interest or a fragment of a biomarker protein), coating the wells of a "96-well" microtiter plate with the antigen, adding an antibody of interest conjugated to a detectable compound such as an enzyme substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the wells, incubating for a period of time, and detecting whether the antigen is present. In ELISA, the antibody of interest does not have to be conjugated to the detectable compound; instead, a second antibody (the second antibody recognizes the antibody of interest) conjugated to the detectable compound can be added to the wells. In addition, antibodies can be coated into the wells instead of coating the wells with antigen. In this case, after the antigen of interest is added to the coated wells, a second antibody conjugated to the detectable compound can be added. Those skilled in the art are familiar with the parameters that can be modified to increase the detection signal and other variations of ELISA known in the art.

[0090] Because multiple biomarkers are used, a threshold can be determined based on the combined concentrations of these biomarkers. This threshold determines whether a patient is classified as successfully treated or unsuccessfully treated. The present invention reflects the insight that successful responses to periodontitis treatment can be distinguished from unsuccessful responses to periodontitis treatment with sufficient accuracy based on the measurement of a combination of biomarkers as described above.

[0091] This insight supports another aspect of the present invention, namely the use of the protein combination of the present invention as a biomarker in a saliva sample of a human patient for assessing whether a periodontitis treatment for the patient is successful or will be successful. For the avoidance of doubt, the protein combination of the present invention is:

[0092] Hemoglobin subunit delta (Hb-delta) and pyruvate kinase (PK); or

[0093] at least two of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8), and keratin-4 (K-4); or

[0094] α-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8).

[0095] Such use may be achieved in a method substantially as described above and below.

[0096] The method of the present invention comprises determining at least one test value reflecting the measured combined concentration of the proteins. The combined concentration value can be any value obtained by inputting the determined concentrations and performing an arithmetic operation on these values. The combined concentration value can be, for example, a simple addition of the concentrations. The combined concentration value can also involve multiplying each concentration by a factor reflecting the desired weight of the concentration and then adding the results. The combined concentration value can also involve multiplying the concentrations by one another, or any combination of multiplication, division, subtraction, exponentiation, and addition. The combined concentration value can also involve raising the concentrations to several powers. Alternatively, the test value reflects a combination of the determined combined concentrations of the proteins and the age of the subject.

[0097] The resulting combined concentration value can be compared to one or more threshold values ​​that reflect, in the same manner, combined concentrations associated with successful treatment of periodontitis. This comparison allows an assessment of whether the test value indicates the presence of successful treatment in the patient whose saliva was tested.

[0098] The threshold value can be, for example, a combined concentration value that is based on the concentration of the same protein determined in a reference sample associated with successful treatment of periodontitis (e.g., in a patient previously diagnosed with periodontitis and who has been treated to reduce the severity of the disease), obtained in the same manner. Typically, therefore, a value reflecting the same or higher combined concentration is indicative of successful treatment of periodontitis in the patient being tested. Similarly, a value reflecting a lower combined concentration in the saliva of a patient with periodontitis being tested is indicative of unsuccessful treatment of periodontitis. However, it will be appreciated that the threshold value can also be calculated (e.g., by using a negative multiplier) such that a test value indicative of successful treatment is below the threshold value, and a test value indicative of unsuccessful treatment is above the threshold value.

[0099] Threshold values ​​can also be determined based on measuring the concentration of the biomarker protein present in a set of samples, the samples including patients known to have been successfully treated and patients known to have been unsuccessfully treated. Thus, the measured concentration values ​​can be statistically analyzed, possibly including machine learning methods, to allow patients classified as successfully treated or unsuccessfully treated to be distinguished with a desired sensitivity and specificity. Thus, the desired threshold value or thresholds can be obtained. Based on the threshold value, the sample to be tested can undergo the same concentration measurement, and then the concentration values ​​can be processed in the same manner as the one or more threshold values ​​are obtained to determine a joint concentration value that can be compared with the threshold value, thereby allowing the test sample to be classified as "yes" or "no" to successful treatment.

[0100] In one interesting embodiment, the combined concentration value is obtained in the form of a score as follows. A numerical value (protein concentration value, e.g., ng / ml) is assigned to each measurement, and these values ​​are used in linear or nonlinear combination to calculate a score between 0 and 1. In cases where the threshold is determined based on a group of subjects as described above, the score between 0 and 1 is typically calculated using a sigmoid function that takes the combined concentration as input (as further shown).

[0101] When the score exceeds a certain threshold, the method indicates successful treatment of periodontitis. The threshold can be selected based on the desired sensitivity and specificity.

[0102] The clinical definition as generally accepted in the art is based on the following:

[0103] Gingival Index (GI)

[0104] The full mouth gingival index will be recorded based on the Lobene Modified Gingival Index (MGI) rated on a scale of 0-4, where:

[0105] 0 = no inflammation,

[0106] 1 = Mild inflammation; slight change in color of any part of the marginal or papillary gingival unit but not the entire marginal or papillary gingival unit, with little or no change in texture.

[0107] 2 = Mild inflammation; but involving the entire marginal or papillary unit,

[0108] 3 = moderate inflammation; pigmentation, redness, edema, and / or hypertrophy of the margin or papillary units,

[0109] 4 = Severe inflammation; marked erythema, edema, and / or hypertrophy of the marginal or papillary gingival units, spontaneous bleeding, hyperemia, or ulceration.

[0110] Probing depth (PD)

[0111] Using a manual UNC-15 periodontal probe, the probing depth was recorded to the nearest mm. The probing depth was the distance from the probe tip (assumed to be at the base of the pocket) to the free gingival margin.

[0112] Gingival recession (REC)

[0113] Gingival recession was recorded to the nearest millimeter using a manual UNC-15 periodontal probe. Gingival recession was measured from the free gingival margin to the cemento-enamel junction. Gingival recession was indicated by positive numbers, and gingival overgrowth was indicated by negative numbers.

[0114] Clinical attachment loss (CAL)

[0115] Clinical attachment loss was calculated as the sum of probing depth + depression at each site.

[0116] Bleeding on probing (BOP)

[0117] After probing, each site was assessed for bleeding on probing, with the site assigned a score of 1 if bleeding occurred within 30 seconds of probing and 0 otherwise.

[0118] The resulting subject groups (patient groups) were defined as follows:

[0119] Healthy group (H): PD ≤ 3 mm in all sites (but up to four 4 mm pockets distal to the last upright molars were allowed), no sites with interproximal attachment loss, GI ≥ 2.0 in ≤ 10% of sites, % BOP score ≤ 10%;

[0120] Gingivitis group (G): GI ≥ 3.0 in >30% of sites, no sites with interproximal attachment loss, no sites with PD > 4 mm, % BOP score > 10%;

[0121] Moderate periodontitis group (MP): ≥8 teeth with 5mm-7mm interproximal PD (equivalent to approximately 2mm-4mm CAL), %BOP score >30%;

[0122] Advanced periodontitis group (AP): ≥12 teeth with interproximal PD ≥7 mm (equivalent to approximately ≥5 mm CAL), %BOP score >30%.

[0123] In one embodiment, the method of the present invention utilizes Figure 1 The system schematically shown in FIG. The system may be a single device with various device components (units) integrated therein. The system may also have various components as independent devices, or some of these components. Figure 1 The components shown are a measuring device (A), a graphical user interface (B) and a computer processing unit (C).

[0124] As mentioned above, the system of the present invention includes a data connection to the interface, whereby the interface itself can be part of the system or can be a remote interface. The latter refers to the possibility of using a different device to provide the actual interface, preferably a handheld device such as a smartphone or tablet. In this case, the data connection will preferably involve wireless data transmission, such as via Wi-Fi or Bluetooth, or through other technologies or standards.

[0125] The measuring device (A) is configured to receive a saliva sample, for example by placing a drop of saliva on a cartridge (A1), which can be inserted into the apparatus (A). The device can be an existing device capable of determining the concentration of at least a combination of biomarker proteins of the present invention from the same saliva sample, i.e.:

[0126] Hemoglobin subunit delta (Hb-delta) and pyruvate kinase (PK); or

[0127] at least two of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8), and keratin-4 (K-4); or

[0128] α-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8).

[0129] The measuring device (A) should be capable of receiving a saliva sample, for example, by placing a drop of saliva on a cartridge (A1), which can be inserted into the device (A). The device can be an existing device capable of determining the concentration of at least two protein biomarkers of the present invention (e.g., HB-δ and PK, or A1AGP, PK, and S100A8) from the same saliva sample.

[0130] The processing unit (C) receives the numerical value of the protein concentration from component (A). The unit (C) is provided with software (typically embedded software) to allow it to calculate a score (S) between 0 and 1. The software also includes the numerical value of a threshold value (T). If the calculated value (S) exceeds (T), the unit (C) will output an indication (I) to the GUI (B) that "the treatment of periodontitis was successful", otherwise the unit (C) outputs "the treatment of periodontitis was unsuccessful". Another embodiment may use a specific value of (S) to indicate the certainty of making the indication (I). This may be "direct" in the sense that, for example, a score S = 0.8 indicates that the certainty of "successful treatment of periodontitis" is 80%. Otherwise, this may be achieved, for example, by limiting the range of R1-R2 so that when R1 < S < R2, the indication (I) displays "uncertain".

[0131] For example, a specific calculation of scores can be achieved through logistic regression using the sigmoid function:

[0132]

[0133] Where N is the number of proteins / biomarkers used, c0, c1, etc. are coefficients (values), and B1, B2, etc. are the concentrations of the respective proteins.

[0134] Coefficient C i This can be determined through a training program:

[0135] N1 subjects who had a successful response to periodontitis treatment (as determined by the dentist via current standards) and N2 subjects who had an unsuccessful response to periodontitis treatment were selected.

[0136] A saliva sample is obtained from each subject and the protein concentration of the biomarker panel described above is determined (the saliva sample can be from patients who have not yet received treatment [to predict response], or patients who have already received treatment [to assess response]).

[0137] The score S of a successful response is defined as 1, and the score S of an unsuccessful response is defined as 0.

[0138] A sigmoid function was fitted to the score versus protein concentration values.

[0139] Note that alternatively, any existing regression or machine learning method (e.g., linear regression, neural networks, support vector machines, etc.) can be used, where patients who successfully respond to treatment have a higher score S and patients who do not successfully respond to treatment have a lower score S.

[0140] In particular, such procedures have been applied using clinical studies to subjects who have demonstrated a successful or unsuccessful response to periodontitis treatment, with successful responses identified by a dental professional through clinical assessment. Here, patients with a post-treatment bleeding on probing percentage of 30 or less (BOP ≤ 30%) are considered high responders to treatment / having a successful treatment outcome. Patients with a post-treatment BOP > 30% are considered low responders / having an unsuccessful treatment response.

[0141] As used herein, "successful treatment" generally means a bleeding on probing percentage of 30 or less (BOP ≤ 30%) following treatment of periodontitis.

[0142] A multi-class classifier that indicates a "success" status may be used.

[0143] The performance of various biomarker combinations was evaluated by leave-one-out cross validation to obtain the preferred biomarker combinations of the present invention.

[0144] With reference to the above system, in another aspect, the present invention also provides a system for assessing whether a human patient has been or will be successfully treated for periodontitis, the system comprising:

[0145] - a detection device capable of and suitable for detecting the following proteins in a saliva sample of a human patient:

[0146] Hemoglobin subunit delta (Hb-delta) and pyruvate kinase (PK); or

[0147] at least two of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8), and keratin-4 (K-4); or

[0148] α-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8).

[0149] As mentioned above, such devices are known and readily available to the skilled person. Typically, there is provided:

[0150] - a container for receiving an oral sample from a subject, wherein a detection device is disposed in the container;

[0151] - a processor capable and adapted to determine an indication of a successful response to a periodontitis treatment based on the determined concentration of said protein.

[0152] Optionally, the system includes a user interface (or a data connection to a remote interface) capable of presenting information, particularly a graphical user interface (GUI); a GUI is a user interface that allows a user to interact with an electronic device through graphical icons and visual indicators such as auxiliary symbols, rather than a text-based user interface, typed instruction labels or text navigation (any such interface is not excluded by the present invention); GUIs are generally well known and are typically used in handheld mobile devices such as MP3 players, portable media players, gaming devices, smart phones and smaller home, office and industrial control devices; as mentioned above, the interface can also optionally be selected to enable input of information such as the subject's age, gender, BMI (body mass index).

[0153] The present invention also provides, alone or as part of the above system, a kit for detecting at least two periodontal disease biomarkers in a saliva sample of a human patient, the kit comprising one or more detection agents for detecting:

[0154] Hemoglobin subunit delta (Hb-delta) and pyruvate kinase (PK); or

[0155] at least two of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8), and keratin-4 (K-4); or

[0156] α-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8).

[0157] Typically, the kit includes two or three detection agents, each for a different biomarker. More typically, the detection agents in the kit include or consist of the following detection agents:

[0158] a first detection agent for detecting hemoglobin subunit delta (Hb-δ), and a second detection agent for detecting pyruvate kinase (PK),

[0159] a first detection agent for detecting keratin-4 (K-4), a second detection agent for detecting alpha-1-acid glycoprotein (A1AGP), and a third detection agent for detecting pyruvate kinase (PK); or

[0160] a first detection agent for detecting keratin-4 (K-4), a second detection agent for detecting alpha-1-acid glycoprotein (A1AGP), and a third detection agent for detecting matrix metalloproteinase-8 (MMP-8); or

[0161] a first detection agent for detecting keratin-4 (K-4), a second detection agent for detecting pyruvate kinase (PK), and a third detection agent for detecting matrix metalloproteinase-8 (MMP-8); or

[0162] a first detection agent for detecting keratin-4 (K-4), a second detection agent for detecting alpha-1-acid glycoprotein (A1AGP), a third detection agent for detecting pyruvate kinase (PK), and a fourth detection agent for detecting matrix metalloproteinase-8 (MMP-8); or

[0163] A first detection agent for detecting alpha-1-acid glycoprotein (A1AGP), a second detection agent for detecting pyruvate kinase (PK), and a third detection agent for detecting S100 calcium binding protein A8 (S100A8).

[0164] As discussed above with respect to the methods of the invention, the kit may include more detection agents, such as detection agents for other proteins. In a preferred embodiment, as mentioned, the detection agents available in the kit consist of detection agents for detecting the three proteins constituting the biomarker panel of the invention.

[0165] Preferably, the kit comprises a solid support, such as a chip, microtiter plate or beads or resin containing the detection agent. In some embodiments, the kit comprises a mass spectrometry probe, such as a ProteinChip TM .

[0166] The kit may also provide a specific wash solution for unbound detection agent or biomarker and / or detection agent (sandwich-type assays).

[0167] In one interesting aspect, the identification of biomarker panels according to the present invention is applied to monitoring the status of a human patient with periodontal disease during a period of treatment. Thus, the present invention also provides an in vitro method for determining a change in the periodontal disease status of a human patient with periodontitis due to treatment of the disease during a treatment interval from a first time point t1 to a second time point t2, the method comprising detecting the concentration of the following proteins in at least one saliva sample obtained from the patient at t1 and at least one saliva sample obtained from the patient at t2:

[0168] Hemoglobin subunit delta (Hb-delta) and pyruvate kinase (PK); or

[0169] at least two of alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8), and keratin-4 (K-4); or

[0170] alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8);

[0171] And compare the concentrations, whereby preferably a difference of one, two, three or four concentrations reflects a change in state. Such a difference may be referred to as a concentration difference, thereby allowing a direct comparison without first generating a number between 0 and 1, or any other classification. It should be understood that the measurements received at two time points may also be processed in the same manner as when determining the patient's state as described above.

[0172] The present invention also provides a method for diagnosing whether a human patient has been successfully or will be successfully treated for periodontal disease, comprising detecting the presence of a protein of the present invention in the patient's saliva. The presence of a successful therapy in the patient's body is assessed based on the concentration of the protein in the sample. Optionally, the method of this aspect includes a further step of treating the patient's periodontal disease. The optional treatment step may include applying a known therapeutic agent or dental procedure, or a combination of a therapeutic agent and a dental procedure. Known therapeutic agents include administering antimicrobial-containing agents, such as mouthwashes, chips, gels, or microspheres. A typical antimicrobial agent for treating gingivitis and periodontitis is chlorhexidine. Other therapeutic agents include antibiotics, typically oral antibiotics, and enzyme inhibitors, such as doxycycline. Known non-surgical treatment procedures include scaling and root planing (SRP). Known surgical treatments include surgical pocket reduction, flap surgery, gum grafting, or bone grafting.

[0173] The present invention also provides a method for detecting the protein of the present invention in a patient, comprising:

[0174] (a) obtaining a saliva sample from a human patient; and

[0175] (b) detecting the presence of the protein of the present invention in the saliva sample by contacting the saliva sample with a detection agent for the protein and detecting the binding between each protein and the detection agent.

[0176] The invention will be further illustrated with reference to the following non-limiting examples.

[0177] Example

[0178] In a clinical study, 71 subjects underwent repeated clinic visits at two independent clinical sites for periodontitis treatment, including:

[0179] 17 had low / unsuccessful responses

[0180] 54 people had high / successful responses

[0181] We obtained an area under the receiver-operator-characteristic curve value of >0.75 to detect successful treatment outcomes.

[0182] In statistics, a receiver operating characteristic (ROC) curve is a graph of the performance of a binary classifier system as its discrimination threshold is varied. 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 detection sensitivity, recall, or probability in machine learning. The false positive rate is also known as the probability of false detection or false alarm and can be calculated as (1-specificity). Therefore, the ROC curve is sensitivity as a function of false detection. Generally, if the probability distributions for both detection and false alarm are known, the ROC curve can be generated by plotting the value of the cumulative distribution function of the probability of detection (the area under the probability distribution from negative infinity to the discrimination threshold) on the y-axis and the value of the cumulative distribution function of the probability of false alarm on the x-axis for each value of the threshold. The accuracy of a test depends on the degree to which the test divides the group being tested into those with or without the disease in question. Accuracy is measured by the area under the ROC curve. An area of ​​1 indicates a perfect test; an area of ​​0.5 indicates a worthless test. The guide for classifying the accuracy of diagnostic tests is the traditional academic point system:

[0183] ——0.90-1=Excellent (A)

[0184] 0.80-0.90 = Good (B)

[0185] ——0.70-0.80=Common (C)

[0186] ——0.60-0.70=Poor (D)

[0187] ——0.50-0.60=Failure (F)

[0188] Various biomarker combinations were evaluated by logistic regression and leave-one-out cross validation (LOOCV) to obtain the biomarker combination of the present invention. It can be seen that the protein biomarker combination of the present invention has an AUC>0.75 to detect successful treatment results.

[0189] Based on the above, in the results of the above clinical studies, it is believed that a ROC AUC value greater than 0.75 represents an ideal accuracy for providing the test according to the present invention.

[0190] The protein biomarkers studied were:

[0191] MMP8

[0192] MMP9

[0193] IL-1β

[0194] HGF

[0195] Free light chain (FLC) kappa

[0196] Free light chain (FLC) λ

[0197] A1AGP

[0198] Hb-β

[0199] Hb-δ

[0200] Keratin 4

[0201] Inhibitory proteins

[0202] Pyruvate kinase

[0203] S100A8

[0204] S100A9

[0205] Furthermore, we considered them as additional predictors κ+λ, κ-λ, and κ / λ in the employed logistic regression.

[0206] Additionally, we included age as a predictor.

[0207] This yields a total of 4204 possible non-redundant groups with up to 4 protein biomarkers (excluding groups with only age). Non-redundant here means not considering groups that include, for example, κ+λ and κ-λ as predictors, as in logistic regression, non-redundant gives the same results as the corresponding group that includes κ and λ as predictors.

[0208] Note that, given the above predictors, not restricting the number of protein markers in a group yields 98,302 possible non-redundant groups (excluding groups with age only).

[0209] For assessing treatment response based on post-treatment marker levels, the AUC LOOCV performance of ˜0.75 was found for 5 groups, which are given in the table below, where each biomarker group is a preferred embodiment of the present invention:

[0210]

[0211] For prediction of treatment success based on pre-treatment marker levels, the following 16 groups were found to provide performance with AUCLOOCV > 0.75:

[0212]

[0213] Each of these groups is a preferred biomarker combination according to the present invention.

[0214] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments.

[0215] For example, different units may contain detectors for different biomarkers. Alternatively, conveniently, the kit of the invention may include a fixed set of detectors for the protein biomarker required in the embodiments, i.e., K-4 in certain embodiments, and a flexible module comprising detectors for other biomarkers, such as A1AGP and / or PK.

[0216] In the process of practicing the claimed invention, those skilled in the art will be able to understand and implement other variations of the disclosed embodiments by studying the drawings, the disclosure and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The fact that certain features of the invention are recited in mutually different dependent claims does not indicate that a combination of these features cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0217] In summary, we disclose here an in vitro method for assessing or predicting a human patient's response to a treatment for periodontal disease. The method is based on the insight of determining a selection of as few as two biomarker proteins. Thus, in a saliva sample from a patient, the concentrations of the proteins described herein are measured. Based on the measured concentrations, a value reflecting the combined concentrations of the proteins is determined. This value is typically used to calculate the probability of successful treatment and is compared to a corresponding threshold value that, in the same manner, reflects the combined concentrations associated with successful treatment of periodontitis. This comparison allows an assessment of whether the test value is indicative of successful treatment of the patient's periodontitis.

Claims

1. A system for assessing or predicting a human patient's response to treatment for a periodontal disease, the periodontal disease being periodontitis, the system comprising: - a measuring device configured to detect the concentration of the following proteins in a saliva sample from said human patient suffering from periodontal disease: (i) hemoglobin subunit delta (Hb-δ) and pyruvate kinase (PK); or (ii) keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), and matrix metalloproteinase-8 (MMP-8); or (iii) keratin-4 (K-4), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8); or (iv) keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8); or (v) α-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8); as well as The processing unit is configured to: - determining at least one test value reflecting the combined concentration determined for said proteins; and - comparing the test value to a threshold value reflecting in the same manner the combined concentration associated with successful treatment of periodontal disease, thereby assessing whether the test value indicates successful treatment of periodontal disease in the patient.

2. The system of claim 1, wherein the system is further configured to evaluate the response of a human patient who has been previously diagnosed with periodontitis and has received treatment for the periodontitis.

3. The system of claim 1 or claim 2, wherein the concentrations of the hemoglobin subunit delta (Hb-delta) and the pyruvate kinase (PK) protein are detected.

4. The system of claim 1 , wherein the system is further configured to predict a human patient's response to a periodontitis treatment, wherein the treatment is proposed or applied no more than 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to the assessment.

5. The system of claim 1 or claim 4, wherein the concentrations of the alpha-1-acid glycoprotein (A1AGP), the pyruvate kinase (PK), and the S100 calcium binding protein A8 (S100A8) proteins are detected.

6. The system of claim 1, 2 or 4, wherein the age of the subject is determined and the test value combined with the age of the subject reflects the combined concentration determined for the protein.

7. The system of claim 1, 2, or 4, wherein the threshold is based on one or more concentrations determined for the protein in one or more reference samples, each sample being associated with successful treatment of periodontitis or unsuccessful treatment of periodontitis.

8. The system of claim 1 , wherein the concentration of the following protein is detected: (i) hemoglobin subunit delta (Hb-δ) and pyruvate kinase (PK); or (ii) alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium binding protein A8 (S100A8); or (iii) keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), and matrix metalloproteinase-8 (MMP-8); or (iv) keratin-4 (K-4), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8); or (v) Keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8).

9. The system of claim 1, 2, 4 or 8, wherein the determined concentration value is arithmetically processed into a number between 0 and 1.

10. A system for assessing or predicting a human patient's response to treatment for periodontal disease, wherein the periodontal disease is periodontitis, the system comprising: - a detection device capable of and suitable for detecting the following proteins in a saliva sample of said human patient: (i) hemoglobin subunit delta (Hb-δ) and pyruvate kinase (PK); or (ii) keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), and matrix metalloproteinase-8 (MMP-8); or (iii) keratin-4 (K-4), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8); or (iv) keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8); or (v) α-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8); - a processor capable and adapted to determine an indication of whether the patient's periodontal disease has been or will be successfully treated based on the determined concentration of the protein.

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

12. The system according to claim 10 or claim 11, further comprising: - a user interface for presenting said indication to a user; and - a data connection between the processor and the user interface for transmitting the indication from the processor to the user interface.

13. A system according to claim 10 or claim 11, wherein the processor is activated by means of an Internet based application.

14. The system of claim 12, wherein the user interface is capable of inputting information regarding the subject's age, and the processor is capable and adapted to determine an indication that the patient has been or will be successfully treated based on the determined concentration of the protein.

15. A kit for detecting at least two biomarkers for successful treatment of a periodontal disease in a saliva sample of a human patient, wherein the periodontal disease is periodontitis, the kit comprising one or more detection agents for detecting the following proteins: (i) hemoglobin subunit delta (Hb-δ) and pyruvate kinase (PK); or (ii) keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), and matrix metalloproteinase-8 (MMP-8); or (iii) keratin-4 (K-4), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8); or (iv) keratin-4 (K-4), alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and matrix metalloproteinase-8 (MMP-8); or (v) alpha-1-acid glycoprotein (A1AGP), pyruvate kinase (PK), and S100 calcium-binding protein A8 (S100A8), The concentration of the detected protein in the saliva sample is used to assess whether the human patient has been or will be successfully treated for periodontal disease.

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

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