Klotho detection

Abstract

The present invention relates to an in vitro method for determining and / or monitoring the health status of an individual by measuring the content of Klotho in saliva or tears. The test kit for performing such an assay is an immunoassay test kit, particularly adapted to perform a double antibody sandwich assay or a competitive assay.

Description

[Technical Field]

[0001] The present invention relates to diagnostic methods for determining Klotho protein levels in an individual, and test kits for carrying out such diagnostic methods.

[0002] Klotho was discovered by Makoto Kuroo in 1997, and its deficiency in mice has been shown to result in the premature appearance of several pathological phenotypes. Klotho-deficient mice exhibited a syndrome similar to human aging, including a shortened lifespan. Klotho has also been shown to be involved in the suppression of several aging phenotypes. Deficiency of Klotho gene expression in mice results in infertility, arteriosclerosis / vascular calcification, skin atrophy, osteoporosis, emphysema, acute and chronic kidney disease, renal fibrosis, diabetes, and cancer (Kuro-o, M. et al., (1997), Nature 390, 45-51). On the other hand, overexpression of Klotho has been shown to extend the lifespan of mice (Kurosu, H. et al., Science 2005, 309, p. 1829). Klotho protein is most abundant in the kidney, brain, and pituitary gland, with lower levels present in skeletal muscle, bladder, ovaries, and testes (Avin, KG et al., (2014), Frontiers in Physiology 5, 189).

[0003] WO2016 / 135295 discloses genetically modified mesenchymal cells expressing Klotho and includes further information about the protein and its association with various diseases, which information is reproduced below to highlight the importance of the protein to human health and well-being.

[0004] The human Klotho gene encodes a type 1 transmembrane protein consisting of 1012 amino acids, but alternative splicing allows the gene to be expressed in a secreted form. Thus, Klotho exists in two forms: membrane-type Klotho and secreted Klotho. Membrane-type Klotho functions as a receptor for hormones that regulate renal phosphate excretion and the synthesis of active vitamin D. On the other hand, secreted Klotho is a humoral factor with pleiotropic activities, including suppression of growth factor signaling, inhibition of oxidative stress, and regulation of ion channels and transporters.

[0005] Fibroblast growth factor 23 (FGF23), a member of the fibroblast growth factor (FGF) family, has been found to be elevated in patients with autosomal dominant hypophosphatemic rickets (ADHR). FGF23 functions as a counter-regulatory hormone for phosphorylated hormones and vitamin D (calcitriol) in a Klotho-dependent manner. Hyperphosphatemia leads to vascular narrowing, myocardial infarction, stroke, and a significant shortening of lifespan in patients with chronic kidney disease (CKD). Lack of FGF-1 signaling in the kidney leads to elevated serum phosphate levels. Secreted and membrane-bound forms of Klotho have been found to form complexes with the FGF-15 receptor, thereby increasing FGF23-dependent signaling (Kurosu et al., 2012). Klotho's function as a cofactor for FGF23 signaling is important for regulating serum phosphate levels.

[0006] Additionally, Klotho functions by regulating various signaling pathways, including insulin-like growth factor 1 (IGF-1). One of Klotho's actions is to enhance cellular resistance to oxidative stress, which is involved in many different pathological processes. Through modulation of cellular responses to oxidative stress, Klotho has been shown to be protective in neurodegenerative diseases such as Alzheimer's disease (Zeldich, E. et al., J Biol Chem 2014, 289(35):24700) and diabetes. Klotho has also been suggested to be an inhibitor of collagen synthesis and, therefore, may be beneficial in fibrotic conditions (Ghosh, A.K. et al., Exp Biol Med 2013, 238(5):461). Expression of Klotho has been shown to silence several types of cancer cells, and is associated with the promotion of cell proliferation and the formation of cancer metastasis (Camili et al., Pigment Cell & Melanoma Res 2011, 24(1), p 75; Wang et al., Am J Cancer Res 2011, 1(1):111, Lee et al., Molecular Cancer 2010, 9:109). Meanwhile, overexpression of Klotho in cancer cells can suppress cell proliferation and promote apoptosis of cancer cells (Chen. B. et al., J of Exp and Clin Cancer Res 2010, 29:99). Furthermore, upregulation of Klotho, indirectly stimulated by administration of renin-angiotensin system inhibitors or their compounds, leads to the suppression of renal fibrosis (Ming Chang Hu et al., Contrib Nephrol 2013, 180:47), and diabetic rat models appear to have naturally low Klotho expression (Meng Fu Cheng et al., Journal of Biomedicine and Biotechnology. 2010, 513853).

[0007] Below we summarize further studies on various specific diseases in which altered Klotho levels are observed.

[0008] Background on Chronic Kidney Disease (CKD) CKD is a global health problem, affecting more than 26 million Americans. Renal Klotho RNA is reduced in CKD patients. This clinical observation has been confirmed in numerous preclinical models, where unilateral nephrectomy and contralateral ischemia-reperfusion injury have been shown to downregulate renal Klotho protein and mRNA expression. A similar decrease in Klotho expression has also been demonstrated in a chronic glomerulonephritis model. Overexpression of Klotho improved renal function and renal histology in this model (Hu, M. C., et al., (2011), Journal of the American Society of Nephrology: 22, 124-136; Haruna, Y., et al., (2007) PNAS, 104, 2331-2336). Coronary artery calcification is highly prevalent in patients with CKD, contributing to increased cardiovascular morbidity and mortality. The Klotho-FGF23 axis plays an important role in vascular calcification (Stompor. T. (2014) World Journal of Cardiology 6, 115-129). It is well known that chronic kidney disease (CKD) is associated with increased cardiovascular morbidity and mortality. A study of 1,120,295 adults in the San Francisco Bay Area found a strong correlation between renal function (estimated glomerular filtration rate, GFR) and cardiovascular events (Go, AS et al., New England Journal of Medicine, 2004; 351: 1296-1305).

[0009] In the Renal Research Institute (RRI)-CKD study, which enrolled 834 adults with moderate to severe CKD (stages 3-5) between June 2000 and February 2006, the authors found that heart rate variability predicted clinical outcomes and cardiovascular disease (CVD) (Chandra, R. et al., (2012) European Dialysis and Transplantation Association - European Renal Association Official Publication 27, 700-709). Therefore, chronic kidney disease (CKD) is a major risk factor for cardiovascular disease, leading to increased morbidity and shortened lifespan. Klotho expression is significantly reduced in the kidneys of CKD patients. Restoring Klotho expression by infusion of mesenchymal stem cell-derived Klotho (MSC-Klotho) may improve kidney function and, consequently, reduce the risk of cardiovascular death.

[0010] Background of cardiovascular disease Cardiovascular disease (CVD) is a widespread disease in the general population and the leading cause of death overall. Klotho has been proposed as an important regulator of CVD pathogenesis. A few clinical studies have observed a correlation between low levels of soluble Klotho and the occurrence and severity of CVD, while high levels are associated with reduced cardiovascular risk. Various polymorphisms in the human Klotho gene have also been associated with the incidence of cardiovascular events. Furthermore, several experimental studies have shown that this protein plays a role in maintaining vascular homeostasis (Yamamoto M. et al.). Klotho mediates anti-inflammatory and anti-aging effects, including ameliorating endothelial dysfunction by promoting NO production, suppressing adhesion molecule expression, inhibiting nuclear element κB, and inhibiting Wnt signaling. Klotho regulates the expression of endothelial NO synthase (eNOS). Six et al. recently observed that the attenuation of FGF23- and phosphate-induced vasoconstriction by Klotho was abolished by the addition of nitro-L-arginine, a competitive inhibitor of NOS. Furthermore, exposure of HUVECs to Klotho increased NO production and induced eNOS phosphorylation and iNOS expression. Interestingly, Klotho can increase H2O2 production in cultured human VSMCs, suggesting that this protein plays a more complex role in regulating vascular health through mediating the ROS / NO balance (Six I, et al. (2014), PLoS One. 2014; 9:e93423).

[0011] Furthermore, this protein is involved in the suppression of vascular calcification and the prevention of cardiac hypertrophy. Its expression in the vascular wall suggests new therapeutic scenarios for vascular disorders. Thus, Klotho protein is associated with CVD and plays a role in maintaining functional vascular integrity (Martin-20 Nunez.M. (2014) World J Cardiol.6(12):1262-1269).

[0012] Background of AD (Alzheimer's disease) Neurodegenerative diseases, particularly Alzheimer's disease (AD), are on the rise in Europe and the United States. According to the Alzheimer's Association, Alzheimer's disease is the sixth leading cause of death in the United States. Someone is diagnosed with Alzheimer's disease every 67 seconds. The cost of treating and caring for these patients is estimated to exceed US$1.1 trillion by 2050. AD is characterized not only by the loss of neurons and synapses, but also by the production of neurotoxic amyloid-β peptides (Aβ-plaques) and their deposition in neurofibrillary tangle formation. Increasing evidence indicates that amyloid deposition is a central feature of the disease. Activated astrocytes initiate an inflammatory response by producing cytokines that promote inflammatory responses, such as IL-6, IL-1, and TNF-α. All of this begins with an inappropriate response to oxidative stress, the accumulation of oxygen free radicals, hyperglycemia, and insulin resistance (Kosales-C'orral, S. et al., (2015) Oxidative medicine and cellular longevity, 985845).

[0013] Recently, it has been shown that the concentration of the anti-aging protein Klotho is significantly lower in the cerebrospinal fluid of patients with AD than in younger patients and elderly patients without AD (Semba, RD et al. (2014) Neuroscience Letters 558, 37-40).

[0014] In the brain, Klotho protein was localized in the choroid plexus and predominantly in the apical plasma membrane of ependymal cells. In the brains of kl- / - mice, a significant decrease in synapses was observed in the hippocampus, suggesting that Klotho functions as a humoral factor in the cerebrospinal fluid. In the kidney, Klotho protein was localized in the distal renal tubules (Li S A. et al., (2004) Cell Structure and Function 29, 91-99).

[0015] Chen et al. demonstrated that loss of Klotho expression resulted in cognitive impairment. They found significant effects of Klotho on oligodendrocyte function, including inducing maturation and myelination of primary rat oligodendrocyte precursor cells (OPCs) in vitro. Klotho promoted OPC maturation. In vivo studies of Klotho knockout mice and control littermates revealed that the knockout mice exhibited significantly reduced expression of key myelin proteins and genes. Immunohistochemistry revealed significantly reduced numbers of total and mature oligodendrocytes in Klotho knockout mice. Ultrastructural analysis showed that Klotho knockout mice exhibited severely impaired myelination of the optic nerve and corpus callosum (Chen, CD et al., (2013) The Journal of Neuroscience 33, 1927-1939).

[0016] Background on Multiple Sclerosis (MS) MS is a complex disease of the CNS characterized by a heterogeneous pathology consisting of both inflammatory and neurodegenerative components. The most common histopathological feature early in the disease is intermittent acute inflammation within patches of white matter, resulting in demyelination. Myelin is crucial for maintaining efficient axonal conduction, and oligodendrocytes, responsible for myelin production and axonal health within the CNS, are damaged or destroyed in MS patients. Endogenous oligodendrocyte progenitor cells (OPCs) are ubiquitously distributed within the human CNS and have been found to be found at high densities within some subacute lesions early in MS.

[0017] Progressive MS is the latest stage of the disease, characterized by gradual worsening of symptoms without remission. Severe neurological impairment dramatically reduces an individual's quality of life, primarily due to the expansion of cortical lesions affecting motor function. Pathologically, axonal degeneration and gray matter neuropathy are widespread. Widespread white and gray matter inflammation has been reported to be associated with widespread microglial activation, as well as the presence of T cells, B cells, and myelin-carrying macrophages. Furthermore, there is a global failure of OPCs to efficiently remyelinate damaged white and gray matter areas, dramatically reducing the chances of recovery (Chang, A. et al., 165-173). Klotho promotes the maturation of OPCs into mature oligodendrocytes (Chen, CD et al., (2013) The Journal of Neuroscience 33, 1927-1939).

[0018] Background of Parkinson's disease (PD) PD is a progressive neurodegenerative disease characterized clinically by resting tremor, bradycardia, cogwheel rigidity, and postural instability. Responsiveness to L-3,4-dihydroxyphenylalanine (L-DOPA) and brain imaging distinguish PD from other disorders. The pathological hallmark of PD is the loss of dopaminergic cells in the substantia nigra and pars compacta, followed by the loss of dopaminergic innervation in the striatum. Motor symptoms are the most obvious consequence of this nigrostriatal neurodegeneration. However, not only basal ganglia disease but also other parts of the central and autonomic nervous systems are affected. As a result, various non-motor symptoms impact patients' quality of life. It is generally believed that the neurodegenerative process in PD begins many years before clinical symptoms appear. The phenotypic overlap between familial and idiopathic PD is sufficient to identify commonly involved pathways. These pathways include mitochondrial dysfunction, oxidative stress, protein misfolding, protein degradation, protein aggregation, and inflammation.

[0019] Kosakai et al. found that the number of tyrosine hydroxylase-positive dopamine neurons in the substantia nigra, pars compacta, and ventral somitic region, as well as dopamine levels in the striatum, were significantly decreased in an age-dependent manner in Klotho-deficient mice. These phenotypic features were completely restored by vitamin D restriction, indicating that the abnormal increase in active vitamin D biosynthesis due to Klotho deficiency induces the degeneration of dopaminergic neurons (Kosakai, A. et al., (2011) Brain research 1382, 109-117).

[0020] Based on the contribution of Klotho to various signaling pathways and its important functions in many organ systems, as well as the observation of reduced levels of this protein in various disease conditions, novel therapies involving Klotho administration may represent a promising therapeutic approach. For example, WO2016 / 135295 provides mesenchymal stem cells that can be administered to patients to enable the treatment of not only the above-mentioned diseases but also other conditions in individuals who would benefit from the administration of Klotho.

[0021] In addition to the therapeutic potential of diseases in which reduced Klotho levels are known, the protein itself has also been considered a kind of marker for the health and well-being of an organism. While considering biological markers for specific diseases is common in the field of diagnostics, monitoring molecules that can indicate health, or at least well-being, in various key health aspects has been largely ignored. Fantuzzi (frontiers in IMMUNOLOGY, July 10, 2014, Volume 5, Article 351) focuses on the "sound of health," or indeed the silence of health, which has not previously resonated sufficiently. He reports that messages of distress have been favored over molecules considered to be markers of metabolic balance and the absence of distress in an organism. Fantuzzi identifies Klotho as one of the candidate health messengers, noting that optimal production, release, and biological activity of this molecule occur in healthy, distress-free states, whereas reduced Klotho levels have been observed under stress. Optimal levels of Klotho in a person, when measured during a regular health checkup, for example, could be an important indicator of good health.

[0022] While administration of Klotho-producing mesenchymal stem cells provides a therapeutic approach to protein expression, particularly for more severe conditions, preventative and therapeutic increases in Klotho levels in individuals can also be achieved through physical training in certain conditions. For example, Matsubara et al. (AM J Physiol Heart Circ Physiol. 306:H348-H355, 2014) reported increased plasma Klotho levels and reduced arterial stiffness in postmenopausal women after aerobic exercise training. Ji et al. (Experimental and Therapeutic Medicine 16:3511-3517, 2018) predicted positive effects of aerobic exercise and the associated promotion of Klotho expression on aging and aging-related diseases. Furthermore, Tang et al. (Journal of Circulating Biomarkers, Volume 7:1-7, 2018) observed increased serum Klotho levels in healthy volunteers after high-intensity physical exercise training. Even a single training session was found to induce the formation of Klotho. Finally, Avin et al. (Frontiers in Physiology, Volume 5, Article 189, June 2014) summarize previously published effects and impressively demonstrate the effect of exercise, particularly in skeletal muscle, on Klotho expression and the resulting increase in plasma Klotho levels.

[0023] These observations correlate the presence and plasma levels of Klotho with the life-prolonging effects of physical activity, especially in patients with certain diseases such as diabetes. Santos-Diaz et al. (Br J Sports Med 2016;0:1-2) showed that even a single exercise session significantly increased serum Klotho levels in both men and women. Mostafizi et al. (Nephro Urol Mon. 2016 Jan;8(1):e30245) reported that the plasma concentration of free Klotho was significantly higher in athletes compared with non-athletes, with no significant differences in other serum components between the two groups.

[0024] The observation that aerobic exercise, particularly strength training, increases serum Klotho levels is a very promising finding. These results suggest that physical training and fitness can significantly improve not only specific conditions but also general health. Furthermore, they suggest that increasing plasma Klotho levels can improve cognitive status or at least slow cognitive decline, especially in elderly individuals and those with neurodegenerative diseases. (Shardell et al., J Gerontol A Biol Sci Med Sci, 2015, 1-6; Cheng et al., Acta Neurobiol Exp 25, 2015, 75: 60-71)

[0025] There is no doubt that Klotho levels are an important indicator of health and disease, and raising low levels to optimal levels can contribute to an individual's longevity. However, currently, measuring an individual's Klotho level typically requires blood sampling at a medical institution and analysis in a clinical laboratory. Measuring Klotho levels is not only important for early diagnosis, but also requires monitoring during and after commonly used drug therapy or Klotho administration for disease treatment, or during health promotion efforts through exercise. In particular, the current state of the art is unsatisfactory for routine monitoring of Klotho levels. Therefore, an object of the present invention is to provide an improved means for measuring a person's Klotho levels in a cost-effective and easily applicable manner, which can be performed, at best, during or immediately after physical exercise to monitor Klotho levels. Summary of the Invention

[0026] In a first aspect, the present invention relates to a method for measuring and / or monitoring the health status of an individual by measuring the content of Klotho in a body fluid, wherein the body fluid is selected from saliva and tears.

[0027] In a second aspect, the present invention relates to a test kit, characterized in that it contains the reagents necessary to perform an immunoassay.

[0028] The present invention solves the above object by providing an improved method for the measurement of Klotho levels in a patient, and in particular provides methods and means in the form of a test kit for carrying out a diagnostic method, which test can be easily performed by an individual or patient at any time when such a measurement is deemed appropriate.

[0029] The method of the present invention is based on the surprising finding by the inventors that Klotho levels in particular in saliva are highly proportional to Klotho levels measured in serum samples, and therefore the method of the present invention is preferably carried out using saliva.

[0030] The purpose of the test of the present invention is to provide a painless, inexpensive, reliable, simple, and safe test suitable for molecular diagnostics compared to methods using serum or urine. The method of the present invention has been compared with HPLC-techniques and comparable results have been obtained. The results already obtained are surprising in that the Klotho levels in serum and saliva are significantly different between patients and healthy volunteers.

[0031] Although the actual values ​​in serum and saliva vary to some extent even within the same individual or group, we found that there is a reliable relationship between Klotho levels in serum and other body fluids, especially saliva (see Figures 1-5). This observation demonstrates that Klotho protein is present in saliva at significant and meaningful levels, which is not the case for many other proteins (Tekus et al., Acta Biol Hung, 2012; 63 (Suppl1):89-98; Ellis et al., NZ Med JK, 2012; 125(1353):47-58; Volkery et al., Vet Rec, 2012; 171(8):195; Holten-Anderson et al., Vet Rec, 2012; 171(8):195. 195; Holten-Anderson et al., Scand J Gastroenterol, 2012; 47(10):1234-41; Rahnama et al., Endokr Res, 2012 Aug 15 epub; Pant Pai et al., Lancet Infect Dis, 2012; 12(5):373-80; Mahboobi et al., J Oral Pathol Med, 2012; 41: 505-16; Yen Bee Ng et al. FEMS Immunol Med Microbiol, 2007; 25 49(2):252-60; Castagnola et al., Acta Otorinologica Italica, 2011; 31: 347-57).

[0032] Taking advantage of this observation, the present invention provides, for the first time, a reliable test method and test kit for monitoring an individual's health status. In certain embodiments of the present invention, this health status is considered to include existing or previous diseases or conditions correlated with low or reduced Klotho levels, or a predisposition to such diseases or conditions. Such diseases or conditions associated with relatively low Klotho levels have been described in some detail in the prior art and in the introduction of this specification. However, this does not exclude additional diseases in which reduced Klotho levels may only be recognized in the future. Therefore, detection of relatively low Klotho levels by the method of the present invention may indicate that a person's health status is unsatisfactory, even if specific disease symptoms have not yet been observed. Therefore, additional diagnostic methods can be applied to detect or rule out serious diseases.

[0033] In a preferred embodiment, the disease or condition associated with or causing a decrease in Klotho levels compared to healthy individuals is selected from at least one of cancer, inflammatory diseases, chronic kidney disease (CKD), neurodegenerative diseases, chronic heart disease, organ fibrosis, arteriosclerosis, dementia, diabetes, erectile dysfunction, autoimmune diseases or autoimmune-related diseases, sepsis, and also premature aging and other age-related diseases.

[0034] Neurodegenerative diseases and prodromal conditions for which low Klotho levels as measured by the methods of the present invention may be indicated include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease. In a further preferred embodiment, the human condition is type 1 diabetes, rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus, autoimmune encephalomyelitis, lupus nephritis, autoimmune nephritis such as IgA nephropathy, osteoarthritis, and osteoarthritis. or activate inflammasomes or their components in a patient. Other disorders autoimmune disease or autoimmune-related disease, including is influenced by.

[0035] The methods of the present invention can be applied either to detect the risk of the presence of such diseases or to monitor the progression of the disease and / or the effectiveness of medical treatment.

[0036] In particular, when the method of the present invention is used in connection with an existing or pre-existing disease, it is preferable to monitor and measure Klotho levels before, during, and / or after treatment. In this context, treatment includes administration of Klotho as well as administration of other therapeutic agents or a combination thereof. As described above, Klotho can be administered as a protein, or Klotho formation can be promoted, for example, by administering stem cells expressing Klotho. Increasing Klotho levels during treatment can significantly improve a person's health.

[0037] A low Klotho level in a healthy person may be a strong indication that the person is not sufficiently physically active. Such lack of exercise may lead to more or less serious health problems and premature aging if the person does not change their lifestyle. Therefore, a further preferred embodiment of the present invention is an in vitro method for measuring Klotho content to monitor the effect of physical activity on an individual's health status. As explained above, physical activity, particularly physical training, muscle training, and endurance training, has been shown to increase Klotho levels, which also reflects an individual's health status and, in particular, an increase in cognitive ability.

[0038] The in vitro method of the present invention can also be used to determine the biological age of an individual. Again, determining such biological age can help improve longevity by addressing possible health problems and unhealthy behaviors, and therefore taking necessary actions that will in turn increase and regulate Klotho levels.

[0039] The actual correlation between serum and saliva immunoassay values ​​can be easily measured and verified for any assay format. For example, the correlation between serum and saliva Klotho levels has been measured to be 3.6:1 when measured by TRF (time-resolved immunofluorescence assay) (Figure 1). Thus, calibration of this test provides a correlation between serum and saliva values ​​for healthy individuals.

[0040] A "normal" value is also the average value for a cohort of individuals whose Klotho levels are considered healthy or within a certain acceptable range of variation. This can be determined by providing test results for a cohort of appropriately similar individuals. The cohort can be selected from individuals of similar age, gender, living in similar circumstances, etc. Additionally, values ​​reported in the literature can be included to select a "normal" value for a particular situation.

[0041] The in vitro method of the present invention preferably involves at least one therapeutic treatment if the detected Klotho level is below a certain threshold, which may be derived, for example, as described above. The therapeutic treatment includes administering Klotho to the individual or increasing the Klotho level of such an individual, whose measured Klotho level corresponds to a serum value of less than 100 pg / ml, based on an appropriate relationship as described above. Preferably, if the Klotho level corresponds to a serum level of less than 200 pg / ml, most preferably less than 300 pg / ml, therapeutic treatment to increase the Klotho level should already be initiated. For this purpose, the Klotho level in saliva is measured to be reduced by approximately 3.6-fold from the value at which treatment is deemed desirable.

[0042] Preferably, administration of Klotho in the context of the present invention involves administering a therapeutically effective number of mesenchymal stem cells containing a Klotho-producing or Klotho protein-encoding region operably linked to a promoter or promoter / enhancer combination, wherein the cells containing the vector express Klotho. Inducing Klotho expression via Klotho-producing viral vectors or CRISPR-Cas9 gene editing to increase Klotho levels in an individual is also contemplated in the context of the present invention. In other embodiments, the Klotho protein is delivered or administered directly to a patient, wherein the Klotho protein is preferably recombinantly produced by biotechnological methods.

[0043] The amount of Klotho administered will depend on the actual levels measured in the individual or patient, and is preferably suitable to raise levels to 340 pg / ml or more when measured in serum, and correspondingly 85 pg / ml or more, preferably 100 pg / ml or more when measured in saliva.

[0044] In another preferred embodiment, instead of administering Klotho protein to a patient or inducing its expression in a patient by biotechnological methods as outlined above, the present invention also comprises subjecting the individual to increased physical activity. In particular, physical training, in particular muscle training, is indicated in such patients when the measured Klotho levels are below values ​​corresponding to "normal" levels measured in groups of healthy people, in particular values ​​as described above.

[0045] According to the present invention, Klotho levels are preferably measured via an immunoassay comprising at least one antibody or aptamer that specifically binds to Klotho. The method of the present invention preferably comprises measuring Klotho levels by a double-antibody sandwich assay or a competitive assay. Both assays are most preferably performed as lateral flow assays.

[0046] The specific design of the assay is not critical, and suitable test formats are available to those skilled in the art. A further subject of the present invention is a test kit for carrying out the method of the present invention, and therefore some specific preferred embodiments are described in more detail below. Such a test kit contains the necessary reactants for carrying out an immunoassay.

[0047] In principle, any immunoassay format can be applied to carry out the method of the present invention. In a preferred embodiment, the test is carried out as a double antibody sandwich assay or a competitive assay. The assay environment, including the solid phase, the antibody, and the label for use in such a method, are well known to those skilled in the art, and the applicable test format is not particularly limited. However, in a highly preferred embodiment of the present invention, the test format provided by the test kit of the present invention is suitable for a lateral flow assay format. This type of test and the corresponding test kit are described in more detail below.

[0048] Lateral flow assay As mentioned above, kits for carrying out the in vitro methods disclosed herein may be based on different principles. One preferred principle is known as a lateral flow immunochromatography assay. Such lateral flow immunochromatography assays can be easily carried out by patients / athletes without the assistance of a doctor or other medically trained personnel.

[0049] Lateral flow tests are simple devices intended to detect the presence (or absence) of a target analyte in a sample without requiring specialized and expensive equipment, but there are also many laboratory-based applications supported by a reader. These tests are typically used for medical diagnostics, either at home, as point-of-care (POC), or in a laboratory setting. A widespread and well-known application is the home pregnancy test.

[0050] This technology is based on a series of capillary beds, such as porous paper strips or sintered polymers. Each of these elements has the ability to spontaneously transport liquid (e.g., saliva). The first element (the sample pad) acts as a sponge, retaining excess sample liquid. Once immersed, the liquid migrates to the second element (the conjugate pad), where the manufacturer stores a so-called conjugate, preferably a dried format of bioactive particles (see below) in a salt-sugar matrix, containing everything necessary to ensure optimal chemical reaction between a control molecule (e.g., Klotho) and its chemical partner (e.g., an antibody) immobilized on the particle surface. The sample liquid dissolves the salt-sugar matrix and the particles simultaneously, and the sample and conjugate flow through the porous structure, mixing by conjugate transport. In this way, the analyte, bound to the particles, travels further through the third capillary bed. This material contains one or more regions (reaction or capture regions, often called stripes) where the manufacturer immobilizes a third molecule. By the time the sample and cadmium mixture reaches these stripes, the analyte is bound to the particles, and a third "capture" molecule binds the complex. After a while, as more liquid passes through the stripes, particles accumulate, causing the stripes to change color. There are usually at least two stripes: one (the control region) captures any particles, thereby indicating that the reaction conditions or technique were successful, and the second contains a specific capture molecule and captures only particles with immobilized analyte molecules. After passing through these reaction regions, the liquid enters a final porous wick (or waste reservoir), which simply serves as a waste container.

[0051] Such test designs can be tailored to the nature of the immunoassay to be performed using the test kit: lateral flow tests can be operated as competitive assays or sandwich assays.

[0052] In principle, any color particle can be used in the conjugate, but latex (blue) or nanometer-sized gold particles (red / black) are most commonly used. Gold particles derive their red color from localized surface plasmon resonance. Fluorescently or magnetically labeled particles can also be used, but these require the use of an electronic reader to evaluate the test results.

[0053] The sample first contacts a conjugate labeled with an antibody against the target analyte or colored particles in the reagent pad. The test line in the capture zone also contains an antibody against the same control, but may bind to a different epitope on the analyte. A positive sample will show the test line as a colored band. An example of a sandwich assay is the sandwich ELISA. While not strictly necessary, most test kits prefer to incorporate a second line containing an antibody that picks up free particles, such as latex or gold, to confirm that the test was performed correctly.

[0054] In a preferred embodiment, a single component of the lateral flow assay is adapted to indicate the presence of Klotho only if a certain threshold value or more of Klotho is present in the sample.

[0055] A preferred test kit of the present invention comprises the following components: 1. Apply the absorbent pad to the test sample (saliva, tears, urine) as a sample pad. 2. Conjugate or Reagent Pad - Contains antibodies specific for the target analyte Klotho, conjugated to labeled, preferably colored particles (usually colloidal gold particles, or latex microspheres). 3. Reaction or capture zone: Membrane - usually a hydrophobic nitrocellulose or cellulose acetate membrane on which anti-target analyte antibodies are immobilized and linearized across the membrane as a capture zone or test line (a control zone containing specific antibodies against the complexed antibodies may also be present). 4. Wick or waste area or reservoir - a further absorbent pad designed to draw and collect the sample across the reaction membrane by tubule action. The stripe components are typically fixed to an inert backing and may be provided in a simple dipstick format or may display capture and control regions in a plastic case with a sample port and reaction window. There are two preferred embodiments of the test kit (lateral flow immunoassay) used in the method of the present invention.

[0056] a. Double antibody sandwich assay In this method, the sample is transferred from the sample pad to the conjugate pad, where the control analyte, Klotho, binds to the conjugate. The sample then continues across the membrane until it reaches the capture zone, where the control / conjugate binds to the immobilized antibody and forms a visible line on the membrane. This control line indicates that the sample migrated across the membrane as intended. Two clear lines on the membrane indicate a positive result; a single line in the control zone indicates a negative result. The double-antibody sandwich assay is best suited for large analytes with multiple antigenic sites, such as bacterial pathogens and viruses. In this invention, an appropriate pair of antibodies is selected that bind to different epitopes on Klotho.

[0057] Such antibodies can be selected from commercially available products or can be produced by raising antibodies, particularly monoclonal antibodies, against different Klotho epitopes using methods known in the art. When a test method or kit suitable for carrying out such a method uses an antibody that specifically binds to Klotho, the term "antibody" does not only refer to antibodies artificially produced by immunization of laboratory animals, such as rabbits, sheep, or goats. In preferred embodiments, the term also includes monoclonal antibodies produced according to hybridoma technology. Furthermore, the term "antibody" also includes antigen-binding fragments of antibodies, such as recombinantly produced antigen-binding fragments. Such constructs can be produced, for example, by phage display technology and techniques derived therefrom.

[0058] b. Competitive assay Competitive assays are primarily used to test for small molecules. Unlike double-antibody sandwich assays, the conjugate pad contains an antibody already bound to the target analyte or its analog. If the target analyte is present in the sample, it will not bind to the conjugate and remain unlabeled. As the sample migrates along the membrane and reaches the capture zone, excess unlabeled analyte binds to the immobilized antibody, preventing capture of the conjugate and resulting in no visible light. Unbound conjugate binds to the antibody in the control zone, forming a visible control line. A single control line on the membrane indicates a positive result. Two visible lines in the capture and control zones indicate a negative result. However, if there is no excess unlabeled target analyte, a weak line may appear in the capture zone, indicating an inconclusive result. Competitive assays are ideal for testing small molecules, such as mycotoxins, that cannot simultaneously bind to multiple antibodies.

[0059] Considering these different test formats that can be used in the method according to the invention, the test kit is characterized in that it comprises a solid phase comprising: a) a sample pad for applying a test sample; b) a conjugate of a Klotho-specific antibody, said conjugate containing a detectable label, or a conjugate or sample pad containing a conjugate of such an antibody / label conjugate and Klotho; c) a reaction or capture area in which an antibody specific to the Klotho protein is immobilized; and d) a wick or waste reservoir for collecting sample solution, sample components, excess reagents or complexes; and optionally further e) Control region: Contains an antibody that specifically binds to the complex of step b), but does not bind to Klotho.

[0060] Lateral flow technology has many variations. Instead of antibodies, the capture area on the membrane can be immobilized with antigens or enzymes depending on the target analyte. It is also possible to apply multiple capture areas to create multiplexed tests.

[0061] In the context of the present invention, the term antibody is meant to encompass Klotho-binding antibody fragments or also aptamers. Thus, the embodiments as outlined above can, in alternative preferred embodiments, also encompass such antibody fragments or aptamers in regions b), c) and e) as described above.

[0062] Lateral flow immunoassays are easy to use, even for untrained operators, and typically provide results within 15 minutes. They are also stable and robust, have a long shelf life, and typically do not require refrigeration. They are also relatively inexpensive to manufacture. These characteristics make them ideal for point-of-care use and for on-site sample testing, not just in laboratories. However, their sensitivity is limited without additional enrichment and incubation steps. While quantitative tests are available, the present invention is primarily directed at qualitative testing of saliva or tears, preferably within a certain range. In this context, preferred test kits are tailored to measure Klotho only when present above a certain concentration. Below this concentration, the test kit will produce a negative result. [Brief explanation of the drawings]

[0063] [Figure 1] Figure 1 shows that the relationship between serum and saliva Klotho levels measured by the TRF method (time-resolved fluoroimmunoassay) is approximately 3.6 to 1. [Figure 2] Figure 2 shows the results of measuring Klotho levels in serum of 467 probes (n=467, minimum 364, 39 pg / ml, range 164-601, measured by Cloud Clone ELISA) from healthy individuals in Munich. [Figure 3] Figure 3 compares data from 467 probes obtained in Munich with a cohort of blood donors from the city of Ulm, which included 96 probes. Serum levels of Klotho were detected by Cloud Clone ELISA. There were differences between age and sex groups, with younger cohorts likely to have higher Klotho values. There were also small differences between female and male probands, with males showing higher values. [Figure 4] Figure 4 shows a comparison of the Munich data and Klotho serum levels in patients with chronic kidney disease stages III and IV and dialysis patients. In patients, not only the present inventors but also others (e.g., Wang et al., BioMed Research International, Vol. 2018, Article ID9481475; Rotondi et al., International Journal of Endocrinology, Vol. 2015, Article ID872193; and Pedersen et al., Clinical Biochemistry, 46 (2013) 1079-1083) have found a strong correlation between renal function and Klotho concentrations in peripheral blood, as shown in the table below.

[0064] [Table 1] [Figure 5] FIG. 5 shows serum Klotho levels measured in normal controls and patients with neurodegenerative diseases.

[0065] For the results shown in the figure above, the amount of Klotho was measured using the SEH757HU enzyme-linked immunosorbent assay from Crowdclone Co., Ltd. The reagents and materials, reagent and sample preparation, assay procedures, and calculations were all performed according to the instructions provided with the test kit. The inventions described in the claims of the original application are set forth below. [1] An in vitro diagnostic method for measuring and / or monitoring the health status of an individual by measuring the Klotho content in saliva or tears. [2] The in vitro method described in [1], wherein the health status of the individual includes an existing or previous disease or condition that correlates with a decrease or decline in Klotho levels, or a predisposition to such a disease or condition. [3] The in vitro method described in [2], wherein the disease or condition is at least one selected from cancer, inflammatory disease, chronic kidney disease (CKD), neurodegenerative disease, chronic heart disease, organ fibrosis, arteriosclerosis, dementia, diabetes, erectile dysfunction, autoimmune disease or autoimmune-related disease, sepsis, premature aging, and other age-related diseases. [4] The in vitro method described in [3], wherein the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease. [5] The in vitro method according to [3], wherein the autoimmune disease or autoimmune-related disease activates inflammation in the patient due to autoimmune nephritis such as type 1 diabetes, rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus, autoimmune encephalomyelitis, lupus nephritis, IgA nephropathy, osteoarthritis, or other diseases. [6] The in vitro method according to any one of [1] to [5], wherein the measuring and / or monitoring of the health status of an individual is carried out in the context of therapeutic treatment of a disease by measuring the content of Klotho before, during and / or after treatment. [7] The in vitro method according to [1], wherein the Klotho content is measured to monitor the effect of physical activity, particularly physical training, muscle training and / or endurance training, on an individual's health status. [8] The in vitro method described in [1] for determining the biological age of the individual. [9] The in vitro method according to any one of [1] to [6], comprising administering Klotho to the individual if the measured Klotho level is equal to or less than a value corresponding to 100 pg / ml measured in serum, preferably equal to or less than a value corresponding to 200 pg / ml measured in serum.

[10] [9] The in vitro method described in [9], wherein administering Klotho comprises administering a therapeutically effective number of mesenchymal stem cells comprising a nucleic acid vector that produces Klotho or contains a region encoding a Klotho protein, said region being operably linked to a promoter or a promoter / enhancer combination, and said vector comprising cells that express Klotho.

[11] The in vitro method according to any one of [1] to [6], comprising increasing the individual's physical activity, in particular physical training such as muscle training and / or endurance training, if the measured Klotho level is equal to or less than a value corresponding to 100 pg / ml measured in serum, preferably equal to or less than a value corresponding to 200 pg / ml measured in serum.

[12] The in vitro method according to any one of [1] to

[11] , wherein the body fluid is saliva.

[13] The in vitro method according to any one of [1] to

[12] , wherein the Klotho content is measured via an immunoassay comprising at least one antibody or aptamer that specifically binds to Klotho, preferably wherein the immunoassay is performed as a double-antibody sandwich assay or a competitive assay, preferably as a lateral flow assay.

[14] A test kit for carrying out the method according to any one of [1] to

[13] , characterized in that it is an immunoassay test kit, and preferably characterized in that the test is carried out as a double antibody sandwich assay or a competitive assay.

[15] Suitable for lateral flow assays, including: a) a sample pad for applying a test sample; b) a conjugate of a Klotho-specific antibody, said conjugate containing a detectable label, or a conjugate or sample pad containing a conjugate of such an antibody / label conjugate and Klotho; c) a reaction or capture area, preferably linear across the width of the solid phase, on which an antibody specific to the Klotho protein is immobilized; and d) a wick or waste reservoir for collecting sample liquid, sample components, excess reagents or complexes; and optionally further e) a control region: comprising an antibody that specifically binds to the complex of step b) but does not bind to Klotho; The test kit according to any one of

[14] to

[16] , comprising a solid phase comprising:

Claims

1. An in vitro method for measuring and / or monitoring the health status of an individual by measuring the Klotho content in saliva.

2. 2. The in vitro method of claim 1, wherein the individual's health status comprises an existing or prior disease or condition, or a predisposition to such a disease or condition, that correlates with a reduced or decreased level of Klotho.

3. 3. The in vitro method of claim 2, wherein the disease or condition is selected from at least one of cancer, inflammatory disease, chronic kidney disease (CKD), neurodegenerative disease, chronic heart disease, organ fibrosis, arteriosclerosis, dementia, diabetes, erectile dysfunction, autoimmune disease or autoimmune-related disease, sepsis, premature aging and other age-related diseases.

4. 4. The in vitro method of claim 3, wherein the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), or Huntington's disease.

5. 4. The in vitro method of claim 3, wherein the autoimmune disease or autoimmune-related disease comprises type 1 diabetes, rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus, autoimmune encephalomyelitis, lupus nephritis, autoimmune nephritis such as IgA nephropathy, osteoarthritis, or other diseases that activate inflammasomes or components thereof in a patient.

6. 6. The in vitro method according to any of claims 1 to 5, wherein said measuring and / or monitoring of the health status of an individual is carried out in the context of therapeutic treatment of a disease by measuring the content of Klotho before, during and / or after treatment.

7. 2. The in vitro method of claim 1, wherein the Klotho content is measured to monitor the effect of physical activity on the health status of an individual.

8. 10. The in vitro method of claim 1 for determining the biological age of the individual.

9. 7. The in vitro method according to any of claims 1 to 6, wherein a measured Klotho level below a value corresponding to 100 pg / ml measured in serum indicates poor health of the individual.

10. 7. The in vitro method according to any one of claims 1 to 6, comprising subjecting the individual to increased physical activity if the measured Klotho level is below a value corresponding to 100 pg / ml measured in serum.

11. 11. The in vitro method according to claim 1, wherein the Klotho content is measured via an immunoassay comprising at least one antibody or aptamer that specifically binds to Klotho.

12. A test kit for carrying out the method according to any one of claims 1 to 11, suitable for a lateral flow assay, comprising: a) a sample pad for applying a test sample; b) a conjugate pad containing labeled particles and Klotho-specific antibodies; A test kit comprising a solid phase comprising: c) a reaction or capture area in which a Klotho-specific antibody is immobilized; and d) a wick or waste reservoir for collecting sample liquid, sample components, excess reagents, or complexes.

13. 8. The in vitro method of claim 7, wherein the Klotho content is measured for monitoring the effect of physical, muscle and / or endurance training on the health status of an individual.

14. 7. The in vitro method according to any of claims 1 to 6, wherein a measured Klotho level below a value corresponding to 200 pg / ml measured in serum indicates poor health of the individual.

15. 11. The in vitro method of claim 10, comprising subjecting the individual to increased physical training, such as strength training and / or endurance training, if the measured Klotho level is below or equal to a value corresponding to 100 pg / ml measured in serum.

16. 7. The in vitro method according to any one of claims 1 to 6, comprising subjecting the individual to increased physical training, such as resistance training and / or endurance training, if the measured Klotho level is below or equal to a value corresponding to 200 pg / ml measured in serum.

17. The in vitro method of claim 11 , wherein the immunoassay is a double-antibody sandwich assay or a competitive assay.

18. 18. The in vitro method of claim 17, wherein the immunoassay is performed as a lateral flow assay.

19. 13. The test kit of claim 12, wherein the reaction or capture area is arranged in a line across the width of the solid phase.

20. the solid phase further comprising e) a control region comprising an antibody that specifically binds to the complex of step b) but does not bind to Klotho; 13. The test kit of claim 12, comprising:

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