Respiratory infection detection and classification

Abstract

The present invention relates to diagnosing and classifying respiratory tract infections based on the determined levels of TK1 protein in a body sample. Specifically, based on the determined levels of TK1 protein, respiratory tract infections can be diagnosed and classified as caused by Mycoplasma pneumoniae.

Description

Technical Field

[0001] The present embodiments relate generally to respiratory tract infections, and particularly to the detection and classification of such respiratory tract infections. Background Art

[0002] The most important mycoplasma infection in medicine is the infection of the lower respiratory tract caused by Mycoplasma pneumoniae, a very small, wall-less bacterium. The disease manifests as an atypical bacterial pneumonia, referred to as mycoplasma pneumonia, with a long incubation period and a wide spectrum of clinical symptoms, but usually presents as a low-intensity, persistent, dry cough. It is estimated that approximately 2 million cases of Mycoplasma pneumoniae infection occur in the United States each year, and 1-10 out of every 50 cases of community-acquired pneumonia are Mycoplasma pneumoniae infections. Therefore, it is generally considered a major health problem worldwide.

[0003] Currently available diagnostic tests for M. pneumoniae infection include culture and serological methods as well as polymerase chain reaction (PCR)-based methods, with a limited number of molecular methods currently in use. Therefore, there is a great need for better diagnostic procedures. Summary of the invention

[0004] Providing improved detection and triage of respiratory infections is an overall goal.

[0005] This and other objectives are met by the embodiments as disclosed herein.

[0006] The invention is defined in the independent claim. Further embodiments of the invention are defined in the dependent claims.

[0007] The present embodiment relates to the use of the determined thymidine kinase 1 (TK1) substance level in a body sample to predict and diagnose the presence of mycoplasma pneumonia caused by Mycoplasma pneumonia in a patient. The determined TK1 substance level can also be used to classify the patient's respiratory infection as mycoplasma pneumonia caused by Mycoplasma pneumonia, or viral pneumonia caused by a virus or bacterial pneumonia caused by bacteria other than Mycoplasma pneumonia.

[0008] The present embodiment thus provides a technique for detecting and classifying Mycoplasma pneumoniae infections which are otherwise difficult to diagnose. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The embodiments and further objects and advantages thereof may best be understood by referring to the following description taken in conjunction with the accompanying drawings, in which:

[0010] Figure 1Serum thymidine kinase 1 (STK1) protein concentrations in healthy subjects (n=144) and humans with acute bacterial (n=98), viral (n=64), or Mycoplasma pneumoniae (n=25) infections are illustrated. The Y axis shows the 10-log values ​​of STK1 concentrations, and significant P values ​​are used for pairwise comparisons between healthy and different patient groups.

[0011] Figure 2 STK1 protein concentrations in healthy subjects (n=144) and humans with acute bacterial (n=60) or Mycoplasma pneumoniae (n=25) lower respiratory tract infections are illustrated. The Y axis shows the 10-log values ​​of STK1 concentrations, and significant P values ​​are used for pairwise comparisons between healthy and different patient groups.

[0012] Figure 3 The difference between healthy subjects and patients with acute infection with M. pneumoniae is shown. Results from patients with clinical diagnosis and possible etiology of M. pneumoniae infection are shown. The P values ​​given in the figures are the statistical differences between the receiver operating characteristic (ROC) curves of STK1 concentrations.

[0013] Figure 4 Results of ROC analysis illustrating the distinction between bacterial and M. pneumoniae etiologies of lower respiratory tract infections.

[0014] Figure 5 Results of ROC analysis illustrating the distinction between viral and M. pneumoniae etiologies of lower respiratory tract infection.

[0015] Figure 6 Results of ROC analysis illustrating the distinction between bacterial and M. pneumoniae etiologies of lower respiratory tract infections.

[0016] Figure 7 The results of ROC analysis for the discrimination between bacterial and M. pneumoniae etiologies of lower respiratory tract infections compared with CRP (C-reactive protein), PCT (procalcitonin) and blood neutrophil count are illustrated.

[0017] Figure 8 The results are presented for serum TK1 (S-TK1), plasma HNL (P-HNL dimer), plasma C-reactive protein (P-CRP), serum procalcitonin (S-PCT), and the ratio between serum TK1 and plasma HNL (TK1 / HNL dimer).

[0018] Fig. 9A and 9B Serum TK1 (S-TK1) (9A) and plasma HNL (P-HNL dimer) protein concentrations of healthy subjects (n=144) and humans with bacterial pneumonia (n=86) or mycoplasma pneumonia (n=28) are shown. The Y axis shows the 10-log value of the concentration. DETAILED DESCRIPTION

[0019] The present embodiments relate generally to respiratory tract infections, and particularly to the detection and classification of such respiratory tract infections.

[0020] The present invention is based on experimental results showing that respiratory tract infections caused by Mycoplasma pneumoniae, particularly Mycoplasma pneumoniae, lead to significant increases in thymidine kinase 1 (TK1) protein, particularly serum TK1 (STK1 or S-TK1) protein levels in human patients. However, this increase in STK1 protein levels is not seen in healthy people or patients with respiratory tract infections caused by other pathogens, such as viral pneumonia caused by viruses and bacterial pneumonia caused by bacteria other than Mycoplasma pneumoniae.

[0021] Therefore, TK1 protein levels, such as STK1 protein levels, can be used to identify or diagnose patients with respiratory tract infections caused by Mycoplasma pneumoniae, thereby distinguishing these Mycoplasma pneumoniae patients from other patients with respiratory tract infections caused by pathogens other than Mycoplasma pneumoniae.

[0022] This means that TK1 protein assay can be used as a valuable biomarker for otherwise difficult to diagnose Mycoplasma pneumoniae infections, optionally together with other biomarkers, such as C-reactive protein (CRP), procalcitonin (PCT), calprotectin and / or human neutrophil lipocalin (HNL), such as HNL in blood (B-HNL) and / or plasma (P-HNL).

[0023] In one embodiment, TK1 is used as a biomarker together with at least one biomarker selected from CRP, PCT, calprotectin and HNL. In a preferred embodiment, TK1 is used as a biomarker together with at least one biomarker selected from CRP, PCT and HNL, more preferably CRP, PCT and P-HNL.

[0024] Thus, in one embodiment, TK1 is used together with CRP. In another embodiment, TK1 is used together with PCT, and in another embodiment, TK1 is used together with HNL, such as B-HNL and / or P-HNL, preferably P-HNL. In yet another embodiment, TK1 is used together with calprotectin. TK1 may also be used together with at least two other biomarkers, such as TK1 and CRP, TK1 and PCT, TK1 and calprotectin, or TK1 and HNL, such as B-HNL and / or P-HNL, preferably P-HNL. In a further embodiment, TK1 is used together with at least three other biomarkers, such as TK1, CRP and PCT; TK1, CRP and calprotectin; TK1, CRP and HNL, such as B-HNL and / or P-HNL, preferably P-HNL; TK1, PCT and calprotectin; TK1, PCT and HNL, such as B-HNL and / or P-HNL, preferably P-HNL; or TK1, calprotectin and HNL, such as B-HNL and / or P-HNL, preferably P-HNL. In other embodiments, TK1 is used together with at least four other biomarkers, such as TK1, CRP, PCT and calprotectin, TK1, CRP, PCT and HNL, such as B-HNL and / or P-HNL, preferably P-HNL; TK1, CRP, calprotectin and HNL, such as B-HNL and / or P-HNL, preferably P-HNL; TK1, PCT, calprotectin and HNL, such as B-HNL and / or P-HNL, preferably P-HNL. In another embodiment, TK1 is used together with CRP, PCT, calprotectin and HNL, such as B-HNL and / or P-HNL, preferably P-HNL.

[0025] For example, when TK1 is used together with at least one other biomarker, the level of TK1 and the level of at least one other biomarker are compared to corresponding thresholds, and then the estimation, diagnosis or classification is based on the comparison of each biomarker (TK1 and at least one other biomarker) with its respective threshold.

[0026] In an alternative embodiment, a value is calculated based on the level of TK1 and the level of at least one other biomarker, and the value is compared to a threshold value. For example, the value can be calculated based on the function f(), f(TK1, (CRP, PCT, calprotectin and / or HNL)), where TK1, CRP, PCT, calprotectin and HNL represent the respective levels or amounts determined for a particular biomarker. A typical example of a function f() that can be used is a ratio or quotient between two biomarker levels, such as TK1 / HNL, such as TK1 / P-HNL, or HNL / TK1, such as P-HNL / TK1. Another example of a function f() is the product of two or more biomarker levels.

[0027] TK1 has been used as a serum biomarker for the prognosis and monitoring of hematological malignancies for many years. TK1 is an enzyme that phosphorylates thymidine (dThd) to deoxythymidine monophosphate (dTMP) using adenosine triphosphate (ATP) as a phosphate donor. dTMP is further phosphorylated and forms deoxythymidine triphosphate (dTTP), which is incorporated into deoxyribonucleic acid (DNA) during the S phase of the cell cycle.

[0028] STK1 has been identified by enzyme activity assays or, more recently, by immunoassays.

[0029] Clinical studies using the radioactive TK1 enzyme activity assay (TK-REA) have demonstrated that serum TK1 measurements have prognostic value in several malignancies, such as non-Hodgkin lymphoma, Hodgkin's disease, and various forms of leukemia.

[0030] In an early study of TK-REA, several different types of malignancies were investigated, as well as other clinical conditions, including viral infections, mononucleosis, and Mycoplasma pneumoniae [1]. In patients with mononucleosis, TK1 levels were found to be dramatically increased in all patients with acute symptoms of the disease. In terms of TK1 levels in the serum of patients with viral diagnoses, 83% had levels below the cutoff level for healthy individuals, while 17% of the sera showed elevated levels of TK1 activity. The presence of elevated TK1 activity has been associated with infection with rubella, measles, cytomegalovirus, and in some cases herpes simplex virus (HSV) or varicella zoster virus (VZV). However, no increase in TK1 activity levels was reported in subjects with Mycoplasma pneumoniae infection [1]. Elevated serum TK1 activity in patients with viral hepatitis, especially hepatitis A infection, was significantly correlated with aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and lactate dehydrogenase (LDH), but not with other liver blood biomarkers.

[0031] TK1 activity in the serum of patients with acute bacterial infections has not been studied much, and there are few publications on this topic. In [2,3], the activity of TK1 in the serum of 100 healthy subjects was measured, with a mean value of 4 U / L, while in 100 patients with unidentified bacterial infections, the median value was 10 U / L, which is similar to that found in 40 women with benign breast cancer. Correspondingly, the mean value in 100 patients with unidentified viral infections was 54 U / L [2,3].

[0032] One aspect of the embodiments relates to a method for predicting the presence of Mycoplasma pneumoniae in a patient or subject with a respiratory tract infection. The method comprises determining the level of a TK1 substance in a body sample obtained from the patient or subject using an anti-TK1 antibody or a fragment thereof. The method further comprises estimating the likelihood that the respiratory tract infection is mycoplasma pneumoniae caused by Mycoplasma pneumoniae based on the level of the TK1 substance in the body sample.

[0033] In one embodiment, the method further comprises determining the level of at least one other biomarker in a body sample or another body sample obtained from the subject. In this embodiment, the at least one other biomarker is selected from CRP, PCT, calprotectin and HNL. In this embodiment, estimating the likelihood comprises estimating the likelihood that the respiratory tract infection is mycoplasma pneumonia caused by Mycoplasma pneumonia based on the level of TK1 substance determined in the body sample and based on the level of at least one other biomarker determined in the body sample or another body sample.

[0034] Another aspect of the embodiments relates to a method for diagnosing Mycoplasma pneumonia in a patient or subject. The method comprises determining the level of TK1 substance in a body sample obtained from the patient or subject using an anti-TK1 antibody or fragment thereof. The method also comprises diagnosing the patient or subject with Mycoplasma pneumonia based on the level of TK1 substance in the body sample.

[0035] In one embodiment, the method further comprises determining the level of at least one other biomarker in a body sample or another body sample obtained from the subject. In this embodiment, the at least one other biomarker is selected from the group consisting of CRP, PCT, calprotectin and HNL. In this embodiment, diagnosing the subject comprises diagnosing the subject as having Mycoplasma pneumonia based on the level of TK1 substance in the determined body sample and based on the level of at least one other biomarker in the determined body sample or another body sample.

[0036] In one embodiment, the estimation and diagnosis steps in the above embodiments are performed based on a comparison between the determined TK1 substance level and a threshold value. In a specific embodiment, if the determined TK1 substance level is above the threshold value, the estimation step includes estimating a high probability that the respiratory tract infection is mycoplasma pneumonia caused by Mycoplasma pneumonia, otherwise estimating a low probability that the respiratory tract infection is mycoplasma pneumonia caused by Mycoplasma pneumonia. Therefore, in one embodiment, if the determined TK1 substance level does not exceed the threshold value, the method includes estimating a high probability that the respiratory tract infection is caused by a pathogen other than Mycoplasma pneumonia, such as viral pneumonia caused by a virus or bacterial pneumonia caused by bacteria other than Mycoplasma pneumonia.

[0037] Another aspect of the embodiment relates to a method for classifying a respiratory infection in a subject. The method includes determining the level of a TK1 substance in a body sample obtained from the subject using an anti-TK1 antibody or a fragment thereof. The method also includes classifying the respiratory infection as mycoplasma pneumonia caused by Mycoplasma pneumoniae, or classifying the respiratory infection as viral pneumonia caused by a virus or bacterial pneumonia caused by bacteria other than Mycoplasma pneumoniae based on the level of the TK1 substance in the determined body sample.

[0038] In one embodiment, the method further comprises determining the level of at least one other biomarker in a body sample or another body sample obtained from the subject. In this embodiment, the at least one other biomarker is selected from the group consisting of CRP, PCT, calprotectin and HNL. In this embodiment, classifying the respiratory tract infection comprises classifying the respiratory tract infection as mycoplasma pneumonia caused by Mycoplasma pneumonia based on the level of TK1 substance in the determined body sample and based on the level of at least one other biomarker in the determined body sample or another body sample, or classifying the respiratory tract infection as viral pneumonia caused by a virus or bacterial pneumonia caused by bacteria other than Mycoplasma pneumonia.

[0039] In a specific embodiment, the method includes determining the level of HNL in a body sample or another body sample obtained from a subject. The method also includes calculating a ratio or quotient between the level of TK1 substance determined in the body sample and the level of HNL determined in the body sample or another body sample. In this specific embodiment, classifying the respiratory tract infection includes classifying the respiratory tract infection as mycoplasma pneumonia caused by Mycoplasma pneumonia if the quotient or ratio exceeds a threshold value, otherwise classifying the respiratory tract infection as bacterial pneumonia caused by bacteria other than Mycoplasma pneumonia.

[0040] Another aspect of the embodiment relates to a method for classifying a respiratory infection in a patient or subject. The method comprises determining the level of a TK1 substance in a body sample obtained from the patient or subject using an anti-TK1 antibody or a fragment thereof. The method further comprises classifying the respiratory infection as mycoplasma pneumonia caused by Mycoplasma pneumonia if the level of the TK1 substance in the body sample exceeds a threshold value, otherwise classifying the respiratory infection as viral pneumonia caused by a virus or bacterial pneumonia caused by bacteria other than Mycoplasma pneumonia.

[0041] Viral pneumonia is pneumonia caused by a virus. Viruses are the most common cause of pneumonia in children, while in adults, bacteria are a more common cause. Common causes of viral pneumonia include influenza A and B viruses, respiratory syncytial virus (RSV), human parainfluenza virus, respiratory syncytial virus, rhinovirus, human bocavirus, and parainfluenza virus. Rare viruses that commonly cause pneumonia include adenovirus, metapneumovirus, severe acute respiratory syndrome (SARS) virus, and Middle East respiratory syndrome (MERS) virus. Viruses that primarily cause other illnesses but sometimes cause pneumonia include herpes simplex virus (HSV), varicella-zoster virus (VZV), measles virus, rubella virus, cytomegalovirus (CMV), smallpox virus, and dengue virus.

[0042] Bacterial pneumonia is a type of pneumonia that is caused by a bacterial infection. Bacterial pneumonia can be caused by both gram-positive and gram-negative bacteria. Streptococcus pneumoniae is the most common bacterial cause of pneumonia in all age groups, except newborns. Streptococcus pneumoniae is a gram-positive bacterium that is often found in the throat of people who do not have pneumonia. Other important gram-positive causes of pneumonia are Staphylococcus aureus and Bacillus anthracis. Gram-negative bacteria are rarely considered as causes of pneumonia. Haemophilus influenzae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Bordetella pertussis, and Moraxella catarrhalis are the most common gram-negative causes of pneumonia. Bacterial pneumonia can also be caused by atypical bacteria, such as Coxiella burnetii, Chlamydia pneumoniae, and Legionella pneumophila.

[0043] Bacterial pneumonia as used herein refers to pneumonia caused by bacteria other than Mycoplasma pneumoniae.

[0044] Mycoplasma pneumonia is a type of pneumonia caused by Mycoplasma pneumoniae. Mycoplasma pneumoniae is spread by respiratory droplet transmission. Once attached to the mucous membranes of the host organism, Mycoplasma pneumoniae extracts nutrients, grows, and reproduces by binary fission. Sites of attachment include the upper and lower respiratory tracts, causing pharyngitis, bronchitis, and pneumonia. Infections caused by this bacterium are sometimes called atypical pneumonia because of their long course, lack of sputum production, and numerous extrapulmonary symptoms. Mycoplasma pneumonia can be complicated by Stevens-Johnson syndrome, autoimmune hemolytic anemia, cardiovascular disease, encephalitis, or Guillain-Barré syndrome.

[0045] In one embodiment, classifying the respiratory tract infection comprises classifying the respiratory tract infection as mycoplasma pneumonia caused by Mycoplasma pneumoniae if the determined level of TK1 substance in the body sample exceeds a threshold value, otherwise classifying the respiratory tract infection as bacterial pneumonia caused by bacteria other than Mycoplasma pneumoniae.

[0046] The threshold used in the method generally depends on the method used to measure TK1 in the body sample, for example based on a specific anti-TK1 antibody or antibodies used in the measurement. The threshold can be determined as disclosed in the Examples section by determining the level of TK1 in body samples collected from patients diagnosed with Mycoplasma pneumonia and optionally from body samples collected from healthy subjects and / or patients diagnosed with viral pneumonia or bacterial pneumonia other than Mycoplasma pneumonia. The threshold can then be determined from such measurements. For example, Figure 1 and 2 The serum TK1 (STK1) substance levels determined in healthy volunteers and different patient groups using the TK1 enzyme-linked immunosorbent assay (ELISA) (AroCell TK 210 ELISA) are described. In this case, a suitable threshold value can be selected in the range of 0.5 to 1.0 μg / L, such as 0.6 to 1.0 μg / L, preferably 0.7 to 0.9 μg / L, such as 0.7 μg / L or 0.8 μg / L.

[0047] In one embodiment, the method further comprises obtaining a body sample from the patient.

[0048] The body sample is preferably a body fluid sample containing TK1 substance. Non-limiting but preferred examples of such body fluid samples include serum samples, plasma samples, blood samples, synovial fluid samples, lymph fluid samples, urine samples and saliva samples, preferably serum samples, plasma samples or blood samples, more preferably serum samples or plasma samples, such as lithium heparin plasma samples.

[0049] In one embodiment, determining the level of the TK1 substance comprises determining the level of the STK1 substance in a serum or plasma sample obtained from a patient having anti-TK1 antibodies or fragments thereof.

[0050] TK1 in the human body exists in various forms, depending on the presence of certain molecules, such as the presence or absence of adenosine triphosphate (ATP); on the concentration of the protein, i.e. high or low concentration; on the type of protein, i.e. native or recombinant TK1; and on the location of the protein, i.e. in the serum or in the cytoplasm.

[0051] Generally, cytoplasmic and recombinant human TK1 exist as tetramers in the presence or high concentration of ATP, and exist as dimers in the absence or low concentration of ATP. The tetramer forms of cytoplasmic and recombinant human TK1 have high TK1 activity, while the dimer forms have lower TK1 activity. Cytoplasmic TK1, also known as cellular TK1, is TK1 that exists in cells and can be isolated from such cells.

[0052] In stark contrast, human STK1 can be in the form of high molecular weight complexes, such as oligomers or containing such oligomers, with TK1 activity as well as dimer and tetramer forms with very low or even absent TK1 activity. Oligomerization appears to be associated with the formation of disulfide cross-links that occur in the blood.

[0053] As used herein, TK1 substances refer to various forms of TK1, such as dimers, tetramers, oligomers, and complexes containing TK1. TK1 substances are preferably serum TK1 substances, i.e., STK1 substances present in the patient's blood, plasma or serum. STK1 substances can then contain the above-mentioned forms of STK1, such as dimers, tetramers, oligomers, and complexes containing STK1. TK1 substances also include complexes having at least one TK1 protein unit and other molecules and / or macromolecules.

[0054] If at least one other biomarker is determined, then the level of at least one other biomarker in the body sample in addition to the TK1 substance or in a body sample of the same type as the TK1 substance can also be determined. In another embodiment, the at least one other biomarker is determined in another body sample from the subject. For example, serum TK1 substance can be determined in a serum sample from the subject, and at least one other biomarker, such as HNL, can be determined in a plasma sample from the subject.

[0055] The level of at least one other biomarker can be determined according to techniques well known in the art, including immunoassays using antibodies that specifically bind to the corresponding biomarker. Illustrative but non-limiting examples of such immunoassays include ELISA or particle enhanced turbidimetric immunoassay (PETIA).

[0056] In one embodiment, determining the level of a TK1 substance comprises contacting the body sample with an anti-TK1 antibody or fragment thereof. This embodiment also includes detecting or determining the amount of the anti-TK1 antibody or fragment that is bound to the TK1 substance.

[0057] Contacting the body sample with the antibody or fragment thereof can be achieved by adding the antibody or fragment thereof to the body sample and incubating the body sample with the antibody or fragment thereof. The antibody or fragment thereof thereby binds to the TK1 substance, forming a complex between the antibody or fragment thereof and the TK1 substance. In such an embodiment, measuring the amount of the antibody or fragment bound to the TK1 substance may include measuring or quantifying the complex between the antibody or fragment thereof and the TK1 substance, thereby measuring or quantifying the amount of the antibody or fragment bound to the TK1 substance.

[0058] In one embodiment, determining the level of a TK1 substance comprises determining the level of a TK1 substance in a body sample based on the determined amount of bound anti-TK1 antibodies or fragments and a standard correlation between the amount of bound anti-TK1 antibodies or fragments and the level of the TK1 substance. Thus, in this embodiment, a standard correlation is established between the amount of bound anti-TK1 antibodies or fragments and a predetermined concentration or level of a TK1 substance, such as a predetermined concentration or level of recombinant TK1. This standard correlation can then be established by determining the respective amounts of bound anti-TK1 antibodies or fragments of different predetermined concentrations of recombinant TK1 to obtain a correlation between them.

[0059] In one embodiment, the method comprises correlating the measured amount of the antibody or fragment that binds to the TK1 substance with the level of the TK1 substance. This can be done by using a predetermined or standard correlation between the measured amount of the antibody or fragment that binds to a reference TK1 substance and the concentration of the reference TK1 substance. A typical reference TK1 substance that can be used when generating such a predetermined or standard correlation is recombinant human TK1.

[0060] Thus, a predetermined or standard correlation can be generated by adding the antibody or fragment thereof to different samples containing different concentrations of a reference TK1 substance, preferably recombinant human TK1. The amount of the antibody or fragment that binds to the reference TK1 substance, preferably recombinant human TK1, is then measured in the different samples, thereby obtaining a standard curve, function or relationship between the concentration of the reference TK1 substance, preferably recombinant human TK1, and the measured amount of the antibody or fragment that binds to the reference TK1 substance, preferably recombinant human TK1.

[0061] This predetermined or standard correlation, such as a standard curve, function or relationship, can then be used to map or convert the measured amount of antibody or fragment that binds to the TK1 substance in the body sample to the concentration of the TK1 substance in the body sample.

[0062] In one embodiment, the body sample is treated before or during incubation of the body sample with the antibody or fragment thereof.Such sample treatment can be used to stabilize selected TK1 forms in the body sample and / or to break down larger TK1 complexes or oligomers into smaller complexes or oligomers.

[0063] Thus, in one embodiment, a sample diluent or pre-treatment buffer is added to the body sample prior to (or in association with) adding the antibody or fragment thereof to the body sample, preferably prior to adding the antibody or fragment thereof to the body sample.

[0064] In one embodiment, the sample dilution buffer comprises ATP, preferably at a concentration selected in the interval of 1.5 mM to 50 mM. As mentioned previously, ATP stabilizes the tetrameric form of TK1, which has high enzymatic TK1 activity.

[0065] In another embodiment, the sample dilution buffer comprises a reducing agent. The reducing agent can then destroy the disulfide crosslinks in the larger TK1 complex and oligomers to obtain smaller TK1 forms, such as tetramers. Various reducing agents capable of destroying disulfide bonds can be used according to the embodiment, including but not limited to dithioerythritol (DTE), dithiothreitol (DTT), dithiobutylamine (DTBA), tris (2-carboxyethyl) phosphine) (TCEP) and combinations thereof. The amount of the reducing agent is generally selected within the range of 0.1 mM to 10 mM.

[0066] In one embodiment, the sample dilution buffer may comprise ATP and a reducing agent.

[0067] In one embodiment, the anti-TK1 antibody is a monoclonal anti-TK1 antibody. In another embodiment, the anti-TK1 antibody is a polyclonal anti-TK1 antibody.

[0068] In one embodiment, the anti-TK1 antibody is specific for an epitope or peptide consisting of an amino acid sequence from the C-terminal region of TK1, preferably human TK1.

[0069] The peptide is preferably selected from a portion of TK1 ranging from amino acid position 200 to the end of TK1, i.e., amino acid position 234 in humans (SEQ ID NO: 28). In a specific embodiment, the peptide is selected from a portion of TK1 protein ranging from amino acid position 205, preferably 210 to amino acid position 230, preferably 225.

[0070] The peptide is preferably an N-mer, wherein N is an integer in the range of 8-20, preferably an integer in the range of 10-15.

[0071] The peptide preferably consists of N consecutive amino acids in the C-terminal region of the TK1 protein.

[0072] In one embodiment, the peptide consists of the following amino acid sequence GEAVAARKLF (SEQ ID NO: 1). In another embodiment, the peptide consists of the following amino acid sequence NCPVPGKPGE (SEQ ID NO: 2). In another embodiment, the peptide consists of the following amino acid sequence PVPGKPGEAV (SEQ ID NO: 3). In yet another embodiment, the peptide consists of the following amino acid sequence NCPVPGKPGEAV (SEQ ID NO: 4).

[0073] The monoclonal anti-TK1 antibody specific for the epitope consisting of GEAVAARKLF (SEQ ID NO: 1) comprises a variable heavy (VH) domain complementarity determining region 1 (CDR1) having the amino acid sequence DYEMH (SEQ ID NO: 5), a VH domain CDR2 having the amino acid sequence AIHPGYGGTAYNQKFKG (SEQ ID NO: 6), a VH domain CDR3 having the amino acid sequence FITKFDY (SEQ ID NO: 7), a variable light (VL) domain CDR1 having the amino acid sequence KSSQSLLDSDGKTFLN (SEQ ID NO: 8), a VL domain CDR2 having the amino acid sequence LVSKLDS (SEQ ID NO: 9), and a VL domain CDR3 having the amino acid sequence WQGTHFPWT (SEQ ID NO: 10).

[0074] Monoclonal anti-TK1 antibodies specific for the epitopes NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3) and NCPVPGKPGEAV (SEQ ID NO: 4) comprise a VH domain CDR1 having the amino acid sequence DYEMH (SEQ ID NO: 5), a VH domain CDR2 having the amino acid sequence AILPGSGGTAYNQKFKG (SEQ ID NO: 17), a VH domain CDR3 having the amino acid sequence LITTFDY (SEQ ID NO: 18), a VL domain CDR1 having the amino acid sequence KSSQSLLDSDGKTYLN (SEQ ID NO: 19), a VL domain CDR2 having the amino acid sequence LVSKLDS (SEQ ID NO: 9), and a VL domain CDR3 having the amino acid sequence WQGTHFPWT (SEQ ID NO: 10).

[0075] In another embodiment, the anti-TK1 antibody is specific for a conformation-dependent epitope of human TK1. The monoclonal anti-TK1 antibody specific for this conformation-dependent epitope comprises a VH domain CDR1 having the amino acid sequence SGYSWH (SEQ ID NO: 11), a VH domain CDR2 having the amino acid sequence YIHYSGSTTYNPSLKG (SEQ ID NO: 12), a VH domain CDR3 having the amino acid sequence WGTGHWYFDV (SEQ ID NO: 13), a VL domain CDR1 having the amino acid sequence RSSTGAVTTTNYAN (SEQ ID NO: 14), a VL domain CDR2 having the amino acid sequence GTNNRVP (SEQ ID NO: 15), and a VL domain CDR3 having the amino acid sequence ALWYSNHWV (SEQ ID NO: 16).

[0076] The above three examples of monoclonal anti-TK1 antibodies that can be used according to the embodiments are further disclosed in WO 2015 / 094106.

[0077] Therefore, in one embodiment, the monoclonal antibody or fragment thereof is selected from a monoclonal antibody or fragment thereof specific to GEAVAARKLF (SEQ ID NO: 1) of human TK1, a monoclonal antibody or fragment thereof specific to at least one of NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3) and NCPVPGKPGEAV (SEQ ID NO: 4) of human TK1, and a monoclonal antibody or fragment thereof specific to a conformation-dependent epitope of human TK1.

[0078] In another embodiment, the anti-TK1 antibody is specific for an epitope or peptide consisting of KPGEAVAARKLFAPQ (SEQ ID NO: 20).

[0079] Anti-TK1 antibodies specific for this epitope are further disclosed in WO 95 / 29192.

[0080] At least one additional amino acid, such as a cysteine ​​residue, may be added to the N-terminus or C-terminus, preferably the N-terminus, of the peptide to serve as a coupling to other molecules, such as a carrier protein.

[0081] In another embodiment, the anti-TK1 antibody is specific for an epitope or peptide consisting of an amino acid sequence from the active site of TK1. The peptide is preferably selected from a portion of TK1 from amino acid position 150 to amino acid position 190 in human TK1. In a specific embodiment, the peptide is selected from a portion of TK1 from amino acid position 155, preferably 160, more preferably 161 to amino acid position 185, preferably 183.

[0082] The peptide is preferably an M-mer, wherein M is an integer in the range of 10 to 40, preferably in the range of 20 to 30, more preferably 23 or 24.

[0083] The peptide preferably consists of M consecutive amino acids in the active site of the TK1 protein.

[0084] At least one additional amino acid, such as a cysteine ​​residue, may be added to the N-terminus or C-terminus, preferably the N-terminus, of the peptide to serve as a coupling to other molecules, such as a carrier protein.

[0085] In one embodiment, the peptide consisting of the amino acid sequence from the active site of TK1 has an amino acid sequence corresponding to amino acids 161 to 183 in human TK1, ie, has an amino acid sequence of AYTKRLGTEKEVEVIGGADKYHS (SEQ ID NO: 21).

[0086] Anti-TK1 antibodies specific for this epitope are further disclosed in WO 2008 / 142664.

[0087] In a further embodiment, the antibody or fragment thereof is a monoclonal antibody or fragment thereof as disclosed in WO 2019 / 201901.

[0088] For example, the monoclonal antibody can be mAb 6C6, mAb 4H4, or mAb 23C11.

[0089] mAb 6C6 VH Domain (SEQ ID NO: 22):

[0090] METGLRWLLLVAVLKGVQCQEQLEESGGDLVKPEGSLTLTCTASRFSFSSSYWICWVRQAPGKGLEWIACIYAGDSGSSYYASWAKGRFTVSKTSSTTVTLQTTSLTAADTATYFCARASVGAAYDYFALWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCWNSYGEP

[0091] mAb 6C6 VL Domain (SEQ ID NO: 23):

[0092] MDTRAPTQLLGLLLLWLPGARCALVMTQTPASVEAAMGGTVTIKCQASEDVSSHLAWYQQRPGQPPKLLIYGASDLASGVPSRFTGSGSGTQFTLAISDLECADAATYYCQGYYYISDSPYVFGGGTEVVVVKGDPVAPTVLIFPPAADQVATGTVGTTVTVCVANKYFPDVQTVTADCVTSQNSQNRGTSTDCVTSQNSFQ

[0093] VH domain of mAb 4H4 (SEQ ID NO: 24):

[0094] METGLRWLLLVAVLKGVQCQSLEESGGGLVQPEGSLTLTCTASGFSFSSGYDMCWVRQTPGKGLEWIACISVDSDGVTYYASWAKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGYESSSGVYIPYFTLWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCVNSY

[0095] VL domain of mAb 4H4 (SEQ ID NO: 25):

[0096] MDMRAPTQLLGLLLLWLPGARCADIVLTQTPASVEAAVGGTVTIKCQASQSIYSYLAWYQHKPGQPPKLLIYKASTLASGVPSRFKGSGSGTEYTLTISDLECADAATYYCQHYYYSSTSGGGVFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYLSDGNSQVTSGTQNTRGIVGTQFQNTQFGTVGTSQFGTQFGTVGTSQFQNGFWGTVTSGVPSRFKGSGTEY

[0097] VH domain of mAb 23C11 (SEQ ID NO: 26):

[0098] METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSNYYMSWVRQAPGKGLEWIGIIYGDDNTYCANWTKGRFTISKTSTTVDLTITSPTTEDTATYFCARGPDYIAAKMDIWGPGTLVTVSLGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSG

[0099] mAv 23C11 VL domain (SEQ ID NO: 27):

[0100] MDTRAPTQLLGLLLLWLPGARCDVVMTQTPASVEAAVGGTVTIKCQASQSISGYLSWYQQKPGQRPKLLIYRASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQCT YGSSTFSSYGNAFGGGTEVVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYADCTQNSGVTQNTRQFPSQNSGTVQNKYADCTQNSGVTSGQNTRGQFQNTQFQNSGTVK

[0101] In one embodiment, the level of TK1 substance in a body sample is determined using a kit. The kit preferably comprises a first or so-called capture anti-TK1 antibody fixed to a support or intended to be fixed to a support and a second or so-called detection anti-TK1 antibody. The first and second anti-TK1 antibodies may be selected from the illustrative examples of monoclonal and polyclonal anti-TK1 antibodies described above.

[0102] In a specific embodiment, the kit comprises a first monoclonal antibody or a first fragment thereof having specificity for an epitope selected from the group consisting of: i) GEAVAARKLF (SEQ ID NO: 1) of human TK1, ii) NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3) and NCPVPGKPGEAV (SEQ ID NO: 4) of human TK1, and iii) a conformation-dependent epitope of human TK1. The kit also comprises a second monoclonal antibody or a second fragment thereof having specificity for an epitope selected from the group consisting of: i) GEAVAARKLF (SEQ ID NO: 1) of human TK1, ii) at least one of NCPVPGKPGE (SEQ ID NO: 2), PVPGKPGEAV (SEQ ID NO: 3) and NCPVPGKPGEAV (SEQ ID NO: 4) of human TK1, and iii) a conformation-dependent epitope of human TK1.

[0103] In one embodiment, the first antibody or its first fragment is a so-called capture antibody that is fixed to a support or is intended to be fixed to a support, and the second antibody or its second fragment is a so-called detection antibody. In another embodiment, the second antibody or its second fragment is a capture antibody that is fixed to a support or is intended to be fixed to a support, and the first antibody or its first fragment is used as a detection antibody.

[0104] In one embodiment, the first and second anti-TK1 antibodies are specific for different epitopes in human TK1.

[0105] In another embodiment, the first and second anti-TK1 antibodies are specific for the same epitope in human TK1. This is possible because multiple copies of the same epitope may be present in a high molecular weight complex of multiple TK1 protein units. Thus, the TK1 substance may be a multivalent complex of multiple, i.e., at least two, TK1 protein units.

[0106] Therefore, the TK1 substance used herein includes TK1 protein, complexes having multiple TK1 protein units, such as dimers, trimers, tetramers, etc. TK1 substances also include complexes having at least one TK1 protein unit and other molecules and / or macromolecules.

[0107] In one embodiment, one of the first and second anti-TK1 antibodies is specific for a peptide consisting of an amino acid sequence from the active site of TK1, and the other of the first and second anti-TK1 antibodies is specific for a peptide consisting of an amino acid sequence from the C-terminal region of TK1.

[0108] In another embodiment, one of the first and second anti-TK1 antibodies is specific for a peptide consisting of a first amino acid sequence of the C-terminal region of TK1, and the other of the first and second anti-TK1 antibodies is specific for a peptide consisting of a first amino acid sequence of the C-terminal region of TK1 or a second different amino acid sequence of the C-terminal region of TK1.

[0109] In another embodiment, one of the first and second anti-TK1 antibodies is specific for a peptide consisting of an amino acid sequence of the C-terminal region of TK1, and the other of the first and second anti-TK1 antibodies is specific for a conformation-dependent epitope of human TK1.

[0110] Antibody fragments as used herein may be selected from single chain antibodies, Fv fragments, scFv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, single domain antibodies (sdAb), scFv-Fc fragments, di-scFv fragments and CDR regions.

[0111] The antibody or fragment thereof specifically binds to the TK1 substance, in particular, specifically binds to the TK1 protein.

[0112] The specificity of an antibody can be determined based on affinity and / or avidity. The affinity represented by the equilibrium constant (KD) of antigen-antibody dissociation is a measure of the binding strength between an antigenic determinant and an antigen binding site on an antibody. The smaller the KD value, the stronger the binding strength between an antigenic determinant and an antibody. Alternatively, affinity can also be expressed as an affinity constant (KA), i.e. 1 / KD. It will be clear to those skilled in the art that affinity can be determined in a manner known per se, depending on the specific antigen of interest.

[0113] Avidity is a measure of the strength of binding between an antibody and its associated antigen. Avidity is related to the affinity between an antigenic determinant and its antigen binding site on the antibody and the number of associated binding sites present on the antibody.

[0114] Typically, antibodies will be present at 10 -5 Up to 10 -12 mol / L (M) or less, preferably 10 -7 Up to 10 -12 M or less, more preferably 10 -8 Up to 10 -12 The dissociation constant (KD) of M binds to its antigen, i.e. the binding constant (KA) is 10 5 Up to 10 12 M -1 or greater, preferably 10 7 Up to 10 12 M -1 or greater, more preferably 10 8 Up to 1012 M -1 .

[0115] Typically, any value greater than 10 -4 The KD value of M (or any value less than 10 4 M -1 KA values ​​of 0.1% or less were considered to indicate nonspecific binding.

[0116] Preferably, the anti-TK1 antibodies or fragments of the embodiments will bind to the serum form of human TK1 with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 5 nM.

[0117] Specific binding of an antibody or fragment thereof to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competition assays, and different variants thereof known per se in the art.

[0118] In one embodiment, the kit is a sandwich assay kit. In a specific embodiment, the kit is an ELISA kit, preferably a sandwich ELISA.

[0119] In the following discussion, it is assumed that the first anti-TK1 antibody or the first fragment is a capture antibody, and the second anti-TK1 antibody or the second fragment serves as a detection antibody. However, the embodiment is not limited thereto, and the capture antibody and the detection antibody may be switched.

[0120] Sandwich ELISA can be used to detect TK1 protein in a biological sample by preparing the surface of a support (e.g., a solid support) to which a first anti-TK1 antibody is bound as a so-called capture antibody. In a preferred embodiment, a known amount of the first anti-TK1 antibody is bound to the surface of the support. Any non-specific binding sites on the surface are optionally but preferably blocked. The biological sample is then applied to the surface so that any TK1 protein present therein will be captured by the immobilized first anti-TK1 antibody. Unbound material is preferably removed by one or more washing steps. A second anti-TK1 antibody, usually denoted a detection antibody, is then added and allowed to bind to any TK1 protein captured by the first anti-TK1 antibody.

[0121] The amount of bound second anti-TK1 antibody is then determined by direct or indirect detection methods. For example, a label or enzyme can be directly attached to the second anti-TK1 antibody, or indirectly attached by a linkage, such as a biotin-streptavidin or biotin-avidin linkage. Alternatively, a second antibody that is labeled or attached to an enzyme and specifically binds to the second anti-TK1 antibody can be used.

[0122] Thus, in one embodiment, the second anti-TK1 antibody has covalently attached biotin. Alternatively, the second anti-TK1 antibody has covalently attached streptavidin or avidin.

[0123] The kit preferably also includes horseradish peroxidase (HRP)-labeled streptavidin or HRP-labeled avidin. Alternatively, the kit also includes HRP-labeled biotin. The kit also includes an HRP substrate, such as 3,3',5,5'-tetramethylbenzidine (TMB) substrate, 3,3'-diaminobenzidine (DAB) substrate or 2,2'-azidobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate. In this case, the level of TK1 protein in the sample can be determined by spectrophotometry, which detects that HRP converts the chromogenic substrate into a detectable colored product.

[0124] In one embodiment, the kit further comprises a microtiter plate (MCP) as a support on which the first anti-TK1 antibody is immobilized or intended to be immobilized.

[0125] A suitable ELISA kit that may be used in accordance with the embodiments is the AroCell TK 210 ELISA produced by AroCell AB, Uppsala, Sweden.

[0126] The kit does not necessarily have to be an ELISA kit. In another embodiment, the kit uses affinity chromatography, wherein the first anti-TK1 antibody is bound to a stationary phase, such as a gel matrix or beads in a column. For example, the gel matrix or beads can be made of agarose, such as

[0127] In this case, the TK1 protein present in the biological sample will be trapped in the column by binding to the immobilized first anti-TK1 antibody. After washing, the bound TK1 protein can be eluted and detected using a second anti-TK1 antibody. For example, the amount of eluted TK1 protein can be determined using Western blotting and a second anti-TK1 antibody for TK1 detection using direct or indirect detection methods.

[0128] The support may also be magnetic beads, e.g. Magnetic beads.

[0129] The kit does not necessarily have to contain two anti-TK1 antibodies, but may contain only one type of anti-TK1 antibody.

[0130] Furthermore, the kit does not necessarily have to contain a so-called capture anti-TK1 antibody. In sharp contrast, a plurality, ie at least two different anti-TK1 antibodies, can be used to determine the level of a TK1 substance without immobilizing at least one anti-TK1 antibody.

[0131] In one embodiment, the patient is a human patient, preferably an adult patient.

[0132] In one embodiment, the method further comprises selecting a treatment for the patient based on the estimated likelihood, diagnosis, or classification obtained based on the determined level of TK1 substance in a body sample from the patient.

[0133] Therefore, the selection of the best or at least appropriate treatment for the patient is based on the determined level of TK1 substance in the body sample, and thereby on whether the patient's respiratory infection is predicted to be caused by Mycoplasma pneumonia, i.e. whether the patient is likely to suffer from Mycoplasma pneumonia, or caused by a virus or other bacteria, i.e. whether the patient is likely to suffer from viral pneumonia or bacterial pneumonia caused by bacteria other than Mycoplasma pneumonia. This means that patients with high determined levels of TK1 substance and therefore predicted to suffer from Mycoplasma pneumonia can choose a treatment suitable for combating Mycoplasma pneumonia infection, while patients with relatively low levels of TK1 substance can choose a treatment suitable for combating viral or other bacterial infections. Treatments suitable for Mycoplasma pneumonia include antibiotics that target bacterial rRNA in ribosomal complexes, including macrolides, tetracyclines, ketolides and fluoroquinolones. The difficulty in eradicating Mycoplasma pneumonia infection is due to the ability of the bacterium to persist in an individual and the lack of a cell wall in Mycoplasma pneumonia, which makes many antibiotics that target the bacterial cell wall ineffective in treating the infection. Thus, M. pneumoniae exhibits resistance to antimicrobial agents such as β-lactams, glycopeptides, sulfonamides, trimethoprim, polymyxin, nalidixic acid, and rifampicin.

[0134] As previously described, the TK1 protein assay according to the embodiments can be combined with at least one other biomarker of Mycoplasma pneumoniae, including but not limited to CRP, PCT, calprotectin, and B-HNL and / or P-HNL. The method may then include at least one additional step of determining the level of CRP, PCT, calprotectin, and / or B-HNL and / or P-HNL in a body sample obtained from the subject. In such embodiments, estimating the likelihood includes estimating the likelihood that the respiratory infection is Mycoplasma pneumoniae caused by Mycoplasma pneumoniae based on the determined TK1 substance level and the determined CRP, PCT, calprotectin, and / or B-HNL and / or P-HNL levels. Accordingly, in such embodiments, diagnosing the subject includes diagnosing the subject as having Mycoplasma pneumoniae based on the determined TK1 substance level and the determined CRP, PCT, calprotectin, and / or B-HNL and / or P-HNL levels.

[0135] Example

[0136] Example 1

[0137] The sandwich AroCell TK 210 ELISA, produced by AroCell AB, Uppsala, Sweden, was used as an alternative method for the determination of TK1 activity. This ELISA is a quantitative enzyme immunoassay based on the sequential addition of sample, biotinylated anti-TK1 monoclonal antibody, streptavidin-labeled enzyme conjugate, and substrate to microtiter wells coated with another monoclonal anti-TK1 antibody. [4] .

[0138] The aim of this study was to investigate whether acute infection with Mycoplasma pneumoniae, more specifically lower respiratory tract infection, alters serum TK1 protein concentrations compared to those caused by other viral and bacterial infections. Inclusion criteria for patients in the study were fever >38°C and signs and symptoms of acute infection. One control group consisted of 144 apparently uninfected healthy subjects and a second control group consisted of 98 patients with a clinical diagnosis of bacterial infection. Lower respiratory tract infection was verified by lung X-ray examination as well as results of CRP, white blood cell count and, in some cases, microbiological tests. In the pneumonia group, the diagnosis was confirmed by a positive chest X-ray and supported by a positive polymerase chain reaction (PCT) test for Mycoplasma pneumoniae in lower respiratory tract samples. [5] .

[0139] The results clearly showed that serum TK1 protein levels were significantly increased in patients with acute M. pneumoniae infection compared to healthy subjects and subjects with acute viral infection and lower respiratory tract infection caused by bacteria. Therefore, these results suggest that TK1 protein determination is a valuable biomarker to supplement or replace other biomarkers, such as CRP and B-HNL, for difficult-to-diagnose M. pneumoniae infection.

[0140] Materials and methods

[0141] Patients and diagnosis of different forms of infection

[0142] 162 patients with acute infection symptoms were clinically diagnosed with bacterial (n = 98) or viral (n = 64) infection. The 98 patients with clinical diagnosis of bacterial infection included 55 women (median age, 37 years; range, 18 to 84 years) and 43 men (median age, 48 years; range, 20 to 90 years).

[0143] Inclusion criteria were a fever of at least 38°C and signs and symptoms of acute respiratory tract infection. In addition to clinical findings, the judges knew the CRP, white blood cell count, X-ray results, and in some cases, microbiological test results. Based on this information, the patient was diagnosed with a bacterial or viral source of infection, i.e., a clinical diagnosis of infection. Lower respiratory tract infection was confirmed by chest X-ray. Exclusion criteria were known chronic viral infections, such as human immunodeficiency virus (HIV) infection.

[0144] AroCell TK 210 ELISA Procedure

[0145] TK1 protein levels in serum samples were measured using the AroCell TK 210 ELISA according to the manufacturer's instructions. [4,6] . In brief, serum samples, calibrators, and controls were pre-incubated with sample dilution buffer (SDB) at room temperature for 1 hour. The antibody pre-coated plates were washed with wash buffer before adding the incubated serum samples, calibrators, and controls. After incubation on a shaking platform for 2 hours, the plates were washed 4 times with wash buffer. The plates were then incubated with biotinylated anti-TK1 antibodies on a shaking platform for 1 hour. After another wash cycle, the plates were incubated with enzyme-labeled streptavidin for 30 minutes. After the last wash, substrate solution was added and the color intensity was measured at 450nM by a spectrophotometer. Serum TK1 protein levels in serum samples were calculated using calibrators as standards and SDB as blanks with the 4-PL curve fitting program. Each sample was analyzed in duplicate and the mean value was expressed as μg / L.

[0146] Statistical methods

[0147] Data are presented as median and interquartile range. Group comparisons were performed by the nonparametric Mann-Whitney test for independent groups. To evaluate the clinical performance of the biomarker assay, receiver operating characteristic (ROC) analysis was performed, and comparison of the area under the curve (AUC) was analyzed by c-statistics.

[0148] For statistical calculations, MedCalc statistical software version 16.4.3 (MedCalc Software bvba, Ostend, Belgium [https: / / www.medcalc.org]; 2016) was used.

[0149] result

[0150] Figure 1The concentrations of TK1 protein in healthy subjects and patients with bacterial, viral or M. pneumoniae infections are shown. In pairwise comparisons between the healthy and patient groups, significant differences were observed with a P value < 0.0001. When the patient group was restricted to patients with upper respiratory tract infections caused by bacteria or M. pneumoniae, the differences between the two groups remained highly significant for both healthy and bacterially caused pneumonia, see Figure 2 .

[0151] ROC curve analysis was performed to evaluate the potential clinical performance of the TK1 biomarker in distinguishing the M. pneumoniae patient group from the healthy control group. Figure 3 The area under the curve (AUC) value was 0.98, and the sensitivity and specificity were 95% and 94%, respectively.

[0152] When the M. pneumoniae group was compared with patients with acute bacterial infection, the AUC value was 0.84, with a sensitivity and specificity of 64% and 90%, respectively. Figure 4 .

[0153] Figure 5 The ROC curve analysis of the M. pneumoniae group compared with patients with acute infections caused by viral agents is shown. In this experiment, the AUC value was 0.77, with a sensitivity of 68% and a specificity of 81%. In contrast, when the M. pneumoniae group was compared with patients with bacterial lower respiratory tract infections, the AUC value was 0.81, with a sensitivity and specificity of 91% and 68%, respectively, see Figure 6 .

[0154] CRP and procalcitonin (PCT) as well as neutrophil counts have been used as biomarkers in patients with bacterial infections. Figure 7 In the present study, ROC curve analysis was presented in which these biomarkers were compared with TK1. From these results, it can be clearly seen that TK1 is superior to any other biomarker in distinguishing between bacterial and M. pneumoniae infections, with significant differences ranging from P values ​​of <0.02 to <0.0002.

[0155] The mechanism leading to the upregulation of serum TK1 in M. pneumoniae infection is unclear. M. pneumoniae is a wall-less bacterium and therefore lacks lipopolysaccharide antigens. Therefore, the mechanism is different from that of most other bacteria. M. pneumoniae has been shown to induce ATP efflux from host cells, which can activate inflammasomes through the P2X7 receptor and subsequently secrete IL-1β. Other studies have also shown that cellular adhesion of M. pneumoniae to host cells is closely associated with the induction of inflammatory responses. Therefore, there is evidence that M. pneumoniae induces host responses by a mechanism that is different from other bacteria.

[0156] The results are shown in Figures 1 to 7In the study, it was strongly suggested that serum TK1 protein determination could serve as a sensitive blood biomarker for distinguishing M. pneumoniae infection from other causes of infection, such as viral or bacterial pathogens. TK1 was clearly superior to CRP, PCT and neutrophil count as a biomarker. Therefore, the use of TK1 concentration as a biomarker, especially in the setting of lower respiratory tract diseases, could significantly improve in vitro diagnostics for a large number of patients seeking primary healthcare.

[0157] Example 2

[0158] Correctly diagnosing acute infections with bacteria, mycoplasmas, or viruses is a clinical challenge that has a great impact on treatment decisions. Current diagnostic tests for respiratory Mycoplasma pneumoniae infections, such as PCR and serology, are either somewhat unreliable or slow and do not fully meet the clinical needs for accurate and rapid diagnosis. The purpose of this example is to examine a panel of candidate biomarkers and their ability to distinguish Mycoplasma pneumoniae respiratory infections from respiratory infections caused by bacteria or viruses.

[0159] Materials and methods

[0160] The study group included 144 healthy controls with a mean age of 43.6±12.8 years, including 57 males (age 41.3±12.7 years) and 87 females (age 45.0±12.8 years); and 156 patients with confirmed etiology of acute respiratory tract infection. Inclusion criteria were fever >38°C and signs and symptoms of acute respiratory tract infection. Clinical diagnosis of the cause of infection was supplemented by objective microbiological / serological tests. Blood was drawn before the start of antibiotic treatment. The study was approved by the Uppsala Ethics Committee.

[0161] Of these 156 patients, 86 had bacterial infections, male n=41 (54 years, IQ range 41-71), female n=45 (43 years, IQ range 29-66); 42 had viral infections, male n=23 (49 years, IQ range 39-73), female n=19 (40 years, IQ range 25-57); and 28 had Mycoplasma pneumoniae infections, male n=14 (age 42.5 years, IQ range 34-61) and female n=14 (age 36.5 years, IQ range 22-44). The estimated duration of mycoplasma infection was 168 hours, 130-196 hours (median and IQ range).

[0162] Biomarker testing

[0163] HNL was measured in EDTA plasma (Diagnostics Development, Uppsala, Sweden) by the HNL assay, which was configured to specifically capture dimers of the molecule, subsequently referred to as P-HNL dimers. Calprotectin was measured in EDTA plasma (Gentian AS, Norway). PCT (Thermo Fisher Scientific Frederick, MD, USA) and TK1 (AroCell AB, Uppsala, Sweden) were measured in serum. All biomarkers were run according to the manufacturer's instructions. For all assays, the imprecision of replicate samples was between 4-10% CV.

[0164] Statistics

[0165] Data are expressed as median and interquartile range or full range as indicated. Group comparisons were performed by nonparametric Mann-Whitney test or Kruskal-Wallis analysis of variance for independent groups. The clinical performance of biomarker testing was evaluated by receiver operating characteristic (ROC) analysis. The diagnostic performance of the biomarker combination was expressed as the area under the ROC curve (AuROC) and calculated by logistic regression analysis. For the calculation of statistical data, MedCalc statistical software version 19.1.5 was used (MedCalc Software bvba, Ostend, Belgium; http: / / www.medcalc.org; 2020).

[0166] result

[0167] Fig. 9A and 9B The distribution of TK1 and HNL dimer in plasma / serum in patients with acute respiratory tract infection caused by M. pneumoniae or bacteria is shown. S-TK1 was significantly increased in patients with M. pneumoniae infection compared with the findings in healthy subjects. The concentration of S-TK1 was increased (p<0.001), while the concentration of P-HNL dimer was significantly decreased (p<0.001) in M. pneumoniae infection compared with bacterial infection.

[0168] ROC curve analysis was used to estimate the difference between respiratory tract infections caused by Mycoplasma pneumoniae and those caused by bacteria. Figure 8 As shown, all biomarkers showed significant differences. The AUCs for P-HNL dimer and S-TK1 were 0.82 and 0.79, respectively.

[0169] Based on the above results, ratios were constructed between the selected biomarkers. For distinguishing M. pneumoniae from bacterial respiratory infections, the ratios between the results for TK1 and HNL dimer in serum and plasma, respectively, increased the AUC of these biomarkers to 0.95.

[0170] Clinical diagnosis of mycoplasma pneumonia from pneumonia caused by bacteria is a challenge and currently relies on clinical signs and symptoms, PCR-based diagnosis and serology. Mild mycoplasma pneumonia may develop into severe disease, so early detection and treatment are important. In this example, the results of different selected blood biomarkers are presented together with their ability to distinguish mycoplasma pneumonia from lower respiratory tract infections caused by bacteria. In the distinction from bacterial infections, the dimer forms of TK1 and HNL seem to be particularly interesting. The concentration of TK1 is increased in Mycoplasma pneumonia infection, which increases their diagnostic performance. HNL dimers are lower in Mycoplasma pneumonia compared to bacterial infections. However, in the logistic regression analysis, both biomarkers significantly increased the diagnostic performance, resulting in a very high AUC (0.95) and an 88% percentage of correctly classified patients. The ratio between TK1 and P-HNL dimers appears to be a superior diagnostic ratio for distinguishing Mycoplasma pneumonia from bacterial infections, and is consistent with the results of logistic regression.

[0171] The above embodiments are to be understood as several illustrative examples of the present invention. It will be appreciated by those skilled in the art that various modifications, combinations and variations may be made to the embodiments without departing from the scope of the present invention. In particular, different partial solutions in different embodiments may be combined in other configurations where technically possible.

[0172] References

[0173] [1]Gronowitz et al., Application of an in vitro assay for serum thymidinekinase:results on viral disease and malignancies in humans. Int J Cancer.1984,33:5-12

[0174] [2]Svobodova et al., Prognostic importance of Thymidine kinase incolorectal and breast cancer. Anticancer Research 2007,27:1907-1910

[0175] [3]Topolcan and Holubec, The role of thymidine kinase in cancerdisease. Expert Opin Med. Diagn. 2008, 2: 129-141

[0176] [4] Kumar et al., Aclinical evaluation of the TK 210ELISA in sera from breast cancer patients demonstrates high sensitivity and specificity in all stages of disease. Tumour Biol 2016,37:11937-11945

[0177] [5]Venge et al., Human Neutrophil Lipocalin as a Superior DiagnosticMeans To Distinguish between Acute Bacterial and Viral Infections. ClinVaccine Immunol. 2015,22:1025-1032

[0178] [6] AroCell TK 210 ELISA, Thymidine Kinase 1 (TK1), Enzyme Linked ImmunoSorbet assay, Instructions for Use, 31-11TF710B1-04, Issue date May 19, 2017, www.e-labeling.eu / ARO1001-15-4. Sequence Listing <110> arosel <120> Respiratory infection detection and classification <130> HSJ104088P.WOP <150> US 62 / 843,652 <151> 2019-05-06 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <400> 1 Gly Glu Ala Val Ala Ala Arg Lys Leu Phe 1 5 10 <210> 2 <211> 10 <212> PRT <213> Homo sapiens <400> 2 Asn Cys Pro Val Pro Gly Lys Pro Gly Glu 1 5 10 <210> 3 <211> 10 <212> PRT <213> Homo sapiens <400> 3 Pro Val Pro Gly Lys Pro Gly Glu Ala Val 1 5 10 <210> 4 <211> 12 <212> PRT <213> Homo sapiens <400> 4 Asn Cys Pro Val Pro Gly Lys Pro Gly Glu Ala Val 1 5 10 <210> 5 <211> 5 <212> PRT <213> Artificial sequence <220> <223> VH CDR regions of monoclonal anti-TK1 antibodies <400> 5 Asp Tyr Glu Met His 1 5 <210> 6 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR regions of monoclonal anti-TK1 antibodies <400> 6 Ala Ile His Pro Gly Tyr Gly Gly Thr Ala Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 7 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VH CDR regions of monoclonal anti-TK1 antibodies <400> 7 Phe Ile Thr Lys Phe Asp Tyr 1 5 <210> 8 <211> 16 <212> PRT <213> Artificial sequence <220> <223> VL CDR regions of monoclonal anti-TK1 antibodies <400> 8 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Phe Leu Asn 1 5 10 15 <210> 9 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR regions of monoclonal anti-TK1 antibodies <400> 9 Leu Val Ser Lys Leu Asp Ser 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR regions of monoclonal anti-TK1 antibodies <400> 10 Trp Gln Gly Thr His Phe Pro Trp Thr 1 5 <210> 11 <211> 6 <212> PRT <213> Artificial sequence <220> <223> VH CDR regions of monoclonal anti-TK1 antibodies <400> 11 Ser Gly Tyr Ser Trp His 1 5 <210> 12 <211> 16 <212> PRT <213> Artificial sequence <220> <223> VH CDR regions of monoclonal anti-TK1 antibodies <400> 12 Tyr Ile His Tyr Ser Gly Ser Thr Thr Tyr Asn Pro Ser Leu Lys Gly 1 5 10 15 <210> 13 <211> 10 <212> PRT <213> Artificial sequence <220> <223> VH CDR regions of monoclonal anti-TK1 antibodies <400> 13 Trp Gly Thr Gly His Trp Tyr Phe Asp Val 1 5 10 <210> 14 <211> 14 <212> PRT <213> Artificial sequence <220> <223> VL CDR regions of monoclonal anti-TK1 antibodies <400> 14 Arg Ser Ser Thr Gly Ala Val Thr Thr Thr Asn Tyr Ala Asn 1 5 10 <210> 15 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VL CDR regions of monoclonal anti-TK1 antibodies <400> 15 Gly Thr Asn Asn Arg Val Pro 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial sequence <220> <223> VL CDR regions of monoclonal anti-TK1 antibodies <400> 16 Ala Leu Trp Tyr Ser Asn His Trp Val 1 5 <210> 17 <211> 17 <212> PRT <213> Artificial sequence <220> <223> VH CDR regions of monoclonal anti-TK1 antibodies <400> 17 Ala Ile Leu Pro Gly Ser Gly Gly Thr Ala Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 18 <211> 7 <212> PRT <213> Artificial sequence <220> <223> VH CDR regions of monoclonal anti-TK1 antibodies <400> 18 Leu Ile Thr Thr Phe Asp Tyr 1 5 <210> 19 <211> 16 <212> PRT <213> Artificial sequence <220> <223> VL CDR regions of monoclonal anti-TK1 antibodies <400> 19 Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn 1 5 10 15 <210> 20 <211> 15 <212> PRT <213> Homo sapiens <400> 20 Lys Pro Gly Glu Ala Val Ala Ala Arg Lys Leu Phe Ala Pro Gln 1 5 10 15 <210> twenty one <211> twenty three <212> PRT <213> Homo sapiens <400> twenty one Ala Tyr Thr Lys Arg Leu Gly Thr Glu Lys Glu Val Glu Val Ile Gly 1 5 10 15 Gly Ala Asp Lys Tyr His Ser 20 <210> twenty two <211> 185 <212> PRT <213> Artificial sequence <220> <223> VH domain of anti-TK1 antibody <400> twenty two Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Glu Gln Leu Glu Glu Ser Gly Gly Asp Leu Val Lys 20 25 30 Pro Glu Gly Ser Leu Thr Leu Thr Cys Thr Ala Ser Arg Phe Ser Phe 35 40 45 Ser Ser Ser Tyr Trp Ile Cys Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60 Leu Glu Trp Ile Ala Cys Ile Tyr Ala Gly Asp Ser Gly Ser Ser Tyr 65 70 75 80 Tyr Ala Ser Trp Ala Lys Gly Arg Phe Thr Val Ser Lys Thr Ser Ser 85 90 95 Thr Thr Val Thr Leu Gln Thr Thr Ser Leu Thr Ala Ala Asp Thr Ala 100 105 110 Thr Tyr Phe Cys Ala Arg Ala Ser Val Gly Ala Ala Tyr Asp Tyr Phe 115 120 125 Ala Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro 130 135 140 Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro 145 150 155 160 Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu 165 170 175 Pro Val Thr Val Thr Trp Asn Ser Gly 180 185 <210> 23 <211> 236 <212> PRT <213> Artificial sequence <220> <223> VL domain of anti-TK1 antibody <400> 23 Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Leu Val Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Ala Ala Met Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Glu Asp Val Ser Ser His Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln 50 55 60 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asp Leu Ala Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Thr Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Ala 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gly 100 105 110 Tyr Tyr Tyr Ile Ser Asp Ser Pro Tyr Val Phe Gly Gly Gly Thr Glu 115 120 125 Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro 130 135 140 Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val 145 150 155 160 Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly 165 170 175 Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser 180 185 190 Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr 195 200 205 Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr 210 215 220 Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225 230 235 <210> 24 <211> 187 <212> PRT <213> Artificial Sequence <220> <223> VH domain of anti-TK1 antibody <400> 24 Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro 20 25 30 Glu Gly Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Phe Ser 35 40 45 Ser Gly Tyr Asp Met Cys Trp Val Arg Gln Thr Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Ala Cys Ile Ser Val Asp Ser Asp Gly Val Thr Tyr Tyr 65 70 75 80 Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr 85 90 95 Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr Ala Thr 100 105 110 Tyr Phe Cys Ala Arg Gly Tyr Glu Ser Ser Ser Gly Val Tyr Ile Pro 115 120 125 Tyr Phe Thr Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly 130 135 140 Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly Asp 145 150 155 160 Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu 165 170 175 Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly 180 185 <210> 25 <211> 238 <212> PRT <213> Artificial sequence <220> <223> VL domain of anti-TK1 antibody <400> 25 Met Asp Met Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Ala Asp Ile Val Leu Thr Gln Thr Pro Ala 20 25 30 Ser Val Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala 35 40 45 Ser Gln Ser Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln His Lys Pro Gly 50 55 60 Gln Pro Pro Lys Leu Leu Ile Tyr Lys Ala Ser Thr Leu Ala Ser Gly 65 70 75 80 Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu 85 90 95 Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln 100 105 110 His Tyr Tyr Tyr Ser Ser Thr Ser Gly Gly Gly Val Phe Gly Gly Gly 115 120 125 Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile 130 135 140 Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val 145 150 155 160 Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val 165 170 175 Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln 180 185 190 Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr 195 200 205 Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln 210 215 220 Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225 230 235 <210> 26 <211> 179 <212> PRT <213> Artificial Sequence <220> <223> VH domain of anti-TK1 antibody <400> 26 Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro 20 25 30 Gly Thr Pro Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser 35 40 45 Asn Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 50 55 60 Trp Ile Gly Ile Ile Tyr Gly Asp Asp Asn Thr Tyr Cys Ala Asn Trp 65 70 75 80 Thr Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu 85 90 95 Thr Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala 100 105 110 Arg Gly Pro Asp Tyr Ile Ala Ala Lys Met Asp Ile Trp Gly Pro Gly 115 120 125 Thr Leu Val Thr Val Ser Leu Gly Gln Pro Lys Ala Pro Ser Val Phe 130 135 140 Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu 145 150 155 160 Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp 165 170 175 Asn Ser Gly <210> 27 <211> 239 <212> PRT <213> artificial sequence <220> <223> VL structure of anti-TK1 antibody <400> 27 Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Leu Pro Gly Ala Arg Cys Asp Val Val Met Thr Gln Thr Pro Ala Ser 20 25 30 Val Glu Ala Ala Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser 35 40 45 Gln Ser Ile Ser Gly Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 50 55 60 Arg Pro Lys Leu Leu Ile Tyr Arg Ala Ser Thr Leu Glu Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys 100 105 110 Thr Tyr Gly Ser Ser Thr Phe Ser Ser Tyr Gly Asn Ala Phe Gly Gly 115 120 125 Gly Thr Glu Val Val Val Lys Gly Asp Pro Val Ala Pro Thr Val Leu 130 135 140 Ile Phe Pro Pro Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile 145 150 155 160 Val Cys Val Ala Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu 165 170 175 Val Asp Gly Thr Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro 180 185 190 Gln Asn Ser Ala Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu 195 200 205 Thr Ser Thr Gln Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr 210 215 220 Gln Gly Thr Thr Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys 225 230 235 <210> 28 <211> 234 <212> PRT <213> Homo sapiens <400> 28 Met Ser Cys Ile Asn Leu Pro Thr Val Leu Pro Gly Ser Pro Ser Lys 1 5 10 15 Thr Arg Gly Gln Ile Gln Val Ile Leu Gly Pro Met Phe Ser Gly Lys 20 25 30 Serving Three Glu With Arg Arg Val Arg Arg Phe Gln Ile Ala Gln Tyr 35 40 45 Lys Cys Leu Val Ile Lys Tyr Ala Lys Asp Thr Arg Tyr Ser Ser Ser Ser 50 55 60 Phe Cys Thr His Asp Arg Asn Thr Met Glu Ala Leu Pro Ala Cys Leu 65 70 75 80 Leu Arg Asp Val Val Gln Glu Al Leu Gly Val Val Ile Gly Ile 85 90 95 Asp Glu Gly Gln Phe Phe Pro Asp Ile Val Glu Phe Cys Glu Ala Met 100 105 110 Ala Asn Ala Gly Lys Thr Val Ile Val Ala Ala Leu Asp Gly Thr Phe 115 120 125 Gln Arg Lys Pro Phe Gly Ala Ile Leu Asn Leu Val Pro Leu Ala Glu 130 135 140 Ser Val Val Lys Leu Thr Ala Val Cys Met Glu Cys Phe Arg Glu Ala 145 150 155 160 Only Tyr Thr Lys Arg Leu Gly Thr Glu Lys Glu Val Glu Val Ile Gly 165 170 175 Gly Ala Asp Lys Tyr His Ser Val Cys Arg Leu Cys Tyr Phe Lys Lys 180 185 190 Ala Ser Gly Gln Pro Ala Gly Pro Asp Asn Lys Glu Asn Cys Pro Val 195 200 205 Pro Gly Lys Pro Gly Glu Ala Val Ala Ala Arg Lys Leu Phe Ala Pro 210 215 220 Gln Gln Ile Leu Gln Cys Ser Pro Ala Asn 225 230

Claims

1. Use of an anti-TK1 antibody in the preparation of a kit for predicting the presence of mycoplasma pneumonia in a subject with respiratory tract infection, the use comprising: determining the level of TK1 substance in a body fluid sample obtained from the subject using the anti-TK1 antibody, wherein the body fluid sample is selected from the group consisting of a serum sample, a plasma sample, and a blood sample; as well as The possibility that the respiratory tract infection is mycoplasma pneumonia caused by Mycoplasma pneumonia is estimated based on the determined level of the TK1 substance in the body fluid sample.

2. The use according to claim 1, further comprising comparing the determined level of TK1 substance in the body fluid sample with a threshold value, wherein estimating the likelihood comprises estimating the likelihood that the respiratory tract infection is mycoplasma pneumonia caused by Mycoplasma pneumoniae based on the comparison.

3. The use according to claim 2, wherein estimating the likelihood comprises: If the determined level of the TK1 substance in the body fluid sample exceeds the threshold value, it is estimated that the respiratory tract infection is likely to be mycoplasma pneumonia caused by Mycoplasma pneumoniae with a high probability, otherwise it is estimated that the respiratory tract infection is likely to be mycoplasma pneumonia caused by Mycoplasma pneumoniae with a low probability.

4. The use according to claim 1, wherein the anti-TK1 antibody is used together with at least one other biomarker to prepare the kit, wherein the at least one other biomarker is selected from the group consisting of C-reactive protein (CRP), procalcitonin (PCT), calprotectin and human neutrophil lipocalin (HNL), and the use further comprises: determining the level of at least one other biomarker in the bodily fluid sample or another bodily fluid sample obtained from the subject, wherein Estimating the likelihood includes a likelihood that the respiratory tract infection is mycoplasma pneumonia caused by Mycoplasma pneumonia based on the determined level of TK1 substance in the body fluid sample and based on the determined level of the at least one other biomarker in the body fluid sample or the other body fluid sample.

5. Use of an anti-TK1 antibody in the preparation of a kit for diagnosing mycoplasma pneumonia in a subject, the use comprising: determining the level of TK1 substance in a body fluid sample obtained from the subject using the anti-TK1 antibody, wherein the body fluid sample is selected from the group consisting of a serum sample, a plasma sample, and a blood sample; as well as The subject is diagnosed with Mycoplasma pneumonia based on the determined level of TK1 substance in the body fluid sample. 6 . The use according to claim 5 , further comprising comparing the determined level of TK1 substance in the body fluid sample with a threshold value, wherein diagnosing the subject comprises diagnosing the subject as having Mycoplasma pneumonia based on the comparison.

7. The use according to claim 6, wherein diagnosing the subject comprises: If the determined level of TK1 substance in the body fluid sample exceeds the threshold value, the subject is diagnosed with Mycoplasma pneumonia.

8. The use according to claim 5, wherein the anti-TK1 antibody is used together with at least one other biomarker to prepare the kit, wherein the at least one other biomarker is selected from the group consisting of C-reactive protein (CRP), procalcitonin (PCT), calprotectin and human neutrophil lipocalin (HNL), and the use comprises: determining the level of at least one other biomarker in the bodily fluid sample or another bodily fluid sample obtained from the subject, wherein Diagnosing the subject comprises: diagnosing the subject as having Mycoplasma pneumonia based on the determined level of TK1 substance in the body fluid sample and based on the determined level of the at least one other biomarker in the body fluid sample or the other body fluid sample.

9. Use of an anti-TK1 antibody in the preparation of a kit for classifying a respiratory tract infection in a subject, the use comprising: determining the level of TK1 substance in a body fluid sample obtained from the subject using the anti-TK1 antibody, wherein the body fluid sample is selected from the group consisting of a serum sample, a plasma sample, and a blood sample; and Based on the determined level of TK1 substance in the body fluid sample, the respiratory tract infection is classified into 1) mycoplasma pneumonia caused by Mycoplasma pneumoniae, or 2) viral pneumonia caused by a virus and bacterial pneumonia caused by bacteria other than Mycoplasma pneumoniae.

10. The use according to claim 9, wherein the anti-TK1 antibody is used together with at least one other biomarker to prepare the kit, wherein the at least one other biomarker is selected from the group consisting of C-reactive protein (CRP), procalcitonin (PCT), calprotectin and human neutrophil lipocalin (HNL), and the use comprises: determining the level of at least one other biomarker in the bodily fluid sample or another bodily fluid sample obtained from the subject, wherein Classifying the respiratory tract infection includes: classifying the respiratory tract infection as mycoplasma pneumonia caused by Mycoplasma pneumonia based on the determined level of TK1 substance in the body fluid sample and based on the determined level of at least one other biomarker in the body fluid sample or the other body fluid sample, or classifying the respiratory tract infection into viral pneumonia caused by a virus and bacterial pneumonia caused by bacteria other than Mycoplasma pneumonia.

11. The use according to claim 9, further comprising: determining the level of human neutrophil lipocalin (HNL) in the body fluid sample or another body fluid sample obtained from the subject; as well as calculating a quotient between the determined level of TK1 substance in the body fluid sample and the determined level of HNL in the body fluid sample or the further body fluid sample, wherein, Classifying the respiratory tract infection includes: if the quotient exceeds a threshold, classifying the respiratory tract infection as mycoplasma pneumonia caused by Mycoplasma pneumoniae, otherwise classifying the respiratory tract infection as bacterial pneumonia caused by bacteria other than Mycoplasma pneumoniae.

12. Use of an anti-TK1 antibody in the preparation of a kit for classifying a respiratory tract infection in a subject, the use comprising: determining the level of TK1 substance in a body fluid sample obtained from the subject using the anti-TK1 antibody, wherein the body fluid sample is selected from the group consisting of a serum sample, a plasma sample, and a blood sample; and If the determined level of the TK1 substance in the body fluid sample exceeds a threshold value, the respiratory tract infection is classified as 1) mycoplasma pneumonia caused by Mycoplasma pneumoniae, otherwise it is classified as 2) viral pneumonia caused by a virus and bacterial pneumonia caused by bacteria other than Mycoplasma pneumoniae.

13. The use according to claim 12, wherein classifying the respiratory tract infection comprises: If the determined level of the TK1 substance in the body fluid sample exceeds the threshold value, the respiratory tract infection is classified as mycoplasma pneumonia caused by Mycoplasma pneumoniae, otherwise the respiratory tract infection is classified as bacterial pneumonia caused by bacteria other than Mycoplasma pneumoniae.

14. The use according to any one of claims 1-3, 5-7 and 9-13, further comprising obtaining the body fluid sample from the subject.

15. The use according to claim 14, wherein determining the level of the TK1 substance comprises: The anti-TK1 antibody is used to determine the level of serum TK1 (STK1) substance in a serum or plasma sample obtained from the subject.

16. The use according to any one of claims 1-3, 5-7 and 9-13, wherein determining the level of the TK1 substance comprises: contacting the body fluid sample with the anti-TK1 antibody; as well as The amount of anti-TK1 antibody bound to the TK1 substance is determined.

17. The use according to claim 16, wherein determining the level of the TK1 substance comprises: Based on the determined amount of bound anti-TK1 antibody and a standard correlation between the amount of bound anti-TK1 antibody and the level of TK1 substance, the level of TK1 substance in the body fluid sample is determined.

18. The use according to any one of claims 1-3, 5-7 and 9-13, wherein the anti-TK1 antibody is a monoclonal anti-TK1 antibody.

19. The use according to any one of claims 1-3, 5-7 and 9-13, wherein the anti-TK1 antibody is specific to an epitope consisting of an amino acid sequence of the C-terminal region of TK1.

20. The use according to claim 19, wherein the epitope is an N-mer, wherein N is an integer in the range of 8 to 20.

21. The use according to claim 19, wherein the epitope is selected from the group consisting of GEAVAARKLF SEQ ID NO: 1, NCPVPGKPGE SEQ ID NO: 2, PVPGKPGEAV SEQ ID NO: 3 and NCPVPGKPGEAV SEQ ID NO:

4.

22. The use according to claim 19, wherein the anti-TK1 antibody is a monoclonal anti-TK1 antibody specific for the epitope consisting of GEAVAARKLF SEQ ID NO: 1 and having the following: a variable heavy (VH) domain complementarity determining region 1 (CDR1) having the amino acid sequence DYEMH SEQ ID NO: 5; a VH domain CDR2 having the amino acid sequence AIHPGYGGTAYNQKFKG SEQ ID NO: 6; a VH domain CDR3 having the amino acid sequence FITKFDY SEQ ID NO: 7; a variable light (VL) domain CDR1 having the amino acid sequence KSSQSLLDSDGKTFLN SEQ ID NO: 8; a VL domain CDR2 having the amino acid sequence LVSKLDS SEQ ID NO: 9; and A VL domain CDR3 having the amino acid sequence WQGTHFPWT SEQ ID NO:

10.

23. The use according to claim 19, wherein the anti-TK1 antibody is a monoclonal anti-TK1 antibody specific for the epitopes NCPVPGKPGE SEQ ID NO: 2, PVPGKPGEAV SEQ ID NO: 3 and NCPVPGKPGEAV SEQ ID NO: 4 and having the following: a variable heavy (VH) domain complementarity determining region 1 (CDR1) having the amino acid sequence DYEMH SEQ ID NO: 5; a VH domain CDR2 having the amino acid sequence AILPGSGGTAYNQKFKG SEQ ID NO: 17; a VH domain CDR3 having the amino acid sequence LITTFDY SEQ ID NO: 18; a variable light (VL) domain CDR1 having the amino acid sequence KSSQSLLDSDGKTYLN SEQ ID NO: 19; a VL domain CDR2 having the amino acid sequence LVSKLDS SEQ ID NO: 9; and A VL domain CDR3 having the amino acid sequence WQGTHFPWT SEQ ID NO:

10.

24. The use according to any one of claims 1-3, 5-7 and 9-13, wherein the anti-TK1 antibody is a monoclonal anti-TK1 antibody that is specific for a conformation-dependent epitope of TK1 and has: a variable heavy (VH) domain complementarity determining region 1 (CDR1) having the amino acid sequence SGYSWH SEQ ID NO: 11; a VH domain CDR2 having the amino acid sequence YIHYSGSTTYNPSLKG SEQ ID NO: 12; a VH domain CDR3 having the amino acid sequence WGTGHWYFDV SEQ ID NO: 13; a variable light (VL) domain CDR1 having the amino acid sequence RSSTGAVTTTNYAN SEQ ID NO: 14; a VL domain CDR2 having the amino acid sequence GTNNRVP SEQ ID NO: 15; and A VL domain CDR3 having the amino acid sequence ALWYSNHWV SEQ ID NO:

16.

25. The use according to any one of claims 1-3, 5-7 and 9-13, wherein determining the level of the TK1 substance comprises determining the level of the TK1 substance in the body fluid sample obtained from the subject using a kit comprising: a first anti-TK1 antibody immobilized or intended to be immobilized to a support; and Secondary anti-TK1 antibody.

26. The use according to claim 25, wherein The first anti-TK1 antibody is a monoclonal anti-TK1 antibody as defined in claim 22 or 23; and The second anti-TK1 antibody is a monoclonal anti-TK1 antibody as defined in claim 22 or 23 .

27. The use according to claim 25, wherein the kit is an enzyme-linked immunosorbent assay (ELISA) kit.

28. The use according to claim 25, wherein the kit is a sandwich ELISA kit.

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