Matrix metalloproteinase 1 monoclonal antibody

Abstract

To provide a monoclonal antibody of matrix metalloproteinase 1.SOLUTION: A monoclonal antibody has a heavy chain variable region with an amino sequence comprising i) CDR1 selected from the group consisting of SEQ ID NOs: 1, 7 and 13, ii) CDR2 selected from the group consisting of SEQ ID NOs: 2, 8 and 14, and iii) CDR3 selected from the group consisting of SEQ ID NOs: 3, 9 and 15. The monoclonal antibody also has a light chain variable region with an amino sequence comprising i) CDR1 selected from the group consisting of SEQ ID NOs: 4, 10 and 16, ii) CDR2 selected from the group consisting of SEQ ID NOs: 5, 11 and 17, and iii) CDR3 selected from the group consisting of SEQ ID NOs: 6, 12 and 18. A polynucleotide encoding the monoclonal antibody and a complementary polynucleotide sequence thereof are provided as well. A detection kit and a detection method are also provided, wherein the detection kit contains the monoclonal antibody of the matrix metalloproteinase 1.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present disclosure relates to MMP-1 and methods for detecting oral cancer, in particular to MMP-1 monoclonal antibodies, detection kits containing MMP-1 monoclonal antibodies, and detection methods using the detection kits. [Background technology]

[0002] According to World Health Organization statistics, more than 529,000 new cases of oral cancer are diagnosed each year. Due to rising incidence rates, the number of cases is expected to reach 856,000 per year by 2035. In Taiwan, according to 2016 statistics on causes of death from the National Health Administration, Ministry of Health and Welfare (NHHA), oral, oropharyngeal, and hypopharyngeal cancers were the fourth leading cause of death among all malignant tumors in men. Approximately 7,000 people are newly diagnosed with the disease each year, and approximately 3,000 people die from it. Between 2012 and 2016, the survival rates for patients with stage I–IV oral cancer were 79.9%, 71.0%, 56.5%, and 35.6%, respectively. Early detection improves survival rates.

[0003] Currently, oral mucosal screening is an important method for the clinical evaluation of oral cancer, and the results of tissue biopsies from lesions are used as the basis for diagnosis. However, oral mucosal screening currently requires specialized oral health professionals (e.g., dentists, oral hygienists, dental therapists, oral health therapists, etc.), and in many areas, the diagnosis of oral cancer is delayed due to a shortage of specialized oral health professionals. Therefore, if it were possible to analyze the presence or absence of tumor markers (biomarkers) in samples, the rate of early diagnosis of oral cancer could be improved.

[0004] Currently, there are no tumor markers for the routine identification of oral cancer. A 2016 report in the Proceedings of the National Academy of Sciences of the United States of America (PNAS) showed that matrix metalloproteinase 1 (MMP-1) was quantified by mass spectrometry and showed an 83-fold difference in MMP-1 expression between patient and control saliva samples (the differences for other molecules ranged from -1.3 to 5.5 fold), making MMP-1 the most promising tumor marker for oral cancer.

[0005] Since existing quantitative analytical methods for MMP-1 using mass spectrometry are not suitable for routine quantitative analysis of a large number of samples, the development of a detection kit for MMP-1 for oral cancer screening is anticipated. Summary of the Invention [Means for solving the problem]

[0006] Some embodiments of the present disclosure provide a monoclonal antibody comprising a heavy chain variable region sequence and a light chain variable region sequence. The heavy chain variable region sequence comprises i) a CDR1 selected from the group consisting of SEQ ID NOs: 1, 7, and 13, ii) a CDR2 selected from the group consisting of SEQ ID NOs: 2, 8, and 14, and iii) a CDR3 selected from the group consisting of SEQ ID NOs: 3, 9, and 15. The light chain variable region sequence comprises i) a CDR1 selected from the group consisting of SEQ ID NOs: 4, 10, and 16, ii) a CDR2 selected from the group consisting of SEQ ID NOs: 5, 11, and 17, and iii) a CDR3 selected from the group consisting of SEQ ID NOs: 6, 12, and 18.

[0007] In some embodiments, the heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 1, 2, and 3, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 4, 5, and 6.

[0008] In some embodiments, the heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 7, 8, and 9, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 10, 11, and 12.

[0009] In some embodiments, the heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 13, 14, and 15, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 16, 17, and 18.

[0010] Some embodiments of the present disclosure provide polynucleotides that encode the aforementioned amino acid sequences or have a sequence complementary to a nucleotide sequence encoding the aforementioned monoclonal antibodies.

[0011] Some embodiments of the present disclosure provide a detection kit. The detection kit includes monoclonal antibody A, which includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes the amino acid sequences of SEQ ID NOs: 1, 2, and 3. The light chain variable region includes the amino acid sequences of SEQ ID NOs: 4, 5, and 6.

[0012] In some embodiments, the detection kit further comprises an enzyme immunoassay reagent kit, colloidal gold immunoassay test strips, or a combination thereof.

[0013] In some embodiments, the enzyme immunoassay reagent kit comprises monoclonal antibody B. Monoclonal antibody B comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequences of SEQ ID NOs: 7, 8, and 9. The light chain variable region comprises the amino acid sequences of SEQ ID NOs: 10, 11, and 12.

[0014] In some embodiments, monoclonal antibody B is conjugated to a chromophore.

[0015] In some embodiments, the colloidal gold immunoassay test strip comprises monoclonal antibody C. Monoclonal antibody C comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises the amino acid sequences of SEQ ID NOs: 13, 14, and 15. The light chain variable region comprises the amino acid sequences of SEQ ID NOs: 16, 17, and 18.

[0016] In some embodiments, the colloidal gold immunoassay test strip further comprises monoclonal antibody A, wherein the monoclonal antibody A in the colloidal gold immunoassay test strip is linked to gold particles.

[0017] Some embodiments of the present disclosure provide a method for detecting matrix metalloproteinase 1 in vitro, which method comprises detecting matrix metalloproteinase 1 in a sample using the above-mentioned detection kit.

[0018] In some embodiments, the sample comprises a bodily fluid or blood.

[0019] In some embodiments, the bodily fluid comprises an oral secretion or a respiratory secretion.

[0020] For a clearer understanding of the foregoing and other objects, features, advantages and embodiments of the present disclosure, reference is made to the accompanying drawings as follows: [Brief explanation of the drawings]

[0021] [Figure 1A] FIG. 1 shows a comparative graph of the binding ability test of a self-produced monoclonal antibody against MMP-1 using enzyme immunoassay in one embodiment of the present disclosure. [Figure 1B] FIG. 1 shows a comparative graph of the binding ability test of a self-produced monoclonal antibody against MMP-1 using enzyme immunoassay in one embodiment of the present disclosure. [Figure 2A]FIG. 1 shows the concentration profiles in normal human saliva samples measured by enzyme immunoassay in one embodiment of the present disclosure, where four optimal pairs of monoclonal antibodies were used as capture and detection antibodies in the enzyme immunoassay, respectively. [Figure 2B] FIG. 1 shows the concentration profiles in saliva samples from oral cancer patients measured by enzyme immunoassay in one embodiment of the present disclosure, where four optimal pairs of monoclonal antibodies were used as capture and detection antibodies in the enzyme immunoassay, respectively. [Figure 3A] FIG. 1 shows a schematic diagram of a colloidal gold immunoassay test strip in one embodiment of the present disclosure. [Figure 3B] FIG. 1 shows a schematic diagram of a colorimetric card showing the grades obtained with a colloidal gold immunoassay test strip in one embodiment of the present disclosure. [Figure 4] FIG. 1 shows a scatter plot of a cross-comparison between a colloidal gold immunoassay test strip and an enzyme immunoassay reagent kit in accordance with one embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0022] To provide a detailed and complete description of the present disclosure, embodiments and specific embodiments of the present disclosure are presented with exemplary descriptions, but these are not the only modes for implementing or using the specific embodiments of the present disclosure. The embodiments disclosed herein may be advantageously combined with or substituted for one another, and one embodiment may be added to another without further description. In the following description, numerous specific details will be set forth in detail to enable a thorough understanding of the following embodiments. However, embodiments of the present disclosure may be practiced without these specific details.

[0023] As used herein, unless the context specifically dictates otherwise, the terms "indefinite article" and "definite article" may mean one or more. It will be further understood that the terms "comprise," "contain," "have," and similar terms as used herein refer to stated features, regions, integers, steps, operations, elements, and / or components, but do not exclude other features, regions, integers, steps, operations, elements, and / or components.

[0024] Although a series of operations or steps are described below to illustrate the methods disclosed herein, the order of the operations or steps is not to be construed as limiting. For example, some operations or steps may be performed in a different order and / or concurrently with other steps. Also, not all illustrated operations, steps, and / or features may be required to practice embodiments of the present disclosure. Furthermore, each of the operations and steps described herein may include multiple substeps and actions.

[0025] As used herein, a "CDR" (complementarity determining region) is a region that contacts an antigen with an antibody and is a part of the variable region of an antibody. Generally, there are three CDRs in the variable region of an antibody: CDR1, CDR2, and CDR3.

[0026] As used herein, a "derived sequence" refers to a sequence that is modified at the 3' or 5' end of a nucleotide sequence.

[0027] Some embodiments of the present disclosure provide a monoclonal antibody that recognizes matrix metalloproteinase 1 (MMP-1), comprising a heavy chain variable region sequence and a light chain variable region sequence. The heavy chain variable region sequence comprises i) a CDR1 selected from the group consisting of SEQ ID NOs: 1, 7, and 13, ii) a CDR2 selected from the group consisting of SEQ ID NOs: 2, 8, and 14, and iii) a CDR3 selected from the group consisting of SEQ ID NOs: 3, 9, and 15. The light chain variable region sequence comprises i) a CDR1 that is an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 10, and 16, ii) a CDR2 selected from the group consisting of SEQ ID NOs: 5, 11, and 17, and iii) a CDR3 selected from the group consisting of SEQ ID NOs: 6, 12, and 18.

[0028] In some embodiments, the heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 1, 2, and 3, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 4, 5, and 6. In some embodiments, the heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 7, 8, and 9, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 10, 11, and 12. In some embodiments, the heavy chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 13, 14, and 15, and the light chain variable region sequence comprises the amino acid sequence of SEQ ID NOs: 16, 17, and 18.

[0029] In some embodiments of the present disclosure, a polynucleotide is provided that encodes the amino acid sequence described above or has a sequence complementary to a nucleotide sequence encoding the monoclonal antibody described above. In some embodiments, the polynucleotide may further comprise a derivative sequence.

[0030] In some embodiments of the present disclosure, a detection kit is provided. The detection kit may be used to detect clinical samples such as body fluids (e.g., oral or respiratory secretions) or blood. The detection kit comprises monoclonal antibody A. The heavy chain variable region of monoclonal antibody A comprises the amino acid sequences of SEQ ID NOs: 1, 2, and 3, and the light chain variable region of monoclonal antibody A comprises the amino acid sequences of SEQ ID NOs: 4, 5, and 6. In some embodiments, the heavy chain variable region of monoclonal antibody A comprises the amino acid sequences of CDR1, SEQ ID NO: 1, CDR2, SEQ ID NO: 2, and CDR3, SEQ ID NO: 3, and the light chain variable region of monoclonal antibody A comprises the amino acid sequences of CDR1, SEQ ID NO: 4, CDR2, SEQ ID NO: 5, and CDR3, SEQ ID NO: 6.

[0031] In some embodiments, the detection kit comprises an enzyme immunoassay reagent kit, a colloidal gold immunoassay test strip, or a combination thereof.

[0032] In some embodiments, the enzyme immunoassay reagent kit uses a sandwich enzyme-linked immunosorbent assay (sandwich ELISA) with monoclonal antibody A as the capture antibody. In one embodiment, the immunoassay reagent kit includes monoclonal antibody A and monoclonal antibody B, where monoclonal antibody A functions as the capture antibody and monoclonal antibody B is conjugated to a chromophore and functions as the detection antibody. The heavy chain variable region of monoclonal antibody B comprises the amino acid sequences of SEQ ID NOs: 7, 8, and 9, and the light chain variable region of monoclonal antibody B comprises the amino acid sequences of SEQ ID NOs: 10, 11, and 12. In one embodiment, the heavy chain variable region of monoclonal antibody B comprises the amino acid sequences of CDR1, SEQ ID NO: 7, CDR2, SEQ ID NO: 8, and CDR3, SEQ ID NO: 9, and the light chain variable region of monoclonal antibody B comprises the amino acid sequences of CDR1, SEQ ID NO: 10, CDR2, SEQ ID NO: 11, and CDR3, SEQ ID NO: 12. In certain embodiments, the chromophore comprises a fluorescent group or a chemiluminescent group (eg, horseradish peroxidase (HRP)).

[0033] It is noteworthy that some embodiments of the present disclosure disclose the use of human recombinant MMP-1 (full-length MMP-1) as an immunogen for the in-house production of 11 types of monoclonal antibodies and for selecting monoclonal antibody pairs (capture antibody: monoclonal antibody A and detection antibody: monoclonal antibody B) that can be used in enzyme immunoassay reagent kits, which overcome limitations inherent to monoclonal antibodies conjugated to chromophores, such as significantly reduced binding ability to MMP-1, high background values ​​that often occur in the detection of clinical samples, and lack of sensitivity, and have better sensitivity than other pairs (which may not even be able to detect MMP-1). Furthermore, it should be emphasized that such pairs are not related to the ability of the monoclonal antibodies to bind to MMP-1. It is worth mentioning that the monoclonal antibody pairs of some embodiments of the present disclosure have higher sensitivity than other antibody pairs, such as commercially available antibodies used as capture antibodies (the immunogen is amino acids 20 to 469 of MMP-1), other monoclonal antibodies produced using protease-inactive MMP-1 as the immunogen, and other polyclonal antibodies produced using MMP-1 fragments (e.g., any fragment of amino acids 100 to 300 of MMP-1, or a protease-inactive fragment) as the immunogen.

[0034] In some embodiments, the colloidal gold immunoassay test strip comprises an overlapping colloidal gold pad and an assay plate, the colloidal gold pad being sprayed with a colloidal gold pad solution containing a detection antibody-gold particle conjugate, and the surface of the assay plate having a nitrocellulose membrane onto which a solution containing a capture antibody is applied as a measurement line. In one embodiment, the colloidal gold immunoassay test strip comprises monoclonal antibody A and monoclonal antibody C, where monoclonal antibody A is used as a detection antibody linked to gold particles, and monoclonal antibody C is used as a capture antibody. The heavy chain variable region of monoclonal antibody C comprises the amino acid sequences of SEQ ID NOs: 13, 14, and 15, and the light chain variable region of monoclonal antibody C comprises the amino acid sequences of SEQ ID NOs: 16, 17, and 18. In one embodiment, the heavy chain variable region of monoclonal antibody C comprises the amino acid sequences of CDR1, SEQ ID NO: 13, CDR2, SEQ ID NO: 14, and CDR3, SEQ ID NO: 15, and the light chain variable region comprises the amino acid sequences of CDR1, SEQ ID NO: 16, CDR2, SEQ ID NO: 17, and CDR3, SEQ ID NO: 18.

[0035] It is noteworthy that in some embodiments of the present disclosure, monoclonal antibody pairs (capture antibody: monoclonal antibody C and detection antibody: monoclonal antibody A) that can be used in colloidal gold immunoassay test strips are selected from monoclonal antibody pairs that can be used in immunoassay reagent kits, and the detection antibodies are successfully linked to gold particles, overcoming the problem that some monoclonal antibodies cannot be linked to gold particles, and some combinations have better sensitivity than other combinations. Furthermore, it has been found that a pair with the best sensitivity in an enzyme immunoassay reagent kit may not have the best sensitivity in a colloidal gold immunoassay test strip. In other words, when an antibody pair in one detection system is applied to another detection system, it is still necessary to verify the advantages and disadvantages of sensitivity through actual testing, and there is not necessarily a positive correlation with existing known detection systems.

[0036] Some embodiments of the present disclosure provide a method for detecting MMP-1. This method includes detecting MMP-1 in a sample using the aforementioned detection kit. For example, the presence / absence or content of MMP-1 can be determined by the presence / absence of color development or quantification of the color development value (e.g., optical density value (OD value)) using an enzyme immunoassay reagent kit, a colloidal gold immunoassay test strip, or a combination thereof. In some embodiments, the sample includes a body fluid (e.g., oral secretions or respiratory secretions) or blood. In some embodiments, the detection kit can use an enzyme immunoassay reagent kit for quantification and a colloidal gold immunoassay test strip for rapid screening, thereby simultaneously obtaining both qualitative and quantitative results.

[0037] Since MMP-1 can be used as a detection index for oral cancer, the MMP-1 detection kit and detection method provided by some embodiments of the present disclosure can be simultaneously used for screening for oral cancer, which does not need to be performed by a professional oral health care professional, thereby increasing the demand for screening, accelerating the diagnosis of oral cancer patients, and improving the possibility of treatment. [Example]

[0038] To further illustrate the monoclonal antibodies, polynucleotides, detection kits, and detection methods for MMP-1 provided by various embodiments of the present disclosure, the following examples were carried out. It should be noted that the following embodiments are provided for illustrative purposes only and are not intended to limit the present disclosure.

[0039] Embodiment 1. Monoclonal antibody development process First, we used recombinant human MMP-1 protein, which retains protease activity, as an immunogen to develop mouse MMP-1 monoclonal antibody cell lines through the mouse immune response. Eleven mouse monoclonal antibody cell lines were selected, and the cell fluids were collected and purified to obtain the respective monoclonal antibodies. These were named strains 1-8A12, 4-26, 6-2, 20-4, 30-22, 31-34, 44-28, 57-41, 61-24, 73-1, and 79-4.

[0040] Embodiment 2-1. Enzyme immunoassay reagent kit for detecting MMP-1: Testing the ability of each monoclonal antibody to bind to MMP-1 To test whether each monoclonal antibody could specifically bind to MMP-1, we serially diluted the 11 monoclonal antibody lines and then tested the binding of the monoclonal antibodies to human recombinant MMP-1 using direct ELISA. The results are shown in Figure 1A.

[0041] Figure 1A shows that each monoclonal antibody could specifically bind to MMP-1, and these monoclonal antibodies had different binding abilities to MMP-1, with the order of binding ability, from strongest to weakest, being 6-2 strain > 73-1 strain > 1-8A12 strain > 4-26 strain > 20-4 strain > 31-34 strain > 44-28 strain > 61-24 strain > 30-22 strain > 57-41 strain > 79-7 strain.

[0042] Furthermore, to test whether the monoclonal antibodies could be used for antibody detection in enzyme immunoassays, i.e., whether the monoclonal antibodies still had the ability to bind to MMP-1 after being conjugated to a chromophore, we conjugated each monoclonal antibody to HRP and then performed an MMP-1 binding assay using the same method as in Figure 1A. The results are shown in Figure 1B.

[0043] Figure 1B shows that each monoclonal antibody conjugated to HRP still had the ability to specifically bind to human recombinant MMP-1. However, compared with Figure 1A, the monoclonal antibodies conjugated to HRP showed slight changes in binding ability to human recombinant MMP-1, with the order from strongest to weakest being 73-1-HRP > 4-26-HRP = 6-2-HRP > 44-28-HRP > 31-34-HRP > 1-8A12-HRP > 20-4-HRP > 61-24-HRP > 57-41-HRP > 30-22-HRP > 79-7-HRP.

[0044] Embodiment 2-2. Enzyme immunoassay reagent kit for detecting MMP-1: screening of antibody pairs Paired tests were performed using the 11 monoclonal antibody strains described above as the capture antibody and the detection antibody (linked to HRP) in an enzyme immunoassay reagent kit. While 11 × 10 = 110 paired combinations could theoretically be generated, three monoclonal antibody strains were eliminated because they were extremely ineffective in the paired tests. In fact, paired tests of a total of 92 combinations were completed, and the optimal pair capable of detecting MMP-1 was selected.

[0045] The test process for each pair, using the 1-8A12 strain as an example, is as follows: (1) A sandwich ELISA was performed using the 1-8A12 strain as the capture antibody and 4-26-HRP as the detection antibody. The capture antibody had four concentrations, and the detection antibody had six concentrations. One set of pairs was tested under 24 conditions. The difference in OD (sample value - background value) between a solution containing human recombinant MMP-1 (sample value) and a solution not containing human recombinant MMP-1 (background value) was calculated under these 24 conditions. The higher the value, the higher the antibody pair's ability to detectably bind to MMP-1. (2) Pair tests for 92 combinations were completed according to Step 1 above. The results are shown in Table 1 below. The values ​​in Table 1 are the maximum difference in OD values ​​for each pair in the pair test.

[0046] [Table 1]

[0047] Table 1 shows that the monoclonal antibody pair with a stronger signal (larger difference) in the paired test did not have an absolute correlation with the MMP-1 binding ability of each monoclonal antibody. In other words, the optimal antibody pair suitable for an enzyme immunoassay reagent kit cannot be determined by the MMP-1 binding ability of each monoclonal antibody, but must be selected in an actual paired test.

[0048] Embodiment 2-3. Enzyme immunoassay reagent kit for detecting MMP-1: concentration standard curves for each antibody pair According to the results of Example 2-2, sandwich ELISA was performed for 12 antibody pairs (shown in Table 2), and the range of detectable human recombinant MMP-1 concentration standard curves for each antibody pair was obtained. The results are shown in Table 3.

[0049] [Table 2]

[0050] [Table 3]

[0051] The results in Table 3 showed that when the tested concentration range of human recombinant MMP-1 was 0.01 to 2.5 ng / ml, the OD value of each pair increased with increasing MMP-1 concentration.

[0052] Next, the concentration range of the standard curve detectable by each antibody pair was evaluated using the recovery rate, and a recovery rate of 80 to 120 was set as the reliable range. Concentrations within this reliable range are marked with an asterisk ( *The results showed that the concentration ranges of the standard curves for the 12 antibody pairs were different, and some pairs had negative values ​​at low concentrations, resulting in high background values ​​and no detection of low-concentration samples.

[0053] Embodiment 2-4. Enzyme immunoassay reagent kit for detecting MMP-1: Detection of endogenous MMP-1 in clinical saliva samples Next, to confirm whether the antibody pairs can detect endogenous MMP-1 in clinical saliva samples in addition to human recombinant MMP-1, the 12 antibody pairs of Examples 2-3 were used to detect eight types of saliva samples with known endogenous MMP-1 contents (the contents were analyzed by multiplex LC-MRM-MS). The results are shown in Table 4.

[0054] [Table 4]

[0055] The results showed that all 12 antibody pairs could detect endogenous MMP-1 in saliva samples, and the MMP-1 contents detected by most of the pairs were consistent with the actual contents, indicating that the 12 antibody pairs also had sufficient specificity for detecting MMP-1 in saliva samples. According to the results in Table 4, Nos. 2, 4, 5, and 10, which had good sensitivity, were selected for further evaluation.

[0056] To evaluate the feasibility of clinical application, 15 clinical saliva samples from normal healthy individuals (healthy controls, HC) and 25 clinical saliva samples from oral cancer patients (oral squamous cell tumor, OSCC) were collected and then diluted 5-fold for enzyme immunoassay experiments. The results are shown in Figure 2A (normal human group) and Figure 2B (oral cancer patient group). Additional data analysis was performed using Figure 2A and Figure 2B.

[0057] The results showed that the measured values ​​of the four antibody pairs were significantly correlated in both the normal and oral cancer patient groups (Spearman's ρ = 0.953-0.988). Furthermore, receiver operating characteristic (ROC) analysis showed that the four antibody pairs were suitable for detecting endogenous MMP-1 (area under the ROC curve (ROC AUC) = 0.937-0.967). The sensitivity and specificity of the four pairs obtained by ROC analysis are summarized in Table 5.

[0058] [Table 5]

[0059] According to Table 5, the antibody pair No. 4 with high sensitivity was selected for the preparation of the MMP-1 enzyme immunoassay reagent kit.

[0060] Embodiment 2-5. Enzyme immunoassay reagent kit for detecting MMP-1: Functional test Functional testing was performed on the MMP-1 enzyme immunoassay reagent kit using antibody pair number 4 according to the Global Laboratory Standards for a Healthier World published by the American Clinical Laboratory Standards Committee. The results were as follows:

[0061] 1. Sensitivity analysis (see standard Ep17-A2): The limit of blank (LoB) was 57.40 pg / ml and the limit of detection (LoD) was 117.02 pg / ml. 2. Linear analysis (see standard Ep06-A): The best nonlinear polynomial fit was a cubic regression, and concentrations were linearly distributed between 140 and 8,000 pg / ml (nonlinearity ≤ 5%). 3. Precision analysis (see standard Ep05-A3): The mean CV(%) for repeat precision was 4.809%, and the mean CV(%) for intra-laboratory precision was 9.569%.

[0062] Embodiment 3-1. Colloidal gold immunoassay test strip for detecting MMP-1: Selection of antibody matching combination Next, we further tested our self-produced monoclonal antibodies to find a suitable antibody combination for a colloidal gold immunoassay test strip for detecting MMP-1. See FIG. 3A, which is a schematic diagram of a colloidal gold immunoassay test strip 1 including a colloidal gold pad 110 and an assay plate 120. The colloidal gold pad 110 and the assay plate 120 may be stacked on top of each other, for example, the colloidal gold pad 110 on top of the assay plate 120, or the assay plate 120 on top of the colloidal gold pad 110. A colloidal gold pad solution 111 containing a detection antibody-gold particle complex was sprayed onto the colloidal gold pad 110, and the surface of the assay plate 120 was covered with a nitrocellulose membrane. A solution containing a capture antibody was coated onto the nitrocellulose membrane to form a measurement line 122. Furthermore, a solution containing an internal control antibody was added onto the nitrocellulose membrane, which could function as an internal control line 121. During the test, a sample was added to the colloidal gold pad 110 containing the detection antibody-gold particle complex, and the sample was transferred by immunochromatography from the colloidal gold pad 110 to the nitrocellulose membrane on the assay plate 120. If MMP-1 bound to the detection antibody-gold particle complex, the MMP-1 bound to the detection antibody-gold particle complex would be captured by the capture antibody on the measurement line 122 and agglutinated to form an orange to red signal (the color of the gold particles). Therefore, the test result could be determined by whether the measurement line 122 was colored.

[0063] To select antibody matching combinations, the monoclonal antibodies 1-8A12, 6-2, 20-4, 31-34, and 73-1 strains used in the four antibody pairs (Nos. 2, 4, 5, and 10) that had good sensitivity in embodiments 2-4 were paired and tested as follows.

[0064] First, five types of antibodies were reacted with nanoscale gold particles (diameter less than 100 nm) to form "detection antibody-gold particle complexes." However, the 1-8A12 and 73-1 strains did not react sufficiently with the gold particles, so the gold particles could not be linked to the detection antibodies. Therefore, only the 6-2-gold particles, 20-4-gold particles, and 31-34-gold particles were paired with human recombinant MMP-1, for a total of 10 combinations. The results are shown in Table 6.

[0065] [Table 6]

[0066] According to Table 6, combination C could achieve good sensitivity, so combination C was selected for the next clinical sample testing.

[0067] Embodiment 3-2. Colloidal Gold Immunoassay Test Strips for MMP-1 Detection: Testing of Clinical Samples To evaluate the feasibility of detecting clinical samples using colloidal gold immunoassay test strips containing Combination C, 215 saliva samples were tested and compared with each other by enzyme immunoassay.

[0068] See Figure 3B for the colloidal gold immunoassay test strip. The reactivity of the sample was scored using a colorimetric card. The colorimetric card was divided into 0 and 5 points according to the color intensity of the measurement line 122. Using the colorimetric card, a total of 12 scores were obtained: 0, 0-1 (0.5), 1, 1-2 (1.5), 2, 2-3 (2.5), 3, 3-4 (3.5), 4, 4-5 (4.5), and 5, greater than 5 (5.5). An XY distribution diagram was then created between the colorimetric card scores and the MMP-1 concentrations measured by enzyme immunoassay. The results are shown in Figure 4.

[0069] Figure 4 shows that the results of the colloidal gold immunoassay test strips and the enzyme immunoassay were positively correlated, with a correlation analysis result of R = 0.871 (p < 0.0001). This means that the colloidal gold immunoassay test strips containing combination C can be used to detect MMP-1 in clinical saliva samples.

[0070] In summary, embodiments of the present disclosure disclose an effective detection method for detecting endogenous MMP-1 in clinical samples. The detection method includes at least an enzyme immunoassay reagent using monoclonal antibody pair No. 4 (capture antibody: strain 31-34, detection antibody: strain 73-1) and a colloidal gold immunoassay test strip using monoclonal antibody pair Combination C (capture antibody: strain 1-8A12, detection antibody: strain 31-34). The amino acid sequence, main characteristics, and corresponding SEQ ID NO of each monoclonal antibody are shown in Table 7 below.

[0071] [Table 7]

[0072] Embodiment 4. Comparison with the results of the control group In the development process of Examples 2 and 3, several control groups were also used as capture antibodies for each detection, and the self-produced 1-8A12 was used as the detection antibody, and the detection results were compared and analyzed simultaneously. The control antibodies, including a total of 16 types of antibodies, are shown in Table 8, including a commercially available antibody (No. 7), polyclonal antibodies (Nos. 1 to 4) produced using MMP-1 peptide fragments as the immunogen, polyclonal antibodies (Nos. 5 and 6) produced using protease-inactivated MMP-1 as the immunogen, and other mouse monoclonal antibodies (Nos. 8 to 16) produced using protease-inactivated MMP-1 as the immunogen.

[0073] [Table 8]

[0074] The comparison results showed that most of the antibodies could efficiently bind to human recombinant MMP-1 in the direct ELISA stage, but when the pairing process began, many pairs showed very high nonspecific binding, and their sensitivity was generally insufficient. Among pairs using a control group as a capture antibody and a self-produced antibody as a detection antibody, the best pair was the commercially available antibody MAB901 and 1-8A12-HRP, but its concentration standard curve range (Table 9A) and efficiency in detecting MMP-1 in saliva samples (Table 9B) were inferior to those of the self-produced antibody pair. Therefore, the pairs selected in the embodiments of the present disclosure were more sensitive than antibody pairs using a capture antibody with a commercially available antibody, a monoclonal antibody produced using protease-inactivated MMP-1 as the immunogen, or a polyclonal antibody using an MMP-1 fragment as the immunogen.

[0075] [Table 9]

[0076] [Table 10]

[0077] Sequence Listing [Table 11]

[0078] [Table 12]

[0079] [Table 13]

[0080] [Table 14]

[0081] Table 15

[0082] Table 16

Claims

1. A heavy chain variable region sequence comprising i) a CDR1 of SEQ ID NO: 7, ii) a CDR2 of SEQ ID NO: 8, and iii) a CDR3 of SEQ ID NO: 9, and a light chain variable region sequence comprising i) a CDR1 of SEQ ID NO: 10, ii) a CDR2 of SEQ ID NO: 11, and iii) a CDR3 of SEQ ID NO: 12; A monoclonal antibody that binds to human matrix metalloproteinase 1, comprising:

2. A polynucleotide encoding the amino acid sequence of the monoclonal antibody of claim 1.

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