Clara cell protein antibody or antigen binding fragment and application thereof

Abstract

The present invention provides Clara cell protein antibodies or antigen binding fragments and uses thereof, kits containing these antibodies and antibody fragments, and methods of using these antibodies and antibody fragments.

Description

Technical Field

[0001] This invention relates to the field of Clara cell protein antibody application technology, specifically to Clara cell protein antibody and antigen-binding fragments of antibodies, kits containing said antibodies and antibody fragments, and applications using these antibodies and antibody fragments. Background Technology

[0002] Clara cell protein 16 (CC16) is a 15.84 kDa protein secreted by Clara cells, non-ciliated cuboidal epithelial cells lining the bronchioles. CC16 is a secretory globin, a homodimer composed of two anti-aligned polypeptide chains. Each monomeric polypeptide chain consists of 70-77 amino acids linked by two disulfide bonds. It possesses various functions including immunosuppression, anti-inflammation, anti-fibrosis, anti-tumor activity, removal of harmful substances deposited in the respiratory tract, and inhibition of pulmonary surfactant degradation. CC16 and SP-D are early biomarkers of impaired Clara cell, type II alveolar cells, and alveolar membrane integrity caused by silica dust. They can serve as auxiliary indicators for early diagnosis (screening) and late-stage diagnosis of silicosis. Establishing a combined discriminant diagnostic model using CC16 and SP-D is valuable for differentiating healthy individuals, silicosis-exposed individuals, and stage I silicosis patients. (Liu Ping, Chongqing Medical University, Master's Thesis, Electronic Journal, 2008, No. 12). The serum CC16 concentration level in workers exposed to SiO2 dust was lower than that in the control group, suggesting that serum CC16 concentration may reflect the early toxic effects of SiO2 dust on respiratory epithelial cells and has the potential to be a sensitive indicator for detecting early silicosis toxicity (Bernard et al, Eur Respir J, 7:1932-1937, 1994); Studies on serum Clara cell protein levels in silicosis patients found that the silica dust exposure group and the 0... + Serum CC16 levels in the group with stage I silicosis were significantly lower than those in the control group (P<0.01). Serum CC16 levels in the non-smokers of the control group were higher than those in the smokers (P<0.05). Silica dust exposure group, 0 + There was no statistically significant difference in serum CC16 levels between the group and the stage I silicosis group (P>0.05). (Liu Ping et al., Chinese Journal of Occupational Health and Disease, 25(1):18-21, 2007)

[0003] The levels of BALF and plasma CC16 protein in asthmatic patients are significantly reduced, and the serum CC16 protein level is even lower in patients with a longer disease course (Nie Xiaomeng et al., Clinical Collection. (06):301-303. 2004). Low concentrations of CC16 can serve as an independent predictor of developmental defects in lung function during childhood, as well as accelerated decline in lung function and the occurrence of COPD in adulthood (Stefano Guerra, 3(7): P613-620, 2015). Serum CC16 levels may be an effective biomarker for the diagnosis and prognosis assessment of patients with acute respiratory distress syndrome and normal renal function (Jia Xuan, 51(06):646-649, 2023). In summary, CC16 levels are considered to be associated with the occurrence, progression, and diagnosis of lung-related diseases such as silicosis, chronic obstructive pulmonary disease (COPD), asthma, acute respiratory distress syndrome (ARDS), pneumonia, and pulmonary fibrosis.

[0004] Therefore, we propose a Clara cell protein antibody or antigen-binding fragment and its application. Summary of the Invention

[0005] This invention is at least in part based on the development of Clara cell protein-specific antibodies. These antibodies and their antigen-binding fragments can be used, for example, to quantify CC16 protein in human serum, determine whether a subject has underlying lung-related disease, determine whether a subject should be discharged or to begin or continue treatment, and select subjects to participate in clinical studies.

[0006] This article provides isolated antibodies and their antigen-binding fragments that specifically bind to Clara cell proteins, detection reagents containing these antibodies and antibody fragments, and various methods for using these antibodies and antibody fragments.

[0007] This invention provides Clara cell protein antibodies or antigen-binding fragments, wherein the antibody is a monoclonal antibody, preferably a monoclonal antibody produced by a hybridoma, and the antigen-binding fragment is an antigen-binding fragment of a monoclonal antibody produced by a hybridoma. Further, the antibody of this invention is produced by mouse hybridoma cells, and the antigen-binding fragment is an antigen-binding fragment of an antibody produced by mouse hybridoma cells. In some embodiments, the antibody or a fragment thereof contains one or more complementarity-determining regions of the light or heavy chain of the hybridoma-produced antibody.

[0008] A composition is also provided comprising the above-described Clara cell protein antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

[0009] Hybridoma cells were also provided, which were prepared by the patent applicant, Bio-Sys (Chongqing) Biotechnology Co., Ltd., on May 28, 2023, at No. 2, Building 2, No. 5, Gangcheng East Ring Road, Jiangbei District, Chongqing.

[0010] A method for quantifying Clara cell protein levels in samples from subjects is also provided, the method comprising contacting the sample with an antibody or an antigen-binding fragment thereof described herein. In some embodiments, the method includes using at least one (e.g., two) antibody or antibody fragment described herein.

[0011] It also provides a method for assessing whether a subject is at risk of developing lung-related disease using Clara cell protein antibodies or antigen-binding fragments or combinations thereof.

[0012] The method includes obtaining a sample from the subject and determining the level of Clara cell protein in the sample using the at least one isolated antibody or antigen-binding fragment, wherein if the subject's Clara cell protein level is elevated or decreased relative to a Clara cell protein reference level, then the subject has an increased risk of developing lung-related disease, and substantially the same Clara cell protein level indicates that the subject has a reduced risk of developing lung-related disease.

[0013] It also provides a method for determining whether a hospitalized patient should be discharged or whether a subject should be admitted to or continue hospitalization, the method comprising contacting a sample with an antibody or antigen-binding fragment thereof described herein to determine the CC16 level in the sample, wherein an increase or decrease in the CC16 level compared to a CC16 reference level indicates that hospitalization should be initiated or continued, and a CC16 level within the reference level indicates that the subject may be considered for discharge.

[0014] A method for selecting subjects to participate in clinical studies is also provided, the method comprising obtaining a subject sample, determining the CC16 level in the sample using at least one antibody or fragment thereof described herein, and selecting the subject to participate in the clinical study if the subject's human CC16 level, compared to a CC16 reference level, indicates that he / she should be selected to participate in the clinical study. In some embodiments, the presence of elevated or decreased CC16 levels indicates that the subject should be selected to participate in the clinical study.

[0015] In some embodiments of any of the methods described herein, the subject is undiagnosed or does not present with symptoms of a disease; the subject has been diagnosed with a lung-related disease, such as silicosis, chronic obstructive pulmonary disease, asthma, acute respiratory distress syndrome, pneumonia, or pulmonary fibrosis. In some embodiments of any of the methods described herein, at least two antibodies or fragments thereof described herein are utilized.

[0016] A kit is also provided containing at least one Clara cell protein antibody or antigen-binding fragment or composition provided by the present invention. In some embodiments, the kit is provided in the form of a chemiluminescent immunoassay (CLIA).

[0017] In some embodiments of any of the methods described herein, the reference level is a threshold level of CC16. In some embodiments, the threshold level is the CC16 level in a healthy patient population. In some embodiments of any of the methods described herein, the reference level is the CC16 level present in a subject sample who does not exhibit disease symptoms, has not been diagnosed with a disease, or has not been identified as being at risk of developing a disease.

[0018] The terms “elevated” or “decreased” mean a determined or measured difference, such as a statistically significant difference (e.g., an increase), compared to a reference level (e.g., the CC16 level in subjects without disease, subjects not presenting disease symptoms, or subjects not identified as being at risk of developing disease; or a threshold level for CC16). In some implementations, the reference is a threshold level, and any level above or below it is considered “elevated” or “decreased.”

[0019] In some implementations, the ratio of two CC16 levels in the subject is calculated. This reference ratio can be compared to a parametric ratio of CC16 levels measured in the subject (e.g., any control subject described herein or the same subject). For example, the reference ratio could be the ratio of CC16 levels before and after treatment for a disease (silicosis, chronic obstructive pulmonary disease, asthma, acute respiratory distress syndrome, pneumonia, pulmonary fibrosis); or the ratio of CC16 levels at two different time points during treatment (e.g., inpatient or outpatient treatment) for a disease (silicosis, chronic obstructive pulmonary disease, asthma, acute respiratory distress syndrome, pneumonia, pulmonary fibrosis).

[0020] In this context, "subject" refers to a person.

[0021] In this article, "biological samples" refers to human serum and plasma.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The methods and materials used in this invention are described herein. Other suitable methods and materials known in the art may also be used. The materials, methods, and embodiments described are for illustrative purposes only and are not intended to limit the invention.

[0023] The present invention has at least the following beneficial effects: a Clara cell protein antibody or antigen-binding fragment, which can be used to determine whether a subject has normal Clara cell protein levels, whether a subject needs hospitalization, and the progression of the subject's condition. Attached Figure Description

[0024] Figure 1 Electrophoresis diagram of recombinant CC16 protein;

[0025] Figure 2 The values ​​of OD450nm for biotin coated with 1A5 and 2C8 at different concentrations of CC16 are given.

[0026] Specifically, the present invention relates to the following aspects:

[0027] 1. An antibody against a Clara cell protein or an antigen-binding fragment thereof, wherein the antibody comprises a light chain and a heavy chain, the light chain having an amino acid sequence as shown in SEQ ID NO: 2, and the heavy chain having an amino acid sequence as shown in SEQ ID NO: 3.

[0028]

[0029] 2. A Clara cell protein having the amino acid sequence shown in SEQ ID NO: 1.

[0030]

[0031] 3. A method for quantifying the level of Clara cell protein in a sample from a subject, the method comprising:

[0032] The sample is contacted with at least one antibody or fragment thereof as described in item 1; and the binding of the antibody or fragment thereof to Clara cell protein is detected.

[0033] 4. A method for determining whether a subject has a risk of developing lung-related disease. The method includes:

[0034] The determination includes obtaining a sample from the subject and determining the level of Clara cell protein in the sample using at least one antibody or antibody fragment as described in item 1, wherein if the subject's Clara cell protein level is elevated or decreased relative to a Clara cell protein reference level, then the subject has an increased risk of developing lung-related disease, and substantially the same Clara cell protein level indicates that the subject has a reduced risk of developing lung-related disease.

[0035] 5. A method for determining whether to discharge a hospitalized patient or to initiate or continue hospitalization for a subject, the method comprising:

[0036] Obtain a sample from the subject; and determine the level of Clara cell protein in the sample using at least one antibody or antibody fragment as described in item 1; an increase or decrease in the level of Clara cell protein in the sample compared to a reference level of Clara cell protein indicates that the subject should be hospitalized or continued, while the same level of Clara cell protein in the sample compared to a reference level of Clara cell protein indicates that the subject should be considered for discharge.

[0037] 6. A method for selecting subjects to participate in a clinical study, the method comprising:

[0038] A sample is obtained from the subject; the level of CC16 in the sample is determined using at least one antibody or antibody fragment as described in item 1; the subject is selected to participate in the clinical study when the subject's CC16 level relative to a reference level of CC16 indicates that the subject should be selected for the clinical study.

[0039] 7. The method in item 6, wherein the presence of elevated or decreased levels of CC16 indicates that the subject should be selected for participation in the clinical study.

[0040] 8. The method of items 4-7, wherein the determination is performed using at least two antibodies or fragments thereof as described in item 1.

[0041] 9. The method of any one of items 4-7, wherein the reference level of CC16 is the threshold level of CC16.

[0042] 10. The method of item 9, wherein the threshold level of the Clara cell protein is the average level of the Clara cell protein in a healthy population.

[0043] 11. The method of any one of items 4-7, wherein the reference level is the level of Clara cell protein present in a sample of a subject who has not been diagnosed with lung-related disease.

[0044] 12. The method of any one of items 3-8, wherein the sample comprises serum or plasma.

[0045] 13. A kit comprising at least one antibody or antigen-binding fragment as described in item 1.

[0046] 14. The kit of item 13, wherein the kit contains two antibodies or antigen-binding fragments thereof.

[0047] 15. A kit of any one of items 13-14, wherein the kit is provided as a magnetic microparticle chemiluminescence assay (MCLIA).

[0048] Detailed Explanation

[0049] This article describes antibodies that specifically bind to Clara cell proteins and their antigen-binding fragments, kits containing said antibodies and fragments, and methods of using these antibodies and fragments.

[0050] CC16

[0051] The CC16 gene is located in the 11q12-p13 region of human autosomes, with a total gene segment length of 4995 bp. This segment contains 3 exons and 2 introns. CC16 has several other names. As a protein secreted by Clara cells, it is also called Clara cell secreted protein (CCSP). It is also known as CC10 because its molecular weight, as measured by polyacrylamide gel electrophoresis, is measured on a spectrophotometer. Furthermore, because it is distributed in different organs, it has many other names, such as uteroglobin.

[0052] The complete mRNA sequence of human CC16 can be found in GenBank accession number BC004481.2, and the peptide sequence is available in GenBank accession number AAH04481.1.

[0053] Antibody and antigen binding antibody fragment

[0054] In this invention, an antibody-antigen binding fragment is provided, wherein the antibody is a monoclonal antibody produced by a hybridoma, and the antigen binding fragment is an antigen binding fragment of a monoclonal antibody produced by a hybridoma.

[0055] The process of specifying antibody and antigen-binding antibody fragments. This includes obtaining recombinant Clara cell protein and immunizing mice with isolated recombinant Clara cell protein.

[0056] Based on its amino acid sequence, the molecular weight of Clara cell protein is predicted to be approximately 9.99 kDa. In vivo, the naturally expressed protein undergoes post-translational modifications, increasing its molecular weight to around 16 kDa, which is consistent with the properties of the CC16 protein. Therefore, Clara cell protein antibodies generated from immunization with human Clara cell protein as an antigen have a higher affinity for Clara cell protein than other antibodies, and thus can be used as diagnostic reagents.

[0057] In some embodiments, the compositions described herein contain at least two different Clara cell protein-binding antibodies or Clara cell protein-binding antibody fragments as described herein. As described in more detail in the Examples section below, this combination of antibodies exhibits a higher affinity for the CC16 antigen than any single antibody or other commercially available antibodies. Such compositions containing antibody or antigen-binding fragments described herein can be used with any of the methods described herein.

[0058] Hybridoma

[0059] This document also provides hybridomas capable of producing antibodies that bind to Clara cell proteins. As is known in the art, the term "hybridoma" refers to cells generated by fusing antibody-producing lymphocytes with antibody-non-antibody-producing cancer cells (typically myeloma or lymphoma). After fusion, the hybridoma proliferates and produces specific monoclonal antibodies that would otherwise be produced by the fused lymphocytes. In some embodiments, the provided hybridoma was prepared by Bio-Sys (Chongqing) Biotechnology Co., Ltd., the patent applicant of May 28, 2023, located at No. 2, Building 2, No. 5, Gangcheng East Ring Road, Jiangbei District, Chongqing. In some embodiments, single cells, harvested cells, and cultures comprising cells derived from the hybridoma prepared by Bio-Sys (Chongqing) Biotechnology Co., Ltd., the patent applicant of May 28, 2023.

[0060] Pharmaceutical Compositions and Kits

[0061] This disclosure provides compositions, such as pharmaceutically acceptable compositions, comprising the Clara cell protein antibody or antigen-binding fragment described herein, formulated together with a pharmaceutically acceptable carrier.

[0062] As described herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. Carriers include, but are not limited to, intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).

[0063] The compositions described herein may be in various forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), dispersions or suspensions, liposomes, and suppositories. A suitable form depends on the target administration modality and the therapeutic application.

[0064] Kits: Kits containing assay reagents described herein include at least one (e.g., two) Clara cell protein antibody or antigen-binding fragment described herein. Kits typically contain the following main components: packaging, reagents containing the above-described binding compositions, and instructions.

[0065] The kits provided in this article can be used in any of the following ways. For example, a sample is obtained from a subject, and the sample is brought into contact with the reagents in the kit to determine the level of Clara cell protein in the sample.

[0066] The provided antibodies and fragments are used to determine the level of Clara cell protein in the sample, to estimate the risk of developing lung-related disease, and to determine whether the subject can be discharged or start or continue hospitalization.

[0067] Methods for determining Clara cell protein levels: This article provides methods for determining Clara cell protein levels in a subject sample, the methods comprising obtaining a sample from a subject, contacting the sample with at least one antibody or antibody fragment described herein, and determining the level of Clara cell protein in the sample.

[0068] A method for determining whether a subject is at risk of developing lung-related disease. The method includes:

[0069] The determination includes obtaining a sample from the subject and determining the level of Clara cell protein in the sample using at least one antibody or antibody fragment described herein, wherein if the subject's Clara cell protein level is elevated or decreased relative to a Clara cell protein reference level, then the subject has an increased risk of developing lung-related disease, and substantially the same Clara cell protein level indicates a reduced risk of developing lung-related disease.

[0070] Methods for determining whether a subject should be hospitalized: This article provides methods for determining whether a subject should be hospitalized, including obtaining a sample from the subject, contacting the sample with at least one antibody or antibody fragment described herein, determining the level of Clara cell protein in the sample, and comparing the level of Clara cell protein in the sample to a reference level, wherein a high or low level of Clara cell protein in the sample indicates that hospitalization of the subject should be initiated.

[0071] Methods for determining whether to discharge or continue treatment of hospitalized patients: This article provides methods for determining whether to discharge or continue treatment of hospitalized subjects, the methods comprising obtaining a sample from the subject, contacting the sample with at least one antibody or antibody fragment described herein, determining the level of Clara cell protein in the sample, wherein a high or low level of Clara cell protein in the sample, compared to a reference level of Clara cell protein, indicates that the subject should continue hospitalization; and a level of Clara cell protein in the sample, compared to a reference level of Clara cell protein, indicates that the subject should be discharged.

[0072] Methods for assisting in the diagnosis of subjects

[0073] This article provides methods for assisting in the diagnosis of test subjects, applicable to a wide range of clinical contexts. For example, such methods can be used for general population screening, such as hospital health check-up screenings.

[0074] In some implementations, the methods described herein can be used to determine the likelihood of the presence of a disease in a subject. Increased or decreased levels of Clara cell protein (relative to a reference level) are generally associated with the presence of certain diseases, including but not limited to lung-related diseases.

[0075] Lung-related diseases that can be diagnosed with the aid of the methods described in this article include silicosis, chronic obstructive pulmonary disease (COPD), asthma, acute respiratory distress syndrome (ARDS), pneumonia, and pulmonary fibrosis.

[0076] In some implementations, CC16 levels can be determined multiple times to measure a subject's response to treatment. For example, CC16 levels obtained after treatment administration (e.g., one or more doses or one or more rounds of treatment) can be compared to CC16 levels before the start of treatment (e.g., baseline levels). Changes in CC16 levels indicate whether the treatment is effective; for example, a gradual approach of CC16 levels to a reference level indicates that the treatment is effective.

[0077] Example 1: Generation and Characterization of Anti-CC16 Antibody

[0078] The Clara cell protein antibody in this invention is prepared by immunizing mice with CC16 protein, then fusing spleen cells with myeloma cells to obtain mouse hybridoma cells, and then producing monoclonal antibodies through hybridoma cells.

[0079] Antigen generation: Human CC16 cDNA clone and mRNA (GenBank accession number BC004481.2) were purchased from Origene Technologies, Inc., Rockville, MD. Using the mRNA as a template, human CC16 gene-specific primers were designed, and standard PCR techniques were used to obtain the complete fragment of the human CC16 gene for constructing an expression vector. Six His markers were incorporated into the amino-terminal region of the protein to facilitate protein purification. The integrity of the expression clone was confirmed by DNA sequencing. Recombinant protein was produced through transient transfection and expression in *E. coli*. Cell lysates were purified by passing them through a Ni column to obtain recombinant Clara cell protein. The purified CC16 protein was verified by polyacrylamide gel electrophoresis. Figure 1 ).

[0080] Hybridoma and Monoclonal Antibody Generation: Monoclonal antibodies were generated by immunizing mice with CC16 protein purified as described above. Three Balb / c mice were immunized as follows:

[0081] T1 60 μg / mouse, combined with an equal volume of CFA (complete Freund's adjuvant).

[0082] T1+21 days 60μg / mouse, combined with an equal volume of IFA (incomplete Freund's adjuvant)

[0083] T1+35 days 60μg / mouse, combined with an equal volume of IFA (incomplete Freund's adjuvant)

[0084] T1+42 days 60μg / mouse, combined with an equal volume of IFA (incomplete Freund's adjuvant)

[0085] Following final immunization, monoclonal antibody titers were determined by collecting blood from the tail of each mouse. Splenocytes from mice with the highest antibody titers were fused to create hybridomas. Hybridomas binding to recombinant Clara cell protein were screened, and two stable cell lines were selected for characterization and product development: 1A5 and 2C8.

[0086] The specificity of antibodies 1A5 and 2C8 was determined by indirect ELISA. Both antibodies prepared could recognize denatured and non-denatured native CC16 protein, and showed no cross-reactivity with commercially available antibodies ab307666 and ab239472 (Abcam plc (Abcm)), demonstrating good specificity.

[0087] Table 1. Specificity analysis of 1A5 and 2C8 antibodies.

[0088]

[0089] The ability of the 1A5 and 2C8 antibodies to be used in antibody sandwich enzyme immunoassays was also identified. Antibodies were coated at constant concentrations in 96-well plates and assayed using 3-fold serial dilutions against CC16 (concentrations from 10 ng / ml to 0.0412 ng / ml). The complex was detected using another monoclonal antibody conjugated with biotin. Figure 2 As shown, all antibody combinations achieved similar sensitivity and detected 0.0412 ng / mL of CC16.

[0090] The increased binding affinity between 1A5 and 2C8 was confirmed in a direct comparison of the two antibody pairs with human serum samples. In the experiments summarized in Table 2, antibodies containing ab307666-ab239472 were compared with 1A5-2C8 using the Abcm ELISA. Two serum samples were tested in a 2-fold dilution series, representing high and low concentrations.

[0091] Table 2: Comparison of the sensitivity of ab307666-ab239472 and 1A5-2C8 to human serum samples

[0092]

[0093] Where ND = Not detected, and EUL = Exceeded the limit of detection.

[0094] The detection limit for the ab307666-ab239472 pair is 1:4 dilution of high-concentration samples, which is not very accurate. In contrast, 1A5-2C8 can accurately measure dilutions as low as 1:256, with good accuracy (<15%). The ab307666-ab239472 pair cannot detect even the least diluted low-concentration serum samples.

[0095] Example 2: Analysis of the 1A5-2C8 antibody pairing kit

[0096] Sensitivity: The sensitivity limit was determined by measuring multiple concentrations of the diluted calibrator in 20 replicates. The concentrations of the calibrator tested, excluding the buffer blank, included 0.02, 0.10, 0.50, and 1.0 ng / mL. Functional sensitivity was defined as the lowest concentration that resulted in a CV < 10%. As shown in Table 3 below, all tested concentrations met this standard, with the lowest tested concentration being 0.02 ng / mL.

[0097] Table 3: Summary of Functional Sensitivity Analysis

[0098]

[0099] Accuracy: The accuracy of the test was evaluated according to Clinical and Laboratory Standards Institute (CLSI) guidelines EP5-A2. Three serum samples from subjects at low, medium, and high concentrations were aliquoted into 20 1.5 mL centrifuge tubes and stored at -80°C. Samples were analyzed in duplicate daily for 20 consecutive days, and the results are shown in Table 4. The intra-round CVA and total CVA were all <10% at low concentrations (4 ng / mL), medium concentrations (50 ng / mL), and high concentrations (100 ng / mL). Grubbs' test was used to identify outliers, and no accuracy deviations were found within the tested concentration range.

[0100] Table 4 Summary of Accuracy Analysis

[0101]

[0102] Evaluation of interfering substances (sensitivity to abnormal substances in the sample): Six serum samples were collected. Each serum sample was divided into 12 aliquots, and 5% interfering substance solution was added to each aliquot to achieve hemoglobin concentrations of 1.25, 2.5, and 5 mg / mL; triglyceride concentrations of 2.5, 5, and 10 mg / mL; and bilirubin concentrations of 0.05, 0.1, and 0.2 mg / mL. 5% process water, 5% ethanol, and 5% 0.02M PBS buffer were added as controls to prevent interference. Analysis was performed using a Clara Cell Protein Assay Kit. The results are summarized in Table 5. Serum samples showed minimal interference from jaundice (bilirubin <0.2 mg / mL), hemolysis (hemoglobin <5 mg / mL), and lipemia (triglycerides <10 mg / mL), with deviations within ±10%.

[0103] Table 5 Summary of abnormal substance test results in the samples

[0104]

[0105]

[0106]

[0107] Determination of the normal concentration reference interval: 200 serum samples from healthy individuals were recruited and tested. They were evenly distributed in terms of sex and age (18 to 72 years). The number of individuals in each age group and the sex distribution were even. The t-test confirmed no difference. The reference values ​​were calculated using the percentile method after statistical analysis. Two-sided cutoff values ​​of P2.5 and P97.5 were used. The reference interval was 3.7 ng / mL to 23.2 ng / mL.

[0108] Clara cell protein concentrations under fasting versus non-fasting conditions: Serum samples were collected from 20 healthy individuals at 8:00 AM after an overnight fast, with a mean CC16 level of 12.3 g / mL (median: 11.9 ng / mL; range: 7.7–13.4 ng / mL). After consuming a standardized breakfast, a second blood sample was collected at 11:00 AM, with a mean CC16 level of 13.2 ng / mL (median: 14.7 ng / mL; range: 8.6–14.8 ng / mL). A third and final blood sample was collected at 2:00 PM, with a mean CC16 level of 13.5 ng / mL (median: 13.7 ng / mL; range: 10.4–17.5 ng / mL). Paired t-tests were used to determine if differences existed. The relative changes in Clara cell protein concentration over time were compared with the RCV to determine the effect of food on the concentration of the analyte. It was found that the average Clara cell protein concentration at 11:00 am and 2:00 pm was <10% higher than the average fasting value at 7:00 am.

[0109] Comparison of Clara cell protein concentrations in normal and disease states: Normal concentrations determined from 200 healthy donors were compared with several different groups: 200 healthy volunteers screened for the absence of inflammatory diseases (BNP, PCT, CRP, and IL-6), 100 patients diagnosed with silicosis, 70 patients diagnosed with chronic obstructive pulmonary disease (COPD), 100 patients diagnosed with pneumonia (PNA), and 60 patients diagnosed with pulmonary fibrosis. Table 6 lists the median, 95% confidence interval, and interquartile range (IQR) of Clara cell protein concentrations for each group. In the analyses described below, patients with silicosis, COPD, PNA, and pulmonary fibrosis had lower Clara cell protein concentrations than healthy individuals, while patients with acute respiratory distress syndrome (ARDS) had higher Clara cell protein concentrations than healthy individuals.

[0110] Table 6: CC16 concentrations in various disease states

[0111]

[0112] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0113] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0114] sequence list

[0115] <110> Bioscience (Tianjin) Diagnostics Co., Ltd.

[0116] tic Technology Co., Ltd)

[0117] <120> Human CC16 and its monoclonal antibody and detection methods

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[0178] SerLeuLysLeuSerCysAlaAlaSerGlyPheThrPheSerSerTyr 202530

[0180] ValMetSerTrpValArgGlnThrProGluLysArgLeuGluTrpVal 354045

[0182] AlaThrIleSerGlyGlyGlyGlyTyrThrTyrTyrProAspSerVal 505560

[0184] LysGlyArgPheThrIleSerArgAspAsnAlaLysAsnThrLeuTyr 65707580

[0186] LeuGlnMetSerSerLeuArgSerGluAspThrAlaLeuTyrTyrCys 859095

[0188] AlaArgGlnGluLeuArgArgGluTyrPheAspTyrTrpGlyGlnGly 100105110

[0190] ThrThrLeuThrValSerSerAlaLysThrThrProProSerValTyr 115120125

[0192] ProLeuAlaProGlySerAlaAlaGlnThrAsnSerMetValThrLeu 130135140

[0194] GlyCysLeuValLysGlyTyrPheProGluProValThrValThrTrp 145150155160

[0196] AsnSerGlySerLeuSerSerGlyValHisThrPheProAlaValLeu 165170175

[0198] GlnSerAspLeuTyrThrLeuSerSerSerValThrValProSerSer 180185190

[0200] ThrTrpProSerGluThrValThrCysAsnValAlaHisProAlaSer 195200205

[0202] SerThrLysValAspLysLysIleValProArgAspCysGlyCysLys 210215220

[0204] ProCysIleCysThrValProGluValSerSerValPheIlePhePro 225230235240

[0206] ProLysProLysAspValLeuThrIleThrLeuThrProLysValThr 245250255

[0208] CysValValValAspIleSerLysAspAspProGluValGlnPheSer 260265270

[0210] TrpPheValAspAspValGluValHisThrAlaGlnThrGlnProArg 275280285

[0212] GluGluGlnPheAsnSerThrPheArgSerValSerGluLeuProIle 290295300

[0214] MetHisGlnAspTrpLeuAsnGlyLysGluPheLysCysArgValAsn 305310315320

[0216] SerAlaAlaPheProAlaProIleGluLysThrIleSerLysThrLys 325330335

[0218] GlyArgProLysAlaProGlnValTyrThrIleProProProLysGlu 340345350

[0220] GlnMetAlaLysAspLysValSerLeuThrCysMetIleThrAspPhe 355360365

[0222] PheProGluAspIleThrValGluTrpGlnTrpAsnGlyGlnProAla 370375380

[0224] GluAsnTyrLysAsnThrGlnProIleMetAspThrAspGlySerTyr 385390395400

[0226] PheValTyrSerLysLeuAsnValGlnLysSerAsnTrpGluAlaGly 405410415

[0228] AsnThrPheThrCysSerValLeuHisGluGlyLeuHisAsnHisHis 420425430

[0230] ThrGluLysSerLeuSerHisSerProGlyLys 435440

Claims

1. An antibody against Clara cell protein or an antigen-binding fragment thereof, characterized in that, The antibody comprises a light chain and a heavy chain, wherein the light chain has an amino acid sequence as shown in SEQ ID NO: 2, and the heavy chain has an amino acid sequence as shown in SEQ ID NO:

3.

2. A recombinant human Clara cell protein, characterized in that, It has the amino acid sequence shown in SEQ ID NO:

1.

3. A composition comprising the Clara cell protein antibody or antigen-binding fragment of claim 1 and a pharmaceutically acceptable carrier.

4. The use of the Clara cell protein antibody or antigen-binding fragment of claim 1, or the composition of claim 3, in the preparation of a substance for quantifying Clara cell protein levels in a subject; The determination includes obtaining a sample from the subject and determining the level of Clara cell protein in the sample by binding it to at least one isolated antibody or antigen-binding fragment.

5. The use of the Clara cell protein antibody or antigen-binding fragment of claim 1, or the composition of claim 3, in assessing a subject's risk of developing lung-related disease; the determination comprising obtaining a sample from the subject and determining the Clara cell protein level in the sample using the at least one isolated antibody or antibody fragment, wherein if the subject's Clara cell protein level is increased or decreased relative to a Clara cell protein reference level, then the subject has an increased risk of developing lung-related disease; substantially the same Clara cell protein level indicates a reduced risk of developing lung-related disease in the subject.

6. The use of the Clara cell protein antibody or antigen-binding fragment of claim 1, or the composition of claim 3, in determining the progression of a subject's disease or in scientific research; The determination includes obtaining a sample from the subject and determining the level of Clara cell protein in the sample using the at least one isolated antibody or antigen-binding fragment, wherein if the subject's Clara cell protein level is higher or lower than a Clara cell protein reference level, then the subject's condition has not improved; if the subject's Clara cell protein level is consistent with a Clara cell protein reference level, then the subject's condition has improved.

7. The use of the Clara cell protein antibody or antigen-binding fragment of claim 1, or the composition of claim 3, in the preparation of a diagnostic reagent.

8. The use according to any one of claims 4-7, characterized in that: The subjects were undiagnosed or did not exhibit symptoms of a disease.

9. The use according to claim 6, characterized in that: The subject had been diagnosed with lung-related disease.

10. The use according to claim 9, characterized in that: The lung-related disease is silicosis; wherein the subject has one or more of the following symptoms: dyspnea, cough, expectoration, chest pain, wheezing, hemoptysis, and systemic symptoms.

11. The use according to claim 9, characterized in that: The lung-related disease is chronic obstructive pulmonary disease; wherein the subject has one or more of the following symptoms: persistent airflow limitation and corresponding respiratory symptoms.

12. The use according to claim 9, characterized in that: The lung-related disease is asthma; wherein the subject has one or more of the following symptoms: recurrent wheezing, shortness of breath, chest tightness or cough, often related to exposure to allergens, cold air, physical or chemical irritants, viral upper respiratory tract infections, exercise, etc.

13. The use according to claim 9, characterized in that: The lung-related disease is acute respiratory distress syndrome; wherein the subject has one or more of the following symptoms: acute onset or exacerbation of hypoxic respiratory failure within one week of the presence of risk factors or the appearance of new or worsening of existing respiratory symptoms.

14. The use according to claim 9, characterized in that: The lung-related disease is pneumonia; wherein the subject has one or more of the following symptoms: fever, cough, sputum production, shortness of breath, tachycardia or nonspecific symptoms including headache, nausea, vomiting, anorexia and weight loss, altered mental status; recent onset of cough, sputum production or worsening of pre-existing respiratory symptoms, with or without purulent sputum, chest pain, dyspnea and hemoptysis.

15. The use according to claim 9, characterized in that: The lung-related disease is pulmonary fibrosis; wherein the subject has one or more of the following symptoms: progressively worsening dyspnea and progressively deteriorating lung function.

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