Polypeptides, antibodies based on pdzk1ip1 protein and uses thereof

Abstract

The present application relates to the technical field of antibody immunology detection, and particularly relates to a polypeptide based on PDZK1IP1 protein, an antibody and application thereof. A polypeptide for preparing a tumor marker PDZK1IP1 antibody is disclosed in the first aspect, and the amino acid sequence at positions 68-81 of the PDZK1IP1 protein is selected. A polyclonal antibody based on the polypeptide is disclosed in the second aspect, and is used for preparing or screening a lung nodule detection reagent, and the expression amount of the PDZK1IP1 protein in a subject is reflected by the polyclonal antibody. A kit for detecting lung cancer related PDZK1IP1 protein is disclosed in the third aspect, and the kit comprises latex particles coated with the polyclonal antibody, and a standard product obtained by expression and purification of a recombinant expression vector of the whole sequence of the PDZK1IP1 protein gene. The present application discloses a non-invasive biomarker protein for distinguishing benign and malignant lung nodules for the first time, and develops a detection method and a corresponding diagnostic kit based on the immunoturbidimetry technology, so as to provide a new detection approach of non-invasion, rapidness, sensitivity and high specificity for early lung cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody immunological detection, and in particular to a polypeptide based on PDZK1IP1 protein, an antibody and applications thereof. Background Art

[0002] Globally, lung cancer, with its high morbidity and mortality, is the leading health threat among malignant tumors, and its incidence rate continues to grow. According to the latest data, the five-year survival rate for lung cancer patients in my country is approximately 19.7%. For patients in the early stages of lung cancer (Stage I), the five-year survival rate can significantly increase to 77% to 92%. Therefore, early diagnosis and timely treatment of lung cancer are crucial for improving patient survival and prognosis.

[0003] With the advancement of medical imaging technology, low-dose spiral CT is widely used to detect early-stage lung nodules and is an effective means for early lung cancer screening. However, low-dose spiral CT is plagued by a high false-positive rate, and repeated CT scans can accelerate lung cancer progression. Other early lung cancer screening methods, including PET, MRI, bronchoscopy, and needle aspiration, suffer from limitations in early detection, invasiveness, and high costs. Traditional conventional tumor markers, such as CEA, CYFRA21-1, and NSE, are detected in very low or even absent blood levels in the early stages of cancer. They are currently primarily used for dynamic monitoring of malignant tumors and are difficult to use as a reliable basis for early diagnosis or definitive diagnosis of lung cancer. Lung nodule marker detection techniques that rely on tissue or blood samples offer the highest accuracy for early lung cancer diagnosis, but these methods suffer from sampling limitations, such as inconvenience and invasiveness. Currently, there are relatively few reports on the discovery and application of noninvasive lung nodule tumor markers, primarily due to the limited sensitivity and specificity of currently commercialized lung cancer and lung nodule biomarkers when tested in noninvasive fluids, such as sputum.

[0004] Regarding the above-mentioned related technologies, the inventors believe that developing a non-invasive liquid biopsy technology based on new biomarkers for early screening of lung nodules, so as to conduct regular screening and improve the detection rate of early lung cancer, is a key strategy to reduce the mortality rate of lung cancer. Summary of the Invention

[0005] In order to improve the current problems of low sampling convenience, invasiveness, and insufficient sensitivity and specificity of non-invasive liquid biomarkers in early screening of lung nodules, the first purpose of the present invention is to provide a biomarker PDZK1IP1 protein and its polypeptide antigen screened from the non-invasive fluid of lung cancer subjects, which can be used to assist in the non-invasive screening of benign and malignant lung nodules.

[0006] The second object of the present invention is to provide a polyclonal antibody prepared using a polypeptide antigen based on the PDZK1IP1 protein, which has high specificity and sensitivity to the PDZK1IP1 protein and can be used for in vitro non-invasive detection of benign and malignant lung nodules.

[0007] The third object of the present invention is to provide a kit for detecting lung cancer-related PDZK1IP1 protein, which includes the above-mentioned PDZK1IP1 protein standard and polyclonal antibody, and performs non-invasive in vitro lung nodule screening based on latex-enhanced immunoturbidimetry, with the advantages of high specificity, accuracy and safety, automated analysis, simple operation and low cost.

[0008] In order to achieve the first object of the present invention, the present invention provides a polypeptide for preparing an antibody against the tumor marker PDZK1IP1, which adopts the following technical solution:

[0009] A polypeptide for preparing a tumor marker PDZK1IP1 antibody, wherein the polypeptide is selected from the amino acid sequence at positions 68 to 81 of the PDZK1IP1 protein, and the amino acid sequence of the PDZK1IP1 protein is shown in SEQ ID NO.1.

[0010] The present invention discovered that the PDZK1IP1 protein is highly expressed in early clinical samples of malignant pulmonary nodules. Using PDZK1IP1 as a biomarker for pulmonary nodule screening, the study further investigated its application in the non-invasive in vitro detection of benign and malignant pulmonary nodules. The full sequence of the PDZK1IP1 protein (SEQ ID NO. 1) was analyzed for antigenicity, hydrophilicity, and surface structure potential. The highly antigenic and hydrophilic amino acid sequence at positions 68 to 81 was selected as an immunogenic peptide, enabling the successful preparation of highly specific polyclonal antibodies.

[0011] In some embodiments, the polypeptide is linked to a cysteine ​​at the C-terminus, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.2.

[0012] In the above technical solution, a cysteine ​​is artificially added to the C-terminus of the polypeptide to provide a free thiol group for coupling with the carrier protein.

[0013] In some embodiments, the tumor is lung cancer.

[0014] In order to achieve the second object of the present invention, the present invention provides a polyclonal antibody, which adopts the following technical solution:

[0015] A polyclonal antibody is obtained by immunizing an animal with the above polypeptide.

[0016] The application also provides application of the polyclonal antibody in preparation or screening of a lung nodule diagnostic reagent, wherein the polyclonal antibody is immunologically combined with PDZK1IP1 protein in a subject, and expression of the PDZK1IP1 protein in the subject is reflected by the polyclonal antibody.

[0017] The experimental results show that the polyclonal antibody prepared by the application has a specificity of 93.33% and a sensitivity of 83.33% in lung nodule malignancy identification in the application of PDZK1IP1 protein detection, and can be used for early screening of lung cancer.

[0018] In some embodiments, when the polyclonal antibody is immunologically combined with the PDZK1IP1 protein in the subject, the body fluid sample is at least one selected from serum, plasma, blood and sputum.

[0019] In some embodiments, the body fluid sample is sputum, and the sputum is subjected to separation treatment, wherein the separation treatment comprises steps of separating extracellular vesicles in the sputum and performing protein extraction and purification on the extracellular vesicles.

[0020] In order to further reduce sampling difficulty and avoid trauma in early screening of lung cancer, the application selects sputum as a detection sample, pre-treats the detected sputum sample, and uses extracellular vesicle protein in the sputum as a final detection sample, thereby improving accuracy and sensitivity of the polyclonal antibody in detection of the PDZK1IP1 protein in the sputum.

[0021] In order to achieve the third object of the application, the application provides a kit for detecting lung cancer-related PDZK1IP1 protein.

[0022] The kit for detecting lung cancer-related PDZK1IP1 protein comprises the polyclonal antibody.

[0023] In some embodiments, the kit comprises:

[0024] The first reagent composition comprises a buffer and a preservative.

[0025] The second reagent composition comprises a buffer, latex particles coated with the polyclonal antibody, a stabilizer and a preservative.

[0026] The standard product is obtained by expression and purification of a recombinant expression vector of the full sequence of the PDZK1IP1 protein gene.

[0027] In some embodiments, the kit further comprises a sputum box, a filter and an affinity column, and a pore size of a filter membrane of the filter is 0.22 μm.

[0028] Through the above technical scheme, the kit of the present invention can use sputum as a sample to perform non-invasive in vitro screening for early lung nodules based on latex-enhanced immunoturbidimetry. It has the advantages of high specificity, accuracy and safety, automated analysis, simple operation and low cost, which greatly enhances the convenience of sampling and detection.

[0029] In summary, the present invention provides a polypeptide, antibody and application thereof based on PDZK1IP1 protein, which have the following beneficial effects:

[0030] First, the present invention uses quantitative proteomics technology of clinical samples to clarify that the level of PDZK1IP1 in the sputum of patients with malignant lung nodules is significantly higher than that in patients with benign lung nodules, revealing for the first time the application of PDZK1IP1 as a biomarker for early screening of benign and malignant lung nodules.

[0031] Second, the present invention obtains highly specific immune peptides through sequence analysis verification, and uses the immune peptides to prepare polyclonal antibodies, realizing for the first time the non-invasive screening technology for benign and malignant lung nodules, avoiding the risk of traumatic sampling for patients, and providing a new non-invasive identification biological resource for early screening of lung nodules.

[0032] Third, based on the PDZK1IP1 protein, the present invention constructs a recombinant expression vector of the full sequence of the PDZK1IP1 protein gene, prepares a recombinant protein standard, and prepares a kit for detecting the lung cancer-related marker PDZK1IP1 protein. The latex-enhanced immunoturbidimetry method is used to quantitatively determine the PDZK1IP1 protein in sputum, which can quickly, non-invasively, cost-effectively, highly specifically, accurately and safely distinguish between benign and malignant lung nodules. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The figure shows the results of antigen polypeptide analysis of the PDZK1IP1 protein sequence using the antigen design tool in Example 1. The horizontal axes 1-5 represent five different antigen fragments, and the vertical axis shows the immunogenicity scores of the antigen polypeptide fragments.

[0034] Figure 2 The ROC curve obtained by the treatment in Example 5 is shown, with the horizontal axis representing specificity and the vertical axis representing sensitivity.

[0035] Figure 3 Shown is a scatter plot of the expression level of PDZK1IP1 protein in the sputum of patients with malignant pulmonary nodules and benign pulmonary nodules in Example 5, the horizontal axis shows the two groups of specimens of malignant pulmonary nodules and benign pulmonary nodules, and the vertical axis shows the PDZK1IP1 protein level (ng / ML) in sputum detected by this kit. DETAILED DESCRIPTION

[0036] The first aspect of the present invention discloses a biomarker for preparing or screening a lung cancer detection reagent. The biomarker is the PDZK1IP1 protein, and the expression level of PDZK1IP1 protein in a subject is positively correlated with the presence or severity of malignant lung nodules. Although PDZK1IP1 protein is used as a marker for lung cancer in this embodiment, in other embodiments, PDZK1IP1 protein can be used as a marker for other types of tumors.

[0037] The second aspect of the present invention is based on the human PDZK1IP1 protein and discloses a polypeptide for preparing antibodies against the tumor marker PDZK1IP1. This polypeptide, as an immunogenic peptide, is not limited to preparing polyclonal antibodies, but can also be used to prepare monoclonal antibodies. The prepared polyclonal antibodies are not limited to immunodetection of PDZK1IP1 protein in lung cancer samples, but can also be used to immunodetect PDZK1IP1 protein in other tumor samples.

[0038] The polypeptide is an analysis of the antigenicity, hydrophilicity, surface accessibility and flexibility of the human PDZK1IP1 protein sequence (SEQ ID NO.1). Among the candidate fragments, the polypeptide is analyzed using an antigen design tool to minimize cross-reactions and ensure specificity, and finally the immune peptide with the highest score is screened as the polypeptide antigen.

[0039] The third aspect of the present invention is to provide a method for preparing polyclonal antibodies by immunizing animals with the above-screened polypeptide antigens. The animals used for the immunization can be rabbits, goats, mice and other animals commonly used for antibody preparation.

[0040] A fourth aspect of the present invention is to provide the use of the polyclonal antibody in the preparation or screening of a lung nodule diagnostic reagent, wherein the polyclonal antibody immunobinds to the PDZK1IP1 protein in the subject, and the polyclonal antibody reflects the expression level of the PDZK1IP1 protein in the subject. The immunobinding can be an enzyme-linked immunosorbent assay, immunofluorescence, immunochemiluminescence, immunoturbidimetry, or immunoblotting to detect the expression level of the PDZK1IP1 protein. When detecting the expression level of the PDZK1IP1 protein in the subject, the test sample is taken from the subject's body fluid, which can be at least one of serum, plasma, blood, and sputum.

[0041] The fifth aspect of the present invention is to provide a kit for detecting lung cancer-related PDZK1IP1 protein and a preparation method thereof, wherein the kit comprises:

[0042] The first reagent composition includes a buffer solution and a preservative solution, which can be a mixed liquid prepared by the buffer solution and the preservative solution at a certain final concentration, or the buffer solution and the preservative solution can be packaged and used separately;

[0043] The second reagent composition includes a buffer solution, latex particles coated with the polyclonal antibody, a stabilizer, and a preservation solution. The second reagent composition may be a mixed liquid prepared by mixing the buffer solution, the latex particles coated with the polyclonal antibody, the stabilizer, and the preservation solution at a certain final concentration, or may be a separately packaged and used buffer solution, a suspension of latex particles coated with the polyclonal antibody, a stabilizer, and a preservation solution.

[0044] The standard product is obtained by expressing and purifying the recombinant expression vector of the full sequence of the PDZK1IP1 protein gene.

[0045] The buffer in the first reagent composition and the second reagent composition may be, but not limited to, a phosphate buffer, and the preservation liquid in the first reagent composition and the second reagent composition may be a liquid with an antiseptic function, and may be, but not limited to, sodium azide.

[0046] The particle size of the latex particles is 100nm to 500nm.

[0047] The kit quantitatively determines the expression level of PDZK1IP1 protein in the subject's body fluid by latex-enhanced immunoturbidimetry. The subject's body fluid can be at least one of serum, plasma, blood, and sputum. As a preferred embodiment, the subject's body fluid is sputum, and the sputum is subjected to a separation treatment, and the separation treatment includes the steps of separating extracellular vesicles in the sputum and extracting and purifying proteins from the extracellular vesicles.

[0048] In some embodiments, the kit further comprises a sputum box, a filter, and a CD63 affinity column, wherein the filter membrane has a pore size of 0.22 μm.

[0049] The following will further illustrate embodiments of the present invention in conjunction with the accompanying drawings and specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. It should be noted that many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein. Therefore, the scope of protection of the present invention is not limited by the specific embodiments disclosed below. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0050] Example 1

[0051] This embodiment discloses a PDZK1IP1 protein polypeptide antigen and a screening method thereof, comprising the following steps:

[0052] Combined with the Uniprot database, the NOVOFOCUS antigen design tool was used to analyze peptides. The antigenicity, hydrophilicity, and surface structure possibility of the PDZK1IP1 protein sequence (SEQ ID NO. 1) were analyzed. The sequence with the highest score was selected as the immune peptide. The sequence of the immune peptide corresponds to amino acids 68 to 81 of the PDZK1IP1 protein. Cysteine ​​was artificially added to the end (C-terminus) of the immune peptide to finally obtain a polypeptide antigen for the preparation of the tumor marker PDZK1IP1 antibody. The sequence of the polypeptide antigen is VGNKADGVLVGTDGC (SEQ ID NO. 2), named IPM-1FJJ.

[0053] Figure 1 This is an antigenic peptide analysis diagram of the PDZK1IP1 protein sequence using NOVOFOCUS, wherein the horizontal axis 5 represents the VGNKADGVLVGTDGC (SEQ ID NO. 2) fragment, which has the highest peptide score and shows the highest antigenicity.

[0054] Example 2

[0055] This embodiment discloses a polyclonal antibody and a method for preparing the same, comprising the following steps:

[0056] S1: Synthesize the polypeptide IPM-1FJJ described in Example 1.

[0057] S2: The polypeptide synthesized in S1 is coupled to hemocyanin (KLH).

[0058] S3: Animal immunization: The initial immune antigen peptide-coupled KLH protein solution obtained in S2 is mixed with an equal volume of Freund's complete adjuvant to fully emulsify the antigen and adjuvant;

[0059] Rabbits were injected subcutaneously at multiple points in the abdominal cavity;

[0060] On the 14th day, the booster immunization was conducted with 50% of the initial immunization dose of antigen, which was fully emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points in the abdominal cavity of the rabbits.

[0061] On the 21st day, venous blood was collected and the antibody titer was measured by indirect ELISA;

[0062] On the 28th day, boost immunization until a satisfactory immune effect is achieved;

[0063] For impulse immunization, rabbit blood was obtained after one immunization three days before fusion.

[0064] S4: Purification and preparation of polyclonal antibodies

[0065] S41: Preparation of antiserum

[0066] The rabbit blood obtained in S3 was allowed to stand at room temperature for 1 hour to allow it to coagulate and separate out the serum. The serum was collected and centrifuged at 15,000 g for 10 minutes at 4°C. The supernatant was collected to obtain the antiserum.

[0067] S42: Purification of polyclonal antibodies

[0068] The antiserum obtained from S41 was diluted with TBS buffer at a ratio of 1:3 and filtered, and then loaded onto a protein A column at a rate of 0.5 ml per minute;

[0069] The column was washed with TBS buffer solution, and the polyclonal antibody bound to the column was eluted with pH 2.7 elution buffer at a rate of 2 ml / min;

[0070] The eluate was neutralized with a neutralization buffer and its pH was adjusted to about pH 7.2 to prevent polyclonal denaturation, thereby obtaining rabbit anti-human PDZK1IP1 protein polyclonal antibodies. The titer of the purified polyclonal antibodies was determined to be approximately 1:1.28×10 6 above.

[0071] Example 3

[0072] This embodiment discloses a kit for detecting lung cancer-related PDZK1IP1 protein and a preparation method thereof.

[0073] 1. Preparation of latex particles coated with rabbit anti-human PDZK1IP1 protein polyclonal antibody.

[0074] Rabbit anti-human PDZK1IP1 protein polyclonal antibody was coupled to the surface of latex particles by chemical cross-linking, which specifically includes the following steps:

[0075] S1: 100 mg of latex particles were dissolved in 5 mL of MES buffer (50 mM, pH 6.5), and a cross-linking surfactant SDS (final mass concentration 0.01%) was added to obtain a latex particle solution. The particle size of the latex particles was 100-500 nm.

[0076] S2: The rabbit anti-human PDZK1IP1 protein polyclonal antibody prepared in Example 2 was dissolved in 5 mL of MES cross-linking buffer solution (50 mM, pH 6.5) to a concentration of 1 μg / mL to obtain a rabbit anti-human PDZK1IP1 protein polyclonal antibody solution.

[0077] S3: The latex particle solution was mixed with the rabbit anti-human PDZK1IP1 protein polyclonal antibody solution, 100 mg of a chemical cross-linking agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), was added, and the mixture was reacted at room temperature for 2 h to obtain latex particles coated with the rabbit anti-human PDZK1IP1 protein polyclonal antibody.

[0078] 2. Preparation of recombinant protein standards.

[0079] S1, strain construction:

[0080] According to common genetic engineering methods, a prokaryotic expression plasmid pET-28a(+) with the complete sequence of the PDZK1IP1 protein gene (SEQ ID NO.1), i.e., a recombinant plasmid, was constructed. The recombinant plasmid was named pET-IPM-1FJJ.

[0081] S2, expression and purification of recombinant antigen protein:

[0082] Expression and purification of the recombinant plasmid pET-IPM-1-FJJ in E. coli BL21(DE3): A single E. coli BL21(DE3) colony containing the recombinant plasmid pET-IPM-1-FJJ was inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking at 200 rpm for 7-8 hours. Then, 5 mL of the bacterial suspension was added to 500 mL of LB medium containing 50 μg / mL kanamycin at a 1:100 ratio and further cultured for 2 hours to an OD600 of approximately 0.6. IPTG was added to the bacterial suspension to a final concentration of 0.6 mmol / L, and the culture was cultured with shaking at 16°C overnight. The overnight induced cells were harvested by centrifugation, and the pellet was resuspended in Lysis Buffer. After incubation on ice for 30 minutes, the pellet was sonicated for 15 minutes, and the supernatant was collected by centrifugation. The recombinant protein was purified using Ni-NTA, and impurities were washed away using a 20 mmoL / L imidazole eluent, followed by elution with a 200 mmoL / L imidazole eluent to obtain a PDZK1IP1 recombinant protein mother solution.

[0083] S3, preparation of recombinant protein standards:

[0084] The recombinant protein standard includes the stabilizer bovine serum albumin and the recombinant protein PDZK1IP1. The PDZK1IP1 recombinant protein stock solution obtained by gradient dilution of S2 with bovine serum albumin is used to prepare the recombinant protein standard, hereinafter referred to as the standard. The concentrations of the calibrator are 0 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 150 ng / mL, and 300 ng / mL, respectively.

[0085] 3. Preparation of the first reagent composition and the second reagent composition.

[0086] The first reagent composition is a mixed solution comprising a phosphate buffer solution and sodium azide. The final concentration of the phosphate buffer solution in the first reagent composition is 100 mmol / L, the final concentration of the sodium azide is 0.1%, and the pH of the first reagent composition is 8.

[0087] The second reagent composition is a mixed solution containing phosphate buffer, bovine serum albumin, sodium azide and the prepared rabbit anti-human PDZK1IP1 antibody latex particles. The final concentration of phosphate buffer in the second reagent composition is 100 mMol / L, the final concentration of rabbit anti-human PDZK1IP1 antibody latex particles is 5 mg / mL, the final concentration of bovine serum albumin is 20 g / L, the final concentration of sodium azide is 0.1%, and the pH of the second reagent composition is 8.

[0088] 4. A kit for detecting lung cancer-related PDZK1IP1 protein.

[0089] The kit composition is shown in Table 1, which includes the above-prepared standard, the first reagent composition (hereinafter referred to as reagent 1), the second reagent composition (hereinafter referred to as reagent 2), and the sputum box, 0.22 μm filter and affinity column.

[0090] Table 1: Kit for detecting lung cancer-related PDZK1IP1 protein

[0091]

[0092] The kit specifications given in Table 1 are for detecting 20 people per kit, but this is not the only specification, and production adjustments can be made according to actual conditions.

[0093] Example 4

[0094] This embodiment discloses a method for using the kit for detecting lung cancer-related PDZK1IP1 protein of Example 3.

[0095] 1. Sample collection and processing.

[0096] Sample collection: Take 5 ml of sputum from a patient with lung nodules into a 20 ml plastic container, add 15 ml of phosphate buffered saline (PBS), rotate for 30 s, centrifuge at 800 g at 4°C for 10 minutes, and take the supernatant;

[0097] Sample separation: The supernatant is passed through a 0.22 μm filter, and the extracellular vesicles are separated by extracellular vesicle CD63 affinity method;

[0098] Sample processing: The extracellular vesicles are subjected to protein extraction and purification using RIPA lysis buffer (Radio Immunoprecipitation Assay Lysis buffer) containing protease and phosphatase inhibitors. After lysis on ice for 1 hour, centrifuge at 4°C and 12000 g for 30 minutes, collect the supernatant into a pre-cooled EP tube, and obtain the sample.

[0099] 2. Preparation of kit standard curve and ROC curve.

[0100] (1) Preparation of kit standard curve.

[0101] Prepare a PDZK1IP1 recombinant protein standard using a serial dilution of bovine serum albumin, with concentrations including S5: 160 ng / mL, S4: 80 ng / mL, S3: 40 ng / mL, S2: 20 ng / mL, S1: 10 ng / mL, and S0: 0 ng / mL.

[0102] Use an automatic biochemical analyzer (model: HITACHI7170S automatic biochemical analyzer) to detect the standards in sequence, make a standard curve, and test at 340nm. According to the above S0-S5 standard concentrations, refer to Table 1, prepare test tubes, take 180μL of reagent 1 in each tube, add 3μL of standard to each tube according to the S0-S5 standard concentration, react at 37℃ for 5 minutes, and read the absorbance A1 标 Then add 50 μL of reagent 2 to each tube and keep it at 37℃. After 5 minutes, measure the absorbance A2 again. 标 , calculate the absorbance difference ΔA 标 =A2 标 -A1 标 . Using the ΔA of a series of standard proteins 标 The standard curve of the kit was fitted using the value and concentration as variables.

[0103] (2) Create the ROC curve of the test kit.

[0104] Sputum samples from experimental and control groups were analyzed using the test kit of the present invention using a fully automated biochemical analyzer (model: HITACHI 7170S). ROC curves were generated using MedCalc software. The cutoff value associated with the Youden index is the cutoff value; samples exceeding this value are defined as protein-positive, while samples less than or equal to this value are considered protein antigen-negative.

[0105] 3. Sample testing.

[0106] The samples were tested using a fully automatic biochemical analyzer (model: HITACHI7170S fully automatic biochemical analyzer). According to the number of samples, the corresponding number of test groups were prepared. 180 μL of reagent 1 was taken for each group, and 3 μL of sample was added. The reaction was carried out at 37°C for 5 minutes, and the absorbance A1 was read. 测 Then, 50 μL of reagent 2 was added to each test group and kept warm at 37°C. After 5 minutes, the absorbance A2 was measured again. 测 , calculate the absorbance difference ΔA 测 =A2 测 -A1 测 , and then obtain the concentration value of the sample to be tested through the standard curve, and use the cut-off value as the judgment standard to judge the test result of the sample to be tested.

[0107] Example 5

[0108] This embodiment discloses Example 3, a use of a kit for detecting lung cancer-related PDZK1IP1 protein in differentiating benign and malignant lung nodules.

[0109] According to clinical procedures, sputum samples of 30 patients with benign pulmonary nodules and 30 patients with malignant pulmonary nodules were collected using sterile sputum boxes, 5 ml per person. The collected samples were processed according to the method of use of the kit disclosed in Example 4.

[0110] According to the method of Example 4, the 60 sputum specimens collected and processed were measured using the kit of the present invention using a full-automatic biochemical analyzer (HITACHI7170S full-automatic biochemical analyzer), and the sputum specimens of 30 patients with benign pulmonary nodules and 30 patients with malignant pulmonary nodules were divided into an experimental group. The test values ​​were processed using MedCalc software to obtain the ROC curve ( Figure 2 ), a cut-off value was set through data statistics, and sputum specimens exceeding this value were defined as protein positive (>19.97 ng / mL was considered malignant pulmonary nodules), and samples less than or equal to this value were considered protein antigen negative.

[0111] The clinical tests of 60 samples are shown in Table 2. Among the 30 patients with malignant pulmonary nodules, 25 samples tested positive; while among the 30 patients with benign pulmonary nodules, only 2 samples tested positive. There was a significant difference between the two groups. The specificity of PDZK1IP1 protein detection for distinguishing malignant pulmonary nodules was 93.33% (28 / 30), and the sensitivity was 83.33% (25 / 30). Figure 3 The scatter plot of the immunoturbidimetric detection of PDZK1IP1 protein in the sputum of patients with malignant and benign pulmonary nodules in this example shows that the expression of PDZK1IP1 protein in the sputum of malignant pulmonary nodules is increased compared with that in benign pulmonary nodules, and the difference is significant. Therefore, the composition designed based on the PDZK1IP1 protein marker in this kit is closely related to the early occurrence and development of lung cancer, and can provide a non-invasive method for clinical screening of benign and malignant pulmonary nodules, and provide an effective means for individualized treatment of patients.

[0112] Table 2 Analysis of clinical results of the marker combination lung nodule detection kit Number of cases: 60

[0113]

[0114] The above-mentioned embodiments are the best modes of the present application, which are only used to explain the technical concept and characteristics of the present application, and enable those skilled in the art to understand the content of the present application and implement it, and cannot limit the protection scope of the present application. Any modification made according to the spirit and essence of the main technical solution of the present application should be covered in the protection scope of the present application.

Claims

1. A polypeptide for preparing an antibody against the tumor marker PDZK1IP1, characterized in that: The polypeptide is selected from the amino acid sequence at positions 68 to 81 of the PDZK1IP1 protein, and the amino acid sequence of the PDZK1IP1 protein is shown in SEQ ID NO.1; a cysteine ​​is connected to the C-terminus of the polypeptide, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.

2.

2. Use of a polyclonal antibody prepared from the polypeptide according to claim 1 in the preparation of a diagnostic reagent for pulmonary nodules.

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