ZO-1 protein quantitative kit and application thereof

Abstract

The invention provides a ZO-1 protein quantitative kit and application thereof, and belongs to the technical field of biological science. The ZO-1 protein quantitative kit disclosed by the invention comprises a ZO-1 antibody with a specific antigen epitope binding region and immune PCR amplification, the specific antigen epitope binding region comprises amino acid sequences as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the immune PCR amplification comprises antigen-antibody reaction, PCR extension reaction and qPCR reaction. The invention also provides a ZO-1 protein quantitative detection method, which comprises the ZO-1 protein quantitative kit for detection, and the method has the advantages of short detection time, improved sensitivity and low detection cost, and is suitable for clinical popularization and application.

Description

Technical Field

[0001] The present invention belongs to the field of biological science and technology, and particularly relates to a ZO-1 protein quantification kit and its application. Background Art

[0002] ZO-1 is a core member of the tight junction protein family, mainly located at the tight junction sites between epithelial and endothelial cells. It can ensure the stability of cell-cell tight junctions by interacting with membrane proteins (such as Occludin and Claudin) and cytoplasmic proteins. At the same time, it can interact with a variety of cytoskeletal proteins and signaling proteins, which is crucial for maintaining cell morphology and regulating barrier function. In addition, under inflammatory conditions or injury stimuli, ZO-1 will redistribute or degrade, leading to the loosening or destruction of tight junction structures.

[0003] Ni Jingjing et al. (2021) found in the study "Value of tight junction protein ZO-1 in vascular endothelial cells in the severity and prognosis assessment of sepsis patients" that sepsis patients often have damage to the intestinal and vascular endothelial barrier functions in the early stage. As an important component protein of tight junctions, the dynamic changes of ZO-1 in plasma can, to a certain extent, reflect the degree of barrier damage. Since the changes in ZO-1 appear earlier in the course of the disease, detecting plasma ZO-1 may become an early warning indicator for predicting the progression of severe infections or sepsis, thus providing a basis for clinical intervention and treatment.

[0004] Currently, Western blot and ELISA methods are mainly used to detect ZO-1 protein in clinical practice. These methods have many problems: the detection cycle is long, usually at least 6-8 hours are required to complete the detection, which cannot meet the needs of rapid diagnosis of critically ill patients; the operation steps are cumbersome and require professional technical personnel to operate; the sample demand is large, and Western blot and ELISA usually require about 100 μg of total protein or 10 6 cells; the content of ZO-1 in the plasma of healthy people is extremely low (usually at ng / mL or lower levels), and traditional protein detection methods are difficult to achieve the required sensitivity and specificity.

[0005] The prior art CN103454427A discloses a method for detecting trace proteins in blood using antibody-nucleic acid combined amplification technology. By combining the principle of specific antigen-antibody binding and the principle of specific amplification of quantitative PCR, the specific signal is amplified millions or even tens of millions of times, thus greatly improving the detection sensitivity. At the same time, according to the concentration of the standard product, quantitative detection of trace proteins in the test sample can be achieved. However, this invention has not been verified for its effects, and its practical application value is difficult to confirm. Summary of the Invention

[0006] To solve the above problems, the present invention provides a ZO-1 protein quantification kit and its application. The ZO-1 protein quantification kit disclosed by the present invention includes a ZO-1 antibody with a specific antigen epitope binding region and immunological PCR amplification. The immunological PCR amplification includes antigen-antibody reaction, PCR extension reaction, and qPCR reaction. The present invention also provides a method for detecting ZO-1 protein quantification, including detecting with the ZO-1 protein quantification kit. The detection time of this method is shortened, the sensitivity is improved, and the detection cost is low, which is suitable for clinical promotion and application. In addition, this method can also achieve simultaneous detection of multiple samples, improving the detection efficiency. These characteristics make this method particularly suitable for early warning and dynamic monitoring of organ damage in critically ill patients with infections, providing a more effective basis for clinical diagnosis and treatment decisions for clinicians.

[0007] The specific technical solutions are as follows:

[0008] On the one hand, the present invention provides a ZO-1 protein quantification kit, including a ZO-1 antibody. The ZO-1 antibody includes a specific antigen epitope binding region, and the specific antigen epitopes include the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0009] SEQ ID NO.1: SGETSIVISDVLKGG;

[0010] SEQ ID NO.2: KFESPKFNHNLLPS;

[0011] SEQ ID NO.3: KRVEETPNYHKARK.

[0012] Specifically, the ZO-1 antibody is a monoclonal antibody or a polyclonal antibody.

[0013] Specifically, the specific antigen epitope binding region of the ZO-1 antibody includes the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0014] SEQ ID NO.4: SGETSIVISDVLKGG;

[0015] SEQ ID NO.5: KRVEETPNYHKARK;

[0016] SEQ ID NO.6: KFESPKFNHNLLPS.

[0017] Specifically, the specific antigen epitope binding region of the ZO-1 antibody includes the heavy chain variable region sequence shown in SEQ ID NO.7 and the light chain variable region sequence shown in SEQ ID NO.8.

[0018] SEQ ID NO.7: DVQLQESGPGLVPGASVKMSCTVTGYSITSDCAWNWVRQAPGNKLEWMGFITYSGSYTYYNPSLKSRISITRDTSSSSFFLQLNSVTTEDTATYYCARDYTYYYAMEYWGQGTSVTVSS;

[0019] SEQ ID NO.8: DIQMTQSSSYLSVSLGGRVTISCRSSQSLVHSDGHTWLAWYQQKPGNAPRLLIYYTSRLHTGVPSRFSGSGSGTQFSLKINSLQDVATYYCQQGKTLPWTFGGGTKLELK.

[0020] In the ZO-1 protein quantification kit, the ZO-1 antibody further includes a signal label, and the signal label is selected from any one or more of enzyme label, biotin label, and isotope label, preferably biotin label.

[0021] More specifically, the ZO-1 antibody in the kit is further linked with an oligonucleotide, and the oligonucleotide is selected from one or two of oligonucleotide A and oligonucleotide B; the oligonucleotide A has the nucleotide sequence shown in SEQ ID NO.9, and the oligonucleotide B has the nucleotide sequence shown in SEQ ID NO.10.

[0022] SEQ ID NO.9(A): TCAGGTGGACTTCTCGATGACTGGATTCGATGACTGGACTGA;

[0023] SEQ ID NO.10(B): GTCCTGTAGTACCGTCGTATCTAGTCGATGACTGGACTGAGT.

[0024] In some embodiments, the ZO-1 antibody includes an oligonucleotide-labeled antibody A labeled with oligonucleotide A and an oligonucleotide-labeled antibody B labeled with oligonucleotide B.

[0025] Specifically, the kit further includes immunological PCR amplification, and the immunological PCR amplification includes antigen-antibody reaction, PCR extension reaction, and qPCR reaction.

[0026] The immunological PCR amplification includes antigen-antibody reaction, PCR extension reaction, and qPCR reaction, and the system of the antigen-antibody reaction further includes an extension primer.

[0027] Specifically, the extension primer has the nucleotide sequence shown in SEQ ID NO.11.

[0028] SEQ ID NO.11: ACTCAGTCCAGTCATCGATACTAGATACGACGGTACTACAGGACGCTACTATCAGTCCAG.

[0029] Specifically, the antigen-antibody reaction system includes: the final concentration of ZO-1 antibody is 3-6 pM, and the final concentration of the extension primer is 3-6 pM.

[0030] Preferably, the antigen-antibody reaction system includes: the final concentration of ZO-1 antibody is 5.82 pM, and the final concentration of the extension primer is 5.82 pM.

[0031] Specifically, the molar ratio of oligonucleotide-labeled antibody A to oligonucleotide-labeled antibody B in the ZO-1 antibody is 1-2:1-2.

[0032] Preferably, the molar ratio of oligonucleotide-labeled antibody A to oligonucleotide-labeled antibody B in the ZO-1 antibody is 1:1.

[0033] Specifically, the antigen-antibody reaction system further includes 1-2 μL of the sample to be tested and 5-10 μL of buffer.

[0034] Preferably, the antigen-antibody reaction system further includes 2 μL of the sample to be tested and 5 μL of buffer.

[0035] Among them, the sample to be tested is any one or more of serum, plasma, and bronchoalveolar lavage fluid.

[0036] Preferably, the sample to be tested is plasma, and the plasma is obtained by centrifuging venous blood at 2000-3000 g for 10-15 min.

[0037] In some embodiments, the plasma is obtained by centrifuging venous blood at 2000 g for 10 min.

[0038] Specifically, the buffer includes Tris-HCl, EDTA, NaCl, BSA, salmon sperm DNA, PEG8000, Tween 20, and trehalose.

[0039] More specifically, the final concentration of Tris-HCl in the buffer is 25 mM, the final concentration of EDTA is 4 mM, the final concentration of NaCl is 150 mM, the final concentration of BSA is 0.2%, the final concentration of salmon sperm DNA is 0.02 mg / mL, the final concentration of PEG8000 is 5%, the final concentration of Tween 20 is 0.05%, and the final concentration of trehalose is 5%.

[0040] Specifically, the system for the PCR extension reaction includes the antigen-antibody reaction product, dNTP mix, NEBuffer 2.1, and T4 DNA polymerase. The system for the PCR extension reaction includes 8.5 - 10 μL of the antigen-antibody reaction product, a final concentration of 0.05 - 0.1 mM of dNTP mix, 80 - 90 μL of NEBuffer 2.1, and 3 - 5 μL of T4 DNA polymerase.

[0041] Among them, the antigen-antibody reaction product is obtained from the system for the antigen-antibody reaction described above.

[0042] Specifically, the system for the PCR extension reaction includes 8.5 μL of the antigen-antibody reaction product, a final concentration of 0.05 mM of dNTP mix, 86 μL of NEBuffer 2.1, and 5 μL of T4 DNA polymerase.

[0043] Specifically, the NEBuffer 2.1 includes NaCl, Tris-HCl, MgCl2, and recombinant albumin.

[0044] In immunological PCR amplification, the system for the qPCR reaction includes the extension reaction product, primer mixture, fluorescent probe, and qPCR Master Mix.

[0045] Among them, the extension reaction product is obtained from the system for the PCR extension reaction described above.

[0046] Specifically, the primer mixture includes a forward primer and a reverse primer. The sequence of the forward primer is as shown in SEQ ID NO.12, and the sequence of the reverse primer is as shown in SEQ ID NO.13.

[0047] SEQ ID NO.12 (forward primer): TCAGGTGGACTTCTCGATGA;

[0048] SEQ ID NO.13 (reverse primer): GTCCTGTAGTACCGTCGTAT.

[0049] Specifically, the sequence of the fluorescent probe is as shown in SEQ ID NO.11.

[0050] SEQ ID NO.14 (TaqMan probe): AGGTCGATCTAGCTAGCTAGCTGA.

[0051] Specifically, the system of the qPCR reaction includes 3-4 μL of extension reaction product, the final concentration of the primer mixture is 0.5-1 μΜ, the fluorescent probe is 0.5-1 μΜ, and 4-6 μL of qPCR Master Mix.

[0052] Preferably, the system of the qPCR reaction includes 4 μL of extension reaction product, the final concentration of the primer mixture is 0.5 μΜ, the fluorescent probe is 0.5 μΜ, and 5 μL of qPCR Master Mix.

[0053] On the other hand, the present invention provides the application of the aforementioned ZO-1 protein quantification kit in the quantitative detection of ZO-1 protein.

[0054] In yet another aspect, the present invention provides a method for quantitatively detecting ZO-1 protein, including using the aforementioned ZO-1 protein quantification kit for detection.

[0055] Compared with the prior art, the present invention has the following remarkable effects:

[0056] (1) The ZO-1 protein quantification kit of the present invention has a minimum detection limit for ZO-1 protein quantification of about 10 ng / mL, and even for some samples, a detection range of <10 ng / mL can be achieved; compared with the conventional ELISA method for ZO-1 protein quantification, the protein PCR detection system is superior in terms of detection limit, repeatability, and specificity.

[0057] (2) The ZO-1 protein quantification kit of the present invention, including sample pretreatment and immunological PCR amplification, can complete all detections in about 4-6 hours, with short time consumption and good operability.

[0058] (3) The ZO-1 protein quantification kit constructed by the present invention can achieve ultrasensitive quantification of ZO-1, providing a feasible idea for subsequent detection of other tight junction proteins, and is expected to further improve the early identification, stratification, and prognosis evaluation levels of critically ill patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 It is the analysis result of the physicochemical properties of the ZO-1 protein sequence;

[0060] Figure 2 It is the expression result of the ZO-1 antibody;

[0061] Figure 3 It is the qPCR amplification curves of the positive group and the negative group;

[0062] Figure 4 It is the detection result of the expression level of ZO-1 protein.

[0063] In the accompanying drawings, the positive group represents samples of critically ill infected patients, and the negative group represents samples of healthy volunteers. Detailed implementation manners

[0064] The present invention will be further elaborated in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are conventional conditions if not otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels if not otherwise specified.

[0065] Information about the subjects in the following embodiments:

[0066] The subjects were from Shanghai Children's Medical Center affiliated to Shanghai Jiao Tong University School of Medicine and were divided into the following two groups;

[0067] ① Positive group (samples of critically ill infected patients, n = 30): Plasma specimens were collected within 24 hours after admission, and critically ill infected patients were determined according to the Sepsis-3 criteria for the diagnosis and treatment of sepsis;

[0068] ② Negative group (samples of healthy volunteers, n = 30): Plasma specimens were collected within 24 hours after admission;

[0069] The sources of important reagents or materials in the following embodiments are shown in Table 1:

[0070] Table 1 Sources of reagents or materials

[0071]

[0072] Example 1 Method for quantitative detection of ZO-1 protein

[0073] I. Collection and pretreatment of samples to be tested

[0074] Collection of samples to be tested:

[0075] Venous blood (3 - 5 mL) was collected from all subjects on an empty stomach in the early morning or within 24 hours after admission, and the venous blood was divided into the positive group and the negative group according to the Sepsis-3 criteria for the diagnosis and treatment of sepsis;

[0076] Sepsis is a systemic inflammatory response disorder caused by infection, leading to life-threatening organ dysfunction. Its diagnostic criteria refer to the Sepsis-3 criteria (2016) and meet the following two conditions: patients with suspected or confirmed infection; assessment of organ dysfunction: SOFA score ≥ 2 points (baseline is 0, if the patient has chronic organ dysfunction, dynamic assessment of changes is required) or rapid screening tool qSOFA ≥ 2 points (indicating a high risk of poor prognosis).

[0077] Table 2 SOFA scoring criteria

[0078]

[0079]

[0080] Baseline SOFA ≥ 2 points: indicating sepsis; dynamic increase in SOFA: indicating deterioration of organ function; total score ≥ 8 points: significantly increased mortality.

[0081] Table 3 qSOFA Scoring Criteria

[0082] Index Standard Score Respiratory rate ≥22 times / min 1 point Systolic blood pressure ≤100 mmHg 1 point Change in consciousness state GCS<15 1 point

[0083] qSOFA ≥ 2 points: indicating a high risk of sepsis-related organ dysfunction, and further evaluation of SOFA score and organ function is required.

[0084] Collection of information on samples to be tested:

[0085] Record the demographic information of the subjects (age, gender, BMI, etc.), type of infection, scores such as SOFA / qSOFA, and past medical history.

[0086] Sample pretreatment:

[0087] Centrifuge 2000g of venous blood for 10 minutes, separate the plasma and aliquot it for storage at -80°C for protein PCR detection; the remaining serum or plasma is used to detect changes in liver function, myocardial function, renal function, and inflammatory indicators.

[0088] (1) Liver function detection: Detect enzymes, proteins, bilirubin, etc. through an automatic biochemical analyzer. The detection indicators include: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), total bilirubin (TBIL), direct bilirubin (DBIL), albumin (ALB), globulin (GLB).

[0089] (2) Myocardial function detection: Detect myocardial enzyme spectrum through biochemical analysis and myocardial injury markers through immunological methods. The detection indicators include: creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), troponin (cTnI / cTnT), myoglobin, B-type natriuretic peptide (BNP).

[0090] (3) Renal function detection: Detect metabolites through biochemical analysis and cystatin C, etc. through immunological methods. The detection indicators include: serum creatinine (Cr), blood urea nitrogen (BUN), uric acid (UA), urinary protein, cystatin C.

[0091] (4) Detection of inflammatory indicators: Detect inflammation-related proteins by immunological methods (such as chemiluminescence method). The detection indicators include: C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and procalcitonin (PCT).

[0092] II. Preparation of ZO-1 monoclonal antibody against specific antigen epitope binding region

[0093] 2.1 Selection of specific antigen epitope binding region:

[0094] Screen the antigen epitope sequences for immunization by bioinformatics.

[0095] Analyze the physicochemical properties of the ZO-1 protein sequence by using methods such as BepiPred3, Hopp-Woods hydrophilicity index, Boman index, and charge distribution. The experimental results are as Figure 1 shown. By selecting 13-15 amino acids, preferably containing positively charged amino acids such as lysine K and arginine R, and the sequence is located in the key functional regions PDZ domain and C-terminal region of the protein, the optimized antigen epitope sequences SEQ ID NO.1 (ZO-1-P), SEQ ID NO.2 (ZO-1-C), and SEQ ID NO.3 (ZO-1-A) are finally obtained.

[0096] SEQ ID NO.1: SGETSIVISDVLKGG;

[0097] SEQ ID NO.2: KFESPKFNHNLLPS;

[0098] SEQ ID NO.3: KRVEETPNYHKARK.

[0099] 2.2 Preparation of monoclonal antibody ZO-1:

[0100] (1) Animal immunization: Subcutaneously inject the purified immunogen and complete Freund's adjuvant into New Zealand white rabbits. Incomplete Freund's adjuvant can be used for the 2nd - 5th booster immunization. During the immunization process, monitor the serum Elisa titer of the white rabbits. When it reaches more than 1:50000 (dilution factor), stop immunization (using the ratio of positive signal / negative signal > 2.1 as the judgment standard);

[0101] (2) Antibody sequence amplification: Collect the spleen tissues of immunized white rabbits with a titer greater than 1:50000 (dilution factor), and prepare single-cell suspensions. Use a flow cytometer to sort single B cells, extract RNA from the sorted B cells, and amplify the heavy chain variable region sequence (SEQ ID NO.7) and light chain variable region sequence (SEQ ID NO.8) of the ZO-1 antibody gene by RT-PCR. Conduct bioinformatics analysis on the obtained sequences, and screen out multiple different sequences;

[0102] SEQ ID NO.7: DVQLQESGPGLVPGASVKMSCTVTGYSITSDCAWNWVRQAPGNKLEWMGFITYSGSYTYYNPSLKSRISITRDTSSSSFFLQLNSVTTEDTATYYCARDYTYYYAMEYWGQGTSVTVSS;

[0103] SEQ ID NO.8: DIQMTQSSSYLSVSLGGRVTISCRSSQSLVHSDGHTWLAWYQQKPGNAPRLLIYYTSRLHTGVPSRFSGSGSGTQFSLKINSLQDVATYYCQQGKTLPWTFGGGTKLELK.

[0104] (3) Antibody expression and purification: The selected sequences were constructed into the eukaryotic expression vector pRAPV1 and transiently transfected and expressed in HEK293 cells. The cell culture supernatant was collected, and the antibody was purified by protein A affinity chromatography. The purity of the purified antibody sample was analyzed by SDS-PAGE, the concentration was determined by the UV method, and the antibody activity was detected by ELISA and dot blot methods. The results are as Figure 2 , it can be seen that the ZO-1 antibody was successfully expressed. Clones with good performance were selected for expression and purification on a 10 mL scale, and a rabbit monoclonal antibody against ZO-1 protein was successfully obtained.

[0105] 2.3 Labeling of monoclonal antibody ZO-1:

[0106] Click chemistry antibody labeling:

[0107] The monoclonal antibody ZO-1 was diluted with PBS to a concentration of 2 mg / mL, and Azido-dPEG8-NHS ester was prepared into a 10 mM stock solution with DMSO. Its solution was slowly added to the antibody solution at a 20-30-fold molar mass, and the reaction was carried out at room temperature for 2 hours, during which gentle mixing was required. After the reaction, the unreacted labeling reagent was removed by dialysis at 4°C to obtain the labeled antibody.

[0108] The DBCO-modified oligonucleotides A and B (sequences shown in SEQ ID NO.9 and SEQ ID NO.10) were dissolved in PBS to prepare a 100 μM stock solution, and added to the labeled antibody solution at a 10-20-fold molar excess and reacted at 37°C for 2 hours. Then, it was purified through a molecular sieve column with a 100 kD cut-off at 4°C to remove the unreacted oligonucleotides, and finally an antibody labeled with oligonucleotides was obtained.

[0109] SEQ ID NO.9(A): TCAGGTGGACTTCTCGATGACTGGATTCGATGACTG GACTGA;

[0110] SEQ ID NO.10(B): GTCCTGTAGTACCGTCGTATCTAGTCGATGACTGGA CTGAGT.

[0111] III. Protein PCR System Detection

[0112] 3.1 Antigen-Antibody Reaction

[0113] Add each component according to the reaction system in the following table, and incubate at 37°C for 1 h or overnight at 4°C.

[0114] Table 4 Reaction System

[0115] Component Volume Sample / Positive group sample / Negative group sample (PBS + 0.1% BSA) 2 μL Oligonucleotide-labeled antibody A (100 pM) 0.5 μL Oligonucleotide-labeled antibody B (100 pM) 0.5 μL Extension primer (100 pM) 0.5 μL Buffer A 5 μL

[0116] SEQ ID NO.11 (Extension Primer): ACTCAGTCCAGTCATCGATACTAGATACGACGGTACTACAGGACGCTACTATCAGTCCAG;

[0117] The final concentration of Tris-HCl in the buffer is 25 mM, the final concentration of EDTA is 4 mM, the final concentration of NaCl is 150 mM, the final concentration of BSA is 0.2%, the final concentration of salmon sperm DNA is 0.02 mg / mL, the final concentration of PEG8000 is 5%, the final concentration of Tween 20 is 0.05%, and the final concentration of trehalose is 5%.

[0118] 3.2 Extension Reaction

[0119] Add the above mixture to the following table for reaction.

[0120] Table 5 Extension Reaction System

[0121] Component Volume The above incubation product 8.5 μL NE Buffer 2.1 (1×) 86 μL dNTP mix (10 mM) 0.5 μL <![CDATA[T4 DNA polymerase]]> 5 μL

[0122] After incubating at 37°C for 5 min, finally add T4 DNA polymerase. React at 37°C for 20 min and then at 80°C for 10 min.

[0123] 3.3 qPCR Reaction

[0124] Table 6 qPCR Reaction System

[0125] Component Volume Extension reaction product 4 μL 2×Luna Universal Probe qPCR Master Mix 5 μL 20× Primer mix (10 μM) 0.5 μL 20× Fluorescent probe (10 μM) 0.5 μL

[0126] The conditions for the qPCR reaction are shown in Table 7:

[0127] Table 7 qPCR reaction conditions

[0128]

[0129] The sequences of the primer mixture are shown in SEQ ID NO.12 and SEQ ID NO.13, and the sequence of the fluorescent probe is shown in SEQ ID NO.14.

[0130] SEQ ID NO.12 (forward primer): TCAGGTGGACTTCTCGATGA;

[0131] SEQ ID NO.13 (reverse primer): GTCCTGTAGTACCGTCGTAT;

[0132] SEQ ID NO.14 (TaqMan probe): AGGTCGATCTAGCTAGCTAGCTGA.

[0133] The qPCR amplification curve is as Figure 3 shown, indicating that the minimum detection limit of the protein PCR method of the present invention for ZO-1 protein can reach about 10 ng / mL. After further optimization, a detection range of <10 ng / mL can be achieved for some samples. The detection results of the expression level of ZO-1 protein are as Figure 4 shown. The average plasma ZO-1 level in the positive group (severe infection patients) was 72 ng / mL, which was significantly higher than that in the negative group (28 ng / mL), and there was a good discrimination.

Claims

1. A ZO-1 protein quantification kit, characterized in that: It comprises a ZO-1 antibody, wherein the ZO-1 antibody comprises a specific antigen epitope binding region, and the specific antigen epitope comprises an amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.

3.

2. The ZO-1 protein quantification kit according to claim 1, characterized in that: The ZO-1 antibody is a monoclonal antibody or a polyclonal antibody; the specific antigen epitope binding region of the ZO-1 antibody includes the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.

6.

3. The ZO-1 protein quantification kit according to claim 2, characterized in that: The specific antigen epitope binding region of the ZO-1 antibody includes the heavy chain variable region sequence shown in SEQ ID NO.7 and the light chain variable region sequence shown in SEQ ID NO.

8.

4. The ZO-1 protein quantification kit according to claim 1, characterized in that: The kit also includes immune PCR amplification, which includes antigen-antibody reaction, PCR extension reaction and qPCR reaction.

5. The ZO-1 protein quantification kit according to claim 4, characterized in that: The ZO-1 antibody is further connected to an oligonucleotide, wherein the oligonucleotide is selected from one or both of oligonucleotide A and oligonucleotide B; the oligonucleotide A has a nucleotide sequence as shown in SEQ ID NO.9, and the oligonucleotide B has a nucleotide sequence as shown in SEQ ID NO.10; The ZO-1 antibody includes oligonucleotide-labeled antibody A labeled with oligonucleotide A and oligonucleotide-labeled antibody B labeled with oligonucleotide B.

6. The ZO-1 protein quantification kit according to claim 4, characterized in that: The antigen-antibody reaction system also includes an extension primer; the extension primer is the nucleotide sequence shown in SEQ ID NO.

11.

7. The ZO-1 protein quantification kit according to claim 1, characterized in that: The ZO-1 antibody further comprises a signal label, and the signal label is selected from any one or more of an enzyme label, a biotin label and an isotope label, preferably a biotin label.

8. The ZO-1 protein quantification kit according to any one of claims 1 to 7, characterized in that: It also includes a sample to be tested. Preferably, the sample to be tested is any one or more of serum, plasma and alveolar lavage fluid.

9. Use of the ZO-1 protein quantification kit according to any one of claims 1 to 8 in the quantitative detection of ZO-1 protein.

10. A method for quantitative detection of ZO-1 protein, characterized in that: The method comprises using the ZO-1 protein quantification kit according to any one of claims 1 to 8 for detection.

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