A lung cancer marker detection method based on induced sputum cells combined with flow cytometry

Abstract

The present application relates to the technical field of biological medicine, and provides a lung cancer marker detection method based on induced sputum cells combined with flow cytometry. The lung cancer marker detection method includes TTF-1 protein, CK7 protein and P63 protein. Antibodies for specifically recognizing and detecting the lung cancer marker include TTF-1 antibody, CK7 antibody and P63 antibody. The amino acid sequence of the light chain variable region of the TTF-1 antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 5. The amino acid sequence of the light chain variable region of the CK7 antibody is shown in SEQ ID NO. 9, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 13. The amino acid sequence of the light chain variable region of the P63 antibody is shown in SEQ ID NO. 17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 21. The present application can obtain an induced sputum sample in a non-invasive or minimally invasive manner, can reduce the pain of patients, can avoid misdiagnosis due to high specificity, can facilitate early diagnosis of lung cancer due to high sensitivity, and early diagnosis is a key to improving the cure rate and survival rate.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a lung cancer marker based on induced sputum cells combined with flow cytometry. BACKGROUND

[0002] Primary bronchogenic carcinoma is simply called lung cancer. Early lung cancer often has no obvious symptoms. Most patients are in the advanced stage when they seek medical treatment. Few cases can be diagnosed in the early stage, highlighting the urgency of improving early diagnosis technology. The prognosis of lung cancer is closely related to the timing of diagnosis. The current routine diagnosis methods for lung cancer include imaging, histopathological biopsy and sputum cytology, etc. Imaging such as low-dose spiral CT is a recognized early screening method. The early lung cancer detection rate is significantly higher than that of X-ray, but there are problems of high false positive rate and radiation exposure, which is not suitable for pregnant women or frequent checkers. Histopathological biopsy is to obtain tumor tissue samples and perform pathological analysis to determine the tumor type and molecular characteristics. However, it has the risk of invasiveness, which may cause pneumothorax, bleeding or infection. The operation is complex, and it is difficult to sample small lesions or special parts. It takes a long time, and it takes several days to several weeks from sampling to result, which may delay treatment. Sputum cytology is to collect the sputum coughed out by the patient, and observe the morphological abnormalities of the exfoliated cells under a microscope after staining to determine whether there are cancer cells. This method is suitable for patients who cannot tolerate biopsy, has low cost and simple operation, but has the problems of low sensitivity of traditional induced sputum cytology, low detection rate, inability to detect protein dynamic function state, and dependence on operator experience.

[0003] In addition, although there has been exploration of lung disease protein marker detection, the existing scheme has key shortcomings: non-specificity of single membrane protein marker, false positive interference, such as CEA significantly increased in chronic inflammation (chronic obstructive pulmonary disease, tuberculosis), and narrow dynamic range; technical obstacles for intracellular protein detection, poor sample compatibility, conventional flow cytometry requires fresh tissue or blood samples, low cell activity in sputum, resulting in loss of phosphorylation signal. SUMMARY

[0004] In view of the deficiencies of the prior art, the application provides a lung cancer marker based on induced sputum cells combined with flow cytometry, which aims to solve the problems mentioned in the background art.

[0005] The application provides a lung cancer marker based on induced sputum cells combined with flow cytometry. The lung cancer marker includes TTF-1 protein, CK7 protein and P63 protein. The antibodies for specifically recognizing and detecting the lung cancer marker include TTF-1 antibody, CK7 antibody and P63 antibody.

[0006] The amino acid sequence of the light chain variable region of the TTF-1 antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 5;

[0007] The amino acid sequence of the light chain variable region of the CK7 antibody is shown as SEQ ID NO. 9, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 13;

[0008] The amino acid sequence of the light chain variable region of the P63 antibody is shown as SEQ ID NO. 17, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 21.

[0009] Further, the amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the TTF-1 antibody are shown as SEQ ID NO. 2-4, respectively, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown as SEQ ID NO. 6-8, respectively;

[0010] The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the CK7 antibody are shown as SEQ ID NO. 10-12, respectively, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown as SEQ ID NO. 14-16, respectively;

[0011] The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the P63 antibody are shown as SEQ ID NO. 18-20, respectively, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown as SEQ ID NO. 22-24, respectively.

[0012] Further, based on the detection of lung cancer markers by induced sputum cell combined with flow cytometry, the application is used for preparing a lung cancer early diagnosis detection kit, which comprises fluorescent group labeled TTF-1 antibody, CK7 antibody and P63 antibody.

[0013] Further, the detection kit further comprises cell preservation solution, antibody preservation solution, cell fixation solution and membrane permeation agent.

[0014] Further, the fluorescent group labeled TTF-1 antibody, CK7 antibody and P63 antibody are respectively: fluorescein isothiocyanate labeled anti-TTF-1 monoclonal antibody, phycoerythrin labeled anti-CK7 monoclonal antibody and allophycocyanin labeled anti-P63 monoclonal antibody.

[0015] Further, the detection kit detects positive cells of TTF-1, CK7 and P63 in induced sputum cells of the subject and positive cells of all three indicators of TTF-1 / CK7 / P63 through flow cytometry, and establishes a diagnosis model Q for early diagnosis of lung cancer, and the formula of the diagnosis model Q is:

[0016] Q = 0.25T + 0.36C + 0.26P + 1.8D;

[0017] In the formula, T is the percentage of the number of TTF-1 positive cells in the total number of induced sputum cells; C is the percentage of the number of CK7 positive cells in the total number of induced sputum cells; P is the percentage of the number of P63 positive cells in the total number of induced sputum cells; and D is the percentage of the number of positive cells of all three indicators of TTF-1 / CK7 / P63 in the total number of induced sputum cells.

[0018] Further, the early diagnosis of lung cancer is performed through the diagnosis model Q, and when Q>0.4, the subject is determined to be positive for lung cancer; and when Q≤0.4, the subject is determined to be negative for lung cancer.

[0019] Further, the use method of the detection kit comprises the following steps:

[0020] Step S1: Collect sputum in deep respiratory tract of the subject through high-osmotic saline atomization induction, and the amount of sputum is not less than 2ml;

[0021] Step S2: Add dithiothreitol to the collected sputum, and oscillate at room temperature for 30 minutes to fully digest the sputum;

[0022] Step S3: Filter the digested sputum through two layers of sterile gauze filter screen, transfer the filtered liquid to a centrifugal tube, and centrifuge at a speed of 1800r / min for 10 minutes to make induced sputum cells precipitate at the bottom of the tube;

[0023] Step S4: Discard the supernatant, add an appropriate amount of cell preservation solution, and blow to resuspend the induced sputum cells to obtain induced sputum cell suspension;

[0024] Step S5: Divide the induced sputum cell suspension into several parts, and add fluorescein isothiocyanate-labeled anti-TTF-1 monoclonal antibody, phycoerythrin-labeled anti-CK7 monoclonal antibody and allophycocyanin-labeled anti-P63 monoclonal antibody respectively, vortex, and incubate at 4°C in dark for 30 minutes;

[0025] Step S6: Add flow cytometry staining buffer, centrifuge at a speed of 3000r / min for 10 minutes, discard the supernatant, and repeat the washing for 3 times;

[0026] Step S7: Add an appropriate amount of cell fixation solution, mix uniformly by blowing, and incubate at room temperature for 20 minutes;

[0027] Step S8: After fixation, the induced sputum cells are washed twice with flow cytometry staining buffer, then a membrane permeabilization agent is added, and incubated at room temperature for 15 minutes;

[0028] Step S9: The fluorescence signals of the induced sputum cells are collected by flow cytometry to obtain the percentage of TTF-1 positive cells in the total number of induced sputum cells, the percentage of CK7 positive cells in the total number of induced sputum cells, the percentage of P63 positive cells in the total number of induced sputum cells, and the percentage of TTF-1 / CK7 / P63 triple positive cells in the total number of induced sputum cells, and a diagnostic model Q is established, and the formula of the diagnostic model Q is:

[0029] Q=0.25T+0.36C+0.26P+1.8D;

[0030] Step S10: Early lung cancer diagnosis is performed by the diagnostic model Q, when Q>0.4, the subject is judged to be lung cancer positive; when Q≤0.4, the subject is judged to be lung cancer negative.

[0031] Further, the lung cancer is non-small cell lung cancer.

[0032] Further, the induced sputum cells include macrophages, neutrophils, airway epithelial cells and tumor cells.

[0033] The present application has the following technical effects:

[0034] (1) The lung cancer markers detected based on the induced sputum cells combined with flow cytometry include TTF-1 protein, CK7 protein and P63 protein, and the antibodies specifically recognizing and detecting the lung cancer markers include TTF-1 antibody, CK7 antibody and P63 antibody, which have high specificity and high sensitivity, increase the amount of diagnostic information, and improve the accuracy of diagnosis. The detection of lung cancer markers based on induced sputum cells combined with flow cytometry is applied to the preparation of a lung cancer early diagnosis kit, the induced sputum sample is obtained by non-invasive or minimally invasive method, which can reduce the pain of patients, high specificity can avoid misdiagnosis, high sensitivity is beneficial to early diagnosis of lung cancer, and early diagnosis is the key to improve the cure rate and survival rate.

[0035] (2) Ordinary sputum can only obtain sputum of upper respiratory tract, which may contain a large number of oral cavity miscellaneous bacteria and epithelial cells, while induced sputum can stimulate the lower respiratory tract to produce sputum by means of atomization of hypertonic saline, etc., so that cells closer to the lesion site can be collected, and the detection probability of cells related to lower respiratory tract diseases such as lung cancer can be improved. The cell types in induced sputum cells are diverse, in addition to common macrophages, neutrophils, etc., airway epithelial cells, tumor cells and other cells with diagnostic value can also be obtained. The characteristics and proportion changes of these cells can provide important basis for disease diagnosis, disease assessment and treatment monitoring. Attached Figure Description

[0036] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0037] Figure 1 This is a graph showing the ROC curve analysis results of Embodiment 3 of the present invention. Detailed Implementation

[0038] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0039] 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 terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0040] This invention provides a method for detecting lung cancer biomarkers based on induced sputum cells combined with flow cytometry. The lung cancer biomarkers include TTF-1 protein, CK7 protein, and P63 protein. The antibodies that specifically recognize the lung cancer biomarkers include TTF-1 antibody, CK7 antibody, and P63 antibody.

[0041] The amino acid sequence of the light chain variable region of the TTF-1 antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.5;

[0042] The amino acid sequence of the light chain variable region of the CK7 antibody is shown in SEQ ID NO.9, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.13;

[0043] The amino acid sequence of the light chain variable region of the P63 antibody is shown in SEQ ID NO.17, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.21.

[0044] In some embodiments, the amino acid sequences of the light chain variable regions CDR-L1, CDR-L2 and CDR-L3 of the TTF-1 antibody are shown in SEQ ID NO.2-4, respectively, and the amino acid sequences of the heavy chain variable regions CDR-H1, CDR-H2 and CDR-H3 are shown in SEQ ID NO.6-8, respectively.

[0045] The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the CK7 antibody are shown in SEQ ID NO. 10-12, respectively, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown in SEQ ID NO. 14-16, respectively;

[0046] The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the P63 antibody are shown in SEQ ID NO. 18-20, respectively, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown in SEQ ID NO. 22-24, respectively.

[0047] In some embodiments, the application of the lung cancer marker detection based on induced sputum cells combined with flow cytometry in the preparation of a lung cancer early diagnosis detection kit, the detection kit comprises a fluorescent group labeled TTF-1 antibody, a CK7 antibody and a P63 antibody.

[0048] In some embodiments, the detection kit further comprises a cell preservation solution, an antibody preservation solution, a cell fixation solution and a membrane permeation agent.

[0049] In some embodiments, the fluorescent group labeled TTF-1 antibody, CK7 antibody and P63 antibody are respectively: a fluorescein isothiocyanate labeled anti-TTF-1 monoclonal antibody, a phycoerythrin labeled anti-CK7 monoclonal antibody and an allophycocyanin labeled anti-P63 monoclonal antibody.

[0050] In some embodiments, the detection kit detects the positive cells of TTF-1, CK7 and P63 in the induced sputum cells of the subject by flow cytometry, and the positive cells of the three indicators of TTF-1 / CK7 / P63, and establishes a diagnosis model Q for early diagnosis of lung cancer, and the formula of the diagnosis model Q is:

[0051] Q = 0.25T + 0.36C + 0.26P + 1.8D;

[0052] In the formula, T is the percentage of the number of TTF-1 positive cells to the total number of induced sputum cells; C is the percentage of the number of CK7 positive cells to the total number of induced sputum cells; P is the percentage of the number of P63 positive cells to the total number of induced sputum cells; and D is the percentage of the number of positive cells of the three indicators of TTF-1 / CK7 / P63 to the total number of induced sputum cells.

[0053] In some embodiments, the early diagnosis of lung cancer is performed by calculation through the diagnosis model Q, when Q>0.4, the subject is judged to be positive for lung cancer; and when Q≤0.4, the subject is judged to be negative for lung cancer.

[0054] In some embodiments, the method for using the detection kit comprises the following steps:

[0055] Step S1: Collect sputum from deep respiratory tract of the subject by high-osmotic saline atomization induction, and the amount of sputum is not less than 2 ml;

[0056] Step S2: Add dithiothreitol to the collected sputum, and oscillate at room temperature for 30 minutes to fully digest the sputum;

[0057] Step S3: Filter the digested sputum through two layers of sterile gauze filter screen, transfer the filtered liquid to a centrifuge tube, and centrifuge at 1800 rpm for 10 minutes to make the induced sputum cells precipitate at the bottom of the tube;

[0058] Step S4: Discard the supernatant, add an appropriate amount of cell preservation solution, and blow to resuspend the induced sputum cells to obtain an induced sputum cell suspension;

[0059] Step S5: Divide the induced sputum cell suspension into several parts, and add fluorescein isothiocyanate-labeled anti-TTF-1 monoclonal antibody, phycoerythrin-labeled anti-CK7 monoclonal antibody, and allophycocyanin-labeled anti-P63 monoclonal antibody respectively, vortex, and incubate at 4°C in the dark for 30 minutes;

[0060] Step S6: Add flow cytometry staining buffer, centrifuge at 3000 rpm for 10 minutes, discard the supernatant, and repeat the washing for 3 times;

[0061] Step S7: Add an appropriate amount of cell fixation solution, mix well by blowing, and incubate at room temperature for 20 minutes;

[0062] Step S8: After fixation, wash the induced sputum cells twice with flow cytometry staining buffer, then add a membrane permeabilization agent, and incubate at room temperature for 15 minutes;

[0063] Step S9: Collect the fluorescence signals of the induced sputum cells by flow cytometry to obtain the percentage of TTF-1 positive cells in the total number of induced sputum cells, the percentage of CK7 positive cells in the total number of induced sputum cells, the percentage of P63 positive cells in the total number of induced sputum cells, and the percentage of TTF-1 / CK7 / P63 triple index positive cells in the total number of induced sputum cells, and establish a diagnostic model Q, and the formula of the diagnostic model Q is:

[0064] Q = 0.25T + 0.36C + 0.26P + 1.8D;

[0065] Step S10: Perform early diagnosis of lung cancer by the diagnostic model Q, when Q > 0.4, the subject is judged as lung cancer positive; and when Q≤0.4, the subject is judged as lung cancer negative.

[0066] In some embodiments, the lung cancer is non-small cell lung cancer.

[0067] In some embodiments, the induced sputum cells include macrophages, neutrophils, airway epithelial cells, and tumor cells.

[0068] Example 1:

[0069] 1. Antigen design and preparation:

[0070] (1) Antigen selection:

[0071] Express the key domains (DNA binding domain) through the prokaryotic (E. coli) expression system, purify the recombinant protein (His tag purification), ensure high purity (> 90%) and correct folding, and obtain TTF-1 / CK7 / P63 recombinant protein;

[0072] Or select TTF-1 / CK7 / P63 protein specific epitopes, chemically synthesize and couple carrier proteins to improve immunogenicity, and obtain TTF-1 / CK7 / P63 specific epitope polypeptides.

[0073] (2) Antigen verification:

[0074] Verify the effectiveness of the antigen by ELISA (enzyme-linked immunosorbent assay).

[0075] 2. Rabbit immunization and titer detection:

[0076] (1) Immunization scheme:

[0077] Select healthy adult rabbits (New Zealand white rabbits), 3 per group to reduce individual differences;

[0078] Primary immunization: subcutaneous injection of 100-200 μg antigen (recombinant protein or specific epitope polypeptide) at multiple points on the back, mixed and emulsified with Freund's complete adjuvant; Booster immunization: repeat injection every 2-3 weeks, use Freund's incomplete adjuvant instead, a total of 4-5 times.

[0079] (2) Titer detection:

[0080] ELISA: dynamically monitor serum antibody titer (titer > 1:10 6 is the best blood collection time);

[0081] Western blotting: verify the specific recognition of the antibody to TTF-1 / CK7 / P63 antigen (human lung adenocarcinoma cell lysate as positive control).

[0082] (3) Serum collection and titer detection:

[0083] Ear marginal vein blood collection (5-10 mL each time) to detect antibody titer by ELISA method, titer ≥ 1:104 (OD450nm >1.0) into the stage of exsanguination; heart puncture or carotid artery exsanguination (50-100 mL serum / rabbit).

[0084] 3. Single B cell sequencing to obtain monoclonal antibodies

[0085] High titer rabbit blood B cell sorting:

[0086] Anticoagulated peripheral blood (5-10 mL) was collected, and peripheral blood mononuclear cells (containing B cells) were separated by density gradient centrifugation; the fluorescence-coupled target antigen (TTF-1, CK7 and P63) was incubated and combined with the B cell surface marker, and after sorting by double positive cells (target antigen binding positive + B cell surface general marker positive), the sorted single B cells were directly lysed, and the antibody heavy chain and light chain variable region genes were amplified by RT-PCR (reverse transcription polymerase chain reaction) one-step method;

[0087] The RT-PCR product was cloned into an expression vector (pTT5), and HEK293 cells were transfected to express full-length IgG. After the supernatant was purified by Protein A (A protein), the antigen binding activity was verified by ELISA or Western blotting (compared with the original serum titer), and the positive clone was sequenced to obtain the light and heavy chain complementarity determining region and framework region sequence, and the anti-TTF-1 monoclonal antibody (TTF-1-12B08), anti-CK7 monoclonal antibody (CK7-12H02) and anti-P63 monoclonal antibody (P63-14D11) were obtained.

[0088] The amino acid sequence of the light chain variable region of the anti-TTF-1 monoclonal antibody (TTF-1-12B08) is:

[0089] AYEMTQTPSTVEKAVGGTVTIKCTESADENVLQSWYQQKPGQPPKLLIHGDTAGSKGVPSRFRGSGSGTEYSLTISGVQCADAATYYCSQDQSVDSEILFFGGGTEVVVK (SEQ ID NO. 1);

[0090] Among them, the complementarity determining region, CDR-L1: TESADENVLQS (SEQ ID NO. 2); CDR-L2: GDTAGSK (SEQ ID NO. 3); CDR-L3: SQDQSVDSEILF (SEQ ID NO. 4);

[0091] The amino acid sequence of the heavy chain variable region of the anti-TTF-1 monoclonal antibody (TTF-1-12B08) is:

[0092] QSVEESGGRLVTPGGSLTLTCTVSGIDLSKGKLDWVRQAPGKGLEWIGFQSVECRPFVFGAGKPRFTISKTSSTTIEKLATSLTTEDTATYFCARKPYEFSIDCDKWGPGTLVTVSS (SEQ ID NO. 5);

[0093] wherein the complementarity determining regions, CDR-H1 : KGKLD (SEQ ID NO. 6); CDR-H2: FQSVECRPFVFGAGKP (SEQ ID NO. 7); CDR-H3: KPYEFSIDCDK (SEQ ID NO. 8);

[0094] Amino acid sequence of the heavy chain variable region of the anti-CK7 monoclonal antibody (CK7-12H02):

[0095] AYEMTQTPSTVEKAVGGTVTIKCSQSFGDSYNLFWYQQKPGQPPKLLIHSAKPSAKGVPSRFRGSGSGTEYSLTISGVQCADAATYYCQSLWQNEDYLSLFGGGTEVVVS (SEQ ID NO. 9);

[0096] wherein the complementarity determining regions, CDR-L1 : SQSFGDSYNLF (SEQ ID NO. 10); CDR-L2: SAKPSAK (SEQ ID NO. 11); CDR-L3: QSLWQNEDYLSL (SEQ ID NO. 12);

[0097] Amino acid sequence of the heavy chain variable region of the anti-CK7 monoclonal antibody (CK7-12H02):

[0098] QSLEESGGRLVTPGGSLTLTCKASGFSFSSGYLDCWVRQAPGKGLEWILDISGSYQEMKEYLEPISGRFTISKTSSTTLELLSLTAATVDTATYFCARSPQTYEWENYALWGPGTLVTVSS (SEQ ID NO. 13);

[0099] wherein the complementarity determining regions, CDR-H1 : SGYLDC (SEQ ID NO. 14); CDR-H2: DISGSYQEMKEYLEPISG (SEQ ID NO. 15); CDR-H3: SPQTYEWENYA (SEQ ID NO. 16);

[0100] The amino acid sequence of the light chain variable region of the anti-P63 monoclonal antibody (P63-14D11):

[0101] AYEMTQTPSTVEKAVGGTVTIKCTRSGVSRQGKPWYQQKPGQPPKLLIHSLSLYTFGVPSRFRGSGSGTEYSLTISGVQCADAATYYCVGQFGSLMIDRLFGGGTEVVVS (SEQ ID NO. 17);

[0102] wherein the complementarity determining regions, CDR-L1: TRSGVSRQGKP (SEQ ID NO. 18); CDR-L2: SLSLYTF (SEQ ID NO. 19); CDR-L3: VGQFGSLMIDRL (SEQ ID NO. 20);

[0103] The amino acid sequence of the heavy chain variable region of the anti-P63 monoclonal antibody (P63-14D11):

[0104] QSLEESGGRLVTPGGSLTLTCKASGFSFSIAESAGWVRQAPGKGLEWIISLTSKPSAKDTEPQKEQCRFTISKTSSTTIELASLTAATVDTATYFCARARIKTRYLESFGLWGPGTLVTVSS (SEQ ID NO. 21);

[0105] wherein the complementarity determining regions, CDR-H1: IAESAG (SEQ ID NO. 22); CDR-H2: SLTSKPSAKDTEPQKEQC (SEQ ID NO. 23); CDR-H3: ARIKTRYLESFGL (SEQ ID NO. 24).

[0106] The anti-TTF-1 monoclonal antibody, the anti-CK7 monoclonal antibody and the anti-P63 monoclonal antibody were subjected to gradient reaction with coated different concentrations of TTF-1 antigen, CK7 antigen and P63 antigen, and the detection dose response curve results are shown in Table 1. The IC50 values of the anti-TTF-1 monoclonal antibody (TTF-1-12B08), the anti-CK7 monoclonal antibody (CK7-12H02) and the anti-P63 monoclonal antibody (P63-14D11) obtained in this embodiment are all lower than those of the comparative examples (commercially available antibodies). This indicates that the anti-TTF-1 monoclonal antibody (TTF-1-12B08), the anti-CK7 monoclonal antibody (CK7-12H02) and the anti-P63 monoclonal antibody (P63-14D11) have significantly better sensitivity.

[0107] Table 1 Detection dose response curve

[0108]

[0109] Example 2:

[0110] 1. Inducing and collecting sputum:

[0111] (1) Atomization induction:

[0112] Make the examinee wear a mask, connect the atomizer, start atomization inhalation with 3% hypertonic saline, the time is 5-10 minutes, observe the reaction of the examinee, if there is no discomfort, the concentration of hypertonic saline can be increased to 4%, continue to atomize for 5-10 minutes. If the examinee can still tolerate, it can be further increased to 5%, and atomized for 5-10 minutes. During the whole atomization process, guide the examinee to take deep breath, so that the mist droplets can fully reach the deep part of the respiratory tract.

[0113] (2) Sputum collection:

[0114] After atomization, let the examinee rest for a while, then guide the examinee to effectively cough sputum. The examinee first takes a deep breath, then coughs out the sputum in the deep part of the respiratory tract, avoiding mixing saliva into the sputum. The coughed sputum is collected into a sterile sputum cup, and the sputum volume is required to be not less than 2ml. If the amount of sputum is not enough at one time, the examinee can take deep breath and cough again until enough sputum is obtained.

[0115] 2. Sputum treatment and detection of induced sputum cell viability:

[0116] (1) Preliminary treatment:

[0117] To prevent the destruction of cell components and the growth of bacteria, the collected sputum is immediately treated. Add 4 times volume of 0.1% digestive solution dithiothreitol to the sputum, and gently shake at room temperature for 30 minutes to fully digest the sputum and make it thin.

[0118] (2) Filtration and centrifugation:

[0119] The digested sputum is filtered through two layers of sterile gauze filter to remove impurities and mucus silk. The filtered liquid is transferred to a centrifuge tube and centrifuged at 1800 rpm for 10 minutes to precipitate the induced sputum cells at the bottom of the tube.

[0120] (3) Cell resuspension:

[0121] Discard the supernatant, add an appropriate amount of normal saline or cell preservation solution, and gently blow to resuspend the induced sputum cells to obtain induced sputum cell suspension.

[0122] (4) Cell counting and viability detection:

[0123] The induced sputum cells were counted and viability tested by trypan blue staining to ensure sufficient number and good viability (cell viability greater than 80%).

[0124] 3. Flow cytometry detection of lung cancer markers:

[0125] (1) Cell grouping: The induced sputum cell suspension was evenly divided into several cell tubes, and different fluorescent group labeled antibodies were added respectively. The fluorescently labeled antibodies can specifically bind to the target cell surface, thereby generating a light signal through the laser light source of the flow cytometer and being converted and recognized by the instrument.

[0126] (2) Antibody incubation: Shake for 2 seconds to mix, incubate at 4°C in the dark for 30 minutes. Shake to mix to avoid cell sedimentation and ensure that the antibody contacts the cells fully. Incubate at 4°C in the dark to ensure cell viability and avoid light bleaching.

[0127] (3) Wash the cells: After incubation, add flow cytometry staining buffer to the cell tube and centrifuge at 3000 rpm for 10 minutes. Discard the supernatant and repeat the washing 3 times to remove unbound antibodies.

[0128] (4) Fix the cells: Add an appropriate amount of cell fixation solution to the washed cell tube, mix gently, and incubate at room temperature for 20 minutes to fix the induced sputum cells. The purpose of fixation is to maintain the morphology and structure of the cells and prevent the loss of intracellular components.

[0129] (5) Membrane permeabilization: After fixation, wash the induced sputum cells with flow cytometry staining buffer 2 times, then add an appropriate amount of membrane permeabilization agent and incubate at room temperature for 15 minutes to make the cell membrane permeable so that the antibody can enter the induced sputum cells and bind to intracellular proteins.

[0130] (6) Sample loading: Add the stained induced sputum cells to the sample tube of the flow cytometer, follow the flow cytometer operation instructions for loading, and detect within 1 hour.

[0131] (7) Instrument settings: According to the types and excitation wavelengths of the fluorescent group labeled antibodies used, set the parameters of the laser light source, filter and detector of the flow cytometer. At the same time, adjust the voltage, gain and other parameters to make the fluorescence signal of the negative control in the appropriate range.

[0132] (8) Data acquisition: Start the flow cytometer and collect the fluorescence signal of the induced sputum cells. Generally, 10,000-50,000 induced sputum cells are collected to ensure the accuracy and reliability of the data.

[0133] (9) Calculate the percentage of cells: count the number of TTF-1 positive cells, the number of CK7 positive cells and the number of P63 positive cells, and the percentage of the total number of induced sputum cells, TTF-1 / CK7 / P63 three indicators are the percentage of positive cells in the total number of induced sputum cells.

[0134] Wherein, the kind of fluorescently labeled antibody used, excitation and emission wavelengths are shown in Table 2, reagent components are shown in Table 3.

[0135] Table 2 Monoclonal antibody and the kind of fluorescent group, excitation and emission wavelength

[0136]

[0137] Table 3 Reagent components

[0138]

[0139] Example 3:

[0140] From Jiangxi Province People's Hospital, through ethical review (number: KT884), the experimental subjects were selected: 50 cases of non-small cell lung cancer patients and 50 cases of healthy people, according to the steps of Example 2, the induced sputum cells of the experimental subjects were obtained, and finally the number of TTF-1 positive cells, the number of CK7 positive cells and the number of P63 positive cells were counted, and the percentage of the total number of induced sputum cells was calculated, and the percentage of the total number of induced sputum cells was calculated. The positive cell number of TTF-1 / CK7 / P63 three indicators, the statistical results are shown in Table 4 and Table 5.

[0141] Table 4 Detection statistical results of non-small cell lung cancer patients

[0142]

[0143] Table 5 Detection statistical results of healthy people

[0144]

[0145] Note: Each batch of experiments are set: same type control (IgG1-FITC / IgG1-PE / IgG1-APC) to exclude non-specific binding; uncolored samples for background fluorescence correction; the experiment was repeated 3 times to take the mean value, CV value <5% is considered as valid data.

[0146] Based on the number of TTF-1 positive cells, the number of CK7 positive cells and the number of P63 positive cells of non-small cell lung cancer patients and healthy people, the percentage of the total number of induced sputum cells, the percentage of the total number of induced sputum cells, and the percentage of the total number of induced sputum cells, and the ROC curve analysis results of the diagnostic model Q are as followsFigure 1 As shown.

[0147] The results showed that simultaneous detection of induced sputum cells using a triple marker combination of TTF-1 monoclonal antibody (TTF-1-12B08), anti-CK7 monoclonal antibody (CK7-12H02), and anti-P63 monoclonal antibody (P63-14D11) yielded excellent diagnostic efficacy. After Q-model calculation and ROC curve analysis, the optimal diagnostic threshold was determined to be 0.4. The results demonstrated a high diagnostic efficacy with a specificity of 0.96 (95% CI: 0.865-0.992), a sensitivity of 0.96 (95% CI: 0.865-0.992), and an AUC (area under the curve) of 0.99 (95% CI: 0.98-1.00). These performances were significantly superior to individual detection of the three markers or detection with all three markers being positive: TTF-1: 0.96 (95% CI: 0.93-0.99), CK7: 0.92 (95% CI: 0.87-0.97), and P63: The mean values ​​for TTF-1, CK7, and P63 were 0.97 (95% CI: 0.95-0.99), and for TTF-1 / CK7 / P63, 0.97 (95% CI: 0.94-0.99). This indicates that the combined diagnostic model Q using TTF-1 monoclonal antibody (TTF-1-12B08), anti-CK7 monoclonal antibody (CK7-12H02), and anti-P63 monoclonal antibody (P63-14D11) can significantly distinguish non-small cell lung cancer from healthy individuals, providing a high-precision detection method for non-invasive clinical screening.

[0148] The specific method for early diagnosis of lung cancer is as follows: Statistical analysis is performed on the percentages of TTF-1 positive cells, CK7 positive cells, and P63 positive cells in the total number of induced sputum cells. The percentages of cells with positive TTF-1, CK7, and P63 indicators in the total number of induced sputum cells are also analyzed. A diagnostic model Q is then established for early diagnosis of lung cancer. The formula for diagnostic model Q is as follows:

[0149] Q = 0.25T + 0.36C + 0.26P + 1.8D;

[0150] In the formula: T is the percentage of TTF-1 positive cells out of the total number of induced sputum cells; C is the percentage of CK7 positive cells out of the total number of induced sputum cells; P is the percentage of P63 positive cells out of the total number of induced sputum cells; D is the percentage of cells in which all three indicators (TTF-1, CK7, and P63) are positive out of the total number of induced sputum cells.

[0151] Early diagnosis of lung cancer is performed using the diagnostic model Q. When Q > 0.4, the examinee is judged to be positive for lung cancer; when Q ≤ 0.4, the examinee is judged to be negative for lung cancer.

[0152] In summary, the lung cancer marker based on induced sputum cells combined with flow cytometry detection includes TTF-1 protein, CK7 protein and P63 protein, and the antibody specific to the lung cancer marker includes TTF-1 antibody, CK7 antibody and P63 antibody, which has high specificity and high sensitivity, increases the amount of information of diagnosis, and improves the accuracy of diagnosis. The lung cancer marker based on induced sputum cells combined with flow cytometry detection is applied to the preparation of a lung cancer early diagnosis detection kit, the induced sputum sample is obtained in a non-invasive or minimally invasive manner, which can reduce the pain of patients, high specificity can avoid misdiagnosis, high sensitivity is beneficial to early diagnosis of lung cancer, and early diagnosis is the key to improve the cure rate and survival rate.

[0153] In addition, ordinary sputum can generally only obtain sputum of the upper respiratory tract, which may contain a large number of oral cavity miscellaneous bacteria and epithelial cells, and induced sputum can stimulate the lower respiratory tract to produce sputum by means of atomization of hypertonic saline, so that cells closer to the lesion site can be collected, and the detection probability of cells related to lower respiratory tract diseases such as lung cancer can be improved. The cell types in induced sputum cells are diverse, in addition to common macrophages, neutrophils and the like, airway epithelial cells, tumor cells and the like with diagnostic value can also be obtained. The characteristics and proportion changes of these cells can provide important basis for the diagnosis, disease assessment and treatment monitoring of diseases.

[0154] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of TTF-1 antibody, CK7 antibody and P63 antibody in the preparation of a detection kit for early diagnosis of lung cancer based on induced sputum cell combination with flow cytometry, characterized in that: The amino acid sequence of the light chain variable region of the TTF-1 antibody is shown as SEQ ID NO. 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 5; The amino acid sequence of the light chain variable region of the CK7 antibody is shown as SEQ ID NO. 9, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 13; The amino acid sequence of the light chain variable region of the P63 antibody is shown as SEQ ID NO. 17, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 21; The lung cancer is non-small cell lung cancer.

2. Use according to claim 1, characterized in that: The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the TTF-1 antibody are shown as SEQ ID NO. 2-4, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown as SEQ ID NO. 6-8; The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the CK7 antibody are shown as SEQ ID NO. 10-12, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown as SEQ ID NO. 14-16; The amino acid sequences of CDR-L1, CDR-L2 and CDR-L3 of the light chain variable region of the P63 antibody are shown as SEQ ID NO. 18-20, and the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 of the heavy chain variable region are shown as SEQ ID NO. 22-24.

3. Use according to claim 1 or 2, characterized in that: The detection kit comprises TTF-1 antibody, CK7 antibody and P63 antibody labeled with fluorescent groups.

4. Use according to claim 3, wherein: The detection kit further comprises cell preservation solution, antibody preservation solution, cell fixation solution and membrane permeation agent.

5. The use according to claim 4, characterized in that: The TTF-1 antibody, CK7 antibody and P63 antibody labeled with fluorescent groups are respectively: anti-TTF-1 monoclonal antibody labeled with fluorescein isothiocyanate, anti-CK7 monoclonal antibody labeled with phycoerythrin and anti-P63 monoclonal antibody labeled with allophycocyanin.

6. Use according to claim 5, wherein: The detection kit detects positive cells of TTF-1, CK7 and P63 in induced sputum cells of the subject by flow cytometry, and positive cells of TTF-1, CK7 and P63 three indicators, and establishes a diagnosis model Q for early diagnosis of lung cancer, and the formula of the diagnosis model Q is: Q = 0.25T + 0.36C + 0.26P + 1.8D; In the formula, T is the percentage of the number of TTF-1 positive cells in the total number of induced sputum cells; C is the percentage of the number of CK7 positive cells in the total number of induced sputum cells; P is the percentage of the number of P63 positive cells in the total number of induced sputum cells; and D is the percentage of the number of positive cells of TTF-1, CK7 and P63 three indicators in the total number of induced sputum cells. The induced sputum cells include macrophages, neutrophils, airway epithelial cells and tumor cells.

7. Use according to claim 6, wherein: The lung cancer early diagnosis is performed by the diagnostic model Q calculation, when Q>0.4, the subject is judged as lung cancer positive; when Q≤0.4, the subject is judged as lung cancer negative.

8. Use according to claim 7, wherein: The use method of the detection kit comprises the following steps: Step S1: collecting sputum in deep respiratory tract of the subject by high-osmotic saline atomization induction, and the sputum amount is not less than 2ml; Step S2: adding dithiothreitol to the collected sputum, and oscillating for 30 minutes at room temperature to fully digest the sputum; Step S3: filtering the digested sputum through two layers of sterile gauze filter screen, transferring the filtered liquid to a centrifugal tube, and centrifuging at 1800r / min for 10 minutes to make the induced sputum cells precipitate at the bottom of the tube; Step S4: discarding the supernatant, adding appropriate amount of cell preservation solution, and blowing to resuspend the induced sputum cells to obtain induced sputum cell suspension; Step S5: dividing the induced sputum cell suspension into several parts, respectively adding fluorescein isothiocyanate-labeled anti-TTF-1 monoclonal antibody, phycoerythrin-labeled anti-CK7 monoclonal antibody and allophycocyanin-labeled anti-P63 monoclonal antibody, vortexing, and incubating at 4℃ in dark for 30 minutes; Step S6: adding flow cytometry staining buffer, centrifuging at 3000r / min for 10 minutes, discarding the supernatant, and repeating the washing for 3 times; Step S7: adding appropriate amount of cell fixation solution, blowing to mix uniformly, and incubating at room temperature for 20 minutes; Step S8: after the fixation is completed, washing the induced sputum cells twice with flow cytometry staining buffer, then adding membrane permeabilization agent, and incubating at room temperature for 15 minutes; Step S9: collecting fluorescence signals of the induced sputum cells by flow cytometry to obtain the percentage of TTF-1 positive cell number in total number of induced sputum cells, the percentage of CK7 positive cell number in total number of induced sputum cells, the percentage of P63 positive cell number in total number of induced sputum cells, and the percentage of TTF-1, CK7 and P63 three index materials in total number of induced sputum cells, and establishing a diagnostic model Q, and the formula of the diagnostic model Q is: Q=0.25T+0.36C+0.26P+1.8D; Step S10: the lung cancer early diagnosis is performed by the diagnostic model Q calculation, when Q>0.4, the subject is judged as lung cancer positive; when Q≤0.4, the subject is judged as lung cancer negative. The use method of the detection kit comprises the following steps: Step S1: collecting sputum in deep respiratory tract of the subject by high-osmotic saline atomization induction, and the sputum amount is not less than 2ml; Step S2: adding dithiothreitol to the collected sputum, and oscillating for 30 minutes at room temperature to fully digest the sputum; Step S3: filtering the digested sputum through two layers of sterile gauze filter screen, transferring the filtered liquid to a centrifugal tube, and centrifuging at 1800r / min for 10 minutes to make the induced sputum cells precipitate at the bottom of the tube; Step S4: discarding the supernatant, adding appropriate amount of cell preservation solution, and blowing to resuspend the induced sputum cells to obtain induced sputum cell suspension; Step S5: dividing the induced sputum cell suspension into several parts, respectively adding fluorescein isothiocyanate-labeled anti-TTF-1 monoclonal antibody, phycoerythrin-labeled anti-CK7 monoclonal antibody and allophycocyanin-labeled anti-P63 monoclonal antibody, vortexing, and incubating at 4℃ in dark for 30 minutes; Step S6: adding flow cytometry staining buffer, centrifuging at 3000r / min for 10 minutes, discarding the supernatant, and repeating the washing for 3 times; Step S7: adding appropriate amount of cell fixation solution, blowing to mix uniformly, and incubating at room temperature for 20 minutes; Step S8: after the fixation is completed, washing the induced sputum cells twice with flow cytometry staining buffer, then adding membrane permeabilization agent, and incubating at room temperature for 15 minutes; Step S9: collecting fluorescence signals of the induced sputum cells by flow cytometry to obtain the percentage of TTF-1 positive cell number in total number of induced sputum cells, the percentage of CK7 positive cell number in total number of induced sputum cells, the percentage of P63 positive cell number in total number of induced sputum cells, and the percentage of TTF-1, CK7 and P63 three index materials in total number of induced sputum cells, and establishing a diagnostic model Q, and the formula of the diagnostic model Q is: Q=0.25T+0.36C+0.26P+1.8D; Step S10: the lung cancer early diagnosis is performed by the diagnostic model Q calculation, when Q>0.4, the subject is judged as lung cancer positive; when Q≤0.4, the subject is judged as lung cancer negative.

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