CN110054663B

CN110054663B - CKAP4 epitope peptide, antigen, monoclonal antibody, application of monoclonal antibody and CKAP4 detection test strip

Info

Publication number: CN110054663B
Application number: CN201910256742.3A
Authority: CN
Inventors: 王云龙; 张璐; 李玉林; 程蕾; 王继创; 邓黎黎
Original assignee: HENAN BIOENGINEERING RESEARCH CENTER
Current assignee: HENAN BIOENGINEERING RESEARCH CENTER
Priority date: 2019-04-01
Filing date: 2019-04-01
Publication date: 2023-10-03
Anticipated expiration: 2039-04-01
Also published as: CN110054663A

Abstract

The invention relates to the technical fields of immunology and proteomics, in particular to CKAP4 epitope peptide, antigen, monoclonal antibody, application thereof and a CKAP4 detection test strip. The CKAP4 epitope peptide is polypeptide A or polypeptide B, the amino acid sequence of the polypeptide A is RSHQDFSRQREEL, and the amino acid sequence of the polypeptide B is DSHGPKEDGG. Corresponding antigen and monoclonal antibody are prepared by taking the polypeptide A and the polypeptide B as sources respectively, and can be specifically combined with the CKAP4, the obtained monoclonal antibody can also be used for detecting the CKAP4, and a CKAP4 detection test strip is prepared, so that the quick detection of the CKAP4 in serum can be realized.

Description

CKAP4 epitope peptide, antigen, monoclonal antibody, application of monoclonal antibody and CKAP4 detection test strip

Technical Field

The invention relates to the technical fields of immunology and proteomics, in particular to CKAP4 epitope peptide, antigen, monoclonal antibody, application thereof and a CKAP4 detection test strip.

Background

The incidence of lung cancer is the leading cause of malignant tumor in men and rapidly increases in women, and lung cancer is a major disease which is harmful to life and health. Most patients have developed to middle and late stages when they are diagnosed, and miss the optimal period for the treatment of the disease, so that the treatment difficulty is further increased. Therefore, searching for effective tumor markers for the research of the mechanism of canceration, the early diagnosis of diseases and the screening of high-risk groups become urgent matters for the research of lung cancer.

The existing method for diagnosing lung cancer at early stage in clinic mainly comprises the traditional examination methods such as X-ray chest radiography, sputum shedding cells, fiber bronchoscope and the like, and is applied to screening of lung cancer (especially in high-risk groups) without reducing the death rate of lung cancer of the screened group, and meanwhile, the method is limited due to poor detection sensitivity, pain in process, poor patient tolerance, incapability of early dynamic monitoring and the like.

The lung cancer marker is a macromolecular substance expressed by tumor cells in the processes of tumor generation and development and distributed in tissues, blood and body fluid, has tissue specificity, and the distribution type and concentration change of the lung cancer marker are closely related to the tumor origin and growth of an affected individual, so that the lung cancer marker has the advantages of molecular diagnosis, parting and stage division of lung cancer from the molecular biology direction, and the like, and is widely applied to early diagnosis, clinical parting and prognosis of lung cancer in clinic in recent years. Therefore, the specific markers of the lung cancer are researched and found, an economical detection method which is more suitable for early diagnosis is established, and the method plays a great role in the prevention and treatment of the lung cancer.

CKAP4, also known as P63, cytoskeletal associated membrane protein 4 (CKAP 4), has a molecular weight of 63KD, is a type II transmembrane protein, and is connected between the endoplasmic reticulum and microtubules. Recent studies have shown that the sensitivity of serum carcinoembryonic antigen, cytokeratin 19 fragment and SCC antigen in the serum is 30% -52%, 17% -82% and 24% -39% respectively in stages I-IV of lung cancer. The study indicated that the sensitivity of CKAP4 in serum was 81% in the training group and 69% in the validation group, both higher than that of the serum diagnostic markers currently used in clinical applications.

At present, the development of a rapid diagnosis kit for CKAP4 is at the beginning, wherein the preparation of specific polypeptide or antibody against CKAP4 and the application of the specific polypeptide or antibody to detection by a proper method are key technologies for preparing the rapid diagnosis kit for CKAP 4. An epitope, also known as an antigenic determinant, is the basis of the antigenicity of an antigenic molecule. Therefore, the discovery of the CKAP4 epitope is an important basis for the development of monoclonal antibodies and rapid diagnosis kits, and no literature for researching the CKAP4 epitope is disclosed at present.

Although the epitope can be predicted by using bioinformatics software at present, more and more researches show that the error probability of the software for predicting the epitope is higher, and the prediction of the epitope by the software only aims at the whole length of the target antigen protein, so that the problem of specificity often exists in the real application process, and the problem needs to be verified by experiments.

Disclosure of Invention

According to the invention, 2 epitope peptides are found by researching the epitope of CKAP 4;

in addition, the invention also prepares CKAP4 antigen based on the antigen epitope peptide;

the invention also prepares a monoclonal antibody which can specifically recognize the antigen prepared by coupling the antigen epitope polypeptide and a carrier;

the invention also aims at defining that the monoclonal antibody can be used for preparing a kit/test strip for detecting CKAP 4;

finally, the invention also establishes a detection test strip capable of rapidly detecting the CKAP4 on the basis of the monoclonal antibody.

The CKAP4 epitope peptide adopts the following technical scheme: a CKAP4 epitope peptide, which is a polypeptide a or a polypeptide B, wherein the amino acid sequence of the polypeptide a is RSHQDFSRQREEL, and the amino acid sequence of the polypeptide B is DSHGPKEDGG.

A CKAP4 antigen, wherein the CKAP4 antigen is a CKAP4-a antigen, and the CKAP4-a antigen is prepared by coupling a polypeptide a (amino acid sequence: RSHQDFSRQREEL) to a carrier protein. The carrier protein may be selected from KLH (keyhole limpet hemocyanin), BSA (bovine serum albumin), ovalbumin OVA, etc. Preferably, the carrier protein of the CKAP4-A antigen is BSA.

A CKAP4 antigen, wherein the CKAP4 antigen is a CKAP4-B antigen, and the CKAP4-B antigen is prepared by coupling a polypeptide B (amino acid sequence: DSHGPKEDGG) to a carrier protein. Preferably, the carrier protein of the CKAP4-B antigen is BSA.

A CKAP4 monoclonal antibody, wherein the CKAP4 monoclonal antibody is a monoclonal antibody (derived from polypeptide a) prepared from the CKAP4-a antigen described above.

A CKAP4 monoclonal antibody, said CKAP4 monoclonal antibody being a monoclonal antibody (derived from polypeptide B) prepared from a CKAP4-B antigen as described above.

Use of two monoclonal antibodies as described above in the preparation of a CKAP4 diagnostic kit.

The application of the two monoclonal antibodies in preparing the CKAP4 detection test strip.

A CKAP4 detection test strip, wherein the labeled antibody and the coated antibody of the test strip are monoclonal antibodies derived from different epitope peptides of CKAP4, and the labeled antibody of the test strip is any one of the two monoclonal antibodies (namely, derived from polypeptide A or polypeptide B).

A CKAP4 detection test strip, wherein the labeled antibody and the coated antibody of the test strip are monoclonal antibodies derived from different epitope peptides of CKAP4, and the coated antibody of the test strip is any one of the two monoclonal antibodies (namely, derived from polypeptide A or polypeptide B).

Wherein, the labeled antibody of the test strip is an antibody combined with a marker (preferably a time-resolved fluorescent microsphere, such as Eu-carboxyl fluorescent microsphere) such as fluorescent microsphere/colloidal gold/nano magnetic bead/quantum dot; coated antibodies refer to antibodies coated on nitrocellulose or other solid media as detection lines. Preferably, the labeled antibody of the CKAP4 detection test strip is a monoclonal antibody derived from the polypeptide A, and the coated antibody is a monoclonal antibody derived from the polypeptide B; or the labeled antibody is a monoclonal antibody derived from the polypeptide B, and the coated antibody is a monoclonal antibody derived from the polypeptide A, so that the specificity, accuracy and effectiveness of the detection result of the test strip are ensured.

The beneficial effects of the invention are as follows: the antigen epitope peptide polypeptide A and the polypeptide B have good antigenicity, and antigens prepared by coupling the polypeptide A and the polypeptide B with carrier proteins can be used for producing monoclonal antibodies with good specificity when being used for immunizing animals, so that a kit/test strip for detecting CKAP4 can be prepared.

The invention provides a test strip capable of rapidly (5 min) detecting CKAP4 in serum, which has higher detection efficiency and a linear range of 5-100ng/ml, has a wider linear range compared with a CKAP4 monoclonal antibody in the prior art, has good specificity, and can rapidly and timely help diagnose illness state and monitor prognosis. The CKAP4 detection test strip has good correlation with an immunohistochemical method and reliable detection effect.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.

FIG. 1 is a schematic structural diagram of a CKAP4 test strip according to the present invention;

FIG. 2 is a schematic diagram of the qualitative detection results of the CKAP4 test strip;

FIG. 3 is a standard chart of test strip 1

In fig. 1: 1-sample pad; 2-a bonding pad; 3-nitrocellulose membrane; 4-T line; 5-C line; 6-a water absorbing pad; 7-a bottom plate.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

1. Screening of antigen epitope:

NCBI refers to the amino acid sequence of human CKAP4, and uses DNASsist, DNA STAR, bcePred Prediction Server and other software to analyze the B cell epitope of the CKAP4 protein. And (3) docking the epitope peptide obtained by software analysis with EGFR (epidermal growth factor receptor) during epitope analysis, selecting the polypeptide with the scoring function of more than 130 points as candidate epitope peptide, and further screening by experiments. Proved by a large number of experiments, two CKAP4 epitope polypeptides are finally selected:

amino acid sequence of polypeptide a: RSHQDFSRQREEL

Amino acid sequence of polypeptide B: DSHGPKEDGG

The polypeptides A and B may be prepared by chemical synthesis methods, such as solid phase synthesis methods and the like. And will not be described in detail herein.

Preparation and identification of ckap4 antigen:

2.1 The preparation of CKAP4 antigen comprises the following specific steps:

(1) The BSA solid (4 mg) was weighed and dissolved in 400. Mu.L MES buffer (0.1M, pH 5.0) to prepare a 10mg/mL BSA solution;

(2) Weighing 2mg of polypeptide A (synthesized by Shanghai Yao Biotechnology Co., ltd. According to the provided amino acid sequence) and 2mg of polypeptide B (synthesized by Shanghai Yao Biotechnology Co., ltd. According to the provided amino acid sequence), and respectively dissolving in 500 μl MES buffer (0.1M, pH 5.0) to obtain CKAP4 (A) solution and CKAP4 (B) solution; respectively taking 200 mu L of the BSA solution obtained in the step (1) and placing the 200 mu L of the BSA solution in 4mg/mL CKAP4 (A) and CKAP4 (B) solutions, and uniformly mixing by vortex;

(3) EDC was equilibrated to room temperature, 20mg of EDC was weighed and dissolved in 2mL of purified water to prepare 10mg/mL of EDC solution;

(4) Taking 100 mu L of the prepared EDC solution, immediately placing the EDC solution into a mixed solution of CKAP4 (A), BSA, CKAP4 (B) and BSA respectively, shaking the mixed solution for 2 hours at 25 ℃ by a shaking table, and finally preparing two complete antigens of CKAP4-A (CKAP 4 (A) -BSA) and CKAP4-B (CKAP 4 (B) -BSA) and preserving the complete antigens at the temperature of minus 20 ℃;

(5) And (3) taking part of the coupling product obtained in the step (4), dialyzing by using phosphate buffer solution with the pH of 7.2, replacing the dialyzate every 4 hours, and taking out the product after 4 times of replacement for identification.

2.2 detection of the antigen obtained

2.2.1 scanning ultraviolet spectra of CKAP4 (A), CKAP4 (B), CKAP4-A, CKAP-B and BSA respectively under a micro ultraviolet spectrophotometer, comparing the scanning patterns before and after coupling, and preliminarily judging whether the polypeptide A or the polypeptide B is coupled with the carrier protein (BSA). Samples of polypeptide A or polypeptide B coupled to a carrier were selected for SDS-PAGE gel electrophoresis.

2.2.2 SDS-PAGE electrophoresis identification (5% concentrate gel, 8% separation gel) comprises the following steps:

(1) Preparing relevant reagents and instrument equipment required by SDS-PAGE experiments;

(2) Washing the electrophoresis board by purified water, clamping the electrophoresis board by the board clamp, and detecting leakage by using the purified water; pouring out purified water after no water leaching, and removing residual purified water by using strip absorbent paper;

(3) Preparing a separation gel (selected according to the molecular size of a sample) according to a formula, slowly injecting the separation gel into the bottom along a gap at one side of the electrophoresis plate, preventing bubbles in the separation gel, and sealing the purified water for about 1h at room temperature;

(4) After the gel is solidified, pouring the liquid seal purified water, and removing the purified water from the side, close to the water absorption paper, of the water absorption paper;

(5) Preparing 5% concentrated glue, slowly injecting the top end of the separating glue close to a gap at one side, keeping the comb clean for avoiding bubbles at the lower end of the comb teeth, slowly inserting the concentrated glue (the depth is about 1 cm), and standing for later use after solidification;

(6) Slowly pulling out the comb, assembling the electrophoresis equipment, filling 1 Xof electrophoresis buffer solution, and applying sample by using a self-made slender gun head, wherein the sample application amount is 6 mu L/hole;

(7) Switching on a power supply, starting electrophoresis when bubbles are generated in the electrophoresis buffer solution from bottom to top, and switching off the power supply when the blue dyeing band is about to migrate out of the electrophoresis board;

(8) Taking out the electrophoresis plate, removing the plate clamp, opening the electrophoresis plate, marking the hole site of the sample, slowly taking out gel, washing purified water for three times to remove impurities, immersing in the dyeing liquid, and dyeing for about 40min by a decolorizing shaker;

(9) After the dyeing is finished, washing the dyed gel with purified water, placing the dyed gel in a decolorizing solution, decolorizing for 2 hours by using a decolorizing shaker, replacing old decolorizing solution on time, observing the gel background, cleaning if the gel is transparent, and photographing the gel in a light box.

3. Mouse immunization program

BALB/c female mice were taken 6, about 6-8 weeks old, 3 of which were immunized with CKAP4-A, CKAP4-B each, and subcutaneously injected by multiple immunization. Immunization was performed according to the following protocol:

first, mixing Freund's Complete Adjuvant (FCA) with CKAP4-A/CKAP4-B in equal volume;

the second and third phases are mixed with CKAP4-A/CKAP4-B in equal volume by adopting Freund's Incomplete Adjuvant (FIA);

if the serum antibody titer of the mice is less than 1:104 after the three-phase immunization is continued until the antibody titer is more than 1:104;

3 days before fusion, booster immunization was performed without adjuvant.

Mice were immunized with CKAP4-A/CKAP4-B according to the protocol described above. The highest titer mice in both immunized groups were selected and the next experiment was continued.

Specific immunization protocols are shown in table 1 below:

TABLE 1

CKAP4 monoclonal antibody preparation

To obtain immortalized hybridoma cells that specifically recognize the CKAP4 antigen, cell fusion is first performed, followed by cell cloning. And (3) cloning for 2-4 times, finally screening a monoclonal cell strain which stably secretes the CKAP4 antibody after the measurement by using indirect ELISA, and preparing the monoclonal antibody by using a mouse peritoneal inoculation induction method. Purifying ascites monoclonal antibody by adopting n-octanoic acid-sulfuric acid according to a precipitation method.

4.1 cell fusion

According to the following steps of 1: ratio 6 spleen cells and SP 2/0 (myeloma cell line) solution were mixed, vortexed uniformly, centrifuged at 1600rpm for 5min, blotted with water paper to remove water, and gently shaken to disperse the pellet. Under the water bath condition of 37 ℃, 1mL of preheated PEG2000 (the operation is completed in about 1 min) is added dropwise, and the time is 1min to promote the fusion reaction. Immediately adding preheated 1640 culture solution, centrifuging the fused cells at low rotation speed, discarding the waste liquid, washing for 2 times, and culturing with HATThe nutrient solution slightly blew off the cells. Preparing 96-well plate with feeder cells, dripping blown fusion cells into 100 μl/well, and adding CO ₂ Culturing in an incubator, and changing liquid according to time during culturing.

4.2.1 Positive monoclonal cell selection and cloning

To obtain immortalized hybridoma cells that specifically recognize the CKAP4 antigen, further cell cloning is required after fusion. The positive hybridoma cells initially detected by the indirect ELISA method can lose the capability of secreting the CKAP4 antibody due to factors such as cell mutation after being cultured, and usually need to be cloned for 2-4 times and finally screened to stably secrete the CKAP4 antibody after being measured by the indirect ELISA. The study adopts a limiting dilution method to carry out positive cell cloning, after about 10d, the supernatant of a monoclonal hole is measured by adopting an indirect ELISA method, the hole with high OD value is selected from the supernatant and then cloned (the OD450 value of serum (N) before immunization and serum (P) after immunization is respectively detected, the reciprocal of the maximum dilution multiple of serum containing antibodies is used as an evaluation index, the result is positive when the OD value (N) of serum before immunization/the OD value (P) of serum after immunization is less than or equal to 2.1 (the titer is 1:64000), specific data is shown in the following table 2), the process is repeated for 2 to 3 times, and monoclonal cell strains with positive clones are selected as candidate cell strains. The screened cell strain is subjected to expansion culture, and is subjected to timely passage and cryopreservation, 15d,30d,60d and 90d are respectively cryopreserved and resuscitated, cell activity is detected by staining cell trypan blue and counting cells, and the capacity of the cells to secrete specific antibodies is detected by an indirect ELISA method, so that the stability of the screened cell strain is judged. Finally 8 cell lines are selected, and the numbers of the cell lines are respectively as follows: 2A2, 3C2, 4D5, 4E2, 5C4, 6E1, 7F2, 8D5 for standby.

Table 2 indirect ELISA method for determining serum antibodies

4.2.2 subtype determination of monoclonal cell lines

The supernatants secreted by the stable cell lines were identified separately, and the main procedures were as follows:

(1) Removing all components in the kit, standing for 30min, and balancing to room temperature.

(2) Diluting the concentrated washing solution to the required concentration, taking out the ELISA plate, respectively diluting each monoclonal antibody with sample diluent, taking the sample diluent as a reference, labeling the ELISA plate in detail, adhering a sealing plate film, and reacting for 30min at 37 ℃.

(3) 6 HRP enzyme-labeled secondary antibodies comprising IgG1, igG2a, igG2b, igG3, igA and IgM were prepared, diluted and labeled in detail on an ELISA plate at 100. Mu.L/well, and the plate was adhered to the plate, and reacted at 37℃for 30min.

The results of the mab subtype assays are shown in table 3 below:

TABLE 3 Table 3

Antibody numbering

2A2

3C2

4D5

4E2

5C4

6E1

7F2

8D5

Subtype type

IgG2a

IgG1

IgG2b

IgG1

By measuring the subtype of monoclonal antibodies produced by 8 hybridoma cells, it was found that 2A2 was IgG2a,5C4 was IgG2b, and 6 other monoclonal antibodies each having IgG1 and 6 IgG1 were used as starting materials for the subsequent pairing screening.

4.3 preparation of monoclonal antibody ascites

The monoclonal antibody is prepared by adopting a mouse abdominal cavity inoculation induction method, and the specific steps are as follows:

(1) Liquid paraffin was injected intraperitoneally into the mice, and 0.5 mL/mouse, and male mice (about 12 weeks old) were sensitized to induce ascites.

(2) Positive hybridoma cell lines (in log phase) were collected and injected intraperitoneally into 3 mice/line, 0.2 mL/line, respectively.

(3) After 7-10d, mice with obviously raised abdomen were screened and sacrificed, and ascites was collected by syringe. Standing ascites at 4deg.C for more than 6 hr, centrifuging at 4000rpm for 5min, removing upper layer oil, and slowly sucking out middle clear part to obtain ascites.

4.4 purification and Performance detection of monoclonal antibodies:

4.4.1 purification of ascites monoclonal antibody by precipitation with n-octanoic acid-ammonium sulfate. The concentration of each monoclonal antibody was measured by a micro ultraviolet spectrophotometer, and then the purity of each purified monoclonal antibody was measured by SDS-PAGE electrophoresis. The treated sample is 6 mu L/hole and 3 mu L/hole Marker, the 8% separating gel and 5% concentrating gel are adopted for electrophoresis, after dyeing and decoloring, the observation and sub-packaging marking are carried out, the preservation is carried out at-20 ℃, the protein concentration of 6 monoclonal antibodies is measured after purification, the antibody concentration is more than 5mg/ml, and the requirement of the method on the measured concentration is met. Specific data are shown in table 4 below:

TABLE 4 Table 4

Antibody numbering	3C2	4D5	4E2	6E1	7F2	8D5
							Concentration (mg/ml)	6.23	5.31	5.34	7.42	8.68	9.07

4.4.2 SDS-PAGE identification of purified monoclonal antibodies using 8% separation gel and 5% concentration gel. The results show that the bands of the 6 monoclonal antibodies are all around 150 to 170KD, the molecular weight is consistent with the expected size, and the purity is better.

4.5 monoclonal antibody specificity identification

4.5.1 monoclonal antibodies purified from 6 hybridoma cells (3C 2, 4D5, 4E2, 6E1, 7F2 and 8D 5) were identified by indirect ELISA using CKAP4 (A) -BSA, CKAP4 (B) -BSA, BSA as coating antigen, respectively, and tested 3 times in parallel, while blank and negative controls were set.

The cross-reactions of monoclonal antibodies with protein vectors are shown in table 5 below:

TABLE 5

Note that: positive and negative respectively, the OD value is >0.105 positive, and negative is negative.

Conclusion: as can be seen from the above table, the wells coated with BSA were all negative, while the wells coated with CKAP4 (A) -BSA reacted positively with the monoclonal antibodies 3C2, 6E1 and 7F2, and reacted negatively with 4D5, 4E2 and 8D 5; wells coated with CKAP4 (B) -BSA reacted positively with 4D5, 4E2 and 8D5 and negatively with monoclonal antibodies to 3C2, 6E1 and 7F2, indicating that the obtained monoclonal antibodies were specific antibodies to CKAP4, but not to the carrier BSA; wherein 3C2, 6E1, 7F2 are specific antibodies to the complete antigen of CKAP4-A and 4D5, 4E2 and 8D5 are specific antibodies to the complete antigen of CKAP4-B.

4.5.2 validation of non-specific response of 6 CKAP4 monoclonal antibodies:

the purified antibodies are used for carrying out immune reaction with CEA antigen and CA125 antigen serum, and the result shows that the purified monoclonal antibodies have specificity, and the screened CKAP4 epitope peptides (polypeptide A and polypeptide B) have potential antigenic determinant characteristics. The specific results are shown in Table 6 below:

TABLE 6

Conclusion: the 6 monoclonal antibodies have no specific reaction to interference antigens (CEA antigen and CA125 antigen) and have good effect.

4.6 ELISA method for screening pairing monoclonal antibodies

And carrying out HRP labeling on the prepared CKAP4 monoclonal antibody by adopting a modified sodium periodate method. By adopting a double-antibody sandwich ELISA detection method, referring to the laboratory detection system, the prepared standard substance is taken as a sample to be detected, the obtained monoclonal antibody and the HRP-labeled monoclonal antibody are subjected to pairing screening (self pairing is removed), whether the antibodies are paired or not is judged through the measured OD value, and the high OD value is selected as the optimal pairing. The method mainly comprises the following steps:

(1) CKAP4 monoclonal antibody is used as coating, and the coating amount is as follows: 500 ng/well, sample size: 20. Mu.L/well;

(2) Diluting HRP-labeled antibody 1/2000 with enzyme-labeled diluent, 50 μl/well;

the preferred pairs screened are shown in table 7 below:

TABLE 7

Embodiment two: preparation of CKAP4 test strip (see figure 1 of the drawings)

1.1 preparation of bond pad 2

1.1.1 Preparation of CKAP4 labeled antibody-fluorescent microsphere complex

(1) Adding 15uL of fluorescent microspheres into 600uL of 50mmol/L boric acid buffer, and uniformly mixing by vortex;

(2) Adding 100ug EDC, shaking at 25-30deg.C for 30min,14000r/min, and centrifuging for 20min;

(3) The sediment is resuspended in boric acid buffer solution, washed for 1 time, and centrifuged in the same step (2);

(4) Re-suspending and precipitating by using 1mL 50mmol/L boric acid buffer solution, adding 100ug of labeled antibody (the antibody forming a complex with fluorescent microspheres can be monoclonal antibody or polyclonal antibody), and mixing uniformly by vortex;

(5) Adding 50uL 10% BSA (which can prevent separation of fluorescent microsphere and labeled antibody), shaking and sealing at 25-30deg.C for 1 hr, 14000r/min, and centrifuging for 15min; the precipitate was blown off with 1mL of a protective solution (0.05M ph=8.0 Tris-Hcl, 0.5% (volume fraction) TWEEN-20 and 0.1% BSA, the concentrations of the above components being final concentrations, BSA concentrations being mass percent), sonicated for 30s, stored at 4 ℃ for later use;

(6) Coupling by shaking at 25-30deg.C for 2h, centrifuging in the same step (2), adding 50uL 10% BSA, shaking at 25-30deg.C for 1h,14000r/min, and centrifuging for 15min; the precipitate was blown off with 1mL of a protective solution (0.05M ph=8.0 Tris-Hcl, 0.5% (volume fraction) TWEEN-20 and 0.1% BSA, the concentrations of the above components being final concentrations, BSA concentrations being mass percent) and stored at 4 ℃ for later use with ultrasound for 30 s. The specific preparation method of the protective liquid comprises the following steps: the mixture was mixed well with pH=8.0.2M Tris-HCl 25mL, TWEEN-20.5 mL, BSA 0.1g and purified water 75 mL.

1.1.2 immobilization of CKAP4 labeled antibody-fluorescent microsphere Complex on glass fiber Membrane to give conjugate pad 2

A glass fiber film (10 cm long and 7mm wide) is treated with a treatment solution (0.01M pH=8.0 Tris-HCl, 0.1% (volume fraction) TWEEN-20, 0.1% (mass fraction) BSA and 0.1% (mass fraction) sucrose, the concentrations of the above components are all final concentrations), dried at 37 ℃, then a prepared CKAP4 labeled antibody-fluorescent microsphere solution ((40 uL/cm) is coated on the glass fiber film, the solution is required to be uniformly filled with the glass fiber film, dried at 37 ℃ and a bonding pad 2,4 ℃ aluminum foil bag (containing a drying agent) is prepared for sealing for standby.

1.2 coating T and C wires on nitrocellulose film

The middle adhesive sticker of the bottom plate 7 (PVC plate) is removed, and the NC film (nitrocellulose film 3) is closely attached to the lower end of the upper sticker. Before scribing, the scribing position (X, Y, Z), the scribing amount and the like of the XYZ three-dimensional spot film metal spraying instrument are adjusted, after the scribing is completed, the scribing is simulated, the quality control line C line 5 and the detection line T line 4 are positioned at the middle position of the NC film 3, then goat anti-mouse IgG (1 mg/mL) is used as the quality control line C line 5, the coating antibody (1.5 mg/mL) is used as the detection line T line 4, the coating antibody is used as the antibody of the detection line T line coated on the nitrocellulose film, and the monoclonal antibody or the polyclonal antibody can be used as the antibody of the detection line T line, after the scribing is completed, the NC film 3 is uniformly scribed, the NC film is dried at 37 ℃ for 2 hours, and the NC film is sealed in a self-sealing bag for standby.

1.3 Assembly of test strips

The two adhesive paper labels at the upper end and the lower end of the bottom plate 7 (PVC plate) are removed, the water absorption pad 6, the combination pad 2 of the fluorescent microsphere with the CKAP4 marked antibody and the sample pad 1 are sequentially overlapped and assembled, the overlapping areas are 1mm, the cover of the microcomputer automatic cutting machine is lifted, the assembled coating plate is placed into the material placing platform, the assembled coating plate is cut into test strips with the width of 3.9mm, and the test strips are stored in a sealing mode at the temperature of 4 ℃ (the structure of the assembled test strips is shown in figure 1 of the specification).

1.4 test strip using method

After the test strip is restored to room temperature, adding a sample to be detected (serum sample) into the sample adding hole, and timing for 5min at 70 uL/hole;

1.4.1 quantitative detection: and (3) the test strip is moved to a fluorescence immunochromatography reader for detection, the fluorescence values of the T line and the C line are recorded, calculation is carried out according to a standard curve preset in the fluorescence immunochromatography reader, and the concentration of CKAP4 in a detected sample can be directly read.

1.4.2 qualitative detection: the test paper strip is moved to the ultraviolet lamp for observation, and the result can be directly interpreted: the fluorescent brightness of the line C or the fluorescent brightness of the line T appears, and the judgment result is invalid; the lines C and T both show fluorescence brightness, and the judgment result is positive; the line C has no fluorescence brightness, the line T has fluorescence brightness, and the test strip result is judged to be negative (see figure 2 of the specification).

Example III Performance verification of the test strip prepared by the invention

1.1 establishment of a standard curve:

preparing a series of concentration gradient CKAP4 standard solutions: 0. 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, 150, 200, 300 and 400 (ng/ml), 70uL is dripped on an immunochromatographic test strip, after 5min, the fluorescence intensities of a T line and a C line are read by a fluorescence immunochromatographic reader, and fitting curves are respectively made for the fluorescence intensities of the T/C and the corresponding CKAP4 standard solution concentrations, so that formulas corresponding to the fluorescence intensities and the concentrations are obtained.

1.2 precision detection

1.2.1 in-batch precision detection: the median quality control product (50 ng/mL) is repeatedly detected by 50 CKAP4 detection test strips in the same batch, and the variation coefficient of T-line fluorescence intensity is adoptedCalculating to obtain the precision in the batch。

1.2.2 inter-batch precision: median quality control (50 ng/mL) was measured using 3 batches (20 per batch) of CKAP4 test strips prepared in the same manner using the coefficient of variation of T-line fluorescence intensityAnd calculating to obtain the precision among batches.

2. Test strip for performing the above test (1.1-1.3):

test strip 1: the labeled antibody adopted in the preparation of the test strip is the monoclonal antibody 6E1 prepared in the first embodiment, the coated antibody is the monoclonal antibody 4D5 prepared in the first embodiment, see Table 6 (other monoclonal antibodies in Table 6 can be used for preparing test strips, and the coated antibody and the labeled antibody can be interchanged), and the rest of the preparation steps are the same as those in the second embodiment (the standard curve chart of the test strip 1 is shown in the figure 3 of the specification);

test strip 2: two CKAP4 antibody samples (from which the best paired antibodies were selected) provided by Shanghai Shuobo biotechnology Co., ltd were used as labeled antibodies and coated antibodies, respectively, and the remaining production steps were the same as in example II;

3. detection result: the specific detection results are shown in Table 8 below

TABLE 8

Test paper strip	Intra-batch precision	Precision between batches	Linear range (gradient dilution quality control)	R ²
					Test strip 1	CV＝14.12	CV＝14.88	5-100ng/ml	0.9877
Test paper strip 2	CV＝10.37	CV＝12.69	10-95ng/ml	0.9631

Example methodological alignment

The method (detection is carried out by adopting the test paper strip 1) and Zheng Da-attached hospital immunohistochemical method are used for parallel detection of 45 clinical plasma samples of lung cancer patients, the coincidence rate reaches 91%, and the specific results are shown in the following table 9

TABLE 9

As shown in the above Table 9, the detection method and the immunohistochemical method of the present invention have good correlation, and the detection result is reliable, and can be used for rapid detection of CKAP 4.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Sequence listing

<110> Henan Bioengineering technology research center Co., ltd

Henan bioengineering technology research center

<120> CKAP4 epitope peptide, antigen, monoclonal antibody, application thereof and CKAP4 detection test strip

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Arg Ser His Gln Asp Phe Ser Arg Gln Arg Glu Glu Leu

1 5 10

<210> 2

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Asp Ser His Gly Pro Lys Glu Asp Gly Gly

1 5 10

Claims

1. The CKAP4 epitope peptide is characterized in that the epitope peptide is a polypeptide A or a polypeptide B, the amino acid sequence of the polypeptide A is RSHQDFSRQREEL, and the amino acid sequence of the polypeptide B is DSHGPKEDGG.

2. A CKAP4 antigen, wherein the CKAP4 antigen is a CKAP4-a antigen, and wherein the CKAP4-a antigen is prepared by coupling the polypeptide a of claim 1 to a carrier.

3. A CKAP4 antigen, wherein the CKAP4 antigen is a CKAP4-B antigen, and wherein the CKAP4-B antigen is prepared by coupling the polypeptide B of claim 1 to a carrier protein.