CN116381251A - Tumor marker diagnosis kit and diagnosis method thereof - Google Patents
Tumor marker diagnosis kit and diagnosis method thereof Download PDFInfo
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Abstract
A tumor marker diagnostic kit and a diagnostic method thereof. The invention discloses a kit for detecting Nectin-4, which comprises an effective amount of Nectin-4 recombinant protein, an effective amount of monoclonal antibody specifically combined with Nectin-4 and a matched detection reagent; the sequence of the Nectin-4 recombinant protein is shown as SEQ ID NO. 1; the monoclonal antibodies specifically combined with Nectin-4 are monoclonal antibody 1A2 and monoclonal antibody 5B6; the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1A2 are shown as SEQ ID NO.2 and SEQ ID NO. 3; the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 5B6 are shown as SEQ ID NO.4 and SEQ ID NO. 5. The kit has good sensitivity, specificity and stability, and is suitable for detecting Nectin-4 in serum, cell culture fluid, urine, tissue fluid and the like.
Description
Technical Field
The invention belongs to the technical field of biological diagnosis, and particularly relates to a tumor marker diagnosis kit and a diagnosis method thereof.
Background
Nectin-4 belongs to the Nectin family of Ig superfamily proteins and is a type I membrane protein. Nectin-4 has high expression specificity in tissues such as breast cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic cancer and the like, and has close relation with the occurrence and metastasis of tumors. Nectin-4 can promote proliferation, differentiation, migration, invasion, etc. of tumor cells by activating PI3K/AKT pathway. Therefore, the targeting of Nectin-4 as a detection or treatment target may be an effective strategy for detecting or treating cancers with high Nectin-4 expression. However, there are no good diagnostic materials and diagnostic kits on the market, and there is a need to develop a monoclonal antibody for diagnosing or treating Nectin-4 for detecting Nectin-4 or treating related cancers.
Disclosure of Invention
In order to make up for the defects of the prior art, one of the purposes of the invention is to provide a Nectin-4 recombinant protein, a monoclonal antibody thereof and a preparation method; the invention also provides a kit for detecting Nectin-4, which is prepared by using the monoclonal antibody and the Nectin-4 recombinant protein.
Accordingly, in one aspect the invention discloses a kit for detecting Nectin-4, said kit comprising an effective amount of a Nectin-4 recombinant protein and an effective amount of a monoclonal antibody which specifically binds Nectin-4 and a complementary detection reagent; the monoclonal antibodies specifically combined with Nectin-4 are monoclonal antibody 1A2 and monoclonal antibody 5B6; the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1A2 are shown as SEQ ID NO.2 and SEQ ID NO. 3; the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 5B6 are shown as SEQ ID NO.4 and SEQ ID NO. 5.
Preferably, the amino acid sequence of the Nectin-4 recombinant protein is shown as SEQ ID NO. 1.
Preferably, the Nectin-4 recombinant protein provided by the invention is used as a calibrator, and the concentrations of the calibrator are respectively 0ng/mL, 0.625ng/mL, 1.25ng/mL, 2.5ng/mL, 5ng/mL and 10ng/mL.
Preferably, the monoclonal antibody 1A2 and the monoclonal antibody 5B6 according to the present invention are both rabbit monoclonal antibodies.
Preferably, the kit is a double-antibody sandwich ELISA kit, wherein the coated antibody in the kit is monoclonal antibody 1A2, and the coating concentration of the monoclonal antibody 1A2 is 1 mug/ml; the enzyme-labeled antibody of the kit is an HRP-labeled monoclonal antibody 5B6, and the dilution factor of the HRP-labeled monoclonal antibody 5B6 is 20000 times.
In still another aspect, the invention also discloses an application of the monoclonal antibody 1A2 and the monoclonal antibody 5B6 which specifically bind to Nectin-4 in preparation of a Nectin-4 detection reagent.
In still another aspect, the invention also discloses application of the Nectin-4 recombinant protein in preparation of a Nectin-4 detection reagent.
According to the invention, the Nectin-4 recombinant protein is truncated and expressed through research and analysis on the Nectin-4 protein. The recombinant protein has good immunogenicity (used for preparing rabbit monoclonal antibodies and kit calibrator). Can provide better anti-raw materials for the detection of Nectin-4.
The invention screens 2 specific monoclonal antibodies on the basis of Nectin-4 recombinant protein, and provides a good tool for establishing Nectin-4 detection. The 2 monoclonal antibodies have good specificity and sensitivity.
Based on the research of the Nectin-4 recombinant protein and the monoclonal antibody, the invention develops a kit for detecting the Nectin-4, which has higher sensitivity, accuracy and stability, and is suitable for detecting the Nectin-4 in serum, cell culture fluid, urine, tissue fluid and the like. Therefore, the kit has better applicability.
Therefore, the invention prepares the Nectin-4 recombinant protein and the related monoclonal antibody through the analysis and research of the Nectin-4, and provides good raw materials and means for the further research of the effect of the Nectin-4 in tumors. The diagnosis kit provided by the research provides a good tool for the diagnosis of Nectin-4, provides a good detection means for the diagnosis of Nectin-4 related tumors, and also provides an important reference for the diagnosis and treatment of the subsequent Nectin-4 serving as a tumor marker.
Drawings
FIG. 1Nectin-4 protein signal peptide analysis results.
FIG. 2 shows the results of the removal of the signal peptide Nectin-4 protein transmembrane domain analysis.
FIG. 3 shows SDS-PAGE detection result of Nectin-4 recombinant protein. Wherein 1 is a purified recombinant protein.
FIG. 4 shows SDS-PAGE detection results of two monoclonal antibodies. Wherein 1 and 2 are monoclonal antibody 1A2 and monoclonal antibody 5B6, respectively.
FIG. 5 standard curve of kit.
Detailed Description
Unless defined otherwise, 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 belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1: preparation of Nectin-4 recombinant protein
Human Nectin-4 protein was analyzed (NCBI Reference Sequence: NP-112178.2).
The signal peptide was first analyzed and the result showed (FIG. 1) that the signal peptide was 31 amino acids at the N-terminus (MPLSLGAEMWGPEAWLLLLLLLASFTGRCPA).
The results of the transmembrane region analysis of Nectin-4 protein from which the signal peptide was removed showed (FIG. 2 and Table 1) that the first 318 amino acids at the N-terminus were the outer membrane region, and the other were the transmembrane region or the intracellular region.
TABLE 1 transmembrane assay results
The amino acid sequence of the outer membrane region in Table 1 (shown as SEQ ID NO. 1) was codon optimized. Cloning the nucleotide sequence (SEQ ID NO. 2) subjected to codon optimization into a prokaryotic expression vector pET28a, selecting correct cloning for amplification after sequencing and identification, extracting DNA, converting into escherichia coli BL21, expressing and purifying the Nectin-4 recombinant protein. The purity of the purified protein was measured by SDS-PAGE, and the purity (FIG. 3) was 90% or more (a specific band appeared at about 35 kDa), and the BCA concentration was 1.45mg/ml, and the protein was quantitatively dispensed and stored at-20℃for further use.
Example 2: preparation of anti-Nectin-4 monoclonal antibodies
2.1 immunization: the Nectin-4 recombinant protein prepared in example 1 was used as an antigen, rabbits were immunized by subcutaneous injection on the back, 2ml of blood was taken for later use before immunization, 0.5mg of the antigen was mixed with an equivalent amount of Freund's complete adjuvant for the first immunization (0.2 ml/5 th) and 0.5mg of the antigen was mixed with an equivalent amount of Freund's incomplete adjuvant for the second to fourth immunization (0.2 ml/5 th), two weeks between the immunization, booster immunization was arranged 4 to 5 weeks after the fifth immunization, 0.5mg of the antigen was mixed for intravenous injection (1 ml), and rabbits were killed 4 days after injection, and spleens were taken for later use. In the immune process, the state of the rabbit is observed, and if abnormal conditions occur, the rabbit is treated in time. The results show that the spirit and diet of the rabbits are normal and no abnormal condition exists in the whole immunization process.
2.2 spleen cell isolation: taking spleen aseptically, and removing fat and other tissues around the spleen in a culture dish; after being washed by 1640 culture solution, the spleen is placed on a stainless steel screen, extruded and screened; suspending with 1640 culture solution, centrifuging at 1400rpm for 5min; discarding the supernatant, and repeatedly washing for one time; re-suspending cells with 1640 culture solution, and diluting a small amount of cell suspension; counting viable cell count with trypan blue staining; based on cell count, the contents were taken to be 2X 10 8 The suspension of individual spleen lymphocytes was ready for use. The results showed that the viability of spleen cells was 95% as measured by trypan blue staining count, indicating that the spleen cells had good activity and cell fusion could be continued.
2.3 cell fusion: spleen cells were cell fused with rabbit bone marrow tumor cells 240E (240E cell line is a plasmacytoma). Respectively taking 2×10 8 Spleen lymphocytes and 1X 10 8 Mixing 240E cells, adding 1640 culture solution, centrifuging, and discarding supernatant to make the two cells fully mixed into paste. The mixture was preheated in a 37℃water bath, and a 50% polyethylene glycol solution preheated at 37℃was slowly added. Then, 1640 culture solution preheated at 37 ℃ is added to dilute polyethylene glycol to lose effect. After the addition of 1640 culture solution, the supernatant was centrifuged, and the cell pellet was suspended in HAT culture solution. Through detection, the cell fusion rate is about 65%, which indicates that the cell fusion is better, and the hybridoma cell can be continuously screened.
2.4 hybridoma cell screening: the fused cells are sub-packed with HAT as selective mediumIn 96-well cell culture plate of feeder cells, the feeder cells are placed at 37 ℃ and 5% CO 2 Culturing in an incubator for 2-3 weeks, performing primary detection, taking cell culture hole supernatant, screening positive clones by an indirect ELISA method, transferring hybridoma cells positive to primary detection into a 24-hole cell culture plate, screening hybridoma cells with strong secretory antibody affinity and good cell growth state by an indirect ELISA method after one week, storing one part of hybridoma cells in liquid nitrogen, performing cloning by a limiting dilution method, generally selecting 5 polyclonal cells for monoclonal, subpackaging each polyclonal cell into 3 96-hole cell culture plates, screening positive clones by an indirect ELISA method after three weeks, screening more than 3 (about 10) positive holes for each polyclonal cell by an indirect ELISA method, transferring into a 24-hole cell culture plate, screening hybridoma cells with strong secretory antibody affinity and good cell growth state by an indirect ELISA method after one week, storing one part of hybridoma cells in liquid nitrogen, and performing expanded culture to produce rabbit monoclonal antibodies. Two cell lines from different clones were selected for subcloning. After two subcloning, the hybridoma cell strain which stably secretes the specific antibody is finally obtained. Through screening and subcloning, 2 better hybridoma cell strains are finally screened and named as hybridoma cell 1A2 and hybridoma cell 5B6. Two hybridoma cells were cultured, and the monoclonal antibody was obtained by purification from the supernatant. SDS-PAGE was used to examine (FIG. 4) that the purity was 90% or higher, BCA kit was used to detect the concentration of the two monoclonal antibodies at 2.06mg/ml and 2.12mg/ml, respectively, and the monoclonal antibodies were quantitatively packaged and stored at-20℃for further use.
Example 3: indirect ELISA procedure
3.1 coating: using Nectin-4 recombinant protein coated ELISA plate prepared in example 1, diluting Nectin-4 recombinant protein to 1 μg/ml with coating buffer (pH9.6CBS buffer), mixing, adding ELISA plate, coating at 2-8deg.C overnight in each well of 0.1 ml;
3.2 closing: after 3 washes with PBST buffer, the wells were blocked with 5% skim milk in PBST buffer, 0.2ml per well, 37 degrees for 2h;
3.3 adding samples: after washing 3 times with PBST buffer, samples (diluted 10-fold or 10-fold ratio depending on the concentration and detection result) were added, respectively, at 0.1ml per well, at 37 degrees for 1h;
3.4 adding secondary antibody: after 3 washes with PBST buffer, HRP-labeled goat anti-rabbit IgG secondary antibody (1:5000 fold dilution) was added to each well; 37 degrees for 0.5h;
3.5 color development: after washing 3 times with PBST buffer, TMB chromogenic solution was added, 0.1ml per well, 37℃for 10min;
3.6 termination and determination: adding stop solution (2M sulfuric acid) with volume of 0.05ml per well, and detecting OD450nm immediately after stopping; positive was judged when the P/N value (sample OD450nm value/blank OD value) > 2.1 and OD450nm value > 0.1, otherwise negative.
Example 4: detection of anti-Nectin-4 monoclonal antibodies
4.1 pairing check
4.1.1HRP labeling and detection: the two monoclonal antibodies are subjected to HRP labeling by using a commercial HRP labeling kit, and the dilution of the HRP-labeled monoclonal antibodies is determined by using a direct ELISA method (an ELISA plate coated in example 3 is used; then the HRP-labeled monoclonal antibodies are subjected to gradient dilution, generally the dilution is 10000, 20000 and the like, the HRP-labeled monoclonal antibodies are added to the ELISA plate, 0.1 ml/hole is added, incubation is carried out at 37 ℃ for 30min, then a substrate is added to detect an OD450nm value, the OD450nm value is more than or equal to 1.1 as a judgment standard), and the dilution of the HRP-labeled monoclonal antibodies is 20000 times by detection.
4.1.2 pairing detection: two monoclonal antibodies were used to coat the ELISA plates (1. Mu.g/ml), nectin-4 recombinant protein prepared in example 1 was used as a sample (diluted to 10ng/ml and used), 0.1 ml/well was used, after incubation at 37℃for 1 hour, HRP-labeled monoclonal antibodies were added in a crossover manner, the dilution was determined as 4.1.1.1 dilution, 0.1 ml/well was used, after incubation at 37℃for 30 minutes, the substrate was added to detect OD450nm, and the pairing effect was determined by comparing the value of OD450nm (the higher the OD450nm indicated the better the pairing effect). The results are shown in Table 2, and the two monoclonal antibodies have good pairing property, wherein monoclonal antibody 1A2A2 is used as a coating antibody, and monoclonal antibody 5B6 is used as a detection antibody.
Table 2 results of the monoclonal antibody pairing test (OD 450nm value)
4.2 sequence analysis of monoclonal antibodies
RNA is extracted from two hybridoma cells, and the sequences of the heavy chain variable region and the light chain variable region of the codes of the two monoclonal antibodies are amplified from the RNA by molecular biological technology, and are sequenced. The job may be delegated to a third party company. The sequences of the two monoclonal antibodies are shown in Table 3 after detection and analysis.
TABLE 3 variable region sequences of two monoclonal antibodies
Example 5: preparation of Nectin-4 detection kit
The present inventors developed a Nectin-4 detection kit (double antibody sandwich ELISA kit) based on Nectin-4 of example 1 and monoclonal antibodies 1A2 and 5B6 of example 2.
The preparation and detection of the kit are as follows:
5.1 preparation of calibrator Nectin-4 recombinant protein prepared in example 1 was used as a mother liquor, diluted with 10mM PBS buffer to final concentrations of 0, 0.625, 1.25, 2.5, 5, 10 (units ng/mL), 1.0 mL/tube, and stored at 2-8deg.C for further use.
5.2 preparation and detection of detection kit
5.2.1 monoclonal antibody 1A2 coated ELISA plate: diluting monoclonal antibody 1A2 to 1 mug/ml by using a coating buffer (pH 9.6 CBS buffer), adding an ELISA plate after uniformly mixing, and coating at 2-8 ℃ for overnight at 0.1ml per hole;
5.2.2 blocking: after 3 washes with PBST buffer, the wells were blocked with 5% skim milk in PBST buffer, 0.2ml per well, 37 degrees for 2h;
5.2.3 adding sample: after washing 3 times with PBST buffer, calibrator (undiluted) and sample (diluted according to concentration and detection result) were added, respectively, at 0.1ml per well, at 37 degrees for 1h;
5.2.4 addition of enzyme-labeled antibody (HRP-labeled monoclonal antibody 5B6 prepared in example 4): after 3 washes with PBST buffer, enzyme-labeled antibody (1:20000-fold dilution) was added to each well; 37 degrees for 0.5h;
5.2.5 color development: after washing 3 times with PBST buffer, TMB chromogenic solution was added, 0.1ml per well, 37℃for 10min;
5.2.6 termination: stop solution (2M sulfuric acid) was added at 0.05ml per well and the OD450nm was measured immediately after termination.
5.2.7 results calculation: a standard curve is established according to the concentration of the calibrator and the OD450nm value (figure 5), the OD450nm value of the sample is substituted into the standard curve for calculation, and then the concentration of the sample is converted according to the dilution multiple.
5.3 sample preparation
5.3.1 serum samples: blood samples were collected using pyrogen-free, endotoxin-free tubes or centrifuge tubes, whole blood samples were left at room temperature for 1-2 h or overnight at 4℃and then centrifuged at 3000rpm/min for 20min at 4℃with care to collect the supernatant. The mixture is preserved at-20 ℃ (1-2 weeks) or-80 ℃ for a long time, and repeated freezing and thawing are avoided. Avoiding the use of hemolysis or hyperlipidemia specimens.
5.3.2 plasma samples: EDTA or heparin sodium or citrate sodium is selected as an anticoagulant according to sample requirements, a blood collection tube or a centrifuge tube containing the anticoagulant is used for collecting a blood sample, the blood sample is mixed for about 20min at room temperature, then centrifugation is carried out at 4 ℃ for 20min at 3000rpm/min, and the supernatant is carefully collected to obtain the plasma. The mixture is preserved at-20 ℃ (1-2 weeks) or-80 ℃ for a long time, and repeated freezing and thawing are avoided. Avoiding the use of hemolysis or hyperlipidemia specimens.
5.3.3 urine specimens: urine was collected in a clean vessel and centrifuged at 3000rpm/min at 4℃for 20min, and the supernatant was carefully collected. The mixture is preserved at-20 ℃ (1-2 weeks) or-80 ℃ for a long time, and repeated freezing and thawing are avoided.
5.3.4 saliva specimens: saliva was collected with a clean centrifuge tube, centrifuged at 3000rpm/min for 20min at 4℃and the supernatant carefully collected. The mixture is preserved at-20 ℃ (1-2 weeks) or-80 ℃ for a long time, and repeated freezing and thawing are avoided.
5.3.5 cell specimen
(1) Cell culture supernatant: the cell culture supernatant was taken into a clean centrifuge tube, centrifuged at 3000rpm/min for 20min at 4℃and the supernatant was carefully collected. The mixture is preserved at-20 ℃ (1-2 weeks) or-80 ℃ for a long time, and repeated freezing and thawing are avoided.
(2) Cell lysate: the cell culture broth was collected, centrifuged at 3000rpm for 20min at 4℃and the supernatant was discarded, and washed three times with pre-chilled PBS (0.01M, PH7.4). Appropriate amounts of pre-chilled PBS or cell lysate (protease inhibitor added just prior to use) were added to resuspend the cells. Cells were allowed to lyse well by repeated freeze thawing. Then, the mixture was centrifuged at 10000rpm for 20min at 4℃to remove cell debris, and the supernatant was collected. The mixture is preserved at-20 ℃ (1-2 weeks) or-80 ℃ for a long time, and repeated freezing and thawing are avoided.
5.3.6 tissue specimens: the tissue samples were washed with PBS buffer (0.01M, PH7.4) to wash away residual blood or impurities from the tissue surface. The tissue pieces were weighed, recorded and chopped (pieces as small as possible). Tissue was homogenized by adding the tissue in a certain ratio (typically tissue weight: PBS volume=1:9) to pre-chilled PBS buffer, and placed on ice while homogenizing. The homogenate was pipetted into a centrifuge tube and centrifuged at 10000rpm for 20min at 4℃and the supernatant carefully collected. The mixture is preserved at-20 ℃ (1-2 weeks) or-80 ℃ for a long time, and repeated freezing and thawing are avoided.
Example 6: kit performance evaluation and detection
The kit of example 5 was used for comparison with a certain inlet kit (control kit).
6.1 comparing the lowest detection limit, taking an internal control reference with the concentration of 0ng, respectively detecting for 20 times by using two kits, and taking the concentration value corresponding to the average value plus twice the standard deviation as the lowest detection limit by using the average value and the standard deviation SD of 20 detection results (OD values). The results show (Table 4) that the lowest limit of detection of the present kit is better than that of the control kit.
Table 4 minimum limit of detection comparison
| Name of the name | Average value of | SD | Mean +2SD | Minimum detection limit |
| The kit | 0.0561 | 0.0015 | 0.0591 | 0.11 |
| Control kit | 0.0956 | 0.0045 | 0.1046 | 0.16 |
6.2 specific comparison study possible cross-reactants were taken, detected with two kits respectively, the average of the two parallel measurements (OD values) of each cross-reactant sample was calculated, and the concentration value of each sample was calculated from the corresponding kit calibration curve and compared with the concentration of the cross-reactant itself. The results showed (Table 5) that both kits were better specific, but the kits of the present study were better than the control kit.
TABLE 5 results of specificity comparison studies
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. A kit for detecting Nectin-4, which is characterized by comprising an effective amount of Nectin-4 recombinant protein, an effective amount of monoclonal antibody specifically binding to Nectin-4 and a matched detection reagent; the monoclonal antibodies specifically combined with Nectin-4 are monoclonal antibody 1A2 and monoclonal antibody 5B6; the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 1A2 are shown as SEQ ID NO.2 and SEQ ID NO. 3; the sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody 5B6 are shown as SEQ ID NO.4 and SEQ ID NO. 5.
2. The kit according to claim 1, wherein the amino acid sequence of the Nectin-4 recombinant protein is shown in SEQ ID NO. 1.
3. The kit according to claim 1, wherein the Nectin-4 recombinant protein is used as a calibrator at a concentration of 0ng/mL, 0.625ng/mL, 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL, respectively.
4. The kit of claim 1, wherein the monoclonal antibodies 1A2 and 5B6 are both rabbit monoclonal antibodies.
5. The kit according to claim 1, wherein the kit is a double-antibody sandwich ELISA kit, the coated antibody in the kit is monoclonal antibody 1A2, and the coating concentration of monoclonal antibody 1A2 is 1 μg/ml; the enzyme-labeled antibody of the kit is an HRP-labeled monoclonal antibody 5B6, and the dilution factor of the HRP-labeled monoclonal antibody 5B6 is 20000 times.
6. Use of monoclonal antibody 1A2 and monoclonal antibody 5B6 of claim 1 that specifically bind to Nectin-4 for the preparation of a Nectin-4 detection reagent.
7. Use of a recombinant Nectin-4 protein according to claim 1 for the preparation of a Nectin-4 assay reagent.
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