CN110885793B - Hybridoma cell strain secreting anti-cotinine monoclonal antibody, anti-cotinine monoclonal antibody and application of anti-cotinine monoclonal antibody - Google Patents
Hybridoma cell strain secreting anti-cotinine monoclonal antibody, anti-cotinine monoclonal antibody and application of anti-cotinine monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain secreting an anti-cotinine monoclonal antibody, an anti-cotinine monoclonal antibody and application thereof. The hybridoma cell strain is classified and named as COT mouse monoclonal antibody 9E-8D cell strain, and is preserved in China Center for Type Culture Collection (CCTCC for short) in 11 months and 15 days in 2019, the preservation address is Wuhan university in Wuhan, China, and the preservation number is CCTCC NO: C2019274. The monoclonal antibody secreted by the hybridoma cell strain COT-9E-8D can be specifically combined with cotinine, has high antibody titer, and has good combination specificity and detection sensitivity on cotinine. The anti-cotinine monoclonal antibody has 50% inhibition concentration IC of cotinine50Can reach 40ng/ml, and is superior to available monoclonal antibody resisting cotinine.
Description
Technical Field
The invention belongs to the technical field of cotinine detection, and particularly relates to a hybridoma cell strain secreting a cotinine-resistant monoclonal antibody, a cotinine-resistant monoclonal antibody and application of the cotinine-resistant monoclonal antibody.
Background
Human exposure to environmental tobacco smoke (EST) poses a potential threat to human health, and this potential threat, as well as the level of human exposure to EST, is of increasing concern. Tobacco contains thousands of chemical species, of which about 60 have been identified or suspected to be carcinogenic. These carcinogens not only affect the health of smokers, but also present a significant threat to the health of passive smokers. Monitoring the tobacco content in the population allows direct insight into the level of human exposure in the EST.
Among the many chemical substances contained in tobacco, nicotine is the main addictive substance. Nicotine entering the human body is absorbed through the oral cavity, the throat, the trachea and the alveoli and enters the blood circulation, wherein about 80 percent of the nicotine is firstly metabolized into cotinine in the liver by cytochrome oxidase P450-2A6(CYP450-2A6) and then is further metabolized in the liver, and metabolites are then discharged through urine and the like. Cotinine accounts for 70-80% of all metabolites, and at present, cotinine becomes the most effective biomarker for evaluating the exposure degree of smoke, smoking amount and the influence degree of smoking on human health of a subject.
The cotinine detection methods reported in the literature at present include gas chromatography, liquid chromatography, high performance liquid chromatography and high performance liquid chromatography-mass spectrometry. However, the pretreatment steps of these instrumental analysis methods are very complicated, time-consuming, low-throughput and costly.
The immunoassay is an analytical method established based on the principle of specific interaction of antigen and antibody, wherein an enzyme-linked immunosorbent assay (ELISA) has the advantages of safety, high flux, high sensitivity, low cost and the like, and is easy to detect a large number of samples. However, in order to establish any immunological detection technology, an antibody capable of specifically binding to a substance to be detected (e.g., cotinine) must be obtained, and the antibodies prepared against cotinine at present have the problems of low antibody titer, low detection sensitivity and the like.
Disclosure of Invention
The invention aims to provide a hybridoma cell strain secreting an anti-cotinine monoclonal antibody, an anti-cotinine monoclonal antibody and application thereof.
In order to achieve the above purpose, the technical solution of the present application is as follows:
the hybridoma cell strain secreting the anti-cotinine monoclonal antibody is classified and named as a COT mouse monoclonal antibody 9E-8D cell strain (hereinafter referred to as a hybridoma cell strain COT-9E-8D), and is preserved in China Center for Type Culture Collection (CCTCC) in 11 months and 15 days in 2019, the preservation address is Wuhan university in Wuhan, China, and the preservation number is CCTCC NO: C2019274.
Based on the above, the invention also provides an anti-cotinine monoclonal antibody, which is obtained by secreting the anti-cotinine monoclonal antibody hybridoma cell strain or a passage cell strain thereof.
The monoclonal antibody secreted by the hybridoma cell strain COT-9E-8D can be specifically combined with cotinine, and the antibody titer reaches 1 x 106For cotinine utensilHas good binding specificity and detection sensitivity, and the anti-cotinine monoclonal antibody has 50 percent inhibition concentration IC of cotinine50Can reach 40ng/ml, the detection limit in qualitative detection can reach at least 100ng/ml, and is superior to the prior anti-cotinine monoclonal antibody.
Based on the above, the invention also provides the application of the hybridoma cell strain secreting the anti-cotinine monoclonal antibody or the anti-cotinine monoclonal antibody in detection of cotinine.
Specifically, the invention provides an application of the hybridoma cell strain secreting the anti-cotinine monoclonal antibody or the anti-cotinine monoclonal antibody in preparation of a cotinine detection kit, and an application of the hybridoma cell strain secreting the anti-cotinine monoclonal antibody or the anti-cotinine monoclonal antibody in preparation of a cotinine detection reagent strip.
Further, the invention also provides a cotinine detection kit, and the cotinine detection kit contains the anti-cotinine monoclonal antibody.
Preferably, the cotinine detection kit is a competitive enzyme-linked immunosorbent assay kit. The competitive enzyme linked immunosorbent assay kit can be used for quantitatively detecting cotinine, IC, in a sample to be detected50Can reach 40 ng/ml.
The invention also provides a cotinine detection test strip which comprises a sample pad, a marker pad, a nitrocellulose membrane and a sample sucking pad, wherein the marker pad is coated with the anti-cotinine monoclonal antibody marked by colloidal gold or enzyme.
The cotinine detection test strip can be used for qualitatively detecting cotinine in a sample to be detected, the detection limit can at least reach 100ng/ml, and the detection sensitivity is high.
In the cotinine detection test strip, the detection line of the nitrocellulose membrane is coated with a cotinine bovine serum albumin conjugate, and the quality control line of the nitrocellulose membrane is coated with a secondary antibody specifically bound with the colloidal gold-labeled or enzyme-labeled anti-cotinine monoclonal antibody.
Compared with the prior art, the invention has the beneficial effects that:
the monoclonal antibody secreted by the hybridoma cell strain COT-9E-8D can be specifically combined with cotinine, and the antibody titer reaches 1 x 106Has good binding specificity and detection sensitivity to cotinine, and the anti-cotinine monoclonal antibody has 50 percent of inhibitory concentration IC to cotinine50Can reach 40ng/ml, the detection limit in qualitative detection can reach at least 100ng/ml, and is superior to the prior anti-cotinine monoclonal antibody.
Drawings
FIG. 1 is a graph showing the potency assay for ascites fluid, an anti-cotinine monoclonal antibody of the present invention;
FIG. 2 is a graph showing a standard curve of the anti-cotinine monoclonal antibody of the present invention for cotinine detection;
fig. 3 is a schematic structural diagram of the cotinine detection test strip of the present invention.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the accompanying drawings and the detailed description.
Cotinine complete artificial antigens (cotinine BSA conjugate, referred to as BSA-COT; cotinine OVA conjugate, referred to as OVA-COT) used in the examples of the present invention were purchased from Guangzhou Youkang Multi-Biotechnology, Inc.
EXAMPLE 1 construction of hybridoma cell lines
1. Animal immunization
The antigen and Freund's immunologic adjuvant are mixed uniformly and then injected into the back through subcutaneous multiple points. The primary basic immunization is emulsified by mixing complete Freund's adjuvant and BSA-COT in equal volume, and the immunization dose of each mouse is 50 mu g of antigen; every three weeks, the mice were boosted with Freund's incomplete adjuvant, 25. mu.g of antigen per mouse; on day 7 after 3 immunizations, COT serum titer and inhibition rate were determined by taking blood from the orbital wells of the mice; one mouse with highest potency and best inhibition rate is selected, abdominal cavity impact immunization is carried out by BSA-COT antigen, and cell fusion is carried out after 3 days.
2. Cell fusion:
(1) tumor cell culture: myeloma cells were subcultured 2 days before fusion.1 day before the fusion, the fresh medium was replaced and the cell density was adjusted so that the number of cells at the time of fusion reached 2X 107And (4) respectively.
(2) Obtaining splenocytes: collecting blood of a mouse after 3 days of impact immunization, picking up eyeballs, and separating serum to serve as a positive control; after the neck is pulled and killed, the mouse is placed in 75% alcohol to be soaked for 5min, and the mouse is dissected in an ultra-clean bench to obtain a complete spleen, so that the splenocytes of the mouse are further obtained.
(3) And (3) fusion process: mixing the mouse spleen cells and myeloma cells (SP2/0) at a ratio of 10:1 in a centrifuge tube, centrifuging at 1000r/min for 10min, pouring out the supernatant culture medium, and tapping the bottom to disperse the cells; placing the centrifuge tube in 37 deg.C water bath, adding 1ml PEG dropwise within 1min, shaking while adding, and continuing to shake for 1.5min after adding; then slowly adding 10ml of serum-free culture medium to stop the PEG effect, standing at 37 ℃ for 5min after the addition is finished, and then centrifuging and washing again to remove the residual PEG; and the pelleted cells were broken up with 200ml HAT medium containing 20% fetal bovine serum, and plated onto 10 plates at 200. mu.l per well.
3. Cell screening and cell line establishment
(1) Half HAT exchange was performed on days 5 and 8 after completion of cell fusion, and fused cells were detected when the bottom of the cell plate was full of 1/10.
(2) Limiting dilution cloning method: the subcloning of the cells in the culture wells of good quality by limiting dilution is necessary to obtain a uniform population of single cells and to obtain hybridoma cells with stable genetic preservation. Early hybridoma cells are easy to lose positivity due to loss of chromosomes, and cell strains which can ensure stable antibody secretion for a long time can be screened only through continuous cloning. The specific experimental method comprises the following steps: cells in the wells of the plate were blown out several times using a pipette and counted, and 3 gradients of 100, 10, 1 cells per well were selected for primary cloning. If the hybridoma cells have poor growth ability, clones may be obtained at 100 cells/well; from day 4, the cell population number in the corresponding well was observed every day and counted, and 1-2 wells of monoclonal cells were picked up 7 days later for supernatant titer detection. If less clonal well antibodies are negative, polyclonal positive wells can be selected for recloning. And cloning for 3-4 times to obtain the cotinine-resistant monoclonal antibody hybridoma cell strain COT-9E-8D.
4. Preparation of monoclonal antibodies
Taking several adult female 10-week mice, injecting incomplete adjuvant 0.5ml into abdominal cavity, and inoculating 10 times per mouse after 1 week6And (3) disinfecting the abdomen with alcohol after the abdominal cavity becomes large, extracting about 5-10ml of ascites, and extracting again every 2 days until the mouse dies. The ascites is centrifuged for 10min at 8000r/min, the supernatant is taken and stored for a short period at 4 ℃. Mixing the ascites supernatant with 4 times volume of acetic acid solution (pH 4.0, 0.06M), and adjusting pH to 4.5 with 0.1M sodium hydroxide; adding caprylic acid at room temperature while stirring, stirring for 30min after adding, standing at 4 deg.C for 2h, centrifuging at 8000rpm at low temperature for 30min to remove precipitate, and filtering to remove white floating substance in supernatant; 1/10 volumes of 10 XPBS, adjusted to pH 7.4 with 0.1M sodium hydroxide, were added to the supernatant; precooling the solution at 4 ℃, adding ammonium sulfate powder at 4 ℃ while stirring, and standing at 4 ℃ for 1 h; centrifuging at low temperature (5000rpm, 15min) to obtain precipitate IgG, adding appropriate amount of 1 × PBS, and dialyzing for 2 days; after completion of dialysis, the dialysate was removed, centrifuged at 4000rpm for 15min and then the concentration was determined by BCA. Adding 0.1% sodium azide antiseptic into the supernatant, subpackaging, and storing at 4 deg.C for several months; or adding 50% glycerol, packaging into small tubes, and storing at-20 deg.C.
The results of the antibody titer test are shown in FIG. 1. As can be seen from FIG. 1, the anti-tartrazine monoclonal antibody of the present invention has a potency of 1X 106。
Example 2 anti-cotinine monoclonal antibody for ELISA quantitative detection of cotinine
1. Selection of optimal working antigen-antibody concentration
(1) Coating: cotinine-coatingen (OVA-COT) was diluted with carbonate coating buffer pH 9.6 to obtain 4 concentration gradients: 0.5, 0.25, 0.125 and 0.0625 μ g/ml; the antigen dilution was added to the microplate at 100. mu.l/well and coated at 37 ℃ for 2 h.
(2) Washing: pouring out the antigen diluent in the ELISA plate, patting dry on absorbent paper, adding 200 μ l of PBST cleaning solution, shaking left and right for 30s, pouring out the cleaning solution, patting dry on absorbent paper, and repeatedly washing for 3 times.
(3) And (3) sealing: adding 200 mu l of ELISA blocking solution into an ELISA plate to reduce non-specific reaction, placing the ELISA plate at 37 ℃ for blocking for 1h, then drying and washing for 1 time.
(4) And (3) competitive reaction: the standard was diluted to 100, 0ng/ml with PBS, 50. mu.l was added to each well of the microplate, and 50. mu.l/well of the purified antibody diluted with blocking solution was immediately added, the antibody having the following 4 concentrations of 1/1000,/5000, 1/25000 and 1/125000, and incubated at 37 ℃ for 0.5 h.
(5) Washing: the plate was washed 3 times according to the same washing procedure as in step (2).
(6) Secondary antibody reaction: mu.l of 5000-fold diluted goat anti-mouse IgG (secondary antibody) labeled with horseradish peroxidase (HRP) was added to each well and incubated at 37 ℃ for 0.5 h.
(7) Washing: the plate was washed 4 times.
(8) Color development: 100 μ l of the ELISA developing solution prepared in situ was added, and the reaction was carried out for 20min under dark conditions.
(9) And (4) terminating: the chromogenic reaction was stopped by adding 50. mu.l of ELISA stop solution to each well.
(10) Reading a plate: and detecting the OD value by using an enzyme-labeling instrument, wherein the detection wavelength is 450nm, and the concentration of the antigen and the antibody on the enzyme-labeled hole with the highest selective inhibition rate and the OD value close to 1.0 is the working concentration of the subsequent test.
2. Establishment of a Standard Curve
(1) Coating: cotinine-coated antigen (OVA-COT) was diluted to 0.125. mu.g/ml with carbonate coating buffer pH 9.6, added to the microplate at 100. mu.l/well, and coated at 37 ℃ for 2 h.
(2) Washing: pouring out the coating antigen in the ELISA plate, patting dry on absorbent paper, filling 200 μ l of washing solution into the hole, shaking left and right for 30s, pouring out the washing solution, patting dry on the absorbent paper, and washing repeatedly for 3 times.
(3) And (3) sealing: to reduce non-specific reactions, 200. mu.l of ELISA blocking solution was added to the microplate, blocked at 37 ℃ for 1 hour, spun-dried and washed 1 time.
(4) And (3) competitive reaction: COT standard was diluted to 8 concentration gradients of 100, 50, 25, 12.5, 6.125, 3.0625, 1.53025 and 0ng/ml with PBS, 50. mu.l of each well was added sequentially to the microplate, and then 50. mu.l/well of purified antibody 1/25000 diluted with antibody diluent (5% nonfat dry milk and 0.1% gelatin) was added immediately, and incubated at 37 ℃ for 0.5 h.
(5) Washing: the plate was washed 3 times.
(6) Secondary antibody reaction: mu.l of horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (secondary antibody) was diluted 1:5000 in dilution buffer per well and incubated at 37 ℃ for 0.5 h.
(7) Washing: the plate was washed 4 times.
(8) Color development: 100 μ l of the ELISA developing solution prepared in situ was added, and the reaction was carried out for 20min under dark conditions.
(9) And (4) terminating: mu.l of ELISA stop solution was added to each well to stop the color reaction.
(10) Reading a plate: detecting OD value with enzyme labeling instrument, measuring wavelength at 450nm, drawing standard curve, and calculating IC50。
IC obtained by calculation50The value was 40ng/ml and the standard curve obtained is shown in FIG. 2.
When a sample to be detected with unknown concentration is detected, the same steps are only needed to be carried out for testing, the OD value is obtained, and then the cotinine concentration in the sample to be detected is calculated by taking the obtained standard curve as a reference.
Example 3 preparation of a test strip for cotinine assay
(1) Preparation of colloidal gold-labeled antibody
Taking 1ml of colloidal gold solution (gold particle diameter 30-40nm), adding 6 μ l of 0.2M K2CO3The solution is mixed evenly after the pH value is adjusted to about 7; adding 10 mu g of anti-cotinine monoclonal antibody into the colloidal gold solution, and uniformly mixing for 30min at room temperature by using a mixer; continuously adding 25 mu L of 10% BSA into the colloidal gold solution for sealing, and uniformly mixing for 30min at room temperature by using a mixing machine to obtain a gold-labeled solution; centrifuging the gold-labeled solution at 4 ℃ and 10000rpm for 40 min; carefully remove the supernatant by gun suctionWhen the gold deposit is near the bottom, the 200 mu L of gun head is changed to suck the gold deposit without sucking the gold deposit; adding 200 μ L colloidal gold redissolution into the gold precipitate, mixing well and dissolving (marked as OD10), then diluting colloidal gold-labeled cotinine antibody (OD10) to OD1.5 with the colloidal gold redissolution, spraying onto colloidal gold pad, and drying for use.
(2) Preparation of solid phase antigen and antibody in NC membrane (nitrocellulose membrane)
Cotinine antigen (BSA-COT) was diluted to 1mg/ml with a draw-coating solution, and secondary antibody was diluted to 0.1 mg/ml. Coating the two on NC film with a film-cutting machine, and drying for later use.
(3) Assembly of colloidal gold test paper
Colloidal gold test paper includes that bottom plate, sample fill up, colloidal gold pad, NC membrane and inhale a kind pad, and its assembled mode includes: drying the NC film in a room with the humidity below 30%, taking out the NC film, cutting off the part without scratching the film, uncovering a part of adhesive paper at the middle lower part of the bottom plate, taking out the colloidal gold pad, cutting off the uneven parts at the front end and the rear end, adhering the colloidal gold pad to the lower end of the NC film by using tweezers, and pressing the lower end of the NC film by keeping 1mm of the colloidal gold pad on the NC film; aligning the lower end of a sample pad (30mm x 17mm) with the lower end of the bottom plate, and pressing the upper end of the sample pad (1 mm) on the colloidal gold pad; adhering the lower end of the sample sucking pad to the upper end of the NC membrane, and keeping 1-2mm to press the sample sucking pad on the NC membrane; cover the sticky paper, press the flat part of the tweezers lightly, and cut off the excess sample pad and sticky paper with scissors. Test strips 4.5mm wide were cut using a slitter.
The structure of the obtained test strip is shown in fig. 3.
(4) Sample detection
Cotinine is prepared into samples to be detected with the concentrations of 100, 50 and 0ng/ml by using PBS solution, 100 mul of cotinine is respectively dripped on a sample pad of a detection test strip, and the samples to be detected with the cotinine concentration of 100ng/ml only have C line color development, while the samples to be detected with the cotinine concentrations of 50 and 0ng/ml have C line and T line color development, which shows that when the anti-cotinine monoclonal antibody used in the embodiment is used for qualitatively detecting cotinine, the detection limit can reach 100ng/ml, and the detection limit is superior to all cotinine colloidal gold products on the market.
Claims (9)
1. A hybridoma cell strain secreting an anti-cotinine monoclonal antibody is characterized by being named as a COT mouse monoclonal antibody 9E-8D cell strain in a classified manner, and the preservation number is CCTCC NO: C2019274.
2. The use of the hybridoma cell line secreting an anti-cotinine monoclonal antibody according to claim 1 for preparing a cotinine detection kit.
3. The use of the hybridoma cell line secreting an anti-cotinine monoclonal antibody according to claim 1 for preparing a cotinine detection reagent strip.
4. An anti-cotinine monoclonal antibody secreted by the hybridoma cell line or a passaged cell line secreting an anti-cotinine monoclonal antibody of claim 1.
5. The use of the anti-cotinine monoclonal antibody of claim 4 in the preparation of a cotinine detection kit or a cotinine detection reagent strip.
6. A cotinine detection kit comprising the anti-cotinine monoclonal antibody according to claim 4.
7. The cotinine detection kit of claim 6, being a competitive enzyme linked immunosorbent assay kit.
8. A cotinine detection test strip, which comprises a sample pad, a marker pad, a nitrocellulose membrane and a sample sucking pad, and is characterized in that the marker pad is coated with a colloidal gold label or an enzyme label of the anti-cotinine monoclonal antibody of claim 4.
9. The cotinine test strip of claim 8, wherein the detection line of the nitrocellulose membrane is coated with a cotinine bovine serum albumin conjugate, and the quality control line of the nitrocellulose membrane is coated with a secondary antibody that specifically binds to the colloidal gold-labeled or enzyme-labeled anti-cotinine monoclonal antibody.
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Address after: Room 309-313, floor 3, building 2, building 1, 1180 Bin'an Road, Binjiang District, Hangzhou City, Zhejiang Province Applicant after: Hangzhou Qianyuan Enshi Gene Technology Co., Ltd Address before: Room 309-313, floor 3, building 2, building 1, 1180 Bin'an Road, Binjiang District, Hangzhou City, Zhejiang Province Applicant before: HANGZHOU ENS GENE TECHNOLOGY DEVELOPMENT Co.,Ltd. |
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| GR01 | Patent grant | ||
| GR01 | Patent grant |