CN104130980A - Anti medroxyprogesterone specific monoclonal antibody hybridoma cell strain and application thereof - Google Patents
Anti medroxyprogesterone specific monoclonal antibody hybridoma cell strain and application thereof Download PDFInfo
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Abstract
The invention discloses an anti medroxyprogesterone specific monoclonal antibody hybridoma cell strain and application thereof, and belongs to the technical field of food safety immune detection. The preservation number of monoclonal cell line E# hybridoma cell strain 4F9 is CGMCC No.9305. The cell strain can secrete and produce monoclonal antibodies against medroxyprogesterone, has good specificity and high sensitivity, the medroxyprogesterone 50% inhibitory concentration IC50 is 0.067ng / mL, and the cross reaction rate with other analogues is less than 0.1%. An immune detection kit can be prepared by use of the monoclonal antibodies against the medroxyprogesterone. The antibodies produced by use of the cell strain can be used for multiple purposes, and can provide powerful testing tools and means for guaranteeing the rapid and healthy development of animal husbandry and food safety.
Description
Technical field
The application that the present invention relates to a strain of hybridoma strain and produce anti-medroxyprogesterone monoclonal antibody specific, belongs to food safety technical field of immunoassay.
Background technology
Medroxyprogesterone (Medroxyprogesterone, MP) belongs to progestogen, and effect is similar with natural progesterone, is the Progesterone derivative of synthetic, is therefore called again medroxyprogestrone Acetate.Medical science aspect, is mainly used for dysmenorrhoea, functional amenorrhea, habitual abortion etc., and heavy dose can be used as long-acting Contraceptive.Livestock-raising aspect, is mainly used to promote growth of animal, but long-term a large amount of use meeting works the mischief to animal body.At present, a lot of countries all regulation feeding animals ban use of hormone, and China also puts into effect the use that corresponding policy is forbidden hormones.Existing detection MP method comprises high performance liquid chromatography (HPLC), LC-MS (LC-MS), gas chromatography mass spectrometry (GC-MS) etc.Although these methods are highly sensitive, loaded down with trivial details pre-treatment step, the high cost of detection, all cannot meet the on-the-spot demand of rapid detection in enormous quantities.Compare, enzyme immunoassay technology (ELISA) provides a kind of quick, sensitive, the easy method of MP of detection.In current MP immunologic detection method, key is high specific and the highly sensitive of monoclonal antibody to MP, so that be difficult to obtain accurately objective result.
Summary of the invention
The object of this invention is to provide the anti-medroxyprogesterone monoclonal antibody specific of strain hybridoma cell strain, monoclonal antibody prepared by this cell strain has good specificity and higher detection sensitivity to MP, for the immunological detection method of setting up MP lays the foundation.
Technical scheme of the present invention, the anti-medroxyprogesterone monoclonal antibody specific of one strain hybridoma cell strain, No. E, called after monoclonal cell strain, claim again hybridoma cell strain 4F9, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.9305, and preservation date is on May 28th, 2014.
The application of described anti-medroxyprogesterone monoclonal antibody specific hybridoma cell strain, the anti-medroxyprogesterone monoclonal antibody specific that immunity obtains, 50% inhibition concentration IC of medroxyprogesterone
50for 0.067ng/mL, be all less than 0.1% with other analogue cross reacting rates.
The invention provides the basic preparation process of 4F9 cell strain:
(1) the immunogenic preparation of medroxyprogesterone and qualification: medroxyprogesterone is raw material, replaces by potassium hydroxide and dibromoacetic acid, obtains medroxyprogesterone haptens (MP-2CME), LC-MS qualification result.Utilize mixed anhydride method by itself and carrier proteins bovine serum albumin (BSA) or chicken serum albumin (OVA) coupling, use ultraviolet qualification result;
(2) immunologic process of mouse: antigen is mixed in proportion with Freund's complete adjuvant, completely after emulsification, by the subcutaneous multi-point injection immunity in back 8-10 week BALB/c mouse, every dosage 100 μ L.Immunologic process totally five times: use for the first time Freund's complete adjuvant immunity, booster immunization freund 's incomplete adjuvant, uses normal saline dilution immunogen (not containing adjuvant) to impact immunity for the last time; Detect serum titer and inhibition by indirect ELISA;
(3) cytogamy and cell strain are set up: impact immunity latter the 3rd day, by polyoxyethylene glycol (PEG4000) method, mouse boosting cell and murine myeloma cell are merged, by HAT culture medium culturing, utilize indirect elisa method to detect the cell strain of secretion MP, utilize indirect competitive ELISA method to measure the inhibition of this cell strain, subclone is carried out in the positive cell strain that screening preferably suppresses three times, finally obtains hybridoma cell strain 4F9;
(4) application of hybridoma cell strain: obtained monoclonal antibody is configured in the immunoassay kit that detects MP, also this antibody is made to the colloid gold immune test paper bar for detection of MP.
The anti-medroxyprogesterone monoclonal antibody specific that immunity obtains, the preparation process of immune medroxyprogesterone haptens (MP-2CME) used is as follows: it is A liquid that medroxyprogesterone 0.1mmol is dissolved in 3mL anhydrous dimethyl sulfoxide, it is B liquid that dibromoacetic acid 0.2mmol is dissolved in 1.4mL anhydrous dimethyl sulfoxide, in A liquid, add potassium hydroxide powder 1.5mmol, under room temperature condition, be stirred to solution colour and become micro-yellow, B liquid is dropwise joined in A liquid, yellow is deepened gradually, room temperature reaction 4h, by 50mL frozen water termination reaction, ethyl acetate extraction three times, merge water, water is obtained to yellow mercury oxide with 2mol/L hcl acidifying, wash four times to neutral, after dry, obtain yellow medroxyprogesterone haptens MP-2CME powder.
The preparation process of immune medroxyprogesterone complete antigen used is as follows: take medroxyprogesterone haptens 0.01mmol and be dissolved in the anhydrous N of 600 μ L, dinethylformamide DMF, being placed in 4 DEG C of ice baths stirs, after solution precooling, add 0.025mmol tri-n-butylamine, condition of ice bath stirs 30min, add 0.025mmol isobutyl chlorocarbonate, continue ice bath and stir 1h, small molecules solution C liquid after must activating; Take in the carbonate buffer solution CBS that carrier proteins BSA 0.00016mmol is dissolved in pH 9.6, obtain protein solution; Under condition of ice bath, C liquid is dropwise added drop-wise in protein solution to reaction 8h; After reaction finishes, by solution centrifugal, get supernatant, obtain medroxyprogesterone complete antigen mixed solution.
The anti-medroxyprogesterone monoclonal antibody specific that immunity is obtained is applied to the immunity detection reagent of preparing medroxyprogesterone.
Described test kit comprises colloid gold immune test paper bar and reagent;
Colloid gold immune test paper bar: liner plate 1, sample pad 2, pad 3, absorbent pad 4 and coated film 5; On liner plate 1, be connected and stick sample pad 2, pad 3, coated film 5 and absorbent pad 4 successively;
The assembling of colloid gold immune test paper bar: 4 one sections of sample pad 1, pad 2, coated film 5, absorbent pad are once connected and are attached on liner plate 1, can obtain rapid detection immunity colloidal gold test paper strip; With spray film instrument embedding MP-OVA on the T of coated film line, embedding sheep anti-mouse igg on C line, 37 DEG C of dry for standby;
Reagent: 1) 96 orifice plates of pre-coated medroxyprogesterone coupling OVA coating antigen; 2) medroxyprogesterone high density standard substance: 2ppb; 3) the concentrated standard substance diluent of 10 times of use of dilution: 0.1M phosphate buffered saline buffer; 4) ELIAS secondary antibody: the sheep anti-mouse igg of 3000 times of dilution HRP marks; 5) antibody working fluid: 0.2 μ g/mL medroxyprogesterone monoclonal antibody; 6) substrate solution A liquid: containing the 0.1M PBS of 1mM hydrogen peroxide and 0.2M citric acid; 7) substrate solution B liquid: containing 0.06%(v/v) 3,3', the ethylene glycol of 5,5'-tetramethyl benzidine (TMB); 8) stop buffer: 2M sulfuric acid; 9) concentrated cleaning solution of 20 times of use of dilution: containing 0.5% Tween-20(v/v) 0.2M PBS; 10) concentrating sample of 10 times of use of dilution redissolves liquid: 10%(v/v) the 0.1M PBS of methyl alcohol.
Biomaterial preservation proves: No. E, a strain monoclonal cell strain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preservation date is on May 28th, 2014, deposit number CGMCC No.9305.
Beneficial effect of the present invention: the present invention has successfully synthesized the artificial antigen that can produce specificity medroxyprogesterone antibody, and building-up process is simple, lays the foundation for obtaining medroxyprogesterone cell strain of monoclonal antibody;
The cell strain that the present invention obtains can specific secretion for the monoclonal antibody of medroxyprogesterone, there is good specificity and higher sensitivity.To medroxyprogesterone 50% inhibition concentration IC
50for 0.067ng/mL, the cross reacting rate of other hormones is less than to 0.1%.
The antibody that the cell strain that the present invention obtains produces can be applied to the exploitation of test kit and test strip, and actual production is had to good using value.
Brief description of the drawings
Fig. 1, MP artificial antigen ultraviolet spectrogram;
Fig. 2, use indirect competitive ELISA method are measured monoclonal antibody typical curve;
Fig. 3, immunity colloidal gold test paper strip structure iron;
A, test strip vertical view; B, test strip side-view;
1, plastics lining board; 2, sample pad; 3, pad; 4, absorption pad; 5, coated film.
Embodiment
The present invention connects BSA as immunogen by derivative transformation medroxyprogesterone, immune mouse obtains qualified positive splenocyte, and by cytogamy, HAT selects to cultivate, and ELISA screening cell conditioned medium, finally obtain the monoclonal antibody to medroxyprogesterone high specific and sensitivity.
Embodiment 1
(1) the haptenic preparation of medroxyprogesterone: medroxyprogesterone 34.4mg(0.1mmol) be dissolved in 3mL anhydrous dimethyl sulfoxide, obtain A liquid, dibromoacetic acid 27.8mg(0.2mmol) be dissolved in 1.4mL anhydrous dimethyl sulfoxide, obtain B liquid, in A liquid, add potassium hydroxide powder 208.5mg(1.5mmol), under room temperature condition, be stirred to solution colour and become micro-yellow, B liquid is dropwise joined in A liquid, yellow is deepened gradually, room temperature reaction 4h, by 50mL frozen water termination reaction, ethyl acetate extraction three times, merge water, water is obtained to yellow mercury oxide with 2mol/L hcl acidifying, wash four times to neutral, after dry, obtain yellow medroxyprogesterone haptens (MP-2CME) powder.
Product is dissolved in after methyl alcohol and identifies with LC-MS.
(2) preparation of medroxyprogesterone complete antigen: take medroxyprogesterone haptens 4.02mg(0.01mmol, Mw=402) be dissolved in the anhydrous N of 600 μ L, dinethylformamide (DMF), being placed in 4 DEG C of ice baths stirs, after solution precooling, add 5.94 μ L(0.025mmol, Mw=185.35, ρ=0.78) tri-n-butylamine, condition of ice bath stirs 30min, add 3.24 μ L(0.025mmol, Mw-136.6, ρ=1.053) isobutyl chlorocarbonate, continue ice bath and stir 1h, small molecules solution C liquid after must activating.Take carrier proteins BSA 10.6mg(0.00016mmol, Mw=64000) be dissolved in the carbonate buffer solution (CBS) of pH 9.6, obtain protein solution.Under condition of ice bath, C liquid is dropwise added drop-wise in protein solution to reaction 8h.After reaction finishes, by solution centrifugal, get supernatant, obtain medroxyprogesterone complete antigen mixed solution.
Clip dialysis tubing approximately 8.0 cm, boil about 10min, with deionized water rinsing repeatedly, medroxyprogesterone complete antigen is moved in dialysis tubing, dialyses three days with the phosphate buffered saline buffer (PBS) of 0.01M, pH 7.4, change during this time liquid every day three times, dialysis finishes rear taking-up complete antigen and is divided in the aseptic centrifuge tube of 1mL,-20 DEG C of preservations, for subsequent use, this complete antigen is as immunogen.Carrier proteins is changed to OVA, and method is constant, and synthetic complete antigen is coating antigen.
(3) medroxyprogesterone artificial antigen qualification: ultraviolet spectrophotometry is identified medroxyprogesterone complete antigen.Principle is to utilize differing molecular to have different separately ultraviolet absorption peaks, and in the process of coupling, according to spectrum superposition theorem, coupled product has the absorption peak of two kinds of coupling materials, relatively show that thus whether coupling is successful.MP artificial antigen ultraviolet spectrogram as shown in Figure 1.
(4) animal immune: antigen is mixed with Freund's complete adjuvant ratio, completely after emulsification, by the subcutaneous multi-point injection immunity in back 8-10 week BALB/c mouse, every dosage 100 μ L.Immunologic process totally five times: use for the first time Freund's complete adjuvant immunity, booster immunization freund 's incomplete adjuvant.The booster immunization timed interval is 21-30 days, and the 5th immunity blood sampling in latter 10 days is used indirect ELISA and indirect competitive ELISA method to measure mice serum and tires and suppress, select the mouse that suppresses best, within immune latter 30 days, impact immunity the last time, do not use adjuvant, abdominal injection.
(5) cytogamy: impact immunity after three days, according to conventional PEG(polyoxyethylene glycol, Mw4000) method carries out cytogamy.Under aseptic condition, get mouse spleen grinding and obtain splenocyte counting, count murine myeloma cell simultaneously.Splenocyte and myeloma cell are pressed to 1:10 and mix, merge with 50%PEG.The cell merging is the most at last added to 96 well culture plates with HAT nutrient solution, in incubator, cultivates.
(6) cell screening and cell strain are set up: within the 3rd day after cytogamy, carry out RPMI-1640 nutrient solution and partly change liquid, within the 6th day, use containing 20% foetal calf serum, the RPMI-1640 nutrient solution of 100 × HT of 1% changes liquid entirely, gets cell conditioned medium carry out ELISA detection screening at the 9th day.
Utilize indirect ELISA method screening positive cell hole, to there is positive cell hole indirect competitive ELISA method to detect inhibition, finally obtain that medroxyprogesterone is had to good specificity and sensitivity cell hole, carry out subclone, after within 7 days, cultivating, use the same method and detect.Three subclones and detect after obtain cell strain 4F9.Measure monoclonal antibody typical curve as shown in Figure 2 by indirect competitive ELISA method.
(7) preparation of monoclonal antibody and qualification: mouse peritoneal injection paraffin oil 1mL, every mouse peritoneal injection 1 × 106 hybridoma after 7-10 days, injection starts to collect ascites on the 7th day after oncocyte, after the centrifugal ascites of 5000RPM, by ascites, by caprylic acid-ammonium purifying, the monoclonal antibody of acquisition is placed in-20 DEG C of preservations.
Utilize indirect ELISA and indirect competitive ELISA method, measure the intersection situation of the analogue of monoclonal antibody to medroxyprogesterone and medroxyprogesterone, and obtain the typical curve of this monoclonal antibody.Net result shows only medroxyprogesterone is had to inhibition, IC
50for 0.067ng/mL, the intersection of analogue is all less than to 0.1%.Illustrate that this monoclonal antibody has very high sensitivity and specificity.
4F9 monoclonal antibody is as shown in table 1 to the cross reacting rate of analogue.
Intersection thing: progesterone (progesterone); Methyltestosterone (methyltestosterone); Testosterone (testosterone); Demethyl testosterone (nortestosterone); Dehydroepiandrosterone (dehydroepiandrosterone); Oestrone (oestrone); Dexamethasone (dexamethasone); Diflucortolone (flumethasone); Betamethasone Valerate (betamethasone); Cortisone (cortisone); Estradiol (estradiol); Trihydroxy-oestrin (estriol); Ethinylestradiol (ethinyloestradiol).
IC50:50% inhibition concentration; CR(%): crossing-over rate.
Table 1
| Analogue | IC 50 (ng/mL) | CR (%) |
| Medroxyprogesterone medroxyprogesterone | 0.063 | 100 |
| Progesterone progesterone | 47 | ?<0.1 |
| Methyltestosterone methyltestosterone | >100 | ?<0.1 |
| Testosterone testosterone | >100 | ?<0.1 |
| Demethyl testosterone nortestosterone | >100 | ?<0.1 |
| Dehydroepiandrosterone dehydroepiandrosterone | >100 | ?<0.1 |
| Oestrone oestrone | >100 | ?<0.1 |
| Dexamethasone dexamethasone | >100 | ?<0.1 |
| Diflucortolone flumethasone | >100 | ?<0.1 |
| Betamethasone Valerate betamethasone | >100 | ?<0.1 |
| Cortisone cortisone | >100 | ?<0.1 |
| Estradiol estradiol | >100 | ?<0.1 |
| Trihydroxy-oestrin estriol | >100 | ?<0.1 |
| Ethinylestradiol ethinyloestradiol | >100 | ?<0.1 |
(8) application of monoclonal antibody: because the MP monoclonal antibody obtaining has higher specificity and sensitivity, can prepare according to standard method the immunity detection reagent of medroxyprogesterone.
As shown in Figure 3, the composition of test kit: 1) 96 orifice plates of pre-coated medroxyprogesterone coupling OVA coating antigen; 2) medroxyprogesterone high density standard substance: 2ppb; 3) the concentrated standard substance diluent of 10 times of use of dilution: 0.1M phosphate buffered saline buffer; 4) ELIAS secondary antibody: the sheep anti-mouse igg of 3000 times of dilution HRP marks; 5) antibody working fluid: 0.2 μ g/mL medroxyprogesterone monoclonal antibody; 6) substrate solution A liquid: containing the 0.1M PBS of 1mM hydrogen peroxide and 0.2M citric acid; 7) substrate solution B liquid: containing 0.06%(v/v) 3,3', the ethylene glycol of 5,5'-tetramethyl benzidine (TMB); 8) stop buffer: 2M sulfuric acid; 9) concentrated cleaning solution of 20 times of use of dilution: containing 0.5% Tween-20(v/v) 0.2M PBS; 10) concentrating sample of 10 times of use of dilution redissolves liquid: 10%(v/v) the 0.1M PBS of methyl alcohol.
Testing sample, through pre-treatment, is removed albumen and is disturbed, and joins 96 hole enzyme plates and detects, according to OD value judged result after the multiple solution-treated of sample.
By the monoclonal antibody obtaining for the preparation of colloid gold immune test paper bar.
The assembling of test strip: 4 one sections of sample pad 2, pad 3, coated film 5, absorbent pad are once connected and are attached on liner plate 1, can obtain rapid detection immunity colloidal gold test paper strip.With spray film instrument embedding MP-OVA on the T of coated film line, embedding sheep anti-mouse igg on C line, 37 DEG C of dry for standby.
Testing sample solution is added drop-wise in the sample pad of test strip, after 5min according to the colour developing situation judged result of T line and C line.T line and the C line explanation negative sample that simultaneously develops the color, the colour developing of T line, the C line explanation positive that do not develop the color.
Claims (3)
1. the anti-medroxyprogesterone monoclonal antibody specific of strain hybridoma cell strain, No. E, called after monoclonal cell strain, claim again hybridoma cell strain 4F9, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.9305, and preservation date is on May 28th, 2014.
2. the application of anti-medroxyprogesterone monoclonal antibody specific hybridoma cell strain described in claim 1, is characterized in that: the anti-medroxyprogesterone monoclonal antibody specific that immunity obtains, 50% inhibition concentration IC of medroxyprogesterone
50for 0.067ng/mL, be all less than 0.1% with other analogue cross reacting rates.
3. the application of anti-medroxyprogesterone monoclonal antibody specific hybridoma cell strain according to claim 2, it is characterized in that the anti-medroxyprogesterone monoclonal antibody specific that immunity obtains, the immune haptenic preparation process of medroxyprogesterone used is as follows: it is A liquid that medroxyprogesterone 0.1mmol is dissolved in 3mL anhydrous dimethyl sulfoxide, it is B liquid that dibromoacetic acid 0.2mmol is dissolved in 1.4mL anhydrous dimethyl sulfoxide, in A liquid, add potassium hydroxide powder 1.5mmol, under room temperature condition, be stirred to solution colour and become micro-yellow, B liquid is dropwise joined in A liquid, yellow is deepened gradually, room temperature reaction 4h, by 50mL frozen water termination reaction, ethyl acetate extraction three times, merge water, water is obtained to yellow mercury oxide with 2mol/L hcl acidifying, wash four times to neutral, after dry, obtain yellow medroxyprogesterone haptens MP-2CME powder.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105087500A (en) * | 2015-09-07 | 2015-11-25 | 江南大学 | Ribavirin monoclonal antibody hybridoma cell strain and application thereof |
| CN114836387A (en) * | 2022-05-11 | 2022-08-02 | 江南大学 | 11-alpha hydroxyprogesterone monoclonal antibody hybridoma cell strain and application thereof |
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| CN201804011U (en) * | 2010-10-15 | 2011-04-20 | 无锡安迪生物工程有限公司 | Dual-channel detection card for simultaneously detecting estradiol and medroxyprogesterone |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN201804011U (en) * | 2010-10-15 | 2011-04-20 | 无锡安迪生物工程有限公司 | Dual-channel detection card for simultaneously detecting estradiol and medroxyprogesterone |
Non-Patent Citations (2)
| Title |
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| HONGLI QI,ET AL: "An enzyme-linked immunoassay for the detection of medroxyprogesterone acetate in intestines based on monoclonal antibody", 《FOOD AND AGRICULTURAL IMMUNOLOGY》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087500A (en) * | 2015-09-07 | 2015-11-25 | 江南大学 | Ribavirin monoclonal antibody hybridoma cell strain and application thereof |
| CN114836387A (en) * | 2022-05-11 | 2022-08-02 | 江南大学 | 11-alpha hydroxyprogesterone monoclonal antibody hybridoma cell strain and application thereof |
| CN114836387B (en) * | 2022-05-11 | 2023-08-04 | 江南大学 | 11-alpha hydroxyprogesterone monoclonal antibody hybridoma cell strain and application thereof |
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