CN104130980B - The one anti-medroxyprogesterone monoclonal antibody specific hybridoma cell strain of strain and application thereof - Google Patents
The one anti-medroxyprogesterone monoclonal antibody specific hybridoma cell strain of strain and application thereof Download PDFInfo
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Abstract
The one anti-medroxyprogesterone monoclonal antibody specific hybridoma cell strain of strain and application thereof, belongs to food safety technical field of immunoassay. Monoclonal cell strain E hybridoma cell strain 4F9, does is preserving number? CGMCC? No.9305. Described cell strain can be secreted and produce the monoclonal antibody for medroxyprogesterone, has good specificity and higher sensitivity, to 50% inhibition concentration IC of medroxyprogesterone50For 0.067ng/mL, all it is less than 0.1% with the cross reacting rate of other analogues. The monoclonal antibody of application medroxyprogesterone can prepare the immunity detection reagent of medroxyprogesterone. The antibody that the cell strain utilizing the present invention to obtain produces may be used for multiple use, for the development of the quick health of the safety and livestock industry that ensure food provides strong testing tool and means.
Description
Technical field
The present invention relates to a strain of hybridoma strain and produce the application of anti-medroxyprogesterone monoclonal antibody specific, belong 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 also called medroxyprogestrone Acetate. Medical science aspect, is mainly used for dysmenorrhoea, functional amenorrhoea, habitual abortion etc., and heavy dose can be used as long-acting contraception pin. Livestock-raising aspect, is mainly used to promote growth of animal, but animal body can be caused harm by long-term a large amount of use. At present, a lot of country all regulation feeding animals prohibit the use hormone, China also puts into effect the use that corresponding policy forbids 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, but loaded down with trivial details pre-treatment step, and the high expense of detection, all cannot meet the demand that on-the-spot big batch detects fast. Comparing, enzyme immunoassay technology (ELISA) provides and a kind of detects quick, sensitive, the easy method of MP. In current MP immunologic detection method, it is important to monoclonal antibody to the high specific of MP and highly sensitive so that being difficult to obtain accurately objective result.
Summary of the invention
It is an object of the invention to provide an anti-medroxyprogesterone monoclonal antibody specific hybridoma cell strain of strain, MP is had good specificity and higher detection sensitivity by monoclonal antibody prepared by this cell strain, lays the foundation for setting up the immunological detection method of MP.
The technical scheme of the present invention, the one anti-medroxyprogesterone monoclonal antibody specific hybridoma cell strain of strain, No. E, called after monoclonal cell strain, also known as hybridoma cell strain 4F9, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.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 medroxyprogesterone50For 0.067ng/mL, all it is less than 0.1% with other analogue cross reacting rates.
The present invention provides the basic preparation process of 4F9 cell strain:
(1) preparation of medroxyprogesterone immunogen and qualification: medroxyprogesterone is raw material, is replaced by potassium hydroxide and dibromoacetic acid, obtains medroxyprogesterone haptens (MP-2CME), LC-MS qualification result. Utilize mixed anhydride method by pure to itself and carrier proteins Bovine albumen (BSA) or chicken serum albumin (OVA) coupling, use ultraviolet qualification result;
(2) the immune process of mouse: antigen is mixed in proportion with Freund's complete adjuvant, after complete emulsification, by back subcutaneous multi-point injection immunity 8-10 week BALB/c mouse, every dosage 100 �� L. Immunity process totally five times: first time is immune with Freund's complete adjuvant, booster immunization freund 's incomplete adjuvant, impacts immunity by normal saline dilution immunogen (containing adjuvant) for the last time; Serum titer and suppression is detected by indirect ELISA;
(3) cytogamy and cell strain are set up: impact immunity latter 3rd day, by polyoxyethylene glycol (PEG4000) method, mouse boosting cell and murine myeloma cell are merged, by HAT culture medium culturing, utilize the cell strain of indirect elisa method detection secretion MP, indirect competitive ELISA method is utilized to measure the inhibition of this cell strain, the positive cell strain that screening preferably suppresses carries out three subclones, final acquisition hybridoma cell strain 4F9;
(4) application of hybridoma cell strain: the monoclonal antibody obtained is configured in the immunoassay kit of detection MP, also this antibody is made the colloid gold immune test paper bar for detecting MP.
The anti-medroxyprogesterone monoclonal antibody specific that immunity obtains, the preparation process of the medroxyprogesterone haptens (MP-2CME) that immunity is 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, A liquid adds potassium hydroxide powder 1.5mmol, it is stirred to solution colour under room temperature condition and turns into micro-yellow, B liquid is dropwise joined in A liquid, yellow is deepened gradually, room temperature reaction 4h, by 50mL frozen water termination reaction, extraction into ethyl acetate three times, merge aqueous phase, aqueous phase 2mol/L hcl acidifying is obtained yellow mercury oxide, wash four times to neutral, yellow medroxyprogesterone haptens MP-2CME powder is obtained after dry.
The preparation process of the medroxyprogesterone complete antigen that immunity is used is as follows: takes medroxyprogesterone haptens 0.01mmol and is dissolved in the 600 anhydrous N of �� L, dinethylformamide DMF, it is placed in 4 DEG C of ice baths to stir, 0.025mmol tri-n-butylamine is added after solution precooling, condition of ice bath stirs 30min, add 0.025mmol isobutyl chlorocarbonate, continue ice bath and stir 1h, small molecule solution C liquid after must activating; Take carrier proteins BSA0.00016mmol to be dissolved in the carbonate buffer solution CBS of pH9.6, obtain protein solution; Under condition of ice bath, C liquid is dropwise added drop-wise in protein solution, reaction 8h; Reaction by solution centrifugal, is got supernatant, is obtained medroxyprogesterone complete antigen mixed solution after terminating.
Anti-medroxyprogesterone monoclonal antibody specific immunity obtained is applied to the immunity detection reagent 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 bag tunicle 5; Liner plate 1 is connected successively and sticks sample pad 2, pad 3, bag tunicle 5 and absorbent pad 4;
The assembling of colloid gold immune test paper bar: sample pad 1, pad 2, bag tunicle 5, absorbent pad 4 one sections are once connected and are attached on liner plate 1, can obtain detecting immunity colloidal gold test paper strip fast; On the T line of bag tunicle, embed MP-OVA with spray film instrument, C line embeds sheep anti-mouse igg, 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 enriched standard product diluent of 10 times is diluted: 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 �� g/mL medroxyprogesterone monoclonal antibody; 6) substrate solution A liquid: containing the 0.1MPBS 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) dilute the concentrated cleaning solution of 20 times: containing 0.5%Tween-20(v/v) 0.2MPBS; 10) the concentrated sample diluting 10 times redissolves liquid: 10%(v/v) 0.1MPBS of methyl alcohol.
Biomaterial preservation proves: No. E, a strain monoclonal cell strain, it is 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 CGMCCNo.9305.
The useful effect of the present invention: the present invention has successfully synthesized the artificial antigen that can produce specificity medroxyprogesterone antibody, 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 the secretion of specificity for the monoclonal antibody of medroxyprogesterone, there is good specificity and higher sensitivity. To medroxyprogesterone 50% inhibition concentration IC50For 0.067ng/mL, the cross reacting rate of other hormones is less than 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 has good using value.
Accompanying drawing explanation
Fig. 1, MP artificial antigen ultraviolet spectrogram;
Fig. 2, with indirect competitive ELISA method measure 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, bag tunicle.
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 screens cell conditioned medium, finally obtain the monoclonal antibody to medroxyprogesterone high specific and sensitivity.
Embodiment 1
(1) preparation of medroxyprogesterone haptens: medroxyprogesterone 34.4mg(0.1mmol) it is dissolved in 3mL anhydrous dimethyl sulfoxide, obtain A liquid, dibromoacetic acid 27.8mg(0.2mmol) it is dissolved in 1.4mL anhydrous dimethyl sulfoxide, obtain B liquid, A liquid adds potassium hydroxide powder 208.5mg(1.5mmol), it is stirred to solution colour under room temperature condition and turns into micro-yellow, B liquid is dropwise joined in A liquid, yellow is deepened gradually, room temperature reaction 4h, by 50mL frozen water termination reaction, extraction into ethyl acetate three times, merge aqueous phase, aqueous phase 2mol/L hcl acidifying is obtained yellow mercury oxide, wash four times to neutral, yellow medroxyprogesterone haptens (MP-2CME) powder is obtained after dry.
Product is identified with LC-MS after being dissolved in methyl alcohol.
(2) preparation of medroxyprogesterone complete antigen: take medroxyprogesterone haptens 4.02mg(0.01mmol, Mw=402) the 600 anhydrous N of �� L it are dissolved in, dinethylformamide (DMF), it is placed in 4 DEG C of ice baths to stir, 5.94 �� L(0.025mmol are added after solution precooling, Mw=185.35, ��=0.78) tri-n-butylamine, condition of ice bath stirs 30min, add 3.24 �� L(0.025mmol, Mw-136.6, ��=1.053) isobutyl chlorocarbonate, continue ice bath and stir 1h, small molecule solution C liquid after must activating. Take carrier proteins BSA10.6mg(0.00016mmol, Mw=64000) it is dissolved in the carbonate buffer solution (CBS) of pH9.6, obtain protein solution. Under condition of ice bath, C liquid is dropwise added drop-wise in protein solution, reaction 8h. Reaction by solution centrifugal, is got supernatant, is obtained medroxyprogesterone complete antigen mixed solution after terminating.
Cut and get dialysis tubing and be about 8.0cm, 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, pH7.4, change liquid every day period three times, dialysis takes out, after terminating, the aseptic centrifuge tube that complete antigen is divided in 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 the complete antigen of synthesis is coating antigen.
(3) medroxyprogesterone artificial antigen qualification: medroxyprogesterone complete antigen is identified by ultraviolet spectrophotometry. Principle utilizes differing molecular to have ultraviolet absorption peaks different separately, and in the process of coupling, according to spectrum superposition theorem, coupled product has the absorption peak of two kinds of coupling materials, thus compares and show that whether coupling is successful. MP artificial antigen ultraviolet spectrogram is as shown in Figure 1.
(4) animal immune: antigen is mixed with Freund's complete adjuvant ratio, after complete emulsification, by back subcutaneous multi-point injection immunity 8-10 week BALB/c mouse, every dosage 100 �� L. Immunity process totally five times: first time is immune with Freund's complete adjuvant, booster immunization freund 's incomplete adjuvant. The booster immunization timed interval is 21-30 days, the 5th immunity blood sampling in latter 10 days, it may also be useful to indirect ELISA and indirect competitive ELISA method measure mice serum and tire and suppress, select to suppress best mouse, within immune latter 30 days, impact immunity the last time, do not use adjuvant, abdominal injection.
(5) cytogamy: impact immunity after three days, conveniently PEG(polyoxyethylene glycol, Mw4000) method carries out cytogamy. Get mouse spleen grinding under aseptic condition to obtain splenocyte and count, count murine myeloma cell simultaneously. Splenocyte and myeloma cell are pressed 1:10 mixing, merges with 50%PEG. The cell HAT nutrient solution merged the most at last is added to 96 well culture plates, cultivates in incubator.
(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 6th day, carry out with containing 20% foetal calf serum, the RPMI-1640 nutrient solution of the 100 �� HT of 1% changes liquid entirely, got cell conditioned medium at the 9th day and carries out ELISA detection screening.
Utilize indirect ELISA method screening positive cell hole, to there is positive cell hole indirect competitive ELISA method detection inhibition, finally obtain that medroxyprogesterone is had good specificity and sensitivity cell hole, carry out subclone, detect by same method after within 7 days, cultivating. Cell strain 4F9 is obtained after three subclones and detection. Monoclonal antibody typical curve is measured 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 oncocyte after within the 7th day, start to collect ascites, 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 monoclonal antibody to the intersection situation of medroxyprogesterone and the analogue of medroxyprogesterone, and obtain the typical curve of this monoclonal antibody. Net result shows, only medroxyprogesterone is had suppression, IC50For 0.067ng/mL, the intersection of analogue is all less than 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); Testis ketone (testosterone); Remove methyltestosterone (nortestosterone); Dehydroepiandrosterone (dehydroepiandrosterone); Oestrone (oestrone); Dexamethasone (dexamethasone); Two fluorine compound (flumethasone); Betamethasone Valerate (betamethasone); Cortisone (cortisone); Estradiol (estradiol); Trihydroxy-oestrin (estriol); Ethinylestradiol (ethinyloestradiol).
IC50:50% inhibition concentration; CR(%): crossing-over rate.
Table 1
| Analogue | IC50 (ng/mL) | CR (%) |
| Medroxyprogesterone medroxyprogesterone | 0.063 | 100 |
| Progesterone progesterone | 47 | <0.1 |
| Methyltestosterone methyltestosterone | >100 | <0.1 |
| Testis ketone testosterone | >100 | <0.1 |
| Remove methyltestosterone nortestosterone | >100 | <0.1 |
| Dehydroepiandrosterone dehydroepiandrosterone | >100 | <0.1 |
| Oestrone oestrone | >100 | <0.1 |
| Dexamethasone dexamethasone | >100 | <0.1 |
| Two fluorine compound 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 obtained has higher specificity and sensitivity, it is possible to prepare the immunity detection reagent of medroxyprogesterone according to standard method.
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 enriched standard product diluent of 10 times is diluted: 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 �� g/mL medroxyprogesterone monoclonal antibody; 6) substrate solution A liquid: containing the 0.1MPBS 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) dilute the concentrated cleaning solution of 20 times: containing 0.5%Tween-20(v/v) 0.2MPBS; 10) the concentrated sample diluting 10 times redissolves liquid: 10%(v/v) 0.1MPBS of methyl alcohol.
Testing sample, through pre-treatment, removes albumen interference, joins 96 hole enzyme plate detections, according to OD value judged result after the multiple solution-treated of sample.
By the monoclonal antibody that obtains for the preparation of colloid gold immune test paper bar.
The assembling of test strip: sample pad 2, pad 3, bag tunicle 5, absorbent pad 4 one sections are once connected and are attached on liner plate 1, can obtain detecting immunity colloidal gold test paper strip fast. On the T line of bag tunicle, embed MP-OVA with spray film instrument, C line embeds sheep anti-mouse igg, 37 DEG C of dry for standby.
Testing sample solution is added drop-wise in the sample pad of test strip, according to the colour developing situation judged result of T line and C line after 5min. T line and C line develop the color explanation negative sample simultaneously, and the colour developing of T line, C line do not develop the color explanation positive.
Claims (2)
1. an anti-medroxyprogesterone monoclonal antibody specific hybridoma cell strain of strain, No. E, called after monoclonal cell strain, also known as hybridoma cell strain 4F9, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCCNo.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, it is characterised in that: prepare anti-medroxyprogesterone monoclonal antibody specific, the IC of medroxyprogesterone50For 0.067ng/mL, all it is less than 0.1% with other analogue cross reacting rates.
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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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| Title |
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| An enzyme-linked immunoassay for the detection of medroxyprogesterone acetate in intestines based on monoclonal antibody;Hongli Qi,et al;《Food and Agricultural Immunology》;20110317;第22卷(第2期);全文 * |
| 检测鸡肉中甲羟孕酮酶联免疫试剂盒的研制;陈飞等;《中国畜牧兽医》;20131220;第40卷(第12期);全文 * |
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