CN101907624A - Immune affinity chromatographic column for rhodamine 6G and preparation method and application thereof - Google Patents
Immune affinity chromatographic column for rhodamine 6G and preparation method and application thereof Download PDFInfo
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- CN101907624A CN101907624A CN2010102400369A CN201010240036A CN101907624A CN 101907624 A CN101907624 A CN 101907624A CN 2010102400369 A CN2010102400369 A CN 2010102400369A CN 201010240036 A CN201010240036 A CN 201010240036A CN 101907624 A CN101907624 A CN 101907624A
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Abstract
The invention discloses an immune affinity chromatographic column for directly detecting rhodamine 6G. The immune affinity chromatographic column provided by the invention for extracting and detecting a forbidden rhodamine 6G additive in food comprises specific antibodies of the rhodamine 6G, specific antibodies Sepharose4B coupled with the rhodamine 6G, a plastic column filled with an affinity material, an alumina column, sampling buffer solution, leacheate and eluent. The immune affinity chromatographic column for the rhodamine 6G is convenient to use, has high specificity and high sensitivity, and can quickly detect the residual rhodamine 6G in the food.
Description
Technical field
The invention belongs to the adjuvant check and analysis technical field of violating a ban in the food security field.Particularly, the present invention relates to a kind of rhodamine 6G immune affinity chromatographic column Its Preparation Method And Use.
Background of invention
Rhodamine is the industry and the biological dye of a series of catechols, and has stronger fluorescence, and tens kinds of products such as rhodamine B, rhodamine 6G, rhodamine 110 are arranged.
Rhodamine 6G (Rhodamine 6G) is called rose-red 6G, and is bigger for the toxicity of human body and other biological.By with the contacting of skin, mucous membrane, suction and per os and invade human body.Chemical reaction takes place when it contacts with protein in the cell magma, and cell is lost vigor, dense phenol liquid can make protein coagulating.When acting on protein, or not, do not cause that deep tissue damages necrosis, and be absorbed and cause the whole body poisoning so can continue to the deep tissue infiltration with it in conjunction with (this point and strong acid, highly basic different).Suck the phenol steam of high concentration, can cause central nervous system disorder, often be exposed in the lower air of phenol concentration, also can cause dermatitis, can make the cholesteroderma brown.
Field fast detection method, also be to use non-polar organic solvent with its dissolving and extraction, extract flows through neutral alumina solid phase extraction column absorption rhodamine 6G, with non-polar organic solvent drip washing pillar, elution samples grease and food endogenous chaff interference, with the naked eye can observe the vivid pink band of appearance on pillar, be judged to be suspicious specimen.
The immunoaffinity chromatography technology is a kind of analytical approach that immune response and chromatographic technique are combined, on purifying substances an efficient height, purifying substances purity height, technology fast, can make immunoassay technology (as ELISA etc.) and chromatographic technique obtain complementation aspect specificity, separating power, speed and the sensitivity, avoid the deficiency of the direct working sample of immunoassay.
Immune affinity column is the technology that eighties of last century is applied in analysis field the nineties, but the rhodamine that directly detects in the food samples with immune affinity column series connection alumina column does not appear in the newspapers as yet.Therefore, the inventor has succeeded in developing a kind of have high specific and sensitivity and direct detection rhodamine 6G immune affinity chromatographic column swift to operate by a large amount of tests.
Summary of the invention
The object of the invention provides a kind of immune affinity chromatographic column of direct detection rhodamine 6G.
Principle of the present invention is according to the specific reaction of antigen-antibody and the characteristic that can dissociate, and residual with the rhodamine 6G that rhodamine antibody extracts in the food, the alumina column of connecting then directly detects rhodamine 6G with its fluorescence.
On the one hand, the invention provides a kind of method for preparing the rhodamine 6G immune affinity chromatographic column, this method comprises the following steps:
A) the rhodamine 6G specific antibody is mixed with the Sepherose4B glue of cyanogen bromide-activated, rotary drum stirs, and coupling is spent the night; Sintered glass filter suction filtration coupling glue, the PBS wash-out is binding antibody not, each 5 milliliters, washes 4 times; Seal not binding site with the 1M monoethanolamine, add excess ethyl alcohol amine and hatched 2.5 hours, the suction filtration monoethanolamine is taken out with coupling buffer and to be washed, and then uses HAC, the alternately washing totally 4 times of Tris-Hcl damping fluid, each 5ml;
B) mucilage binding after the coupling is gone in the affinity chromatography plastic column, dress is annotated the 1ml/ post, slightly firmly compresses, and promptly gets described rhodamine 6G immune affinity chromatographic column.
In a preferred embodiment of the invention, preparation method's concrete operations of described rhodamine 6G immune affinity chromatographic column are:
Get Sepherose 4B (100-120 order) weight in wet base 4g (6ml), use 0.1M pH9.5Na
2CO
3Solution soaked 4 hours.Take by weighing cyanogen bromide 0.8g in the ventilating kitchen and be dissolved in 1.2ml Na
2CO
3(2g solution 3ml damping fluid), electromagnetic agitation 10 minutes.Under the ice bath electromagnetic agitation, cyanogen bromide solution is added among the Sepherose4B, finish in 2 minutes, drip 2N NaOH, make the pH value remain on 11, ice bath reaction 10 minutes.
Reacted Sepherose4B is poured in the Buchner funnel, with the Na of precooling
2CO
3Wash in 2-3 minute for solution 4-10 ℃ and finish, take out the liquid of washing 25 times of volumes and prepare crosslinking protein.
Under 4 ℃, antibody (polyclone or the monoclonal antibody) solution that embodiment 1 is prepared mixes with the Sepherose4B glue of cyanogen bromide-activated, and rotary drum stirs, and coupling reaction is spent the night.
Sintered glass filter suction filtration coupling glue, the PBS wash-out is binding antibody not, each 5 milliliters, washes 4 times.Measure antibody content respectively.
(ethanolamine) seals not binding site with the 1M monoethanolamine, adds excess ethyl alcohol amine and hatches 2.5 hours.The suction filtration monoethanolamine is taken out with coupling buffer and to be washed.And then with alternately HAC, the Tris-Hcl damping fluid is washed 4 times, each 5ml.
At last, the mucilage binding after the coupling is gone in the affinity chromatography plastic column, dress is annotated the 1ml/ post, slightly firmly compresses, and promptly obtains rhodamine 6G immune affinity column of the present invention.
On the other hand, the invention provides a kind of rhodamine 6G immune affinity chromatographic column, this immune affinity chromatographic column comprises coupling Sepharose4B that the rhodamine 6G specific antibody arranged and the plastic column that loads this affinitive material, and the alumina column of affinitive material plastic column series connection therewith.
Wherein said rhodamine 6G specific antibody is monoclonal antibody or polyclonal antibody.Rhodamine 6G specific antibody of the present invention prepares by following method: 1) prepare rhodamine 6G and carrier protein couplet thing by the haptenic coupling method of micromolecule; And, 2) with this conjugate as immunogen immune animal (for example, rabbit), separation of serum, purifying obtains polyclonal antibody; Perhaps 3) immune mouse obtains monoclonal antibody by hybridoma technology; 4) conjugate with different carriers albumen is that coating antigen is used to detect the antibody quality.
Wherein said carrier protein includes but not limited to, for example the blue albumen of bovine serum albumin(BSA) (BSA), human serum albumins, ovalbumin and key hole copper (keyhole limpethemocyanin, KLH).The conjugate of described rhodamine 6G and carrier protein adopts diazotising method, glutaraldehyde method, active ester method or multi-anhydride method and the coupling of mixed anhydride method integrated processes.
The preservation condition of immune affinity chromatographic column of the present invention is: and the phosphate buffer of 0.01% merthiolate (PBS, 0.01mol/L, pH7.4), 4 ℃ of preservations.
In addition, for making things convenient for rig-site utilization, immune affinity column of the present invention can further include sample-loading buffer, leacheate and eluent.
In a preferred embodiment of the invention, described sample-loading buffer be PBS (0.02MPB-0.05MNaCl, PH7.2), described leacheate be PBST (0.02MPB-0.05MNaCl-0.2%Tween20, PH7.2); Described affinity chromatographic column eluent is methyl alcohol/1M acetate (V/V is 97/3).
On the other hand, the invention provides a kind of using method of rhodamine 6G immune affinity chromatographic column, this method comprises:
A) immune affinity column and sample-loading buffer, leacheate and the eluent of 4 ℃ of preservations of taking-up are back to room temperature, with the abundant drip washing affinity column of sample-loading buffer, remove and store albumen and the antiseptic that contains among the Buffer;
B) sample to be checked that will handle well be diluted to sample-loading buffer volume required, last sample, after the sample solution drip-dry, leacheate drip washing cylinder discards, rubber pipette bulb dries up solution in the post;
C) rhodamine 6G of usefulness eluent wash-out specificity combination.
D) rhodamine 6G of wash-out flows through the alumina column of series connection, detects by fluorescence.
The present invention uses sausage to carry out recovery experiment, and the result shows the recovery of rhodamine 6G immune affinity chromatographic column between 80%-110%, and the recovery has reached the recovery requirement of analytical approach.
Therefore, on the other hand, the invention provides described rhodamine 6G immune affinity chromatographic column residual purposes of rhodamine 6G in food such as extraction sausage.
In the specific embodiments, described food is sausage.
It is in order further to understand the present invention better that following embodiment is provided, and never content of the present invention and protection domain is constituted any restriction.
Embodiment
Embodiment 1 anti-rhodamine 6G Polyclonal Antibody Preparation
1.1 the immunogenic preparation of rhodamine 6G
Take by weighing rhodamine 6G 50.0mg (about 0.1mmol), be dissolved in the 4ml deionized water, add methyl alcohol 4ml, potassium hydroxide 20mg transfers pH to 3.0, ethyl acetate extraction 60 ℃ of hydrolysis 4 hours with 6M hydrochloric acid, dry up, 0.1M pH 7.4PBS redissolves, and rhodamine 6G solution is dropwise joined in 10ml, the 0.1M pH 7.4 ice-cold carrier protein PB solution, adds EDC 100mg subsequently.Carrier consumption: BSA:50mg, KLH:50mg.Conjugate is crossed the SephadexG-25M chromatographic column and is purified.Measure carrier concn as conjugate concentration with ultraviolet absorption method.The conjugate of purifying adds equivalent glycerine-20 ℃ preservation.
1.2 anti-rhodamine 6G Polyclonal Antibody Preparation
Select 5 of healthy, anosis, male, the purebred New Zealand white rabbit of body weight 2.5kg.Head exempts from syringe the method for taking out to be mixed into water in oil emulsion with Freund's complete adjuvant and immunogene by 1: 1, every rabbit 0.25mg immunogene, and the subcutaneous multi-point injection in back, the injected dose multi-point average distributes.Every two all booster immunizations once, use incomplete Freund, dosage, method are exempted from head.From immunity for the third time, back 10 days of immunity, auricular vein is got blood 1ml, and room temperature is solidified behind the 2h 4 ℃ and is spent the night, and the 8000r/min separation of serum is in order to the detection antibody titer.Tire when no longer raising, do not add back 7 days of adjuvant last immunity (the 8th time), the arteria carotis bloodletting, isolate anti-whole serum, the saturated ammonium sulfate solution precipitation with 40% gets thick polyclonal antibody, separate with the DE-52 ion-exchange chromatography then and remove other haemocyanins, get pure polyclonal antibody.
Measure by indirect ELISA and indirect competitive ELISA, the result shows that the polyclonal antibody that the present invention prepares has very high tiring and good specificity.
1.3 anti-rhodamine 6G MONOCLONAL ANTIBODIES SPECIFIC FOR
1.3.1 immune animal and Fusion of Cells
To 8 ages in week female Balb/c mouse (body weight 18～22g), first immunisation is with 100 μ g rhodamine 6G-KLH and equivalent Fu Shi Freund's complete adjuvant mixing, lumbar injection.First is got spleen and merges with 10 μ g rhodamine 6G-KLH intrasplenic injections after 3 weeks after 3 days.Second batch use 100 μ g rhodamine 6G-KLH and equivalent freund 's incomplete adjuvant mixing again after, the lumbar injection supplementary immunization, the spleen fusion is got according to a conventional method with 10 μ g rhodamine 6G-KLH intrasplenic injections in 3 week backs after 3 days.When microscopy hybridoma clonal growth reaches 1/3～1/2 visual field, get supernatant and screen.
1.3.2 hybridoma screening and antibody test
With rhodamine 6G-BSA is envelope antigen, and the cell hole with the anti-rhodamine 6G antibody of indirect noncompetitive ELISA method screening secretion suppresses ELISA method conclusive evidence with indirect competition.With the positive contrast of the serum of immune mouse, with the negative contrast of the supernatant of Sp2/0 cellular incubation, the criterion in positive cell hole is (A
Test-A
Blank)/(A
Contrast-A
Blank) 〉=2.1.
1.3.3 hybridoma cloning
Adopt accurate counting dilution method in the culture flask, the positive colony cell is blown even, get a droplet to culture flask, accurate counting number of cells under the inverted microscope, dilution is 70/ml, gets 20 times of 1ml dilutions again, inoculation goes in 96 well culture plates to carry out subclone.Till the culture supernatant in all cells hole all is positive.
1.3.4 the production of monoclonal antibody
Adopt in the animal body and induce monoclonal antibody method.The hybridoma that piping and druming is cultivated, the centrifugal 10min of 1000r/min abandons supernatant, with the hybridoma mixing that suspends, and cell number is transferred to 2 * 10 with physiological saline
6/ ml, every Balb/c mouse peritoneal injection 0.5ml hybridoma, and, collect ascites after 10～14 days simultaneously in offside lumbar injection 0.5ml norphytane and incomplete freund adjuvant potpourri (mixing at 1: 1).
1.3.5 Purification of Monoclonal Antibodies
The employing ammonium sulfate precipitation method carries out purifying to ascites or purifies with ion exchange process.
The preparation of embodiment 2 rhodamine 6G immune affinity chromatographic columns
Present embodiment is the preparation of rhodamine 6G immune affinity chromatographic column
Get Sepherose 4B (100-120 order) weight in wet base 4g (6ml), use 0.1M pH9.5Na
2CO
3Solution soaked 4 hours.Take by weighing cyanogen bromide 0.8g in the ventilating kitchen and be dissolved in 1.2ml Na
2CO
3(2g solution 3ml damping fluid), electromagnetic agitation 10 minutes.Under the ice bath electromagnetic agitation, cyanogen bromide solution is added among the Sepherose4B, finish in 2 minutes, drip 2N NaOH, make the pH value remain on 11, ice bath reaction 10 minutes.
Reacted Sepherose4B is poured in the Buchner funnel, with the Na of precooling
2CO
3Wash in 2-3 minute for solution 4-10 ℃ and finish, take out the liquid of washing 25 times of volumes and prepare crosslinking protein.
Under 4 ℃, antibody (polyclonal antibody or the monoclonal antibody) solution that embodiment 1 is prepared mixes with the Sepherose4B glue of cyanogen bromide-activated, and rotary drum stirs, and coupling reaction is spent the night.
Sintered glass filter suction filtration coupling glue, the PBS wash-out is binding antibody not, each 5 milliliters, washes 4 times.Measure antibody content respectively.
(ethanolamine) seals not binding site with the 1M monoethanolamine, adds excess ethyl alcohol amine and hatches 2.5 hours.The suction filtration monoethanolamine is taken out with coupling buffer and to be washed.And then with alternately HAC, the Tris-Hcl damping fluid is washed 4 times, each 5ml.
At last, the mucilage binding after the coupling is gone in the affinity chromatography plastic column, the dress post, the 1ml/ post slightly firmly compresses, and promptly gets described rhodamine 6G immune affinity chromatographic column.
The rhodamine 6G immune affinity column of this preparation is connected with alumina column, promptly directly detected the immune affinity chromatographic column of rhodamine 6G
Preservation condition: the phosphate buffer of 0.01% merthiolate (PBS, 0.01mol/L, pH7.4), 4 ℃ of preservations.
Embodiment 3
Get 12 parts in feminine gender (being defined as the clenbuterol feminine gender in advance after testing) sausage, per 5 parts of 5g; Earlier with after the sausage homogenate, get 9 parts, divide 3 groups, every group is added rhodamine 6G concentration and is respectively 50ug/kg, 80ug/kg, 100ug/kg as positive, other 3 parts as negative control.
With above-mentioned sausage sample 10ml80% dissolve with methanol, centrifuging and taking supernatant, nitrogen dry up the back and dissolve affinity chromatographic column in the preparation with sample-loading buffer.
The immune affinity column that will take out from 4 ℃ of refrigerators and all reagent room temperatures were placed 1 hour, with the abundant drip washing affinity column of sample-loading buffer 5ml * 6 times, removed and stored albumen and the antiseptic that contains among the Buffer.
With negative sausage extract to be checked and each 5ml of interpolation sample extracting solution, sample is 5 times in the circulation, (access the sample effluent and go up this affinity column once more).After the sample solution drip-dry, with leacheate drip washing 5 times, each 1ml, rubber pipette bulb dries up solution in the post.
Add the 1ml eluent earlier and soaked affinity chromatographic column 10 minutes, dry up solution in the post with rubber pipette bulb then; Add the 1ml eluent again and soaked 3 minutes, divide wash-out 4 times with the 2ml eluent at last, promptly each 0.5ml.The shared eluent 4ml of whole elution process.
Eluent flows through the alumina column of series connection, according to rhodamine 6G content in the fluorescent strength determining sausage.
The average recovery rate of measuring sausage interpolation sample is between the 75-110%, shows that this method satisfies the analytical approach requirement.
Claims (6)
1. method for preparing the rhodamine 6G immune affinity chromatographic column, this method comprises the following steps:
A) the rhodamine 6G specific antibody is mixed with the Sepherose4B glue of cyanogen bromide-activated, stir with rotary drum, coupling is spent the night; Sintered glass filter suction filtration coupling glue, the PBS wash-out is binding antibody not, each 5 milliliters, washes 4 times; Seal not binding site with the 1M monoethanolamine, add excess ethyl alcohol amine and hatched 2.5 hours, the suction filtration monoethanolamine is taken out with coupling buffer and to be washed, and then uses HAC, the alternately washing totally 4 times of Tris-Hcl damping fluid, each 5ml;
B) mucilage binding after the coupling is gone in the plastic column, dress is annotated the 1ml/ post, slightly firmly compresses, and promptly gets described rhodamine 6G immune affinity chromatographic column.
C) prepared rhodamine 6G immune affinity chromatographic column is connected with alumina column, and directly detect the immune affinity chromatographic column of rhodamine 6G.
2. according to the rhodamine 6G immune affinity chromatographic column of the described method of claim 1 preparation, this immune affinity chromatographic column comprises that coupling has the Sepharose4B and the plastic column that loads this affinitive material of rhodamine 6G specific antibody, also further comprises the alumina column of series connection.
3. the described rhodamine 6G immune affinity column of claim 2, wherein said rhodamine 6G specific antibody is polyclonal antibody or monoclonal antibody.
4. claim 2 or 3 described clenbuterol immune affinity chromatographic columns, it further comprises sample-loading buffer, leacheate and eluent.
5. the described rhodamine 6G immune affinity chromatographic column of claim 4, wherein said sample-loading buffer is PBS, and described leacheate is PBST, and described eluent is that volume ratio is methyl alcohol/1M acetate mixture of 97: 3.
6. the using method of the described rhodamine 6G immune affinity chromatographic column of claim 2, this method comprises:
A) immune affinity chromatographic column and sample-loading buffer, leacheate and the eluent of 4 ℃ of preservations of taking-up are back to room temperature, with the abundant drip washing affinity column of sample-loading buffer, remove and store albumen and the antiseptic that contains among the Buffer;
B) sample to be checked that will handle well be diluted to sample-loading buffer volume required, last sample, after the sample solution drip-dry, leacheate drip washing cylinder discards, rubber pipette bulb dries up solution in the post;
C) rhodamine 6G of usefulness eluent wash-out specificity combination;
D) rhodamine 6G under the wash-out flow through the series connection alumina column, utilize its fluoroscopic examination rhodamine 6G.
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| CN102087278A (en) * | 2010-12-23 | 2011-06-08 | 江南大学 | Method combining immunoaffinity column, high performance liquid chromatography and mass spectrum for detecting rhodamine B |
| CN102680588A (en) * | 2011-03-09 | 2012-09-19 | 北京康华源医药信息咨询有限公司 | A column chromatographic separation detection technology, and application thereof |
| CN102798710A (en) * | 2012-08-08 | 2012-11-28 | 广州市质量监督检测研究院 | Detection kit for basic red G and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102087278A (en) * | 2010-12-23 | 2011-06-08 | 江南大学 | Method combining immunoaffinity column, high performance liquid chromatography and mass spectrum for detecting rhodamine B |
| CN102680588A (en) * | 2011-03-09 | 2012-09-19 | 北京康华源医药信息咨询有限公司 | A column chromatographic separation detection technology, and application thereof |
| CN102680588B (en) * | 2011-03-09 | 2015-10-28 | 常州博闻迪医药科技有限公司 | A kind of column chromatography for separation detection technique and uses thereof |
| CN102798710A (en) * | 2012-08-08 | 2012-11-28 | 广州市质量监督检测研究院 | Detection kit for basic red G and preparation method thereof |
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Application publication date: 20101208 |