CN100489532C - Method for detecting salinomycin and spcial enzyme-linked immune reagent kit thereof - Google Patents
Method for detecting salinomycin and spcial enzyme-linked immune reagent kit thereof Download PDFInfo
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- CN100489532C CN100489532C CNB2006100072857A CN200610007285A CN100489532C CN 100489532 C CN100489532 C CN 100489532C CN B2006100072857 A CNB2006100072857 A CN B2006100072857A CN 200610007285 A CN200610007285 A CN 200610007285A CN 100489532 C CN100489532 C CN 100489532C
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Abstract
The present invention discloses a method for detecting salinomycin and its special-purpose ELIA kit. Said kit includes salinomycin specific antibody, coating source and enzyme label. The described coating source is conjugate of salinomycin semiantigen and carrier protein or antiantibody, the described enzyme label is enzyme-labelled antiantibody or enzyme-labelled salinomycin semiantigen. When the described coating source is the conjugate of salinomycin semiantigen and carrier protein, the described enzyme label is enzyme-labelled antiantibody, and when the described coating source is antiantibody, the described enzyme label is enzyme-labelled salinomycin semiantigen.
Description
Technical field
The present invention relates to a kind of method and special ELISA reagent kit thereof that detects salinomycin.
Background technology
Salinomycin (Salinomycin Sodium Premix) belongs to a kind of polyethers microbiotic for animals, and most of gram-positive bacterias and part mould are played antibacterial action, uses separately and can bring into play the good preventing effect to chicken coccidiasis.But the use salinomycin can not be excessive, and excessive use will be accumulated in animal body in a large number, and institute's edible animal is poisoned to death, and jeopardizes human health by food chain.In Dec, 2002 China Ministry of Agriculture to announce No. 235 civilian regulation salinomycin high residue amount in the muscle of institute's edible animal be that the high residue amount of 600 μ g/kg in skin, fat is that the high residue amount of 1200 μ g/kg in liver is 1800 μ g/kg.Therefore, it is very important detecting the high residue amount of salinomycin in animal food.
At present, the method that is usually used in the salinomycin residue detection mainly contains microbial method and instrumental method.Though the microorganism detection method is economical, easy and simple to handle, when having other microbial inhibitors to exist in sample, its sensitivity and specificity are restricted; Simple instrument analytical methods such as high efficiency liquid phase chromatographic analysis method, gas spectrum, GC-MS method, though highly sensitive, sample pre-treatment and measurement operation are loaded down with trivial details, the expense height is unwell to the great amount of samples examination, can be used as residual conclusive evidence analysis.
Summary of the invention
The purpose of this invention is to provide a kind of method and special ELISA reagent kit thereof that detects salinomycin.
The enzyme linked immunological kit of detection salinomycin provided by the present invention comprises salinomycin specific antibody and coating antigen and enzyme labeling thing; Described coating antigen is the conjugate or the antiantibody of salinomycin haptens and carrier protein; Described enzyme labeling thing is enzyme mark antiantibody or enzyme mark salinomycin haptens; When described coating antigen was the conjugate of salinomycin haptens and carrier protein, described enzyme labeling thing was an enzyme mark antiantibody; When described coating antigen was the salinomycin antiantibody, described enzyme labeling thing was an enzyme mark salinomycin haptens.
The conjugate of described salinomycin haptens and carrier protein can obtain by salinomycin haptens and carrier protein are carried out coupling with mixed anhydride method or active ester method; Described salinomycin haptens obtains salinomycin and p-aminobenzoic acid by condensation reaction.
The marker enzyme of described enzyme labeling thing is horseradish peroxidase or alkaline phosphatase, wherein preferred alkaline phosphatase; Alkaline phosphate ester enzyme labeling antiantibody can adopt several different methods of the prior art such as glutaraldehyde method or sodium periodate method that enzyme is crosslinked on antiantibody; The salinomycin haptens of alkaline phosphate ester enzyme labeling can adopt mixed anhydride method that alkaline phosphatase and salinomycin hapten conjugation are obtained.Described salinomycin haptens obtains salinomycin and p-aminobenzoic acid by condensation reaction.
Described salinomycin specific antibody can be salinomycin monoclonal antibody or salinomycin polyclonal antibody; They all are that conjugate with salinomycin haptens and carrier protein obtains as immunogene; Polyclonal antibody can be mouse source, Ma Yuan, Yang Yuan, rabbit source or cavy source antibody, and described salinomycin monoclonal antibody is the salinomycin mouse monoclonal antibody, and described salinomycin polyclonal antibody is preferably the salinomycin rabbit polyclonal antibody.Described antiantibody is sheep anti mouse or goat-anti rabbit antiantibody.The used antibody diluent of antibody working fluid is to be the 0.05mol/L of pH value 8.2, contains the phosphate buffer of 5% methyl alcohol.
Described salinomycin mouse monoclonal antibody is preferably the antibody of the monoclonal hybridoma strain A-2-3 CGMCCNo.1609 secretion of salinomycin.
The monoclonal hybridoma strain A-2-3 CGMCC No.1609 of salinomycin has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (being called for short CGMCC) on February 9th, 2006.
Above antibody all can prepare as immunogene according to a conventional method with the conjugate of salinomycin haptens and carrier protein.Described carrier protein can be common carrier albumen such as mouse haemocyanin, bovine serum albumin(BSA), rabbit anteserum albumen, human serum albumins, ovalbumin or hemocyanin; The conjugate of described salinomycin haptens and carrier protein can obtain by salinomycin haptens and carrier protein are carried out coupling with mixed anhydride method; Described salinomycin haptens obtains salinomycin and p-aminobenzoic acid by condensation reaction.
For more convenient on-site supervision and great amount of samples examination, described kit also comprises salinomycin standard solution, developer, stop buffer, concentrated cleaning solution, concentrates redissolution liquid.
Described standard solution is the solution that six concentration gradients contain the salinomycin medicine, and used salinomycin drug dilution liquid is for containing 5 ‰ (mass concentration) N, N '-dimethyl formamide (DMF), the phosphate buffer of 1% calf serum (BSA).
Described concentrated cleaning solution is pH7.4, the phosphate buffer that contains 0.8%～1.2% polysorbas20,1 ‰ thimerosal antiseptics of 0.01mol/L.
Described when marker enzyme is horseradish peroxidase, developer is made up of colour developing liquid A liquid and colour developing liquid B liquid, and colour developing liquid A liquid is hydrogen peroxide or urea peroxide, and colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1～2mol/L sulfuric acid or hydrochloric acid; When marker enzyme was alkaline phosphatase, developer was a 4-nitrophenols phosphate buffer, and stop buffer is a sodium hydrate buffer solution.
Described concentrated redissolution liquid can be and contains 5 ‰ (mass concentration) N, N '-dimethyl formamide (DMF), the phosphate buffer of the 0.01mol/L～0.05mol/L of 1% calf serum (BSA).
It is pH9.6 that described bag is cushioned liquid, the carbonate buffer solution of 0.1mol/L.
Described confining liquid is the aqueous solution that contains 3～10% horse serums, 1% inert protein.
Used bag be can be polystyrene, cellulose, polyacrylamide, tygon, polypropylene, cross-link dextran, glass, silicon rubber, Ago-Gel etc. by the carrier mass of salinomycin antigen or antiantibody, and the form of carrier can be test tube, micro-reaction plate shrinkage pool, globule, sequin etc.
The method of detection salinomycin provided by the present invention may further comprise the steps:
1) sample pre-treatments
Sample pre-treatments: take by weighing 2g animal tissue homogenate and put in the centrifuge tube, adding the 10ml volume ratio is the methyl alcohol of 4:1 and the mixed liquor of 5% physiological saline, the thermal agitation mixing; In 15 ℃, the centrifugal 10min of the above speed of 5000g; Supernatant is moved in another centrifuge tube, in 15 ℃, the centrifugal 5min of 3000g; Get the 5ml supernatant, add the 10ml phenixin, vibration mixing, the centrifugal 10min of the above speed of 5000g; Upper strata liquid is moved into another Guan Zhongyong nitrogen dry up, with the dry residue of concentrated redissolution liquid dissolving of 4 times of 1ml dilutions, water intaking is analyzed mutually.
2) utilize the enzyme linked immunological kit test sample of above-mentioned detection salinomycin.
3) detect data analysis.
Salinomycin is a small-molecule substance, has only immunoreactivity, does not have immunogenicity, can not bring out body and produce immune response, must with the macromolecular carrier albumen coupling after just have immunogenicity.Salinomycin and p-aminobenzoic acid are synthesized the salinomycin haptens by condensation reaction, help to make at the stronger polyclonal antibody of salinomycin antigentic specificity.Again salinomycin is adopted mixed anhydride method and carrier protein couplet to obtain immunogene.It is low or too high all unfavorable to immunity that haptens and the ratio that combines of carrier protein are crossed, and haptens is respectively 11:1,17:1,17:1 with the mol ratio that combines of OVA, RSA and KLH.
But the residual quantity of salinomycin in the kit qualitative and quantitative analysis of the present invention animal tissue sample.Detection principle of the present invention is when wrapping by the conjugate of salinomycin haptens and carrier protein on the capillary strip in advance, after adding series standard product or sample solution and salinomycin antibody working fluid, in the sample on residual salinomycin and the capillary strip coupled antigen of pre-bag quilt compete the antibody of anti-salinomycin, add the enzyme labeling thing and carry out the enzymatic activity amplification, the colour developing back stops; When bag is by antiantibody on the capillary strip, behind the adding salinomycin antibody working fluid, add series standard product or sample solution and enzyme-labelled antigen again, salinomycin that sample is residual and enzyme-labelled antigen are competed anti-salinomycin antibody, colour developing; Colour developing stops the back and measures every hole absorbance (OD value) with microplate reader, and the content of sample absorbance and its residue salinomycin is negative correlation, relatively can draw the content of corresponding residue salinomycin with typical curve.Also can be according to the depth of the sample solution color on the ELISA Plate, with the salinomycin titer color of the series concentration concentration range of salinomycin in the judgement sample relatively.
The enzyme linked immunological kit of detection salinomycin of the present invention is low to the pre-treatment requirement of sample, and sample pretreatment process is simple, simultaneously the fast detecting gross sample; Main agents provides with forms such as working fluid, concentrate or freeze-dried powders, the method of inspection is convenient and easy, show through precision and accuracy test experiments kit, enzyme linked immunological kit of the present invention has characteristics such as high specific, high sensitivity, pinpoint accuracy, pin-point accuracy, will play a significant role in the detection of food and cattle salt mycin residual quantity.Kit of the present invention is simple in structure, easy to use, low price, be easy to carry, and can be used for the detection of salinomycin in the animal derived food; The method of detection salinomycin of the present invention is efficient, accurate, easy, be suitable for the qualitative and quantitative analysis of batch samples screening.
Description of drawings
Fig. 1 is for being the enzyme linked immunological kit salinomycin canonical plotting of coating antigen with salinomycin antigen
Fig. 2 is for being the enzyme linked immunological kit salinomycin canonical plotting of coating antigen with the antiantibody
Embodiment
The method of following embodiment is conventional method if no special instructions.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
Embodiment 1, be the preparation and the detection method thereof of the enzyme linked immunological kit of coating antigen with the conjugate of salinomycin haptens and carrier protein
With the conjugate of salinomycin haptens and carrier protein is that the enzyme linked immunological kit of coating antigen comprises:
(1) is coated with the ELISA Plate of salinomycin and carrier protein couplet thing;
(2) the sheep anti mouse antiantibody working fluid of alkaline phosphate ester enzyme labeling: with antibody diluent the sheep anti mouse antiantibody of alkaline phosphate ester enzyme labeling being diluted to protein concentration is 0.1 μ g/L, 12ml/ bottle, 1 bottle.Enzyme labeling thing dilution is a pH value 8.2, contains 1.2 μ g/L antibody proteins, contains the phosphate buffer of the 0.05mol/L of 5% methyl alcohol.
(3) salinomycin standard solution: with dilution salinomycin is diluted to 6 bottles of series standard product solution, 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81 μ g/L, 3ml/ bottle.Used salinomycin drug dilution liquid is for containing 5 ‰ (mass concentration) N, N '-dimethyl formamide (DMF), 1% calf serum (BSA), the phosphate buffer of 0.05mol/L.
(4) colour developing liquid is 4-nitrophenols phosphate buffer, 8ml/ bottle, 1 bottle.
(5) salinomycin mouse monoclonal antibody working fluid: is protein concentration 0.1 μ g/L with antibody diluent with the antibody dilution of the monoclonal hybridoma strain A-2-3 CGMCC No.1609 of salinomycin secretion, 12ml/ bottle, 1 bottle.Antibody diluent is a pH value 8.2, contains 1.2 μ g/L antibody proteins, contains the phosphate buffer of the 0.05mol/L of 5% methyl alcohol.
(6) concentrated cleaning solution: contain the phosphate buffer (0.01M, pH value 7.4) of 0.9% tween, 1 ‰ (mass concentration) thimerosal antiseptic, 50ml/ bottle, 1 bottle.Be 20 times of normal working concentration.
(7) stop buffer: 2mol/L sodium hydroxide solution, 8ml/ bottle, 1 bottle.
(8) concentrate redissolution liquid: contain 5 ‰ N, N '-dimethyl formamide (DMF), 0.01～0.05mol/L phosphate buffer of 1% calf serum (BSA), 400ml/ bottle, 1 bottle.Be 5 times of normal working concentration.
(9) bag is cushioned liquid: pH9.6, the carbonate buffer solution of 0.1mol/L.
(10) confining liquid: the aqueous solution of 5% horse serum, 1% inert protein.
Wherein, bag is as follows by the preparation method of the sheep anti mouse antiantibody of the ELISA Plate of salinomycin and carrier protein couplet thing, salinomycin specific antibody, alkaline phosphate ester enzyme labeling:
One, the preparation of ELISA Plate
1, the haptenic synthetic method of salinomycin:
Haptenic synthetic: that salinomycin and p-aminobenzoic acid are synthesized the salinomycin haptens by condensation reaction.Concrete steps are: salinomycin and p-aminobenzoic acid are mixed stirring reaction in the 1:1 ratio, and reaction is spent the night under the room temperature.
2, coating antigen: adopt mixed anhydride method that salinomycin haptens and ovalbumin (OVA) coupling are obtained coating antigen.
The concrete preparation method of coating antigen is as follows:
(1) get salinomycin haptens 2g and be dissolved in 30ml, 50% N is in N '-dimethyl formamide solution;
(2) getting the 0.5ml isobutyl chlorocarbonate is dissolved in and is added in the haptens solution stirring at room reaction 4 hours in the no Shui diox of 5ml;
(3) getting egg white 22g is dissolved in the 70ml pH9.6 carbonate buffer solution;
(4) ovalbumin being added drop-wise in the haptens 4 ℃ of stirrings spends the night;
(5) artificial antigen that will react was dialysed 7 days to the phosphate buffer of 0.2M, changed liquid every day 3～4 times.
3, the preparation of ELISA Plate:
Be cushioned liquid with bag salinomycin haptens and ovalbumin conjugate are diluted to 0.06 μ g/ml, every hole adds 100 μ l, 37 ℃ of incubation 2h, the coating buffer that inclines washs 2 times with the concentrated cleaning solution that dilutes 19 times, each 30s, pat dry, in every hole, add 150 μ l confining liquids, 37 ℃ of incubation 1-2h then, the liquid in the hole that inclines, preserve with the vacuum seal of aluminium film dry back.
Two, the preparation of salinomycin mouse monoclonal antibody
1, immunogenic synthetic: that salinomycin and p-aminobenzoic acid are synthesized the salinomycin haptens by condensation reaction.Adopt mixed anhydride method that salinomycin haptens and hemocyanin (KLH) coupling are obtained immunogene again.It is 97.3% that synthetic immunogene adopts immunoelectrophoresis to measure its purity.
The concrete steps that immunogene is synthetic:
(1) get salinomycin haptens 2g and be dissolved in 30ml, 50% N is in N '-dimethyl formamide solution;
(2) getting the 0.5ml isobutyl chlorocarbonate again is dissolved in and is added in the haptens solution stirring at room reaction 4 hours in the no Shui diox of 5ml;
(3) getting hemocyanin (KLH) 32g is dissolved in the 70ml pH9.6 carbonate buffer solution;
(4) hemocyanin being added drop-wise in the haptens 4 ℃ of stirrings spends the night again;
(5) artificial antigen that will react was dialysed 7 days to the phosphate buffer of 0.2M, changed liquid every day 3～4 times.
2, the preparation of salinomycin mouse monoclonal antibody
The animal immune program adopts the Balb/c mouse as immune animal, with salinomycin haptens and hemocyanin conjugate is immunogene, immunizing dose is 100 μ g/, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, At intervals of two to three weeks are got the same dose immunogene and are added equivalent incomplete Freund mixing and emulsifying, and booster immunization once, four exempt from the pneumoretroperitoneum booster immunization once, extracting spleen cell after 3 days.
Immune BALB/c mouse splenocyte is got in Fusion of Cells and cloning, merges in 5:1 ratio and SP2/0 myeloma cell, adopts indirect competitive ELISA to measure cell conditioned medium liquid, screens positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, up to the monoclonal hybridoma strain A-2-3 of the hybridoma cell strain-salinomycin that obtains stably excreting monoclonal antibody CGMCC No.1609.
The monoclonal hybridoma strain A-2-3CGMCC No.1609 that the salinomycin that is in exponential phase is got in cell cryopreservation and recovery makes the cell suspension of 5 * 106/ml with cryopreserving liquid, is sub-packed in frozen pipe, preserves in that liquid nitrogen is medium-term and long-term.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.
MONOCLONAL ANTIBODIES SPECIFIC FOR and purifying adopt in the body and induce method, and Balb/c mouse (8 all ages) abdominal cavity is only injected sterilization paraffin oil 0.5ml/, and pneumoretroperitoneum injection hybridoma was 5 * 106/in 7～14 days, gathered ascites after 7～10 days.Carry out the ascites purifying through sad-saturated ammonium sulfate method, bottle packing ,-20 ℃ of preservations.
Three, the preparation of enzyme labelled antibody
The preparation of antiantibody: with mouse source antibody is that immunogene is carried out immunity to the pathogen-free domestic goat, obtains the sheep anti mouse antiantibody.
Enzyme mark antiantibody preparation: sheep anti mouse antiantibody and alkaline phosphatase are carried out coupling, the preferred glutaraldehyde method of method that adopts, with alkaline phosphatase during with the ratio of 2:1 and antiantibody coupling, 60%～70% enzyme and 8% antiantibody coupling are arranged approximately, and the rate ratio of enzyme labeling thing uses the horseradish peroxidase height.
Alkaline phosphate ester enzyme labeling sheep anti mouse antiantibody concrete steps are as follows:
1) takes by weighing alkaline phosphatase 25mg and be dissolved in 1.25% glutaraldehyde solution, in the room temperature standing over night.
2) reacted enzyme solutions is used the physiological saline wash-out through Sephadex G-25 chromatographic column.Flow speed control is collected brown effluent at 1ml/1min.Greater than 5ml, then be concentrated into 5ml as volume with poly-hexylene glycol.Place in the 25ml small beaker, slowly stir.
3) get sheep anti mouse antiantibody 12.5mg and be diluted to 5ml, dropwise add in the enzyme solutions under stirring with physiological saline.
4) with 1M pH9.5 carbonic acid buffer 0.25ml, continue to stir 3h.
5) add 0.2M lysine 0.25ml, behind the mixing, put room temperature 2h.
6) under agitation dropwise add the equal-volume saturated ammonium sulfate, put 4 ℃ of 1h.
7) 3000rpm centrifugal half an hour, abandon supernatant.Sediment is washed secondary with semi-saturation ammonium sulfate, and last sediment is dissolved in the phosphate buffer of a small amount of 0.15M pH7.4.
8) above-mentioned solution is packed in the bag filter, with the phosphate buffer dialysis of 0.15M pH7.4, detect with Nai Shi reagent after removing ammonium ion, the centrifugal 30min of 10000rpm removes precipitation, and supernatant is enzyme conjugates, after the packing, and stored frozen.
Utilize the method for salinomycin residual in this kit test sample as follows:
One, sample pre-treatments
Muscle, liver: get muscle, liver sample refiner 10000r/min homogenate 1min, taking by weighing 2 ± 0.05g homogenate puts in the centrifuge tube, add 10ml methyl alcohol-5% normal saline solution (methyl alcohol: physiological saline=4:1 (volume ratio)) and mix thermal agitation 10min.In 15 ℃, the centrifugal 10min of the above speed of 5000g.Supernatant moves in another centrifuge tube centrifugal once more 15 ℃, 3000g, 5min.Get the 5ml supernatant, add the 10ml phenixin, mix vibration 10min, the centrifugal 10min of the above speed of 5000g.Upper strata liquid is moved another Guan Zhongyong nitrogen dry up (or in the heart bottle, 50 ℃ of evaporated under reduced pressure), with the dry residue of concentrated redissolution liquid dissolving of 4 times of 1ml dilutions.Water intaking is analyzed mutually.
Two, detection method
In 96 hole ELISA Plate micropores of salinomycin haptens and ovalbumin conjugate bag quilt, add series standard product solution or sample solution 50 μ l, add salinomycin mouse monoclonal antibody working fluid 50 μ l again,, react 30min in 37 ℃ of constant temperature ovens with cover plate film shrouding.Pour out liquid in the hole, every hole adds the concentrated cleaning solution (containing 0.8%～1.2% tween, the phosphate buffer of 1 ‰ thimerosal antiseptics (0.01M PH7.4)) of 19 times of 250 μ l dilutions, pours out liquid in the hole after 30 seconds, so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Every hole adds alkaline phosphate ester enzyme labeling sheep anti mouse antiantibody working fluid 100 μ l with cover plate film shrouding, reacts 30min in 37 ℃ of constant temperature ovens.Pour out liquid in the hole, the repeated washing step.Add substrate colour developing liquid 100 μ l, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 30min.Every hole adds stop buffer (1mol/L NaOH) 50 μ l, and the mixing that vibrates is gently measured every hole absorbance (OD value) with microplate reader.
Three, interpretation of result
Each the concentration standard product solution that is obtained or the mean value (B) of sample absorbance are divided by the absorbance (B of first standard (0 standard)
0) multiply by 100% again, i.e. percentage absorbance.
B is the mean light absorbency value of standard solution or sample solution in the formula, B
0It is the mean light absorbency value of 0 μ g/L standard solution.Natural logarithm value with salinomycin concentration is an X-axis, and the percentage absorbance is a Y-axis, the drawing standard curve map, as shown in Figure 1.The concentration of salinomycin can be read from typical curve in corresponding each sample.Also can use regression equation method, calculate the concentration of salinomycin in the sample solution.Utilize computer professional software, the express-analysis of a large amount of samples of being more convenient for.Whole testing process only needed just can finish in 1.4 hours, and lowest detection is limited to 1.0 μ g/L.
Embodiment 2, with goat-anti rabbit antiantibody as enzyme linked immunological kit of coating antigen and preparation method thereof
Comprise with the enzyme linked immunological kit of goat-anti rabbit antiantibody as coating antigen:
(1) is coated with the ELISA Plate of salinomycin antiantibody;
(2) the salinomycin haptens working fluid of alkaline phosphate ester enzyme labeling: is 0.1mol/L with distilled water with the dilution of the salinomycin haptens of alkaline phosphate ester enzyme labeling, 12ml/ bottle, 1 bottle.
Salinomycin standard solution: with dilution salinomycin is diluted to 6 bottles of series standard product solution, 0 μ g/L, 1 μ g/L, 3 μ g/L, 9 μ g/L, 27 μ g/L, 81 μ g/L, 3ml/ bottle.Used salinomycin drug dilution liquid is for containing 5 ‰ N, N '-dimethyl formamide (DMF), the phosphate buffer of 1% calf serum (BSA) 0.05mol/L
(4) colour developing liquid is 4-nitrophenols phosphate buffer, 8ml/ bottle, 1 bottle.
(5) salinomycin rabbit polyclonal antibody working fluid: is protein concentration 0.1 μ g/L with antibody diluent with the dilution of salinomycin rabbit polyclonal antibody, 12ml/ bottle, 1 bottle.Antibody diluent is the 0.05mol/L that contains pH value 8.2, and 1.2 μ g/L antibody proteins contain the phosphate buffer of 5% methyl alcohol.
(6) concentrated cleaning solution: contain the phosphate buffer (0.01MpH7.4) of 0.9% tween, 1 ‰ thimerosal antiseptics, 50ml/ bottle, 1 bottle.Be 20 times of normal working concentration.
(7) stop buffer: 2mol/L sodium hydrate buffer solution, 8ml/ bottle, 1 bottle.
(8) concentrate redissolution liquid: contain 5 ‰ N, N '-dimethyl formamide (DMF), 0.01～0.05mol/L phosphate buffer of 1% calf serum (BSA), 400ml/ bottle, 1 bottle.Be 5 times of normal working concentration.
(9) bag is cushioned liquid: pH9.6, the carbonate buffer solution of 0.1mol/L.
(10) confining liquid: the aqueous solution of 5% horse serum, 1% inert protein.
Wherein, bag is as follows by the haptenic preparation method of salinomycin of the ELISA Plate of goat-anti rabbit antiantibody, salinomycin specific antibody, alkaline phosphate ester enzyme labeling:
One, the preparation of ELISA Plate
1, the preparation of goat-anti rabbit antiantibody coating antigen: with rabbit source antibody is that immunogene is carried out immunity to the pathogen-free domestic goat, obtains goat-anti rabbit antiantibody.
2, be coated with the ELISA Plate preparation method of goat-anti rabbit antiantibody: the material of ELISA Plate is a polyvinyl chloride, be cushioned liquid with bag goat-anti rabbit antiantibody is diluted to 0.5 μ g/ml, the every hole of ELISA Plate adds 100 μ l, 37 ℃ of incubation 2h, coating buffer inclines, concentrated cleaning solution with 19 times of dilutions washs 2 times, and each 30s pats dry, in every hole, add 150 μ l confining liquids then, 37 ℃ of incubation 1-2h, liquid in the hole of inclining, preserve with the vacuum seal of aluminium film dry back.
Two, the preparation of salinomycin rabbit polyclonal antibody
Adopt new zealand white rabbit as immune animal, with salinomycin haptens and hemocyanin conjugate is immunogene, immunizing dose is 1mg/kg, Freund's complete adjuvant with immunogene and equivalent when head exempts from is mixed and made into emulsifying agent, the subcutaneous multi-point injection of nape portion, getting the same dose immunogene 3～4 weeks adds equivalent incomplete Freund mixing and emulsifying at interval, and booster immunization is once, immunity is 5 times altogether, does not add adjuvant for the last time.Take a blood sample behind last immunity 7～10d, measure serum antibody titer, heart is taken a blood sample, and obtains the rabbit polyclonal antibody of purifying through ammonium sulfate precipitation.
Three, the preparation of enzyme labeling salinomycin antigen: salinomycin and p-aminobenzoic acid by the synthetic salinomycin haptens of condensation reaction, are carried out coupling with the salinomycin haptens with alkaline phosphatase employing mixed anhydride method and obtain enzyme labeling salinomycin antigen.
The concrete preparation method of enzyme labeling salinomycin antigen is as follows:
Get salinomycin haptens 2g and be dissolved in 30ml, 50% N, in N '-dimethyl formamide solution, getting the 0.5ml isobutyl chlorocarbonate again is dissolved in and is added in the haptens solution stirring at room reaction 4 hours in the no Shui diox of 5ml, get alkaline phosphatase 32g and be dissolved in the 70ml pH9.6 carbonate buffer solution, again alkaline phosphatase is added drop-wise in the haptens 4 ℃ of stirrings and spends the night.The artificial antigen that has reacted was dialysed 7 days to the phosphate buffer of 0.2M, change liquid every day 3～4 times.
Utilize the method for salinomycin residual in this kit test sample as follows:
The concrete steps of sample pre-treatments are with the sample pre-treatments step among the embodiment 1.
Detection method:
In the 96 hole ELISA Plate micropores that are coated with goat-anti rabbit antiantibody, add salinomycin polyclonal antibody working fluid 100 μ l, react 30min in 37 ℃ of constant temperature ovens, the concentrated cleaning solution that every hole adds 19 times of 250 μ l dilutions (contains 0.8%～1.2% tween, phosphate buffer (the 0.01M of 1 ‰ thimerosal antiseptics, pH value 7.4)), pour out liquid in the hole after 30 seconds, so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Each 50 μ l of salinomycin working fluid that every hole adds series standard product solution or sample solution and alkaline phosphate ester enzyme labeling with cover plate film shrouding, react 30min in 37 ℃ of constant temperature ovens.Pour out liquid in the hole, every hole adds 250 μ l concentrated cleaning solutions and (contains 0.8%～1.2% tween, the phosphate buffer (0.01M of 1 ‰ thimerosal antiseptics, pH value 7.4)), pour out liquid in the hole after 30 seconds, so repetitive operation is washed plate 5 times altogether, pats dry with thieving paper.Add substrate colour developing liquid 4-nitrophenols phosphate buffer 1 00 μ l, the mixing that vibrates gently, 37 ℃ of constant temperature oven lucifuge colour developing 30min.Every hole adds stop buffer (2mol/L NaOH) 50 μ l, and the mixing that vibrates is gently measured every hole absorbance (OD value) with microplate reader.
The method of interpretation of result is with the interpretation of result method among the embodiment 1, the canonical plotting of this kit, as shown in Figure 2.Interpretation of result shows that the whole testing process of the kit of preparation only needed just can finish in 1.4 hours, and lowest detection is limited to 1.0 μ g/L.
Embodiment 3, kit precision, accuracy and storage life test
1, kit precision test
(1) standard items precision test
Every batch is extracted 10 kits from three batches of kits, respectively extracts 20 micropores from the elisa plate of each kit out, measures the absorbance (0D value) of 9 μ g/L standard solutions, calculates the coefficient of variation.The result is as shown in table 1, shows coefficient of variation scope between 4.8%-16.2%, meets precision and is less than or equal to 20% regulation.
The test of table 1 standard precision
(2) the repeatable test of sample
Each sample adds the salinomycin standard items by 10 μ g/kg concentration, gets each three of the kits of three different batches respectively, and each concentration repeats 5 times, calculates the coefficient of variation respectively.Measurement result is shown in table 3, table 4, and the result shows that the chicken sample coefficient of variation all is lower than 20%, and the Variation Lines number average of chicken gizzard sample is lower than 25%.
The repeatable test of table 2 chicken meat sample
The repeatable test of table 3 chicken gizzard sample
The result shows the Variation Lines number average of chicken meat sample less than 20%, and the Variation Lines number average of chicken gizzard sample is less than 20%.
2, the accuracy determination of kit
The salinomycin standard solution of getting two concentration is respectively 5 μ g/kg (L) and 10 μ g/kg (L), detects salinomycin with kit according to the method for embodiment 1 or embodiment 2 respectively, each concentration do 4 parallel, accuracy in computation respectively.Kit accuracy testing result is as shown in table 4, and the result shows the accuracy of chicken interpolation between 62.4%～80.3%, and chicken gizzard adds accuracy between 71.6%～89.8%.
The test of table 4 accuracy determination
3, kit storage life test
Kit is kept at 2-8 ℃ respectively, after 6 months, maximum absorbance value (zero standard), 50% inhibition concentration, the salinomycin of measuring kit add the practical measurement value, and the result shows that the maximum absorbance value (zero standard), 50% inhibition concentration of kit are all within normal range.Consideration has improper preservation condition and occurs in transportation and use, and the mentioned reagent box was placed 6 days under the condition of 37 ℃ of preservations, carries out the accelerated deterioration experiment, and the result shows that the every index of kit meets the requirements fully.Consider that the freezing situation of kit takes place, kit was put into-20 ℃ of refrigerators freezing 5 days, measurement result shows that also the every index of kit is normal fully.Can draw kit from above result can preserve more than 6 months at least at 2-8 ℃.
Claims (9)
1, a kind of enzyme linked immunological kit that detects salinomycin comprises salinomycin specific antibody, coating antigen, enzyme labeling thing, salinomycin standard solution, developer, concentrated cleaning solution, stop buffer and concentrates redissolution liquid; Described coating antigen is the conjugate or the antiantibody of salinomycin haptens and carrier protein; Described enzyme labeling thing is enzyme mark antiantibody or enzyme mark salinomycin haptens; When described coating antigen was the conjugate of salinomycin haptens and carrier protein, described enzyme labeling thing was an enzyme mark antiantibody; When described coating antigen antiantibody, described enzyme labeling thing is an enzyme mark salinomycin haptens; Described salinomycin haptens is that salinomycin and p-aminobenzoic acid are mixed in the 1:1 ratio, stirring reaction, and reaction is spent the night and is obtained under the room temperature; Described antiantibody is sheep anti mouse or goat-anti rabbit antiantibody; Described concentrated redissolution liquid is for containing quality percentage composition 0.5%N, N '-dimethyl formamide, the phosphate buffer of 0.01～0.05mol/L of 1% calf serum.
2, enzyme linked immunological kit according to claim 1 is characterized in that: described salinomycin specific antibody is salinomycin monoclonal antibody or salinomycin polyclonal antibody; They all are that conjugate with salinomycin haptens and carrier protein obtains as immunogene; Described salinomycin haptens is that salinomycin and p-aminobenzoic acid are mixed in the 1:1 ratio, stirring reaction, and reaction is spent the night and is obtained under the room temperature; Described carrier protein is mouse haemocyanin, bovine serum albumin(BSA), rabbit anteserum albumen, human serum albumins, ovalbumin or hemocyanin.
3, enzyme linked immunological kit according to claim 1 and 2 is characterized in that: the marker enzyme of described enzyme labeling thing is horseradish peroxidase or alkaline phosphatase.
4, enzyme linked immunological kit according to claim 4 is characterized in that: the marker enzyme of described enzyme labeling thing is an alkaline phosphatase.
5, enzyme linked immunological kit according to claim 2 is characterized in that: the antibody of the monoclonal hybridoma strain A-2-3 CGMCC No.1609 secretion that described salinomycin monoclonal antibody is a salinomycin.
6, enzyme linked immunological kit according to claim 1 is characterized in that: described concentrated cleaning solution is a pH value 7.4, the phosphate buffer that contains 0.8%～1.2% polysorbas20,0.1% thimerosal antiseptic of 0.01mol/L; Described percentage composition is the quality percentage composition.
7, enzyme linked immunological kit according to claim 1, it is characterized in that: when marker enzyme is horseradish peroxidase, described developer is made up of colour developing liquid A liquid and colour developing liquid B liquid, colour developing liquid A liquid is hydrogen peroxide or urea peroxide, colour developing liquid B liquid is o-phenylenediamine or tetramethyl benzidine, and stop buffer is 1～2mol/L sulfuric acid, hydrochloric acid solution; When marker enzyme was alkaline phosphatase, developer was a 4-nitrophenols phosphate buffer, and stop buffer is 1～2mol/L sodium hydroxide solution.
8, enzyme linked immunological kit according to claim 1 is characterized in that: it is pH value 9.6 that the used bag of coated elisa plate is cushioned liquid, the carbonate buffer solution of 0.1mol/L; Used confining liquid is the aqueous solution that contains 3～10% horse serums, 1% inert protein; Described percentage composition is the quality percentage composition.
9, a kind of method that detects salinomycin may further comprise the steps:
1) sample pre-treatments: take by weighing 2g animal tissue homogenate and put in the centrifuge tube, adding the 10ml volume ratio is the methyl alcohol of 4:1 and the mixed liquor of 5% physiological saline, mixing; In 15 ℃, the centrifugal 10min of the above speed of 5000g; With supernatant in 15 ℃, the centrifugal 5min of 3000g; Get the 5ml supernatant, add the 10ml phenixin, vibration mixing, the centrifugal 10min of the above speed of 5000g; Upper strata liquid is dried up with nitrogen, and with the dry residue of the described concentrated redissolution liquid dissolving of the claim 1 of 4 times of 1ml dilutions, water intaking is analyzed mutually;
2) utilize the enzyme linked immunological kit test sample of arbitrary described detection salinomycin in the claim 1-8.
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| CNB2006100072857A CN100489532C (en) | 2006-02-17 | 2006-02-17 | Method for detecting salinomycin and spcial enzyme-linked immune reagent kit thereof |
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| CNB2006100072857A CN100489532C (en) | 2006-02-17 | 2006-02-17 | Method for detecting salinomycin and spcial enzyme-linked immune reagent kit thereof |
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| CN100489532C true CN100489532C (en) | 2009-05-20 |
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| CN101236200B (en) * | 2008-02-06 | 2012-09-05 | 中国计量学院 | Chlorpromazine ELISA reagent kit and its detection method |
| CN101433825B (en) * | 2008-12-03 | 2011-11-23 | 中国农业大学 | Method for extracting salinomycin compound from animal sample and special immune affinity sorbent thereof |
| CN107860724A (en) * | 2017-11-09 | 2018-03-30 | 陈军 | A kind of salbutamol detection method based on optical biosensor |
| CN109735502B (en) * | 2018-11-22 | 2023-08-18 | 黄宝兵 | Hybridoma cell strain secreting salinomycin-resistant monoclonal antibody and application thereof |
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