CN1687783A - Affinity chromatography-enzyme linked immunity test method for ractopamine and dedicated kit - Google Patents
Affinity chromatography-enzyme linked immunity test method for ractopamine and dedicated kit Download PDFInfo
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Abstract
This invention uses immunity compatible chromatography post and enzyme linked immunosorbent assay to detect the remain RactopamineHy-drochloridie in food. It belongs to immunology and food safety detect technology. The method is: prepare muti-clone antibody by immunity antigen synthesis, antigen peridium, animal immunity and antigen depuration. Then put the antigen on to agarose gel to prepare immunity compatible chromatography post. You can enrich the RactopamineHy-drochloridie in the sample by compatible post and gather the eluent of the compatible post, then use linked immunosorbent assay to determine the consistency of the RactopamineHy-drochloridie.
Description
Technical field
The invention belongs to the immuno analytical method field.In particular to a kind of Ractopamine (RCT) affinity chromatography-enzyme immunoassay method and dedicated test kit thereof.
Background technology
Ractopamine (Ractopamine) is one of beta 2-adrenergic activator (BAA) member, belongs to catecholamines (adrenaline and norepinephrine BAA) material together with " clenbuterol hydrochloride ".Just find as far back as latter stage 1950's, catecholamines (adrenaline and norepinephrine) can stimulate the acid of adipose tissue release fat, and increase proteins deposited, but up to the initial stage seventies, Catecholamine matter and analog thereof improve the ability of body nutrition deployment and trunk composition, just at first are familiar with by research department of American Cyanamid Company.Just be used for Production of Livestock and Poultry subsequently on a large scale to improve lean meat percentage, reduce fat deposition, and increase the output of milk etc.
BAA starts from the latter stage seventies as the practicality development research of the heavy partitioning agent of nutrition.Over twenties years, headed by U.S. major company of a few family and research department, developed hundreds of similar medicines,, filtered out kind surplus the growth promoting function stronger BAA ten through the hundreds of of rat, ox, sheep, pig, fowl etc. time are exceeded ten thousand (only) animal experiments.Wherein practical BAA has Ractopamine (ractopamine), clenbuterol (clenbuterol), salbutamol (salbutamol), Sai Manteluo (cimaterol), pyridinemethanol class, degreasing BAA.Its Chinese food gram dopamine, clenbuterol, salbutamol are used always aborning because of its oral efficient height.
According to Kuiper (1998) summary, 1989,135 people took place because of the edible incident that contains the beef liver PI of beta 2-adrenergic activator residue in the Spain middle part; The Aragon had 232 people again because of same former thereby PI in 1992; The poisoning of 15 beta 2-adrenergic activators takes place in area, Hispanic Barcelona of nineteen ninety-five; 1990-1991, there is the beef liver PI of 8 families because of edible residual beta 2-adrenergic activator in the area, Lyons, France; 1996, only Italy with regard to edible residual beef that the beta 2-adrenergic activator arranged of 62 causes and beef liver take place the incident of PI; Toxicity symptom has muscular tremor, palpitaition, neuroticism, headache, myalgia, arcus senilis halo vision, feels sick, vomits, has a fever, trembles.Take food and still can detect the beta 2-adrenergic activator in patient's urine after 40 hours.The people that HDH is arranged is to the residual beta 2-adrenergic activator of food particularly sensitivity (Yin Jingdong etc., 1998).Therefore European Union has taked tight measure, comes into the market by the strict control of a series of rules and regulations Ractopamine, clenbuterol.It all is illegal having the beta 2-adrenergic activator residual except that special circumstances in any animal body.
Present many countries have proposed maximum residue limit(MRL) to Ractopamine, are 0.5ng/g as Britain in the maximum residue limit of edible animal tissue, and Dutch regulation hepatic tissue maximum residue limit is 1ng/g (G.A.Mitchell, et al, 1998).And China also is defined as 1ng/g (hepatic tissue) or 1ng/ml (urine).
The application of beta 2-adrenergic activator in animal produces forbidded strictly in No. 3 literary compositions of China Ministry of Agriculture in March, 1997 [(1997) are sent out in agriculture and animal husbandry].But because aspects such as funds, technology, the behavior of abuse beta 2-adrenergic activator fails effectively to be checked always, calendar year 2001, has recurred the incident of Heyuan, Xinyi, Guangdong, tens up to a hundred people's PIs of zhejiang and other places.
Therefore, need set up a kind of sensitivity, special, simple to operate, the detection method that is suitable for batch samples screening, could carry out in time the illegal use of Ractopamine, clenbuterol class material, effectively monitor.
Present detection method mainly contains high performance liquid chromatography-mass spectrometry method (HPLC-MS) and GC-MS(gas chromatography-mass spectrography) (GC-MS).These instrument analysis technologies are generally consuming time, and valency is expensive, the mass detection difficulty, and complex operation all can not adapt to quick, easy, the inexpensive requirement of market surveillance needs.
Summary of the invention
The object of the invention is to provide a kind of sample pre-treatments simple, highly sensitive, high specificity, rapidly and efficiently, be suitable for the Ractopamine detection method of batch samples screening, and development structure is simple, easy to use, low price, portable Ractopamine detection kit.
The inventor mainly adopts affinity chromatography-enzyme immunoadsorption method (IAC-ELISA) to detect RCT; Utilize the immunoaffinity chromatography method with the enriching and purifying in addition of the RCT in the test sample earlier, by the enzyme linked immunological adsorption method it is carried out detection by quantitative again; Finished the present invention.
The invention provides a kind of method of utilizing Ractopamine RCT in immunoaffinity chromatography and the enzyme linked immunological absorption test sample, comprise step:
(1) sample pre-treatments;
(2) preparation enzyme-labelled antigen: the horseradish peroxidase of activation is mixed the preparation enzyme-labelled antigen with the RCT of purifying;
(3) preparation RCT antibody;
(4) gel of preparation coupling RCT antibody;
(5) coated elisa plate;
(6) immunoaffinity chromatography purifying: the gel dress post with coupling antibody, add sample and cross post, wash-out is collected eluent;
(7) enzyme linked immunological absorption detects: eluent is added on the ELISA Plate of antibody sandwich, and washing pats dry behind the incubation; Add enzyme-labelled antigen, washing pats dry behind the incubation; Colour developing, termination; Read the OD value with microplate reader;
(8) drawing standard curve;
(9) according to typical curve the RCT content in the eluent is carried out quantitative Analysis.
Preferably, sample pre-treatments adopts and takes by weighing animal product, adds HCl homogenate, freeze thawing, centrifuging and taking supernatant, add NaOH and reconcile pH value to 11, add isobutyl alcohol, vibration is left standstill, quantitatively draw isobutyl alcohol, water bath method adds phosphate solution dissolving precipitate again, and is frozen to be measured; Or get animals urine, and centrifugal removal impurity, it is to be measured to get supernatant.
Preferably, the preparation enzyme-labelled antigen adopts the horseradish peroxidase with activation to mix with the RCT of purifying, carries out labeled reactant in bicarbonate-carbonic acid buffer, adds not binding site of sodium borohydride reduction afterwards; Wherein prepare RCT antibody adopt with RCT and bovine serum albumin(BSA) BSA or with oralbumin OVA coupling as antigen, prepare polyclone RCT antibody or monoclonal RCT antibody.
Preferably, the method for preparing polyclone RCT antibody adopts the New Zealand white rabbit immunoprophylaxis, and immunizing antigen is BSA-RCT or OVA-RCT, bloodletting; Foreign protein and antibody immunoglobulin in the fractional precipitation serum must contain the solution of polyclone RCT antibody after centrifugal, and be frozen standby.
Preferably, the method for preparing monoclonal RCT antibody adopts the mouse inoculation immunity, and immunizing antigen is BSA-RCT or OVA-RCT, and blood sampling is measured antibody titer, extracting spleen cell; Splenocyte and myeloma cell are merged; The screening hybridoma obtains the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain; In mouse peritoneal, inject hybridoma, gather ascites, stand-by after purifying.
Preferably, the gel of preparation coupling RCT antibody adopts activation gel earlier; With gel after the activation and RCT antibody vibration mixing; Centrifuge washing is with wash-out foreign protein and unconjugated antibody; Wash the unconjugated reactive group of sealing in turn with different pH gradient solutions successively, use phosphate buffer drip washing again, stored refrigerated is standby.
Preferably, coated elisa plate adopts the RCT antibody sandwich on ELISA Plate, and refrigeration is spent the night; Washing pats dry.
Preferably, the immunoaffinity chromatography purifying adopts the gel dress post with coupling antibody, adds sample, is circulated throughout post, uses the glycine solution wash-out, collects eluent.
Preferably, enzyme linked immunological absorption detect adopt with sample eluent be added in bag by after ELISA Plate on, washing pats dry behind the incubation; Add enzyme-labelled antigen, washing pats dry behind the incubation; Then add the colour developing of substrate mixed liquor; Cessation reaction; Read the OD value with microplate reader.
The present invention also provides a kind of kit that is used for described method, it is characterized in that it comprises:
A reagent: standard RCT reagent;
C reagent: enzyme-labelled antigen reagent;
K reagent: the gel of coupling RCT antibody;
Bag is by the ELISA Plate of RCT antibody.
Preferably, described kit also comprises:
B reagent: dilution;
D reagent: enzyme-labelled antigen dilution
E reagent: the phosphate PBS solid that contains Tween-20;
F reagent: o-phenylenediamine reagent;
G reagent: sodium acetate-citrate buffer solution;
H reagent: 30%H
2O
2
I reagent: sulfuric acid solution;
J reagent: glycine solution.
Details are as follows for technical solution of the present invention:
1, sample pre-treatments:
Animal product (comprising liver, lung, the heart, kidney, muscle) takes by weighing 10g, adds 50ml 0.1mol/LHCl and carries out tissue homogenate, twice of freeze thawing, the centrifuging and taking supernatant is regulated pH value to 11 with 10mol/L NaOH, adds isobutyl alcohol 30ml again, vibration 30min, leave standstill 4h, quantitatively draw isobutyl alcohol, water bath method, add 1ml phosphate solution (PBS again, 0.01mol/L pH7.4) the dissolving precipitate is frozen to be measured;
The animal urine sample, behind the centrifugal removal impurity, it is to be measured to get supernatant.
2, preparation enzyme-labelled antigen: the horseradish peroxidase of activation is mixed with the RCT of purifying, in bicarbonate-carbonic acid buffer, carry out labeled reactant, add not binding site of sodium borohydride reduction afterwards;
3, preparation RCT antibody
With RCT and bovine serum albumin(BSA) BSA or with oralbumin OVA coupling as antigen, preparation polyclone RCT antibody or monoclonal RCT antibody;
The method that wherein prepares polyclone RCT antibody:
Prepare antigen with RCT and bovine serum albumin(BSA) (BSA) coupling or with oralbumin (OVA) coupling; Each 1mg/ml BSA-RCT of immunizing dose or OVA-RCT, first immunisation is with the complete freund adjuvant emulsification of 1ml, during booster immunization with the incomplete freund adjuvant emulsification of 1ml; New Zealand white rabbit is raised immunoprophylaxis after several weeks, and each immunity is two weeks at interval, and immunity is 5 times altogether.Directly intramuscular injection the last time, week back blood sampling detects antibody titer.Arteria carotis bloodletting afterwards; With foreign protein and antibody immunoglobulin in the ammonium sulfate precipitation serum of 30%-60%, must contain the solution of polyclone RCT antibody after centrifugal;-70 ℃ of preservations are standby.
The method that wherein prepares monoclonal RCT antibody:
Prepare antigen with RCT and bovine serum albumin(BSA) (BSA) coupling or with oralbumin (OVA) coupling; Adopt mouse as immune animal, immunizing antigen dosage is that 50ug (0.1ml) adds isopyknic complete good fortune formula adjuvant emulsion, carries out first immunisation; After January, get the isodose immunizing antigen and add incomplete good fortune formula adjuvant, booster immunization is carried out in emulsification, two exempt from after, antibody titer, extracting spleen cell are measured in blood sampling in ten days; Splenocyte is merged in 5: 1 ratios and SP2/0 myeloma cell; Adopt limiting dilution assay screening hybridoma, obtain the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain; A certain amount of hybridoma of injection is gathered ascites in mouse peritoneal, and is stand-by after purifying.
4, the sepharose-4B gel of preparation coupling antibody
With cyanogen bromide CNBr activation sepharose-4B Ago-Gel; And with low concentration HCl solution cyclic washing gel; Before coupling antibody, use phosphate buffer balance sepharose-4B gel earlier; With the RCT antibody vibration mixing of gel, antibody is adsorbed onto on the sepharose-4B gel particle of activation with suitably dilution; Centrifuge washing is with wash-out foreign protein and unconjugated antibody; Wash the unconjugated reactive group of sealing in turn with different pH solution successively, use phosphate buffer (to add 0.02%NaN again
3) drip washing, 4 ℃ of preservations are standby.
5, reagent preparation
Standard RCT reagent: the RCT standard items add dilution (PBS phosphate buffer solution pH7.4) mixing, are made into gradient solution.
Enzyme-labelled antigen reagent: enzyme-labelled antigen adds 10ml enzyme-labelled antigen dilution, and (the PBS phosphate buffer solution contains 1% gelatin, pH7.4) dissolving, mixing.
Cleansing solution preparation: in the phosphate that contains 0.1% Tween-20 (PBS) solid, add 200ml distilled water preparation cleansing solution, be used for the washing of ELISA Plate.
The preparation of substrate mixed liquor:, add 6ul 30%H again with 20ml sodium acetate-citrate buffer solution dilution 40mg o-phenylenediamine
2O
2, be mixed with the substrate mixed liquor, matching while using.
6, coated elisa plate: RCT antibody (monoclonal antibody or polyclonal antibody) is coated on the ELISA Plate, and refrigeration is spent the night; Washing pats dry; On ELISA Plate, 4 ℃ of refrigerator overnight are put in the 100ul/ hole with antibody sandwich.Add cleansing solution again, wash 3 times in the 250ul/ hole, each 3min; Be placed on the thieving paper and pat dry.
7, get 5ml coupling antibody sepharose-4B gel dress post, add the cleansing solution balance earlier, until the OD280 of effluent value less than 0.5.Add the equal-volume testing sample solution again, 4 ℃ are circulated throughout post.Use 0.1mol/L pH2.5 glycine solution wash-out afterwards, collect eluent.
8, in ELISA Plate, add testing sample eluent and RCT standard solution; 37 ℃ of incubation 0.5h; Washing afterwards pats dry; Add 100ul enzyme-labelled antigen solution, 37 ℃ of incubation 0.5h; Washing pats dry; Every hole adds 150ul substrate mixed liquor, color development at room temperature 15min; Every hole adds 50ul stop buffer (sulfuric acid solution); Microplate reader reads each hole OD value.
9, drawing standard curve: obtain each gradient solution OD value, drawing standard curve with the RCT standard items;
10, according to typical curve the RCT content in the sample eluent is carried out quantitative Analysis.
The result judges: the OD value in standard control hole raises along with the decline of RCT concentration, with the OD value comparison in sample detection hole OD value and standard control hole, if the RCT content in the identical then test sample of OD value is identical with the RCT concentration of corresponding standard solution.
The establishment of RCT detection kit of the present invention:
Kit comprises: bag is by the ELISA Plate of RCT antibody; A reagent: standard RCT reagent; B reagent: dilution; C reagent: enzyme-labelled antigen reagent; D reagent: enzyme-labelled antigen dilution; E reagent: the phosphate PBS solid that contains Tween-20; F reagent: o-phenylenediamine reagent; G reagent: sodium acetate-citrate buffer solution; H reagent: 30%H
2O
2); I reagent: sulfuric acid solution; J reagent: glycine solution; K reagent: coupling antibody sepharose-4B gel.
Wherein RCT antibody can be polyclone RCT antibody or monoclonal RCT antibody.
Enzyme-linked immuno-sorbent assay (ELISA) is a kind of method for quick, does not need complicated instrument and equipment, and is highly sensitive, high specificity, and sample pre-treatments is simple, is fit to very much the detection of a large amount of samples, is convenient to on-site supervision.Be widely used in each research field at present.Immunoaffinity chromatography purification technique (IAC) is a kind of high efficiency technical of enriching and purifying material, and IAC has improved the sensitivity that detects greatly with combining of ELISA.
Realized detection to Ractopamine with the ELISA method, the research blank in domestic this field has been filled up in the research.Last creativeness combines the specific detection that has realized Ractopamine with IAC technology and elisa technique.
Use the IAC-ELISA detection technique can reach the detectability of 1ng/mL to the Ractopamine in the sample.
The method of the invention advantage is as follows:
1, highly sensitive, detectability is low, recovery height, good reproducibility as a result;
2, high specificity: IAC and ELISA method all are based upon the specificity combination of antibody and antigen, and gained antibody cross reaction rate is extremely low;
3, detection is with low cost, need not large-scale instrument and equipment, is suitable for promoting;
4, sample pretreatment is simple, need not asepticize and handles, and sample extraction is convenient and swift;
5, by the microplate reader interpretation, the result has objectivity and unalterable feature;
6, simple, operating personnel only need the most basic experimental knowledge, can be standardized as product;
7, lack detection time, the whole operation process was less than 2.5 hours;
8, can carry out mass detection.
Description of drawings
Fig. 1: polyclonal antibody detects the canonical plotting of RCT.
Fig. 2: monoclonal antibody detects the canonical plotting of RCT.
Embodiment
Below in conjunction with instantiation, further set forth the present invention.Should be understood that these examples only to be used to the present invention is described and be not used in and limit the scope of the invention.
Embodiment 1:
1, sample pre-treatments:
Pork product (comprising liver, lung, the heart, kidney, muscle) takes by weighing 10g, adds 50ml 0.1mol/LHCl and carries out tissue homogenate, twice of freeze thawing, the centrifuging and taking supernatant is regulated pH value to 11 with 10mol/L NaOH, adds isobutyl alcohol 30ml again, vibration 30min, leave standstill 4h, quantitatively draw isobutyl alcohol, water bath method, add 1ml phosphate solution (PBS again, 0.01mol/L pH7.4) the dissolving precipitate is frozen to be measured;
The pig urine sample, behind the centrifugal removal impurity, it is to be measured to get supernatant.
2, preparation enzyme-labelled antigen: the horseradish peroxidase of activation is mixed with the RCT of purifying, in bicarbonate-carbonic acid buffer, carry out labeled reactant, add not binding site of sodium borohydride reduction afterwards;
3, preparation RCT antibody
RCT and bovine serum albumin(BSA) BSA coupling are prepared antigen; The each 1mg/ml BSA-RCT of immunizing dose, first immunisation is with the complete freund adjuvant emulsification of 1ml, during booster immunization with the incomplete freund adjuvant emulsification of 1ml; New Zealand white rabbit is raised immunoprophylaxis after several weeks, and each immunity is two weeks at interval, and immunity is 5 times altogether.Directly intramuscular injection the last time, week back blood sampling detects antibody titer.Arteria carotis bloodletting afterwards; With foreign protein and antibody immunoglobulin in the ammonium sulfate precipitation serum of 30%-60%, must contain the solution of polyclone RCT antibody after centrifugal;-70 ℃ of preservations are standby.
4, the sepharose-4B gel of preparation coupling antibody
With CNBr activation sepharose-4B gel; And with low concentration HCl solution cyclic washing gel; Before coupling antibody, use phosphate buffer balance sepharose-4B gel earlier; With the polyclone RCT antibody vibration mixing of gel, antibody is adsorbed onto on the sepharose-4B gel particle of activation with suitably dilution; Centrifuge washing is with wash-out foreign protein and unconjugated antibody; Wash the unconjugated reactive group of sealing in turn with different pH solution successively, use phosphate buffer (to add 0.02%NaN again
3) drip washing, 4 ℃ of preservations are standby.
5, reagent preparation
Standard RCT reagent: the RCT standard items add dilution, and (the PBS phosphate buffer solution, pH7.4) mixing is made into gradient solution.
Enzyme-labelled antigen reagent: enzyme-labelled antigen adds 10ml enzyme-labelled antigen dilution, and (the PBS phosphate buffer solution contains 1% gelatin, pH7.4) dissolving, mixing.
Cleansing solution preparation: in the phosphate that contains 0.1% Tween-20 (PBS) solid, add 200ml distilled water preparation cleansing solution, be used for the washing of ELISA Plate.
The preparation of substrate mixed liquor:, add 6ul 30%H again with 20ml sodium acetate-citrate buffer solution dilution 40mg o-phenylenediamine
2O
2, be mixed with the substrate mixed liquor, matching while using.
6, coated elisa plate: the RCT polyclonal antibody is coated on the ELISA Plate, and refrigeration is spent the night; Washing pats dry; On ELISA Plate, 4 ℃ of refrigerator overnight are put in the 100ul/ hole with antibody sandwich.Add cleansing solution again, wash 3 times in the 250ul/ hole, each 3min; Be placed on the thieving paper and pat dry.
7, get 5ml coupling antibody sepharose-4B gel dress post, add the cleansing solution balance earlier, until the OD280 of effluent value less than 0.5.Add the equal-volume testing sample solution again, 4 ℃ are circulated throughout post.Use 0.1mol/L pH2.5 glycine solution wash-out afterwards, collect eluent.
8, in ELISA Plate, add testing sample eluent and RCT standard solution; 37 ℃ of incubation 0.5h; Washing afterwards pats dry; Add 100ul enzyme-labelled antigen solution, 37 ℃ of incubation 0.5h; Washing pats dry; Every hole adds 150ul substrate mixed liquor, color development at room temperature 15min; Every hole adds 50ul stop buffer (sulfuric acid solution); Microplate reader reads each hole OD value.
9, drawing standard curve: obtain each gradient solution OD value, drawing standard curve with the RCT standard items; The results are shown in Figure 1.
10, according to typical curve the RCT content in the sample eluent is carried out quantitative Analysis.
The result judges: the OD value in standard control hole raises along with the decline of RCT concentration, the OD value in sample detection hole OD value and standard control hole is compared, if the RCT content in the identical then test sample of OD value is identical with the RCT concentration of corresponding standard solution, testing result is as follows:
| Sample | Muscle | Liver | Kidney | Urine |
| Testing result (ng/mL) | ????5.4 | ????8.7 | ????7.5 | ????4.8 |
Embodiment 2:
The establishment of RCT detection kit:
Kit comprises: the ELISA Plate of Sheet clone RCT antibody; A reagent: standard RCT reagent; B reagent: dilution; C reagent: enzyme-labelled antigen reagent; D reagent: enzyme-labelled antigen dilution; E reagent: the phosphate PBS solid that contains Tween-20; F reagent: o-phenylenediamine reagent; G reagent: sodium acetate-citrate buffer solution; H reagent: 30%H
2O
2); I reagent: sulfuric acid solution; J reagent: glycine solution; K reagent: the sepharose-4B gel of coupling monoclonal RCT antibody.
Embodiment 3:
The establishment of RCT detection kit:
Kit comprises: bag is by the ELISA Plate of polyclone RCT antibody; A reagent: standard RCT reagent; B reagent: dilution; C reagent: enzyme-labelled antigen reagent; D reagent: enzyme-labelled antigen dilution; E reagent: the phosphate PBS solid that contains Tween-20; F reagent: o-phenylenediamine reagent; G reagent: sodium acetate-citrate buffer solution; H reagent: 30%H
2O
2); I reagent: sulfuric acid solution; J reagent: glycine solution; K reagent: the sepharose-4B gel of coupling polyclone RCT antibody.
Embodiment 4: Application Example 2 kits detect
Detection with pork product and pig urine sample RCT is an example, and the concrete detection step of kit is described.
1, sample pre-treatments:
Take by weighing pork product and (comprise liver, lung, the heart, kidney, muscle) 10g adds 50ml, and 0.1mol/L HCl carries out tissue homogenate, freeze thawing twice, the centrifuging and taking supernatant is regulated pH value to 11 with 10mol/L NaOH, add isobutyl alcohol 30ml again, vibration 30min leaves standstill 4h, quantitatively draw isobutyl alcohol, water bath method adds 1ml phosphate solution (PBS again, 0.01mol/L pH7.4) the dissolving precipitate is frozen to be measured.
The pig urine sample, behind the centrifugal removal impurity, it is to be measured to get supernatant.
2, the preparation of reagent
Standard RCT reagent: A reagent adds B reagent mixing, is made into gradient solution.
Enzyme-labelled antigen reagent: C reagent adds 10ml D agent dissolves, mixing.
E cleansing solution preparation: in E reagent, add 200ml distilled water preparation E cleansing solution, be used for the washing of ELISA Plate.
The preparation of substrate mixed liquor: with 20ml G reagent dilution 40mg F reagent, add 6ul H reagent again, be mixed with the substrate mixed liquor, matching while using.
3, get 5ml K reagent dress post, add E cleansing solution balance earlier, until the OD of effluent
280Value is less than 0.5.Add the equal-volume testing sample solution again, 4 ℃ are circulated throughout post.Use J reagent wash-out afterwards, collect eluent.
4, in the ELISA Plate of Sheet clone RCT antibody, add testing sample eluent and RCT standard solution; 37 ℃ of incubation 0.5h; Washing afterwards pats dry.
ELISA Plate, the 1st row's standard control adds the RCT gradient solution in preceding 11 holes, addition 100ul, the 12nd hole adds blank.All the other each holes then add sample eluent to be measured, addition 100ul.
5, add 100ul enzyme-labelled antigen solution, 37 ℃ of incubation 0.5h; Washing pats dry; Every hole adds 150ul substrate mixed liquor, color development at room temperature 15min; Every hole adds 50ul I reagent and stops; Microplate reader reads each hole OD value.
6, drawing standard curve: obtain each gradient solution OD value, drawing standard curve with the RCT standard items; The detection of the method is limited to 0.2ng/mL.Result such as Fig. 2.
7, according to typical curve the RCT content in the sample eluent is carried out quantitative Analysis.
The result judges: the OD value in standard control hole raises along with the decline of RCT concentration, and the 12nd hole OD value should be the highest; The OD value in sample detection hole OD value and standard control hole is compared, if the RCT content in the identical then test sample of OD value is identical with the RCT concentration of corresponding standard solution, testing result is as follows:
| Sample | Muscle | Liver | Kidney | Urine |
| Testing result (ng/mL) | ????5.8 | ????9.1 | ????7.9 | ????5.2 |
Claims (10)
1, a kind of method of utilizing Ractopamine RCT in immunoaffinity chromatography and the enzyme linked immunological absorption test sample comprises step:
(1) sample pre-treatments;
(2) preparation enzyme-labelled antigen: the horseradish peroxidase of activation is mixed the preparation enzyme-labelled antigen with the RCT of purifying;
(3) preparation RCT antibody;
(4) gel of preparation coupling RCT antibody;
(5) coated elisa plate;
(6) immunoaffinity chromatography purifying: the gel dress post with coupling antibody, add sample and cross post, wash-out is collected eluent;
(7) enzyme linked immunological absorption detects: eluent is added on the ELISA Plate of antibody sandwich, and washing pats dry behind the incubation; Add enzyme-labelled antigen, washing pats dry behind the incubation; Colour developing, termination; Read the OD value with microplate reader;
(8) drawing standard curve;
(9) according to typical curve the RCT content in the eluent is carried out quantitative Analysis.
2, according to the described method of claim 1, wherein sample pre-treatments adopts and takes by weighing animal product, adds HCl homogenate, freeze thawing, the centrifuging and taking supernatant adds NaOH and reconciles pH value to 11, adds isobutyl alcohol, vibration, leave standstill, quantitatively draw isobutyl alcohol, water bath method, add phosphate solution dissolving precipitate again, frozen to be measured; Or get animals urine, and centrifugal removal impurity, it is to be measured to get supernatant.
3, method according to claim 1 wherein prepares enzyme-labelled antigen and adopts the horseradish peroxidase with activation to mix with the RCT of purifying, carries out labeled reactant in bicarbonate-carbonic acid buffer, adds not binding site of sodium borohydride reduction afterwards; Wherein prepare RCT antibody adopt with RCT and bovine serum albumin(BSA) BSA or with oralbumin OVA coupling as antigen, prepare polyclone RCT antibody or monoclonal RCT antibody.
4, method according to claim 3, the method that wherein prepares polyclone RCT antibody adopts the New Zealand white rabbit immunoprophylaxis, and immunizing antigen is BSA-RCT or OVA-RCT, bloodletting; Foreign protein and antibody immunoglobulin in the fractional precipitation serum must contain the solution of polyclone RCT antibody after centrifugal, and be frozen standby; The method that wherein prepares monoclonal RCT antibody adopts the mouse inoculation immunity, and immunizing antigen is BSA-RCT or OVA-RCT, and blood sampling is measured antibody titer, extracting spleen cell; Splenocyte and myeloma cell are merged; The screening hybridoma obtains the monoclonal antibody of complete homogeneity and stable monoclonal hybridoma strain; In mouse peritoneal, inject hybridoma, gather ascites, stand-by after purifying.
5, method according to claim 1, the gel that wherein prepares coupling RCT antibody adopts activation gel earlier; With gel after the activation and RCT antibody vibration mixing; Centrifuge washing is with wash-out foreign protein and unconjugated antibody; Wash the unconjugated reactive group of sealing in turn with different pH gradient solutions successively, use phosphate buffer drip washing again, stored refrigerated is standby.
6, method according to claim 1, wherein coated elisa plate adopts the RCT antibody sandwich on ELISA Plate, and refrigeration is spent the night; Washing pats dry.
7, method according to claim 1, wherein the immunoaffinity chromatography purifying adopts the gel dress post with coupling antibody, adds sample, is circulated throughout post, uses the glycine solution wash-out, collects eluent.
8, method according to claim 1, wherein enzyme linked immunological absorption detect adopt with sample eluent be added in bag by after ELISA Plate on, washing pats dry behind the incubation; Add enzyme-labelled antigen, washing pats dry behind the incubation; Then add the colour developing of substrate mixed liquor; Cessation reaction; Read the OD value with microplate reader.
9, a kind of kit that is used for the described arbitrary method of claim 1-8 is characterized in that it comprises:
A reagent: standard RCT reagent;
C reagent: enzyme-labelled antigen reagent;
K reagent: the gel of coupling RCT antibody;
Bag is by the ELISA Plate of RCT antibody.
10, kit according to claim 9 is characterized in that it also comprises:
B reagent: dilution;
D reagent: enzyme-labelled antigen dilution
E reagent: the phosphate PBS solid that contains Tween-20;
F reagent: o-phenylenediamine reagent;
G reagent: sodium acetate-citrate buffer solution;
H reagent: 30%H
2O
2
I reagent: sulfuric acid solution;
J reagent: glycine solution.
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Cited By (7)
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| CN101509923A (en) * | 2009-02-16 | 2009-08-19 | 安徽农业大学 | Diagnosis sheet for detecting antigen/antibody and method for making same |
| CN100570323C (en) * | 2006-08-08 | 2009-12-16 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of ractopamine column and its production and use |
| CN101241134B (en) * | 2008-01-18 | 2012-05-30 | 华南农业大学 | ELISA kit for detecting ractopamine residue and method of use thereof |
| CN101315379B (en) * | 2008-07-15 | 2012-08-08 | 天津九鼎生物工程有限公司 | Reagent kit for detecting Ractopamine and application thereof |
| CN102778564A (en) * | 2012-05-31 | 2012-11-14 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting ractopamine |
| CN103100237A (en) * | 2012-12-31 | 2013-05-15 | 南宁市蓝光生物技术有限公司 | Mini-type efficient ractopamine immunoaffinity purification enriching column preparation and application |
| CN107192820A (en) * | 2016-11-28 | 2017-09-22 | 天津科技大学 | The preparation method of the immunoaffinity gel detection column of protein anabolic hormone in a kind of detection food |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100570323C (en) * | 2006-08-08 | 2009-12-16 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of ractopamine column and its production and use |
| CN101241134B (en) * | 2008-01-18 | 2012-05-30 | 华南农业大学 | ELISA kit for detecting ractopamine residue and method of use thereof |
| CN101315379B (en) * | 2008-07-15 | 2012-08-08 | 天津九鼎生物工程有限公司 | Reagent kit for detecting Ractopamine and application thereof |
| CN101509923A (en) * | 2009-02-16 | 2009-08-19 | 安徽农业大学 | Diagnosis sheet for detecting antigen/antibody and method for making same |
| CN102778564A (en) * | 2012-05-31 | 2012-11-14 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting ractopamine |
| CN102778564B (en) * | 2012-05-31 | 2014-06-04 | 华中农业大学 | Monoclonal antibody, enzyme linked immunosorbent assay method and kit for detecting ractopamine |
| CN103100237A (en) * | 2012-12-31 | 2013-05-15 | 南宁市蓝光生物技术有限公司 | Mini-type efficient ractopamine immunoaffinity purification enriching column preparation and application |
| CN103100237B (en) * | 2012-12-31 | 2015-12-02 | 南宁市蓝光生物技术有限公司 | Micro high efficiency Ractopamine immunoaffinity purification enriching column preparations and applicatio |
| CN107192820A (en) * | 2016-11-28 | 2017-09-22 | 天津科技大学 | The preparation method of the immunoaffinity gel detection column of protein anabolic hormone in a kind of detection food |
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