CN1687783A - Affinity chromatography-enzyme linked immunity test method for ractopamine and dedicated kit - Google Patents

Abstract

The invention relates to a method for detecting ractopamine RCT. More precisely, it uses an immunoaffinity chromatography column and an enzyme-linked immunosorbent method to detect ractopamine residues in food, and belongs to the technical field of immunology and food safety detection. The method of the invention is as follows: firstly, the polyclonal antibody is prepared through the steps of synthesizing the immune antigen, coating the antigen, animal immunization, antibody purification, etc., and then cross-linking the antibody to the agarose gel to prepare the immunoaffinity chromatography column. After the tested sample is processed, it passes through the affinity column to enrich the ractopamine therein; the eluate from the affinity column is collected for enzyme-linked immunosorbent assay to determine the content of ractopamine therein.

Description

Affinity chromatography-ELISA detection method and special kit for ractopamine

technical field

The invention belongs to the technical field of immune analysis. Specifically, it relates to a ractopamine (RCT) affinity chromatography-ELISA detection method and a special detection kit thereof.

Background technique

Ractopamine is one of the members of β2-adrenergic agonist (BAA), and it belongs to catecholamines (adrenaline and norepinephrine BAA) substances like "leptbuterol". As early as the late 1950s, it was discovered that catecholamines (epinephrine and norepinephrine) could stimulate fatty tissue to release fatty acids and increase protein deposition, but it was not until the early 1970s that catecholamines and their analogs improved the body's nutritional allocation The ability to form and carcass was first recognized by the American Cyamide Research Department. Then it is used on a large scale in livestock and poultry production to increase lean meat percentage, reduce fat deposition, and increase milk production.

The practical development and research of BAA as a nutrient redistribution agent began in the late 1970s. Over the past two decades, led by several large companies and research laboratories in the United States, hundreds of structurally similar drugs have been developed. After hundreds of tests on rats, cattle, sheep, pigs, poultry, etc., more than ten thousand (only) animals More than ten kinds of BAA with strong growth-promoting effect were screened out. Among them, practical BAAs include ractopamine, clenbuterol, salbutamol, cimaterol, pyridinemethanol, and defatted BAA. Among them, ractopamine, clenbuterol, and albuterol are commonly used in production because of their high oral efficiency.

According to Kuiper (1998), in 1989, 135 people were collectively poisoned by eating beef liver containing β2-adrenergic agonist residues in central Spain; in 1992, another 232 people were collectively poisoned by the same reason in northern Spain ; In 1995, 15 cases of β2-adrenergic agonist poisoning occurred in Barcelona, Spain; from 1990 to 1991, 8 families in Lyon, France were collectively poisoned by eating beef liver with residual β2-adrenergic agonists; 1996 In 2010, there were 62 mass poisoning incidents in Italy alone due to consumption of beef and beef liver with residual β2-adrenergic agonists; symptoms of poisoning include muscle tremors, palpitations, nervousness, headache, muscle pain, dizziness, nausea, and vomiting , fever, tremors. Beta2-adrenergic agonists can still be detected in the patient's urine 40 hours after eating. People with a history of heart disease are particularly sensitive to food residues of β2-adrenergic agonists (Yin Jingdong et al., 1998). Therefore, the European Union has taken strict measures to strictly control the entry of ractopamine and clenbuterol through a series of regulations. It is illegal to have β2-adrenergic agonist residues in any animal except in special circumstances.

At present, many countries have proposed the maximum residue limit for ractopamine, such as the maximum residue in animal edible tissue in the UK is 0.5ng/g, and the Netherlands stipulates that the maximum residue in liver tissue is 1ng/g (G.A.Mitchell, et al, 1998). In my country, it is also stipulated as 1ng/g (liver tissue) or 1ng/ml (urine).

In March 1997, the Ministry of Agriculture of my country issued a document No. 3 [Nong Mu Fa (1997)] to strictly prohibit the application of β2-adrenergic agonists in animal production. However, due to financial, technical and other reasons, the abuse of β2-adrenergic agonists has not been effectively curbed. In 2001, more than a dozen collective crimes involving hundreds of people occurred successively in Guangdong Heyuan, Guangdong Xinyi, Zhejiang and other places. poisoning incidents.

Therefore, it is necessary to establish a detection method that is sensitive, specific, easy to operate, and suitable for large-scale sample screening in order to monitor the illegal use of ractopamine and clenbuterol in a timely and effective manner.

The current detection methods mainly include high performance liquid chromatography-mass spectrometry (HPLC-MS) and gas chromatography-mass spectrometry (GC-MS). These instrumental analysis techniques are generally time-consuming, expensive, difficult to detect in large quantities, and cumbersome to operate, all of which cannot meet the requirements of fast, simple, and cheap market supervision.

Contents of the invention

The purpose of the present invention is to provide a ractopamine detection method that is simple in sample pretreatment, high in sensitivity, strong in specificity, fast and efficient, and suitable for large-scale sample screening, and develops ractopamine with simple structure, convenient use, cheap price and portability Detection kit.

The inventor mainly adopts affinity chromatography-enzyme-linked immunosorbent method (IAC-ELISA) to detect RCT; utilize immunoaffinity chromatography method to carry out enrichment and purification to the RCT in the detection sample, then pass ELISA method to its RCT. Carry out quantitative detection; Complete the present invention.

The invention provides a method for detecting ractopamine RCT in a sample by using immunoaffinity chromatography and enzyme-linked immunosorbent adsorption, comprising the steps of:

(1) Sample pretreatment;

(2) Prepare enzyme-labeled antigen: mix activated horseradish peroxidase with purified RCT to prepare enzyme-labeled antigen;

(3) Preparation of RCT antibody;

(4) prepare the gel of coupling RCT antibody;

(5) coated microtiter plate;

(6) Immunoaffinity chromatography purification: the gel coupled with the antibody is packed into a column, the sample is added to pass through the column, eluted, and the eluate is collected;

(7) Enzyme-linked immunosorbent assay: Add the eluate to the antibody-coated ELISA plate, wash and pat dry after incubation; add enzyme-labeled antigen, wash and pat dry after incubation; develop color, terminate; The standard instrument reads the OD value;

(8) Draw a standard curve;

(9) Quantitatively calculate the RCT content in the eluate according to the standard curve.

Preferably, the sample pretreatment is by weighing animal products, adding HCl to homogenate, freezing and thawing, centrifuging to take the supernatant, adding NaOH to adjust the pH value to 11, adding isobutanol, shaking, standing still, quantitatively absorbing isobutanol, water bath Evaporate to dryness, then add phosphate solution to dissolve the precipitate, and freeze it for testing; or take animal urine, centrifuge to remove impurities, and take the supernatant for testing.

Preferably, the enzyme-labeled antigen is prepared by mixing activated horseradish peroxidase with purified RCT, performing a labeling reaction in a carbonic acid buffer system, and then adding sodium borohydride to reduce the unbound site; wherein RCT antibody is prepared using RCT Conjugate bovine serum albumin BSA or ovalbumin OVA as an antigen to prepare polyclonal RCT antibody or monoclonal RCT antibody.

Preferably, the method for preparing the polyclonal RCT antibody is to inoculate and immunize New Zealand white rabbits, the immunization antigen is BSA-RCT or OVA-RCT, and bloodletting; the miscellaneous proteins and antibody immunoglobulins in the serum are fractionated and precipitated, and polyclonal RCT is obtained after centrifugation. The antibody solution was frozen for later use.

Preferably, the method for preparing the monoclonal RCT antibody is to inoculate and immunize mice, the immune antigen is BSA-RCT or OVA-RCT, blood is collected, the antibody titer is determined, and splenocytes are taken; splenocytes are fused with myeloma cells; screening Hybridoma cells, to obtain completely homogeneous monoclonal antibodies and stable monoclonal hybridoma cell lines; intraperitoneally inject hybridoma cells in mice, collect ascites, and use after purification.

Preferably, the gel coupled with the RCT antibody is prepared by first activating the gel; shaking and mixing the activated gel with the RCT antibody; centrifuging and washing to elute foreign proteins and unbound antibodies; alternately using different pH gradient solutions Wash and block unbound active groups, rinse with phosphate buffer, and store in cold storage for later use.

Preferably, the coated microtiter plate is coated with the RCT antibody on the microtiter plate, refrigerated overnight; washed and patted dry.

Preferably, the immunoaffinity chromatography purification is performed by packing the gel coupled with the antibody into a column, adding a sample, circulating through the column, eluting with a glycine solution, and collecting the eluate.

Preferably, the enzyme-linked immunosorbent assay adopts the method of adding the sample eluent to the coated ELISA plate, washing and patting dry after incubation; adding enzyme-labeled antigen, washing and patting dry after incubation; then adding the substrate mixture Color development; stop the reaction; read the OD value with a microplate reader.

The present invention also provides a kit for the method, characterized in that it comprises:

A reagent: standard RCT reagent;

C reagent: enzyme-labeled antigen reagent;

K reagent: gel coupled with RCT antibody;

A microtiter plate coated with RCT antibody.

Preferably, the kit also includes:

B reagent: diluent;

D reagent: enzyme-labeled antigen diluent

E reagent: phosphate PBS solid containing Tween-20;

F reagent: o-phenylenediamine reagent;

G reagent: sodium acetate-citric acid buffer;

H reagent: 30% H ₂ O ₂ ;

I reagent: sulfuric acid solution;

J reagent: glycine solution.

Technical solution of the present invention is described in detail as follows:

1. Sample pretreatment:

Weigh 10g of animal products (including liver, lung, heart, kidney, muscle), add 50ml 0.1mol/L HCl for tissue homogenization, freeze and thaw twice, centrifuge to get the supernatant, adjust the pH value to 11 with 10mol/L NaOH, Then add 30ml of isobutanol, shake for 30min, let it stand for 4h, quantitatively absorb isobutanol, evaporate to dryness in a water bath, then add 1ml of phosphate solution (PBS, 0.01mol/L, pH7.4) to dissolve the eluate, and freeze it for testing ;

Animal urine samples were centrifuged to remove impurities, and the supernatant was taken for testing.

2. Preparation of enzyme-labeled antigen: mix activated horseradish peroxidase with purified RCT, perform labeling reaction in carbonic acid buffer system, and then add sodium borohydride to reduce unbound sites;

3. Preparation of RCT antibody

Coupling RCT with bovine serum albumin BSA or ovalbumin OVA as an antigen to prepare polyclonal RCT antibody or monoclonal RCT antibody;

Wherein the method for preparing the polyclonal RCT antibody:

Coupling RCT with bovine serum albumin (BSA) or ovalbumin (OVA) to prepare antigen; immunization dose 1mg/ml BSA-RCT or OVA-RCT each time, emulsified with 1ml complete Freund's adjuvant for the first immunization , emulsified with 1ml incomplete Freund's adjuvant for booster immunization; New Zealand white rabbits were raised for several weeks and then immunized, with an interval of two weeks between each immunization, and a total of 5 immunizations. After the last direct intramuscular injection, blood was collected to detect the antibody titer one week later. Afterwards, the carotid artery was bled; miscellaneous proteins and antibody immunoglobulins in the serum were fractionally precipitated with 30%-60% ammonium sulfate, and centrifuged to obtain a solution containing polyclonal RCT antibodies; stored at -70°C for later use.

Wherein the method for preparing the monoclonal RCT antibody:

Coupling RCT with bovine serum albumin (BSA) or ovalbumin (OVA) to prepare antigen; using mice as immunized animals, the dose of immunizing antigen is 50ug (0.1ml) plus an equal volume of complete Freudian adjuvant Emulsify for the first immunization; one month later, take the same amount of immune antigen plus incomplete Ford’s adjuvant, emulsify, and carry out booster immunization. After the second immunization, blood is collected ten days to determine the antibody titer, and spleen cells are collected; Fusion with SP2/0 myeloma cells at a ratio of 5:1; hybridoma cells were screened by limiting dilution method to obtain completely homogeneous monoclonal antibodies and stable monoclonal hybridoma cell lines; a certain amount of Ascites was collected for hybridoma cells and purified for use.

4. Preparation of sepharose-4B gel coupled with antibody

Activate sepharose-4B agarose gel with cyanogen bromide CNBr; wash the gel repeatedly with low concentration HCl solution; Shake and mix well to make the antibody adsorb to the activated sepharose-4B gel particles; centrifuge and wash to elute the impurity protein and unbound antibody; wash with different pH solutions in turn to block the unbound active group, and then use phosphate buffer solution (with the addition of 0.02% NaN ₃ ), and stored at 4°C for future use.

5. Preparation of reagents

Standard RCT reagent: RCT standard plus diluent (PBS phosphate buffer solution pH 7.4) and mix well to make a gradient solution.

Enzyme-labeled antigen reagent: add 10ml enzyme-labeled antigen diluent (PBS phosphate buffer solution, containing 1% gelatin, pH7.4) to dissolve the enzyme-labeled antigen, and mix well.

Washing solution preparation: Add 200ml of distilled water to the phosphate (PBS) solid containing 0.1% Tween-20 to prepare a washing solution for washing the microtiter plate.

Preparation of the substrate mixture: dilute 40 mg of o-phenylenediamine with 20 ml of sodium acetate-citric acid buffer solution, and then add 6 ul of 30% H ₂ O ₂ to prepare the substrate mixture, which is ready-to-use.

6. Coating microtiter plate: coat RCT antibody (monoclonal antibody or polyclonal antibody) on microtiter plate, refrigerate overnight; wash and pat dry; coat antibody on microtiter plate, 100ul/well, put Into 4 ℃ refrigerator overnight. Then add washing solution, 250ul/well, wash 3 times, 3min each time; put on absorbent paper and pat dry.

7. Take 5ml of conjugated antibody sepharose-4B gel and pack it into the column, add washing solution to balance until the OD280 value of the effluent is less than 0.5. Then add an equal volume of the sample solution to be tested, and circulate through the column at 4°C. Then eluted with 0.1mol/L pH2.5 glycine solution, and collected the eluate.

8. Add the eluent of the sample to be tested and the RCT standard solution to the microplate; incubate at 37°C for 0.5h; then wash and pat dry; add 100ul enzyme-labeled antigen solution, incubate at 37°C for 0.5h; wash and pat dry; Add 150ul of substrate mixture to each well, and develop color at room temperature for 15min; add 50ul of stop solution (sulfuric acid solution) to each well; read the OD value of each well with a microplate reader.

9. Draw a standard curve: use the OD value of each gradient solution obtained from the RCT standard to draw a standard curve;

10. Quantitatively calculate the RCT content in the sample eluate according to the standard curve.

Result judgment: The OD value of the standard control well increases with the decrease of the RCT concentration. Compare the OD value of the sample detection well with the OD value of the standard control well. If the OD value is the same, the RCT content in the tested sample is the same as the corresponding standard solution. RCTs with the same concentration.

The formation of RCT detection kit of the present invention:

The kit includes: ELISA plate coated with RCT antibody; A reagent: standard RCT reagent; B reagent: diluent; C reagent: enzyme-labeled antigen reagent; D reagent: enzyme-labeled antigen diluent; E reagent: containing Tween- 20 phosphate PBS solid; F reagent: o-phenylenediamine reagent; G reagent: sodium acetate-citric acid buffer; H reagent: 30% H ₂ O ₂ ); I reagent: sulfuric acid solution; J reagent: glycine solution; K reagent: conjugated antibody sepharose-4B gel.

Wherein the RCT antibody can be a polyclonal RCT antibody or a monoclonal RCT antibody.

Enzyme-linked immunosorbent assay (ELISA) is a rapid detection method that does not require complex equipment, has high sensitivity, strong specificity, and simple sample pretreatment. It is very suitable for the detection of a large number of samples and is convenient for on-site monitoring. It has been widely used in various research fields. Immunoaffinity chromatography (IAC) is an efficient technology for enriching and purifying substances. The combination of IAC and ELISA greatly improves the sensitivity of detection.

The detection of ractopamine was realized by ELISA method, and this research filled the research gap in this field in China. Finally, the specific detection of ractopamine was realized by creatively combining IAC technology with ELISA technology.

Using IAC-ELISA detection technology, the detection limit of ractopamine in the sample can reach 1ng/mL.

Method advantage of the present invention is as follows:

1. High sensitivity, low detection limit, high recovery rate and good repeatability of results;

2. Strong specificity: Both IAC and ELISA methods are based on the specific combination of antibodies and antigens, and the obtained antibody cross-reactivity rate is extremely low;

3. The detection cost is low, no large-scale equipment is required, and it is suitable for promotion;

4. The sample pretreatment is simple, without aseptic treatment, and the sample extraction is convenient and fast;

5. Interpreted by a microplate reader, the results are objective and unchangeable;

6. Simple and easy to operate, operators only need the most basic experimental knowledge, and can be standardized into products;

7. The detection time is short, the whole operation process is less than 2.5 hours;

8. Can carry out mass inspection.

Description of drawings

Figure 1: Standard curves for polyclonal antibody detection RCTs.

Figure 2: Standard curves for monoclonal antibody detection RCTs.

Detailed ways

Below in conjunction with specific example, further set forth the present invention. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

Example 1:

1. Sample pretreatment:

Weigh 10g of pork products (including liver, lung, heart, kidney, muscle), add 50ml 0.1mol/L HCl for tissue homogenization, freeze and thaw twice, centrifuge to get the supernatant, adjust the pH value to 11 with 10mol/L NaOH, Then add 30ml of isobutanol, shake for 30min, let it stand for 4h, quantitatively absorb isobutanol, evaporate to dryness in a water bath, then add 1ml of phosphate solution (PBS, 0.01mol/L, pH7.4) to dissolve the eluate, and freeze it for testing ;

Pig urine samples were centrifuged to remove impurities, and the supernatant was taken for testing.

2. Preparation of enzyme-labeled antigen: mix activated horseradish peroxidase with purified RCT, perform labeling reaction in carbonic acid buffer system, and then add sodium borohydride to reduce unbound sites;

3. Preparation of RCT antibody

Coupling RCT with bovine serum albumin BSA to prepare antigen; immunization dose 1mg/ml BSA-RCT each time, emulsified with 1ml complete Freund's adjuvant for the first immunization, emulsified with 1ml incomplete Freund's adjuvant for booster immunization; New Zealand The white rabbits were immunized after being fed for several weeks, with a two-week interval between each immunization, and a total of 5 immunizations. After the last direct intramuscular injection, blood was collected to detect the antibody titer one week later. Afterwards, the carotid artery was bled; miscellaneous proteins and antibody immunoglobulins in the serum were fractionally precipitated with 30%-60% ammonium sulfate, and centrifuged to obtain a solution containing polyclonal RCT antibodies; stored at -70°C for later use.

4. Preparation of sepharose-4B gel coupled with antibody

Activate the sepharose-4B gel with CNBr; wash the gel repeatedly with low-concentration HCl solution; equilibrate the sepharose-4B gel with phosphate buffer before coupling the antibody; shake and mix the gel with an appropriately diluted polyclonal RCT antibody , so that the antibody is adsorbed to the activated sepharose-4B gel particles; centrifugal washing to elute the foreign protein and unbound antibody; sequentially wash with different pH solutions to block the unbound active group, and then use phosphate buffer (addition 0.02% NaN ₃ ), and stored at 4°C for later use.

5. Preparation of reagents

Standard RCT reagent: RCT standard plus diluent (PBS phosphate buffer solution, pH 7.4) and mix well to prepare a gradient solution.

Enzyme-labeled antigen reagent: add 10ml enzyme-labeled antigen diluent (PBS phosphate buffer solution, containing 1% gelatin, pH7.4) to dissolve the enzyme-labeled antigen, and mix well.

Washing solution preparation: Add 200ml of distilled water to the phosphate (PBS) solid containing 0.1% Tween-20 to prepare a washing solution for washing the microtiter plate.

Preparation of the substrate mixture: dilute 40 mg of o-phenylenediamine with 20 ml of sodium acetate-citric acid buffer solution, and then add 6 ul of 30% H ₂ O ₂ to prepare the substrate mixture, which is ready-to-use.

6. Enzyme plate coating: Coat the RCT polyclonal antibody on the plate and refrigerate overnight; wash and pat dry; coat the antibody on the plate, 100ul/well, and put it in a 4°C refrigerator overnight. Then add washing solution, 250ul/well, wash 3 times, 3min each time; put on absorbent paper and pat dry.

7. Take 5ml of conjugated antibody sepharose-4B gel and pack it into the column, add washing solution to balance until the OD280 value of the effluent is less than 0.5. Then add an equal volume of the sample solution to be tested, and circulate through the column at 4°C. Then eluted with 0.1mol/L pH2.5 glycine solution, and collected the eluate.

8. Add the eluent of the sample to be tested and the RCT standard solution to the microplate; incubate at 37°C for 0.5h; then wash and pat dry; add 100ul enzyme-labeled antigen solution, incubate at 37°C for 0.5h; wash and pat dry; Add 150ul of substrate mixture to each well, and develop color at room temperature for 15min; add 50ul of stop solution (sulfuric acid solution) to each well; read the OD value of each well with a microplate reader.

9. Draw a standard curve: use the OD value of each gradient solution obtained from the RCT standard to draw a standard curve; the results are shown in Figure 1.

10. Quantitatively calculate the RCT content in the sample eluate according to the standard curve.

Result judgment: The OD value of the standard control well increases with the decrease of the RCT concentration. Compare the OD value of the sample detection well with the OD value of the standard control well. If the OD value is the same, the RCT content in the tested sample is the same as the corresponding standard solution. The same concentration of RCT, the test results are as follows:

samples muscle liver kidney Urine Test result (ng/mL) 5.4 8.7 7.5 4.8

Example 2:

The composition of the RCT detection kit:

The kit includes: ELISA plate coated with monoclonal RCT antibody; A reagent: standard RCT reagent; B reagent: diluent; C reagent: enzyme-labeled antigen reagent; D reagent: enzyme-labeled antigen diluent; E reagent: containing Tween -20 phosphate PBS solid; F reagent: o-phenylenediamine reagent; G reagent: sodium acetate-citric acid buffer; H reagent: 30% H ₂ O ₂ ); I reagent: sulfuric acid solution; J reagent: glycine solution ; Reagent K: sepharose-4B gel coupled with monoclonal RCT antibody.

Example 3:

The composition of the RCT detection kit:

The kit includes: ELISA plate coated with polyclonal RCT antibody; A reagent: standard RCT reagent; B reagent: diluent; C reagent: enzyme-labeled antigen reagent; D reagent: enzyme-labeled antigen diluent; Phosphate PBS solid at temperature -20; F reagent: o-phenylenediamine reagent; G reagent: sodium acetate-citric acid buffer; H reagent: 30% H ₂ O ₂ ); I reagent: sulfuric acid solution; J reagent: glycine Solution; Reagent K: sepharose-4B gel conjugated polyclonal RCT antibody.

Embodiment 4: application embodiment 2 kit detects

Taking the detection of pork products and pig urine sample RCT as an example, the specific detection steps of the kit are illustrated.

1. Sample pretreatment:

Weigh 10g of pork products (including liver, lung, heart, kidney, muscle), add 50ml, 0.1mol/L HCl for tissue homogenization, freeze and thaw twice, centrifuge to get the supernatant, adjust the pH value to 11. Add 30ml of isobutanol, shake for 30min, let it stand for 4h, absorb isobutanol quantitatively, evaporate to dryness in a water bath, add 1ml of phosphate solution (PBS, 0.01mol/L, pH7.4) to dissolve the eluate, and freeze to be tested.

Pig urine samples were centrifuged to remove impurities, and the supernatant was taken for testing.

2. Preparation of reagents

Standard RCT reagents: mix reagent A and reagent B to form a gradient solution.

Enzyme-labeled antigen reagent: C reagent plus 10ml D reagent to dissolve, mix well.

Preparation of washing solution E: add 200ml of distilled water to reagent E to prepare washing solution E, which is used for washing the microtiter plate.

Preparation of the substrate mixture: dilute 40mg of F reagent with 20ml of G reagent, then add 6ul of H reagent to prepare the substrate mixture, which is prepared immediately.

3. Take 5ml K reagent and pack it into the column, and add E washing solution to balance until the OD ₂₈₀ value of the effluent is less than 0.5. Then add an equal volume of the sample solution to be tested, and circulate through the column at 4°C. Afterwards, it was eluted with reagent J, and the eluate was collected.

4. Add the eluent of the sample to be tested and the RCT standard solution to the ELISA plate coated with the monoclonal RCT antibody; incubate at 37°C for 0.5h; then wash and pat dry.

ELISA plate, the first row of standard control, add RCT gradient solution in the first 11 wells, the addition amount is 100ul, and add the blank control in the 12th well. The sample eluent to be tested was added to the remaining wells, and the amount added was 100ul.

5. Add 100ul enzyme-labeled antigen solution, incubate at 37°C for 0.5h; wash and pat dry; add 150ul substrate mixture to each well, develop color at room temperature for 15min; add 50ul I reagent to each well to terminate; read OD of each well with a microplate reader value.

6. Draw a standard curve: Use the OD value of each gradient solution obtained from the RCT standard to draw a standard curve; the detection limit of this method is 0.2 ng/mL. The result is shown in Figure 2.

7. Quantitatively calculate the RCT content in the sample eluate according to the standard curve.

Result judgment: The OD value of the standard control well increases with the decrease of the RCT concentration, and the OD value of the 12th well should be the highest; compare the OD value of the sample detection well with the OD value of the standard control well, if the OD value is the same, the tested sample The RCT content in the solution is the same as the RCT concentration of the corresponding standard solution, and the test results are as follows:

samples muscle liver kidney Urine Test 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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