CN101368952B

CN101368952B - ELISA adsorption analysis method for measuring clenobuterol hydrochloride content in milk, pork liver, chicken liver and animal feed

Info

Publication number: CN101368952B
Application number: CN2008100461515A
Authority: CN
Inventors: 邓安平; 贺莉; 杨红; 李大伟
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2008-09-24
Filing date: 2008-09-24
Publication date: 2012-07-04
Anticipated expiration: 2028-09-24
Also published as: CN101368952A

Abstract

The invention discloses an enzyme-linked immunosorbent analysis method (ELISA) for measuring the contents of Clenobuterol hydrochloride in the milk, pig liver, chicken liver and animal feeds. The method is characterized in that modified compound of the Clenobuterol hydrochloride can be synthesized and connected with proteins so as to prepare immunogen and envelope antigen. Four polyclonal antibodies of the rabbit anti-Clenobuterol hydrochloride can be obtained through immunized animals. The four polyclonal antibodies can all be used for immunoassay of the Clenobuterol hydrochloride, the standardized curve has a concentration range of 0.01 to 100 ng/mL, IC50 has a range of 0.1 to 0.9ng/mL, wherein, IC50 of the ELISA which has the highest sensitivity is 0.1 to 0.3ng/mL, compared with ELISA in the market, the sensitivity of test kits can be enhanced by five to fifty times; the four antibodies has a cross-reactivity rate of 25.37% to 45.83% with salbutamol, and hardly has cross-reactivity with other nine medicaments which can be added in the animal feeds, thereby presenting strong specificity of the prepared ELISA.

Description

Measure the ELISA adsorption analysis method of clenobuterol hydrochloride content in milk, pork liver, chicken gizzard and the animal feed

Technical field

The present invention relates to a kind of ELISA adsorption analysis method (ELISA) of measuring clenobuterol hydrochloride content in milk, pork liver, chicken gizzard and the animal feed, belong to the research field of food safety supervision or food analysis.

Background technology

Clenobuterol hydrochloride (CL) is commonly called as clenbuterol hydrochloride, chemical name: α-(uncle's fourth is amino) methyl-4-amino-3; 5-dichlorbenzyl alcohol hydrochloride, English name: Clenbuterol hydrochloride is a kind of receptor, medicine of synthetic; It optionally acts on the adrenaline beta 2 receptor, causes sympathetic activation, under therapeutic dose; Has lax tracheal smooth muscle effect; Be used to treat asthma,, also be usually used in postponing childbirth because it has strong relexation to uterine smooth muscle.The eighties in 20th century; Discover a certain amount of clenobuterol hydrochloride is added in the animal feed; Can change the metabolic pathway of nutriment, the promotion animal muscle is synthesizing of skeletal muscle protein particularly, and it is synthetic to suppress fat simultaneously; Thereby promotion growth of animal; Increase lean meat percentage, so clenobuterol hydrochloride is worldwide as animal feed additive be widely used (Kuiper H.A.et al.Illegal use of β-adrenergic agonists:International.Journal of Animal Science1998,76:195-207; Prezelj A.et al.Abuse of clenbuterol and its detection.Current Medicinal Chemistry.2003,10:281-290).Its interpolation brings huge profit to the operator, but because its residual in livestock products, and that gives consumers in general has healthyly also caused very big harm.

The effective dose of using during Domestic Animal is produced generally is 5～10 times of asthma dosage, and the stronger property of medicine is arranged, and the dissolving metabolic rate is low, is difficult for a large amount of metabolism and discharges, and residual quantity is higher.The clenobuterol hydrochloride chemical property is stable, and general high temperature is difficult to decompose, and as a parahormone, is deep in the cell membranes in tissue through body of gland and plays a role, and is difficult to metabolite clearance.Clenobuterol hydrochloride is had an effect in animal vascular system, makes tracheaectasy, influences the glycometabolism in muscle protein metabolism, fat metabolism and the liver, has a strong impact on animal and grows normally.The edible animal food that contains clenobuterol hydrochloride residue of people; Produce to feel sick, phenomenons such as vomiting, dizziness, palpitaition, weak, face and limb muscle vibration; Bigger to hypertension, cardiac harm, be prone to cause tachycardia, ventricular premature beat even threat to life.A lot of food poisonings that caused by clenobuterol hydrochloride (Pulce C.D.et al.Collective human foodpoisonings by CLenbutenol residues in Vealliver.Veterinary and humantoxicology once took place both at home and abroad; .1991,33:480-481; Ke Hua etc. clenobuterol hydrochloride causes the investigation and the urine examination of food poisoning together. Chinese health supervision magazine, 2003,10 (2): 91-93), national governments all forbid clenobuterol hydrochloride is used for as feed addictive the production of meat animals.China Ministry of Agriculture is file publishing repeatedly, forbids in feed, using clenobuterol hydrochloride, and the strict content that detects clenobuterol hydrochloride in the meat products of market (The Ministry of Agriculture of the People's Republic of China, MOA's agriculture and animal husbandry is sent out [1997] No. 3; [2002] No. 193).

The method of measuring clenobuterol hydrochloride content mainly contains two types; One type is chromatogram analysis method, has comprised high performance liquid chromatography, GC-MS, liquid chromatograph mass spectrography method, gas chromatography fourier infrared coupling method, CZE method etc.The HPLC standard method (GB/T5009.192-2003) of clenobuterol hydrochloride in the samples such as the detection blood of China's promulgation, tissue; Be to utilize BOS (deactivating processing) or ODS anti-phase C18 alkyl silica gel post (255nm * 4.6mm through alkali; 5 μ m) sample is separated, the selection wavelength is 244nm, flow velocity 1.0mL/min; Room temperature condition, detection limit 5ng/g.But the chromatography instrument is expensive, sample pretreatment is complicated, time-consuming, it is high to detect cost; Another kind of is immune analysis method; Mainly be ELISA adsorption analysis method and colloid gold immune analytical approach; The quick detection kit of two kinds of immune analysis methods of this of various brands all has sale on market; But the sensitivity of the immune analysis method of the mensuration clenobuterol hydrochloride of seeing at present is not high, compels to wait to improve.

Summary of the invention

The ELISA adsorption analysis method (ELISA) that the objective of the invention is to be directed against the deficiency of prior art and clenobuterol hydrochloride content in a kind of mensuration milk, pork liver, chicken gizzard and the animal feed is provided is characterized in that with specific reaction between antibody and antigen and the antibody be the basic high sensitivity of setting up, the analytical approach of high specific.

The object of the invention is realized that by following technical measures wherein said raw material umber is parts by weight except that specified otherwise.

(1) preparation of clenobuterol hydrochloride trim

1～5mg clenobuterol hydrochloride is dissolved in 0.2～0.6mL water, slowly drips the NaNO that 0.1～0.3mL concentration is 10～16.7mg/mL ₂Solution, with the pH value to 1.5 that hydrochloric acid is regulated mixed liquor, the 4 ℃ of reactions in dark place are spent the night; The above-mentioned solution that takes a morsel is added drop-wise to N, and in the accelerine, solution colour becomes faint yellowly by colourless, shows that diazo-reaction accomplishes; 5～11mg sulfaminic acid amine solvent in 0.1～0.2mL water, slowly is added drop-wise in the diazonium salt solution cessation reaction.

(2) preparation of immunogene and envelope antigen

Take by weighing bovine serum albumin(BSA) 50～100mg and ovalbumin 30～60mg, be dissolved in respectively in the PBS that 5mL concentration is 0.01mol/L pH7.5; Diazotising solution is slowly dropped in the protein solution, add little amount of N aOH solution, make the pH value of mixed liquor remain on 7.5, stir following 4 ℃ of reactions 4 hours; Potpourri is packed in the bag filter, dialysis a couple of days, obtain clenobuterol hydrochloride-protein solution, freeze drying ,-20 ℃ of preservations are for use; Clenobuterol hydrochloride-bovine serum albumin(BSA) and clenobuterol hydrochloride-ovalbumin are used separately as immunogene and envelope antigen.

R＝CH(OH)CH ₂NHC(CH ₃) ₃

(3) clenobuterol hydrochloride Polyclonal Antibody Preparation

Two kinds of respectively immune two rabbits of immunogene: 1～4mg immunogene is dissolved in the physiological saline of 0.5～4mL, adds the complete freund adjuvant of 0.5～3mL, be mixed into water in oil emulsion, every each 0.5～2.0mL emulsion of drawing of rabbit; MSI is gone into the rabbit back, after 1～5 week, rabbit is carried out booster immunization, uses incomplete freund adjuvant; All the other are identical with immunity for the first time, after the immunity for the second time, carry out immunity next time 2～5 weeks, and for the third time, after the 4th immunity; Extract 0.1～0.5mL ear blood in 5～7 days, detect the situation that antibody produces, after the 5th immunity, 5～15 days execution rabbits; Get whole blood, blood is placed in refrigerator spent the night, draw supernatant liquor; Packing stores in low temperature refrigerator, and four kinds of antibody are arranged; Be the antibody I of clenobuterol hydrochloride-bovine serum albumin(BSA) preparation, the antibody I II of II and clenobuterol hydrochloride-ovalbumin preparation, IV.

(4) set up the ELISA adsorption analysis method of measuring clenobuterol hydrochloride content

Gained antibody performance is characterized, under the optimum test condition, set up the ELISA that measures clenobuterol hydrochloride content.

Advantage of the present invention:

1. successfully prepare the polyclonal antibody of anti-clenobuterol hydrochloride and set up the ELISA adsorption analysis method of measuring clenobuterol hydrochloride content in milk, pork liver, chicken gizzard and the animal feed.

2. highly sensitive: compare with most commercial ELISA kit, sensitivity improves 5～50 times.

3. high specificity: the cross reacting rate of four kinds of antibody and salbutamol is 25.37～45.83%, does not almost have cross reaction with other the 9 kinds medicines that can make an addition in the animal feed.

4. sample preparation is simple, test volume is big, testing expense is low.

5. to the mensuration of sample, ELISA and HPLC have good correlativity.

Description of drawings

Fig. 1. be the ultraviolet-visible light spectrogram of clenobuterol hydrochloride (CL), bovine serum albumin(BSA) (BSA) and clenobuterol hydrochloride-bovine serum albumin(BSA) (CL-BSA) cross-linking agent

Learnt that by Fig. 1 CL and BSA have characteristic absorption peak at 298nm and 280nm place respectively, CL-BSA has an acromion at 325nm, and the characteristic peak for the crosslinked formed bigger conjugated system of CL and BSA shows clenobuterol hydrochloride success and protein-crosslinking.

Clenobuterol hydrochloride (CL), ovalbumin (OVA) are similar with Fig. 1 with the ultraviolet-visible light spectrogram of clenobuterol hydrochloride-ovalbumin (CL-OVA) cross-linking agent

Fig. 2. be the typical curve that the ELISA that sets up of four kinds of polyclonal antibodies measures clenobuterol hydrochloride

■ envelope antigen CL-OVA, 1:20,000 (e.g.10ng/well); Antibody I, 1:100,000; Goat-anti rabbit 1gG-horseradish peroxidase (GaRIgG-HRP), 1:20,000; IC ₅₀0.18ng/mL; ● envelope antigen CL-OVA, 1:5,000 (e.g.40ng/well); Antibody I I, 1:10,000; GaRIgG-HRP1:20,000; IC ₅₀0.55ng/mL; ▲ envelope antigen CL-BSA, 1:5,000 (e.g.40ng/well); Antibody I II; 1:10,000; GaRIgG-HRP, 1:20,000; IC ₅₀0.67ng/mL;

Envelope antigen CL-BSA1:2,000 (e.g.1000ng/well); Antibody I V, 1:10,000; GaRIgG-HRP1:20,000; IC500.44ng/mL.

Learnt that by Fig. 2 four kinds of antibody all can be used for the immunoassay of clenobuterol hydrochloride, the concentration range of typical curve is 0.01～100ng/mL, the IC of the ELISA that sensitivity is the highest ₅₀Be 0.18ng/mL, the IC of other three kinds of ELISAs ₅₀Be respectively 0.55,0.67 and 0.44ng/mL.

Fig. 3. be ELISA and HPLC correlation curve to the testing result of clenobuterol hydrochloride in 5 mark-on samples

Learn by Fig. 3, two kinds of method good relationship, regression equation is Y=0.992X+0.469, and related coefficient is 0.989, and n=5 explains that the correlativity of the two is fine

Embodiment

Through embodiment the present invention is carried out concrete description below; Be necessary to be pointed out that at this this enforcement only is used for invention is further specified; But can not be interpreted as the restriction to protection domain of the present invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of foregoing invention.

Embodiment:

1. the preparation of clenobuterol hydrochloride trim

1～5mg clenobuterol hydrochloride is dissolved in 0.2～0.6mL water, slowly drips the NaNO that 0.1～0.3mL concentration is 10～16.7mg/mL ₂Solution, with the pH value to 1.5 that hydrochloric acid is regulated mixed liquor, the 4 ℃ of reactions in dark place are spent the night; The above-mentioned solution that takes a morsel is added drop-wise to N, and in the accelerine, solution colour becomes faint yellowly by colourless, shows that diazo-reaction accomplishes; 5～11mg sulfaminic acid amine solvent slowly is added drop-wise in the diazonium salt solution cessation reaction in 0.1～0.2mL water.

2. the preparation of immunogene and envelope antigen

Take by weighing bovine serum albumin(BSA) 50～100mg and ovalbumin 30～60mg, be dissolved in respectively in the PBS that 5mL concentration is 0.01mol/L pH7.5; Diazotising solution is slowly dropped in the protein solution, add little amount of N aOH solution, make the pH value of mixed liquor remain on 7.5, stir following 4 ℃ of reactions 4 hours; Potpourri is packed in the bag filter, dialysis a couple of days, obtain clenobuterol hydrochloride-protein solution, freeze drying ,-20 ℃ of preservations are for use; Clenobuterol hydrochloride-bovine serum albumin(BSA) (CL-BSA) and clenobuterol hydrochloride-ovalbumin (CL-OVA) are used separately as immunogene and envelope antigen.

The ultraviolet-visible light spectrogram of CL, BSA and CL-BSA cross-linking agent is as shown in Figure 1, and the ultraviolet-visible light spectrogram of CL, OVA and CL-OVA cross-linking agent is similar with Fig. 1.

3. clenobuterol hydrochloride Polyclonal Antibody Preparation

Two kinds of respectively immune two rabbits of immunogene: 1～4mg immunogene is dissolved in the physiological saline of 0.5～4mL, adds the complete freund adjuvant of 0.5～3mL, be mixed into water in oil emulsion, every each 0.5～2.0mL emulsion of drawing of rabbit; MSI is gone into the rabbit back, after 1～5 week, rabbit is carried out booster immunization, uses incomplete freund adjuvant; All the other are identical with immunity for the first time, after the immunity for the second time, carry out immunity next time 2～5 weeks, and for the third time, after the 4th immunity; Extract 0.1～0.5mL ear blood in 5～7 days, detect the situation that antibody produces, after the 5th immunity, 5～15 days execution rabbits; Get whole blood, blood is placed in refrigerator spent the night, draw supernatant liquor; Packing stores in low temperature refrigerator, and four kinds of antibody are arranged; Be the antibody I of CL-BSA immunogen preparing, the antibody I II of II and CL-OVA immunogen preparing, IV.

4. the optimization experiment condition is set up the ELISA adsorption analysis method (ELISA) of measuring clenobuterol hydrochloride

Gained antibody performance is characterized, under the optimum test condition, set up the ELISA adsorption analysis method of measuring clenobuterol hydrochloride content in milk, pork liver, chicken gizzard and the animal feed.

(1) solution preparation

(a) sodium carbonate-sodium bicarbonate buffer liquid

Take by weighing 2.606g Na ₂CO ₃10H ₂O, 3.434g NaHCO ₃, after the dissolving of 800mL ultrapure water mixing, regulate the pH value, add water to 1L, be made into 0.05mol/L, the sodium carbonate of pH=9.6-sodium bicarbonate buffer liquid;

(b) phosphate buffer (storing solution, PBS * 10)

Take by weighing 21.961g Na ₂HPO ₄12H ₂O, 6.031g NaH ₂PO ₄2H ₂O, 87.666g NaCl adds the 800mL ultrapure water and mixes heating for dissolving; NaOH with 1mol/L regulates pH=7.5, adds ultrapure water to 1L, is made into to contain 0.15mol/L NaCl, the 0.1mol/L phosphate buffer (storing solution) of pH=7.5;

(c) casein solution

Take by weighing the casein heating for dissolving in the PBS of 0.01mol/L, be made into 1.0% casein solution;

(d) phosphate buffer-tween storing solution (the 0.1mol/L phosphoric acid buffer storing solution that contains 1%Tween20, PBST * 10, pH=7.5);

(e) preparation of clenobuterol hydrochloride standard solution (0.5mg/mL)

0.005g CL is dissolved in the 5mL pure water;

(f) preparation (1mg/mL) of CL-OVA and CL-BSA cross-linking agent

Claim CL-OV or CL-BSA cross-linking agent 5mg with microbalance, add the dissolving of 5mL ultrapure water;

(g) substrate solution (20mL pure water; The 1mL sodium-acetate buffer; 200 μ L tetramethyl benzidines (TMB) (1%); 20 μ L hydrogen peroxide (5%));

(i) sodium-acetate buffer

Take by weighing 3.450g CH ₃COONa3H ₂O with the dissolving of 100mL ultrapure water, uses 1mol/L citric acid (21.031g C again ₆H ₈O ₇H ₂O is dissolved in the 100mL water) regulate pH=5.8 after, the water constant volume is made into the 0.1mol/L sodium-acetate buffer to 250mL again;

(ii) TMB: take by weighing 0.0717g TMB, use the 7.17mL dmso solution, mixing is made into 1%, w/v;

(iii) hydrogen peroxide: the hydrogen peroxide of getting 20 μ L30% adds in the 100 μ L ultrapure waters, and mixing is made into 5%;

(h) H ₂SO ₄Solution: pipette the dense H of 25mL ₂SO ₄, be dissolved in the ultrapure water of 475mL, be made into 5%H ₂SO ₄Solution.

(2) key instrument

Wash plate machine: A5082, Tecan, Austria; ELIASA: A2082, Tecan, Austria; Efficient liquid

Chromatography: Alltech-001

(3) indirect competitive ELISA step

(a) use the envelope antigen wrapper sheet, every hole 200 μ L, 4 ℃ are spent the night;

(b) PBST damping fluid (PBST storing solution 1:10 dilution) full hole washing is three times;

(c) add casein solution and blockade, every hole 280 μ L, room temperature was placed 1 hour;

(d) the PBST damping fluid is washed plate three times;

(e) every successively hole adds the standard solution of 100 μ L and the certain dilution polyclonal antibody of 100 μ L, and room temperature was placed 1 hour;

(f) the PBST damping fluid is washed plate three times;

(g) add ELIAS secondary antibody (goat-anti rabbit 1gG-horseradish peroxidase, GaRIgG-HRP), every hole 200 μ L, room temperature was placed 1 hour;

(h) the PBST damping fluid is washed plate three times;

(i) add substrate solution colour developing, every hole 200 μ L, jolting 15～20 minutes;

(j) add 5%H ₂SO ₄Solution, every hole 80 μ L, cessation reaction;

(k) measure absorbance with ELIASA, make typical curve, carry out interpretation of result and discussion.

(4) ELISA optimization of experimental conditions

The present invention has done optimization to the various combination of envelope antigen and antibody, the concentration of envelope antigen, the dilutability of antibody, the dilutability of ELIAS secondary antibody etc., and Optimization result is as shown in table 1.

(5) typical curve of ELISA and sensitivity

The concentration of clenobuterol hydrochloride standard standard solution is: 0,0.01,0.03,0.1,0.3,1.0,10,100ng/mL, by the storing solution 1.0mg/mL of clenobuterol hydrochloride through the pure water dilution and get.Logarithm with clenobuterol hydrochloride concentration is a horizontal ordinate, with relative signal B/B ₀* 100% is typical curve (B for ordinate ₀: clenobuterol hydrochloride mark liquid concentration is the pairing absorbance of 0ng/mL; B: the absorbance that other each concentration are corresponding).Fig. 2 is the typical curve that the ELISA that sets up of four kinds of polyclonal antibodies measures Clenizole Hydrochloride, the IC of table 1 under optimum experimental condition ₅₀Value, IC ₅₀Between 0.1～0.9ng/mL; The ELISA sensitivity that antibody I is set up is the highest, IC ₅₀Be 0.1～0.3ng/mL.

(6) specificity of ELISA

The ELISA specificity can be represented with cross reacting rate.Cross reacting rate (CR%)=(IC of clenobuterol hydrochloride ₅₀The IC of/test substances ₅₀) * 100%.Cross reacting rate is more little, and the specificity of ELISA is high more.

Select salbutamol and other the 9 kinds medicines that can make an addition in the animal feed to carry out the cross reaction experiment in the present invention, the structure of cross reaction thing and cross reaction experiment are shown in table 2, table 3.The cross reacting rate of four kinds of antibody and salbutamol is 25.37～45.83%, does not almost have cross reaction with other the 9 kinds medicines that can make an addition in the animal feed; The ELISA high specificity of being set up is described.

5.ELISA to clenobuterol hydrochloride Determination on content in the mark-on sample

Selected 5 kinds of negative samples: milk I, milk II, pork liver I, chicken gizzard I and animal feed I carry out the mark-on experiment, get 1～5g sample; The Clenizole Hydrochloride storing solution that adds 1～5 μ L, (1) milk sample: measure 1～5mL milk sample in centrifuge tube, add 1 times of pure water dilution, whirlpool appearance whirlpool 5 minutes; Adding concentration is 0.1mol/L hydrochloric acid 1～5mL, and water bath chader vibrates half an hour, hold over night, ultrasonic extraction next day 15 minutes; 50 ℃ of water-bath heating, centrifugal, it is to be measured to get supernatant liquor; (2) liver sample: the liver washing that market is bought is dehematized, and dries, and gets an amount of liver sample chopping; Homogenate 5 minutes takes by weighing 1～5g pulpous state sample, and adding concentration is 0.1mol/L watery hydrochloric acid 1～5mL; Water bath chader vibrates half an hour, hold over night, ultrasonic extraction next day 15 minutes; Centrifugal, it is to be measured to get supernatant liquor; (3) feed sample: get the feed sample, grind to form fine powder, claim 1～5g sample, adding concentration is 0.1mol/L watery hydrochloric acid 1～5mL, and water bath chader vibrates half an hour, ultrasonic extraction next day 15 minutes, and centrifugal, it is to be measured to get supernatant liquor; Mark-on sample extraction liquid is directly measured with ELISA by 1:400～1:1000 dilution back, and the recovery of standard addition of clenobuterol hydrochloride is 92.2～97.0%, and relative standard deviation is 1.3～5.3%, and illustration method accuracy and precision are all relatively good.

6.ELISA comparison with HPLC

The HPLC condition of clenobuterol hydrochloride is: chromatographic condition: Hypersil Gold post, column temperature are room temperature, and moving phase is methyl alcohol: 0.01mol/L NaH ₂PO ₄The WS=35:65; Flow 1mL/min, sample introduction 20 μ L, ultraviolet detection wavelength: 240nm; The concentration of clenobuterol hydrochloride standard solution is respectively: 0.5,1.0,2.0,5.0,10.0 and 20 μ g/mL, sample extraction liquid directly measure after with the membrane filtration of 0.45 μ m.

The reliability of the ELISA that is set up is further verified with HPLC, 5 kinds of mark-on samples, i.e. milk I, mark-on concentration 1 μ g/mL; Milk II, mark-on concentration 2 μ g/mL; Pork liver I, mark-on concentration 4 μ g/g; Chicken gizzard I, mark-on concentration 5 μ g/g; Animal feed I, mark-on concentration 3 μ g/g; The mark-on sample is measured with the 0.1mol/L hcl as extraction agent and with ELISA and HPLC; Measuring the result is horizontal ordinate with ELISA, HPLC be ordinate map both correlation curve, as shown in Figure 3; Regression equation is Y=0.992X+0.469; Related coefficient is 0.989, and n=5 explains that the correlativity of the two is fine.

7.ELISA clenobuterol hydrochloride content in the mensuration authentic sample

From the supermarket, buy 6 milk appearance, from the market of farm produce, buy 3 pork liver appearance and 3 chicken gizzard appearance, buy 4 animal feed appearance from the agricultural materials market, use the highest ELISA of sensitivity that is set up that above-mentioned 16 samples are measured, it is as shown in table 4 to measure the result.The result shows that 75% all contains clenobuterol hydrochloride in the feed sample, and maximum concentration reaches 64.03ng/g; All find in milk, pork liver and the chicken gizzard sample to contain clenobuterol hydrochloride, maximum concentration is respectively 5.33ng/mL, 2.28ng/g and 0.74ng/g.

Table 1ELISA optimum experimental condition and IC ₅₀Value

The chemical constitution of table 2 cross reacting material

The cross reaction experimental result of four kinds of antibody of table 3

Table 4ELISA measures clenobuterol hydrochloride content in the authentic sample

Claims

1. measure the ELISA adsorption analysis method of clenobuterol hydrochloride content in milk, pork liver, chicken gizzard and the animal feed, it is characterized in that this method may further comprise the steps:

(1) preparation of clenobuterol hydrochloride trim

1～5mg clenobuterol hydrochloride is dissolved in 0.2～0.6mL water, slowly drips the NaNO that 0.1～0.3mL concentration is 10～16.7mg/mL ₂Solution, with the pH value to 1.5 that hydrochloric acid is regulated mixed liquor, the 4 ℃ of reactions in dark place are spent the night; The above-mentioned solution that takes a morsel is added drop-wise to N, and in the accelerine, solution colour becomes faint yellowly by colourless, shows that diazo-reaction accomplishes; 5～11mg sulfaminic acid amine solvent slowly is added drop-wise in the diazonium salt solution cessation reaction in 0.1～0.2mL water;

(2) preparation of immunogene and envelope antigen

Take by weighing bovine serum albumin(BSA) 50～100mg and ovalbumin 30～60mg, be dissolved in respectively in the PBS that 5mL concentration is 0.01mol/L pH7.5; Diazotising solution is slowly dropped in the protein solution, add little amount of N aOH solution, make the pH value of mixed liquor remain on 7.5, stir following 4 ℃ of reactions 4 hours; Potpourri is packed in the bag filter, dialysis a couple of days, obtain two kinds of clenobuterol hydrochloride-protein solutions, freeze drying ,-20 ℃ of preservations are for use; Clenobuterol hydrochloride-bovine serum albumin(BSA) and clenobuterol hydrochloride-ovalbumin are used separately as immunogene and envelope antigen;

(3) clenobuterol hydrochloride Polyclonal Antibody Preparation

Two kinds of respectively immune two rabbits of immunogene: 1～4mg immunogene is dissolved in the physiological saline of 0.5～4mL, adds the complete freund adjuvant of 0.5～3mL, be mixed into water in oil emulsion, every each 0.5～2.0mL emulsion of drawing of rabbit; MSI is gone into the rabbit back, after 1～5 week, rabbit is carried out booster immunization, uses incomplete freund adjuvant; All the other are identical with immunity for the first time, after the immunity for the second time, carry out immunity next time 2～5 weeks, and for the third time, after the 4th immunity; Extract 0.1～0.5mL ear blood in 5～7 days, detect the situation that antibody produces, after the 5th immunity, 5～15 days execution rabbits; Get whole blood, blood is placed in refrigerator spent the night, draw supernatant liquor; Packing stores in low temperature refrigerator, and four kinds of antibody are arranged; Be the antibody I of clenobuterol hydrochloride-bovine serum albumin(BSA) preparation, the antibody I II of II and clenobuterol hydrochloride-ovalbumin preparation, IV;

Gained antibody performance is characterized, under the optimum test condition, set up the ELISA that measures clenobuterol hydrochloride content;

(5) ELISA is to clenobuterol hydrochloride Determination on content in the mark-on sample

Selected 5 kinds of negative samples: milk I, milk II, pork liver I, chicken gizzard I and animal feed I carry out the mark-on experiment, get 1～5g sample; The clenobuterol hydrochloride storing solution that adds 1～5 μ L, (1) milk sample: measure 1～5mL milk sample in centrifuge tube, add 1 times of pure water dilution, whirlpool appearance whirlpool 5 minutes; Adding concentration is 0.1mol/L hydrochloric acid 1～5mL, and water bath chader vibrates half an hour, hold over night, ultrasonic extraction next day 15 minutes; 50 ℃ of water-bath heating, centrifugal, it is to be measured to get supernatant liquor; (2) liver sample: the liver washing that market is bought is dehematized, and dries, and gets an amount of liver sample chopping; Homogenate 5 minutes takes by weighing 1～5g pulpous state sample, and adding concentration is 0.1mol/L watery hydrochloric acid 1～5mL; Water bath chader vibrates half an hour, hold over night, ultrasonic extraction next day 15 minutes; Centrifugal, it is to be measured to get supernatant liquor; (3) feed sample: get the feed sample, grind to form fine powder, claim 1～5g sample, adding concentration is 0.1mol/L watery hydrochloric acid 1～5mL, and water bath chader vibrates half an hour, ultrasonic extraction next day 15 minutes, and centrifugal, it is to be measured to get supernatant liquor; Mark-on sample extraction liquid is directly measured with ELISA by 1:400～1:1000 dilution back, and the recovery of standard addition of clenobuterol hydrochloride is 92.2～97.0%, and relative standard deviation is 1.3～5.3%, and illustration method accuracy and precision are all relatively good;

(6) comparison of ELISA and HPLC

The HPLC condition of clenobuterol hydrochloride is: chromatographic condition: Hypersil Gold post, column temperature are room temperature, and moving phase is methyl alcohol: 0.01mol/L NaH ₂PO ₄The WS=35:65; Flow 1mL/min, sample introduction 20 μ L, ultraviolet detection wavelength: 240nm; The concentration of clenobuterol hydrochloride standard solution is respectively: 0.5,1.0,2.0,5.0,10.0 and 20 μ g/mL, sample extraction liquid directly measure after with the membrane filtration of 0.45 μ m;

The reliability of the ELISA that is set up is further verified with HPLC, 5 kinds of mark-on samples, i.e. milk I, mark-on concentration 1 μ g/mL; Milk II, mark-on concentration 2 μ g/mL; Pork liver I, mark-on concentration 4 μ g/g; Chicken gizzard I, mark-on concentration 5 μ g/g; Animal feed I, mark-on concentration 3 μ g/g; The mark-on sample uses concentration as the 0.1mol/L hcl as extraction agent and with ELISA and HPLC mensuration, and the mensuration result is horizontal ordinate with ELISA, HPLC be ordinate map both correlation curve; Regression equation is Y=0.992X+0.469; Related coefficient is 0.989, and n=5 explains that the correlativity of the two is fine.