CN102608310A - Salbutamol enzyme linked immunoassay kit and use method thereof - Google Patents

Abstract

The invention relates to a salbutamol enzyme linked immunoassay kit and a use method of the salbutamol enzyme linked immunoassay kit. The salbutamol enzyme linked immunoassay kit comprises the following components: a coated microplate, calibrator, enzyme label working solution, substrate solution A, substrate solution B, stop buffer, concentrated wash solution and concentrated extracting solution. The kit prepared according to the invention can be used for quickly quantifying and detecting salbutamol residual amount in samples such as urine or serum, muscle or tissue, feed, milk and the like, and has the advantages of wide use range, convenience in use and accurate detection. The sensitivity of the kit prepared according to the invention is 0.1 ppb; for the precision, the within-run coefficient of variation (CV%) is less than 10 percent, and the between-run coefficient of variation (CV%) is less than 15 percent; the accuracy is represented as recovery rate, and the recovery rate should be 70-110 percent. The range of IC50 is 0.6-1.0 ppb, and the absolute value of the linearly dependent coefficient r is not less than 0.9900.

Description

A kind of salbutamol enzyme linked immunological kit and method of application thereof

Technical field

The invention belongs to the detection kit field, relate to the drug-testing kit, especially a kind of salbutamol enzyme linked immunological kit and method of application thereof.

Background technology

(Salbutamol, it is anti-depressant a kind of SAL) to belong to β-2, is a kind of medicine that can combine with beta-2 adrenergic receptor, is mainly used in treatment asthma, bronchial spasm etc. clinically for salbutamol.SAL is used as feed addictive, can significantly promote growth of animal, increase feed conversion rate and lean meat percentage, long-term excess to use, then can in animal tissue, accumulate, the people can cause poisoning after having eaten this animal product.

What Chang Zuowei growth accelerator used in β-2 excitant is that (Clenbuterol, CL), but along with the increasing to the CL inspecting force, illegal user begins to turn to other substitutes to Clenbuterol.Because the growth promoting function of SAL and CL is more or less the same; Its elimination time ratio Clenbuterol in animal body is short, toxicity a little less than, therefore, salbutamol (Salbutamol; Sal) be continue Clenbuterol (Clenbuterol, a kind of β class growth of animal agonist of CL) the most often being abused afterwards.

2. the growth promoting function of salbutamol

Experiment confirm, salbutamol can cause that the fiber finer intracellular organic matter increases through the synthetic of stimulating protein in animal body, and volume increases and protein degradation slows down, and salbutamol can reduce the fat content of rouge body simultaneously, reduces the fat deposition of non-rouge body portion.The infiltration rate of salbutamol in the livestock and poultry body is also very fast, reaches the top of blood concentration about 1.5h.Distribution half-life is 3.29h.The salbutamol that experiment showed, to pig has significant effect for reducing fat, and fattening pig is more effective than lean meat pig.

3. the residual harm that human body is produced of salbutamol animal food

The salbutamol part is degraded in liver, and part is discharged from urine with original shape.Some big its disappear to protein, lipometabolic effect immediately after the drug withdrawal.Therefore, salbutamol usually adopts to mix as the heavy partitioning agent of nutrition raises administration, raises the pig 1-3 month with 2-8mg/ ton feed, reaches somatotrophic effect, but also can cause drug accumulation simultaneously, mainly be in internal organ, hair, retina the residence time more of a specified duration.Salbutamol can appear in urine and each organ of health after the medication, in liver, can keep ten surplus day, and in retinal tissue, can keep 4 months at least.The people is in edible muscle twitches, flesh carbuncle, headache, dizzy, oversensitive, palpitaition, the tachycardia of occurring after containing the higher animal tissue of salbutamol; Even feel sick, toxicity symptom such as vomiting; The sustainable 1-6 of this type of symptom days, generally eat in the back 15 minutes-6h and symptom occurs, symptom duration 90 minutes-6 days; Though symptom is reversible, the people that cardiovascular disease is arranged is still had bigger harm.

4. salbutamol detection method progress

Technological abroad at present for the residue detection of salbutamol; Mainly contain immuno analytical methods such as physico-chemical analysis technology such as thin-layer chromatography technology, high performance liquid chromatography, chemoluminescence method, gas chromatography-spectral analysis method and radioimmunoassay, fluorescence immunoassay, enzyme immunity, and all can reach higher sensitivity.But the physico-chemical analysis method is generally consuming time, cost is high, complex operation, sample pre-treatments complicated, be difficult to carry out mass detection; And immuno analytical method is the analytical technology that the specificity association reaction with antigen, antibody is the basis; It relates to the combined action between stereochemistry, electric charge, hydrogen bond and the dipole between antigen and antibody; Thereby have unrivaled selection of conventional physico-chemical analysis method and sensitivity highly, be highly suitable for the analysis of trace components in the complex matrices.In addition, that immune analysis method also has is easy, characteristics fast, thereby one of technology of beyond doubt tool development and application potential is great innovation and the breakthrough aspect the left drug analysis.

4.1 chromatography

Chromatographic technique mainly contains thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) for detecting the classical technology of salbutamol; High performance liquid chromatography/triode display (HPLC/PDA); High performance liquid chromatography/fluorescence (HPLC/FLU), liquid chromatograph mass spectrography (LC/MS), gas chromatography-mass spectrography (GS-MS); Gas chromatography-matter is with (GC-FTIP), methods such as Capillary Electrophoresis.These methods are sensitive, accurate, the conclusive evidence method that the Chang Zuowei salbutamolum residue detects.The research report that domestic and international application HPLC carries out the salbutamol analysis is a lot.Domestic RPLC-the fluorescence detection, RPLC-outer detection method, homophase benzene successfully set up got SAL methods such as high performance liquid chromatography.Just because of the accuracy of HPLC survey method, thereby often be used to the false-positive affirmation work of salbutamol in the selective examination work.Like countries such as European Union, the U.S. GS-MS method being used for salbutamol as " conclusive evidence method " detects.China also detects demonstration really with HPLC and GC/MS as salbutamol.Though these methods have sensitivity, characteristic of accurate, sample preparation is loaded down with trivial details time-consuming, and cost is high, and needs the instrument and equipment that comes complicated operation through the professional of specialized training, has therefore limited its widespread use.

4.2 biology sensor detection method

Biosensor technique is that a biology sensor outreaches computer, and current technology can be used for detecting samples such as urine, serum.This detection method has shortcomings such as sensitivity is low, low precision.

4.3 immunological detection

Immunoassay is the analytical technology that is the basis with antigen and antibody specificity, reversible association reaction.Immune response relates to the combined action between stereochemistry, electric charge, hydrogen bond and the dipole between antigen and antibody molecule; Thereby has conventional physico-chemical analysis unrivaled selectivity of technology and very high sensitivity; Be highly suitable for the analysis of trace components in the complex matrices, like the analysis and the detection of animal tissue's veterinary drug residue.Immuno analytical method have sensitivity, special, can detect the characteristics of a large amount of samples fast and once, accuracy of detection can reach the ng level.Soil has been played radioimmunoassay technique (RIA), enzyme immunological technique (EIA), and the test strips detection technique, wherein most widely used is the EIA enzyme immunoassay technology.Many testing agencies such as European Union, the U.S. all classify it as main " screening method " at present, are used for the illegal detection of using of salbutamol.

In a word; In various detection methods; What be fit to be applied to large quantities of test sample is the ELISA detection method, and it has fast, sensitive, simple to operate and characteristics that the one-time detection sample size is big, and can directly detect urine, blood sample; With HPLC, GC/MS the higher rate that conforms to is arranged, and very be fit to the detection of living animal.And HPLC and GC/MS method all need expensive instrument, complicated sample handling procedure and special operative skill; And it is more time-consuming; Therefore be not suitable for being used as the detection of gross sample, but its validity, accuracy and susceptibility make it still have the irreplaceable advantage of ELISA method.At present, when detecting, generally use the ELISA kit and carry out screening, with methods such as HPLC and GC/MS positive is confirmed again, thereby can guarantee the reliability of testing result.

Enzyme linked immunosorbent assay (ELISA) (ELISA) method has the characteristics easy, quick, sensitive, that cost is low as a kind of screening technique, and therefore, during the extensive screening of monitoring and basic unit detected at the scene, immunological detection method was actual, has broad application prospects.My company passes through mixed anhydride method with salbutamol and ovalbumin (OVA) synthetic immunogen; Prepare polyclonal antibody antibody with coupling immunogen immune sheep; And use the ammonium sulfate precipitation method preliminary purification; Be further purified with the ELISA method with affinity chromatography and identify, enzyme-labelled antigen adopts the glutaraldehyde two step method with horseradish peroxidase with the clenobuterol hydrochloride haptens is crosslinked forms.Set up direct competitive enzyme linked immunosorbent assay analysis method (ELISA) with gained antibody and enzyme-labelled antigen and detect salbutamol, lowest detection is limited to 0.1ng/ml.

Summary of the invention

The objective of the invention is to overcome the weak point of prior art, provide a kind of usable range wide, easy to use, detect salbutamol enzyme linked immunological kit and method of application thereof accurately.

The objective of the invention is to realize like this:

A kind of salbutamol enzyme linked immunological kit, form as follows:

Comprise encapsulating plate, calibration object, enzyme labeling thing working fluid, substrate solution A, substrate solution B, stop buffer, concentrated cleaning solution, concentrated extracting solution,

The said plate that encapsulates is coated with desertification butylamine alcohol polyclonal antibody at the bottom of for the hole; Package amount is 1 μ g/ml, encapsulates temperature and spends the night for 4 ℃;

Said calibration object is the serial calibration object of variable concentrations;

Said enzyme labeling thing working fluid contains the clenobuterol hydrochloride haptens of underlined HRP, for the sodium periodate method with HRP with the clenobuterol hydrochloride hapten;

Said substrate solution A is that hydrogen peroxide urea solution, B are TMB solution;

Said stop buffer is a 2M sulfuric acid.

And said concentrated cleaning solution is the 20X concentrated cleaning solution, before using with distilled water according to 1: the dilution proportion of 19v/v, use behind the mixing.

And, said concentrated extracting solution 20X concentrated extracting solution, before using with distilled water according to 1: the dilution proportion of 19v/v, use behind the mixing.

And, said encapsulate plate to encapsulate step following:

A) antibody sandwich: encapsulate the concentration coated elisa plate with desertification butylamine alcohol polyclonal antibody the best, the 100ul/ hole, 4 ℃ are spent the night, and best package amount is 1 μ g/ml, and 0.05M pH9.6 CBS is used to encapsulate solid phase carrier as dilution;

B) wash plate: liquid in the hole is dried, fully wash 4-5 time with cleansing solution 250 μ l/ holes, each 10s at interval claps dried with thieving paper;

C) sealing: the 1%BSA+10%sucrose confining liquid is added ELISA Plate, the 100ul/ hole, 37 ℃ of incubations 2 hours dry liquid in the hole, clap with thieving paper and do, and vacuum drying adds the aluminium foil bag Vacuum Package.

A kind of method of application of salbutamol enzyme linked immunological kit, step is following:

(1) required reagent is taken out from cold storage environment, place more than the equilibrium at room temperature 30min, note to shake up before every kind of liquid reagent uses;

(2) taking-up needs the microwell plate of quantity, and no microwell plate is put into aluminide-coating bag, is stored in 2-8 ℃;

(3) cleansing solution also need be risen again before use;

(4) numbering: the corresponding micropore of sample and calibration object is numbered according to the order of sequence, and it is parallel that each sample and calibration object are done 2 holes, and write down the position that calibration hole and sample aperture belong to;

(5) add calibration object/sample: add calibration object/sample 50 μ l in the micropore of correspondence, add enzyme labeling thing 50 μ l/ holes immediately, the mixing that vibrates gently is with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of shrouding film shrouding;

(6) wash plate: carefully open the shrouding film, liquid in the hole is dried, fully wash 4-5 time with cleansing solution 250 μ l/ holes, each 10s at interval claps dried with thieving paper;

(7) colour developing: add colour developing liquid 100 μ l/ holes, substrate A liquid and 1: 1 mixing of substrate B liquid, the mixing that vibrates gently behind shrouding film shrouding, is put in 25 ℃ of lucifuge environment and is reacted 15min;

(8) measure: add stop buffer 50 μ l/ holes, the mixing that vibrates is gently set ELIASA in the 450nm place, measures every hole OD value;

(9) result: the mean value of calibration that is obtained and sample absorbance multiply by 100 again divided by the absorbance of first calibration.

Advantage of the present invention and good effect are:

1, the kit of the present invention preparation fast quantification that can be used for salbutamolum residue amount in the samples such as urine appearance or serum, muscle or tissue, feed, milk detects, have usable range wide, easy to use, detect advantage accurately.

2, the sensitivity of the kit of the present invention's preparation is 0.1ppb, precision: variation within batch coefficient (CV%), CV%＜10%; Interassay coefficient of variation (CV%), CV%＜15%; Accuracy is represented accuracy with the recovery, and the recovery should be between 70%-110%.The IC50 scope: between 0.6-1.0ppb, linearly dependent coefficient | r|: be not less than 0.9900.

3, this kit success with salbutamol antigen and carrier protein OVA, make SAL and OVA coupling synthesize immunizing antigen SAL-OVA through mixed anhydride method.

4, the present invention has obtained highly purified polyclonal antibody through immune sheep and purification technique.

5, the present invention adopts the glutaraldehyde two step method with horseradish peroxidase and the crosslinked enzyme-labelled antigen that forms of clenobuterol hydrochloride haptens.

6, exploitation of this kit and foundation rely on ripe ELISA liquid system platform, through consulting a large amount of lists of references, and with reference to domestic and international similar kit, through a large amount of experiments and the optimization of system, have finally established the production and the reaction conditions of kit.

Description of drawings

Fig. 1 is a Monoclonal Antibody program of the present invention;

Fig. 2 suppresses curve for salbutamol of the present invention;

Fig. 3 suppresses curve for salbutamol urine matrix of the present invention.

Embodiment

Below in conjunction with embodiment, the present invention is further specified, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.

The measuring principle of kit of the present invention is: kit adopts the direct competitive ELISA method; Preparatory coated antibody on the ELISA Plate capillary strip; Salbutamol and enzyme-labelled antigen residual in the sample are competed desertification butylamine alcohol antibody; With the tmb substrate colour developing, the sample absorbance becomes negative correlation with the content of its contained residue salbutamol, multiply by the content that its corresponding extension rate can draw salbutamol in the sample more again with typical curve.

A kind of salbutamol enzyme linked immunological kit, it is formed as follows:

This kit is by encapsulating plate, calibration object, high concentration calibration object, enzyme labeling thing working fluid, substrate solution A, substrate solution B, stop buffer, 20X concentrated cleaning solution, and the 20X concentrated extracting solution is specifically seen table 1.

Table 1 kit constituent table

Test sample is handled:

Urine (extension rate: 1 times) can directly be carried out check and analysis.If urine sample is muddy shape, 4000 rev/mins, centrifugal 5 minutes or filter after get supernatant and detect.For fear of high background value, available sample extracting solution is done 5 times of dilutions.

The method of application of this kit is following: (operating under the room temperature 20-25 ℃ of condition)

1) required reagent is taken out from cold storage environment, place room temperature (20-25 ℃) more than the balance 30min, note to shake up before every kind of liquid reagent uses.

2) taking-up needs the microwell plate of quantity, and no microwell plate is put into aluminide-coating bag, is stored in 2-8 ℃.

3) cleansing solution also need be risen again before use.

4) numbering: the corresponding micropore of sample and calibration object is numbered according to the order of sequence, and it is parallel that each sample and calibration object are done 2 holes, and write down the position that calibration hole and sample aperture belong to.

5) add calibration object/sample: add calibration object/sample 50 μ l in the micropore of correspondence, add enzyme labeling thing 50 μ l/ holes immediately, the mixing that vibrates gently is with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of shrouding film shrouding.

6) wash plate: carefully open the shrouding film, liquid in the hole is dried, fully wash 4-5 time with cleansing solution 250 μ l/ holes, each 10s at interval claps dried (clapping the bubble that is not eliminated after doing can poke with clean rifle head) with thieving paper.

7) colour developing: add colour developing liquid (substrate A liquid and 1: 1 mixing of substrate B liquid) 100 μ l/ holes, the mixing that vibrates gently behind shrouding film shrouding, is put in 25 ℃ of lucifuge environment and is reacted 15min.

8) measure: add stop buffer 50 μ l/ holes, the mixing that vibrates is gently set ELIASA in the 450nm place (suggestion detects with dual wavelength 450/630nm, in 5min, runs through data), measures every hole OD value.(if no ELIASA does not then add stop buffer and can judge with ocular estimate)

9) result

The mean value of calibration that is obtained and sample absorbance multiply by 100 again divided by the absorbance of first calibration (0 calibration).Therefore 0 calibration equals 100% and provide absorbance with the form of number percent.

It is the semilog coordinate system curve map of a corresponding salbutamol concentration that the calibration value that calculates plots, and the concentration of corresponding each sample can be read from calibration curve.

In order to obtain the actual concentrations of the salbutamol in the sample, the concentration value of reading from typical curve must multiply by corresponding dilution factor.

10) the directly inhibition ELISA method bioassay standard curve of the present invention's employing confirms as follows

Suppressing with the salbutamol standard items of anti-SAL monoclonal antibody to variable concentrations, with the standard items of 100ppb, carry out 3 times of dilutions, is matrix with 0.01MPBS.Measure according to the ELIS running program.With salbutamol concentration is horizontal ordinate, and variable concentrations mark article OD value B/B0 value is an ordinate, draws the typical curve equation,

Concentration value	Absorbance	Entry name
				100	0.321	OD
33.3	0.356	OD
			11.1	0.452	OD
3.7	0.956	OD
			1.23	1.211	OD
0.41	1.521	OD
			0.137	1.686	OD
0.045	1.876	OD
			0	1.871	OD

Conclusion (of pressure testing): visible by Fig. 2, be horizontal ordinate with salbutamol concentration, variable concentrations mark article OD value B/B0 value is an ordinate, draws the typical curve equation, the linear within the specific limits inhibition of standard items.

Detect instance:

This kit can be used for the direct detection of pig urine, if the pig urine samples is muddy, answers 4000 rev/mins, centrifugal 5 minutes or filter after get the supernatant detection.For fear of high background value, available sample extracting solution is done 5 times of dilutions.

According to the operation of kit, to containing the test of many times of carrying out of series concentration SAL standard items, the competition curve of acquisition; Can know that by Fig. 3 this curve becomes the S type, the range of linearity can produce 50% and suppress between 0.1-100ppb when SAL marks article concentration at 1.4ppb; Linearly dependent coefficient is 0.9697; Minimum detectability (IC10) is about 0.1ppb, and this result shows that anti-SAL monoclonal antibody also can present compatibility preferably in urine matrix.

Main material preparation method involved in the present invention is following:

1. enzyme conjugates

Enzyme in the enzyme-labelled antigen is a horseradish peroxidase, and horseradish peroxidase-clenobuterol hydrochloride label adopts the preparation of glutaraldehyde two step method.

Glutaraldehyde activation horseradish peroxidase: take by weighing the 10mg horseradish peroxidase and be dissolved in 0.5ml 1.25% glutaraldehyde solution; Room temperature lucifuge stirring reaction 10 hours; Then with reactant liquor in 4 degree to the PBS of 0.1mol/l PH 6. dialysis 72 hours, during change liquid 6 times.

The preparation of horseradish peroxidase-clenobuterol hydrochloride label (CLEN-HRP): the clenobuterol hydrochloride that takes by weighing 5mg is dissolved among the PBS of 1ml0.1mol/l PH 6., adds above-mentioned horseradish peroxidase through the glutaraldehyde activation then, adds the carbonate buffer solution of 0.1ml0.2mol/Lph9.5 again; Spend lucifuge stirring reactions 20 hours in 4; Add 0.1ml 0.2mol/L L-lysine solution again, last sephadex G 75 pillar chromatographies are collected the eluent that 402nm and 242nm wavelength have maximum absorption band; Merge and collect liquid; 4 degree are to the PBS of 0.1mol/l PH 6. dialysis 48 hours, during change liquid 4 times, the glycerine that adds equivalent is preserved subsequent use in-20 degree.

2. immunogene and coating antigen

The summary of the preparation principle of immunogene and coating antigen: salbutamol (SAL) be a micromolecular compound, only has reactionogenicity and lacks immunogenicity, genus haptens.The conjugates that haptens and carrier protein couplet form not only has immunogenicity, and itself special reactionogenicity is arranged.With salbutamol antigen and carrier protein OVA, make SAL and the synthetic immunizing antigen SAL-OVA of OVA coupling through mixed anhydride method, carry out scanning of ultraviolet-visible absorbing wavelength and infrared spectrum behind the purifying and identify whether coupling is successful, successfully promptly can be used as immunogene.

2.1 preparation flow

A) preparation of salbutamol succinic anhydride: adopt the succinic anhydride method that 200mgSal is added after the evaporation of 25ml methyl alcohol does, add the 40ml absolute ethyl alcohol again, stirring at room.Add after the 90mg succinic anhydride continues to be stirred to milky, stirred again two hours, filter the back salbutamol-succinic anhydride (SAL-HS).

B) coupling of salbutamol-succinic anhydride and carrier protein: adopt carbodlimide method, salbutamol-succinic anhydride is mixed stirring at room 3h with the EDC mol ratio at 1: 1; Add carrier protein OVA (mol ratio is 20: 1) again; Room temperature effect 1h, and constantly stir, PH keeps 7.0-7.4; Reaction finishes back dialysis 48h, during change liquid (PBS) 6-8 time.Immunizing antigen (SAL-HS-OVA).

2.2SAL-OVA the authentication method of artificial antigen

2.2.1 ultraviolet spectrophotometer method

The standard solution of accurately joining SAL and OVA with physiological saline.Take by weighing a certain amount of SAL-OVA and be dissolved in the physiological saline, survey the concentration of its protein with the Broafdr method.Concentration according to protein in this concentration adjustment SAL-OVA solution makes it consistent with OVA.Use the Uv3000 ultraviolet scanner, scan respectively in the scope of (200-360) at wavelength.The substitution formula calculates the coupling rate of SAL-OVA.

2.2.2SDS polypropylene phthalein amine gel electrophoresis

After pressing slab-electrophoresis 10% gel formula and in mould, pouring into separation gel, add one deck water seal on the separation gel surface carefully and live the glue face, place 1h after; Sop up water, perfusion concentrates glue, inserts that to remove comb behind the suitable comb placement hl subsequent use; Select SAL-OVA as identifying sample, from the SAL-OVA solution of 1mg/ml, draw the sample buffer that 1ul adds equivalent, 100 ℃ of water-baths application of sample after 30 minutes; Simultaneously carry out electrophoresis with OVA as contrast, electrophoresis is used coomassie brilliant blue staining after finishing; On decolorization swinging table, decolour the analysis result of taking a picture at last with destainer.

3 Polyclonal Antibody Preparation flow processs

3.1 above-mentioned synthetic SAL-OVA as immunogene, is diluted to 1mg/ml, with the freund 's incomplete adjuvant mixed in equal amounts; Adopt intracutaneous injection inoculation sheep; Whenever immunity was all around once taken a blood sample to test clearly at every turn and is tired in immune back 7 days, observation antiserum titre growth pattern; When reaching satisfied tiring, jugular vein blood sampling separation of serum.

3.2 purifying antibody

Antibody globulin with in the saturated ammonium sulfate salting out method purification serum is further purified with affinity chromatography, uses the spectrophotometer measurement protein content, and detects its ELISA and tire.

3.2 the CHARACTERISTICS IDENTIFICATION of anti-SAL polyclonal antibody

3.2.1 titration

Antibody behind the purifying from 100 * continuously doubling dilutions, is added in the ELISA Plate that is encapsulated by SAL-OVA, add the anti-sheep IgG of rabbit of HRP mark again.If positive control, each two hole of negative control.Testing result is positive more than or equal to the twice of negative control with 450nm OD value.With positive Kongzui highly diluted multiple for tiring.

3.2.2 specific assay

Use SAL, BSA, KLH, OVA coated elisa plate respectively, contain the PBS sealing of 50g/L skimmed milk power after, add dilution back antibody, the reaction back adds the anti-sheep IgG of HRP mark rabbit, the colour developing of tmb substrate system is measured absorbance under the 450nm with ELIASA.

3.2.3 molecular weight determination

Suitably carry out polyacrylamide gel electrophoresis after the dilution with the ascites behind the purifying, the 100g/L separation gel, electrophoresis is accomplished poststaining, decolouring, observations.

The preparation preparation of 4 reagent

4.1 standard solution: accurately take by weighing salbutamol 1mg, be mixed with the mother liquor of 1mg/ml, be diluted to final required concentration (0.1ppb-8.1ppb), can with PBS again.

4.2 enzyme-labelled antigen solution: with the enzyme dilution enzyme-labelled antigen is diluted to 1: 1000, can.

4.3 substrate A: the acetic acid-citrate buffer solution with 0.1M is diluted to 0.07% with urea peroxide, can.

4.4 substrate B: the acetic acid-citrate buffer solution with 0.1M is diluted to 0.07% with TMB, can.

4.5 stop buffer: 2M sulfuric acid.

4.6 cleansing solution: get the 50ml concentrated cleaning solution and add deionized water 950ml dilution, use behind the mixing.Put 4 ℃ of preservations.

4.7 extract: get the 50ml concentrated extracting solution and add deionized water 950ml dilution, use behind the mixing, put 4 ℃ of preservations.

4.8 encapsulate plate to encapsulate step following:

A) encapsulate: antibody is encapsulated the concentration coated elisa plate with the best, the 100ul/ hole, 4 ℃ are spent the night, and best package amount is 1 μ g/ml, and 0.05M pH9.6 CBS is used to encapsulate solid phase carrier as dilution.

B) wash plate: liquid in the hole is dried, fully wash 4-5 time with cleansing solution 250 μ l/ holes, each 10s at interval claps dried with thieving paper.

C) sealing: the 1%BSA+10%sucrose confining liquid is added ELISA Plate, the 100ul/ hole, 37 ℃ of incubations 2 hours dry liquid in the hole, clap with thieving paper and do.Vacuum drying adds the aluminium foil bag Vacuum Package.

Salbutamol ELISA diagnostic kit analytical performance of the present invention is following:

1, finished product calibrating

1.1 PE

Each component should meet outward appearance and sense organ requirement separately, and loading amount is accurate, and put correctly the position, and assembly is complete.Label is clear, and is complete, and print What is correct.

1.2 experiment calibrating

1.2.1 sensitivity: represent with LDL.Measure 20 hole zero standards, the OD mean value that record records, the minimum medicine of sample when adding 3 times of standard deviations can reach 20 parts of blank samples mensuration averages is dense to be detectability.

1.2.2 accuracy: the recovery is measured the recovery with additive process, and selected 1ppb, two of 5ppb add concentration.Each concentration is done 5 parallel appearance, does 2 holes, is no less than revision test 3 times for every kind.The recovery is 70～110%.

1.2.3 specificity: other drug (seeing instructions) cross reacting rate is met the requirements.

1.2.4 accuracy: selection standard curve 50% inhibition concentration (IC ₅₀) near the standard solution replication of concentration.This reagent is selected 5ppb standard items replication for use.

Variation within batch coefficient: CV＜10%

Interassay coefficient of variation: CV＜15%

1.2.5 it is linear: as the series standard article to be measured,, calculated its R value with the mapping of 4p simulation curve.R＞0.9900.

1.2.6 stability: stability: 37 ℃ of stable experiments of acceleration are answered and were stablized in 5 days.The OD value of 0 titer, IC ₅₀, represent concentration the interpolation recovery, quality-control product measured value all in tolerance.

2 preserve and the terms of validity: in 2-8 ℃ of drying, keep in Dark Place, examine and determine certainly qualified from the term of validity be 6 months.

Claims (5)

1. salbutamol enzyme linked immunological kit is characterized in that: form as follows:

Comprise encapsulating plate, calibration object, enzyme labeling thing working fluid, substrate solution A, substrate solution B, stop buffer, concentrated cleaning solution, concentrated extracting solution,

The said plate that encapsulates is coated with desertification butylamine alcohol polyclonal antibody at the bottom of for the hole; Package amount is 1 μ g/ml, encapsulates temperature and spends the night for 4 ℃;

Said calibration object is the serial calibration object of variable concentrations;

Said enzyme labeling thing working fluid contains the clenobuterol hydrochloride haptens of underlined HRP, for the sodium periodate method with HRP with the clenobuterol hydrochloride hapten;

Said substrate solution A is that hydrogen peroxide urea solution, B are TMB solution;

Said stop buffer is a 2M sulfuric acid.

2. salbutamol enzyme linked immunological kit according to claim 1 is characterized in that: said concentrated cleaning solution is the 20X concentrated cleaning solution, before using with distilled water according to 1: the dilution proportion of 19v/v, use behind the mixing.

3. salbutamol enzyme linked immunological kit according to claim 1 is characterized in that: said concentrated extracting solution 20X concentrated extracting solution, before using with distilled water according to 1: the dilution proportion of 19v/v, use behind the mixing.

4. salbutamol enzyme linked immunological kit according to claim 1 is characterized in that: said encapsulate plate to encapsulate step following:

A) antibody sandwich: encapsulate the concentration coated elisa plate with desertification butylamine alcohol polyclonal antibody the best, the 100ul/ hole, 4 ℃ are spent the night, and best package amount is 1 μ g/ml, and 0.05M pH9.6 CBS is used to encapsulate solid phase carrier as dilution;

B) wash plate: liquid in the hole is dried, fully wash 4-5 time with cleansing solution 250 μ l/ holes, each 10s at interval claps dried with thieving paper;

C) sealing: the 1%BSA+10%sucrose confining liquid is added ELISA Plate, the 100ul/ hole, 37 ℃ of incubations 2 hours dry liquid in the hole, clap with thieving paper and do, and vacuum drying adds the aluminium foil bag Vacuum Package.

5. the method for application of a salbutamol enzyme linked immunological kit as claimed in claim 1, it is characterized in that: step is following:

(1) required reagent is taken out from cold storage environment, place more than the equilibrium at room temperature 30min, note to shake up before every kind of liquid reagent uses;

(2) taking-up needs the microwell plate of quantity, and no microwell plate is put into aluminide-coating bag, is stored in 2-8 ℃;

(3) cleansing solution also need be risen again before use;

(4) numbering: the corresponding micropore of sample and calibration object is numbered according to the order of sequence, and it is parallel that each sample and calibration object are done 2 holes, and write down the position that calibration hole and sample aperture belong to;

(5) add calibration object/sample: add calibration object/sample 50 μ l in the micropore of correspondence, add enzyme labeling thing 50 μ l/ holes immediately, the mixing that vibrates gently is with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of shrouding film shrouding;

(6) wash plate: carefully open the shrouding film, liquid in the hole is dried, fully wash 4-5 time with cleansing solution 250 μ l/ holes, each 10s at interval claps dried with thieving paper;

(7) colour developing: add colour developing liquid 100 μ l/ holes, substrate A liquid and 1: 1 mixing of substrate B liquid, the mixing that vibrates gently behind shrouding film shrouding, is put in 25 ℃ of lucifuge environment and is reacted 15min;

(8) measure: add stop buffer 50 μ l/ holes, the mixing that vibrates is gently set ELIASA in the 450nm place, measures every hole OD value;

(9) result: the mean value of calibration that is obtained and sample absorbance multiply by 100 again divided by the absorbance of first calibration.

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