CN104655847A - Enzyme linked immunosorbent assay kit (ELISA kit) for detecting adprin and detection method thereof - Google Patents

Abstract

The invention provides an enzyme linked immunosorbent assay kit (ELISA kit) for detecting adprin and a detection method thereof. The ELISA kit is sensitive, accurate and quick in detection, is simple in operation, is strong in specificity and is suitable for detection for a large scale of samples. The ELISA kit includes: an ELISA plate coated by an adprin antigen, an adprin standard sample, an adprin antibody operating fluid, an adprin enzyme-labeled secondary antibody operating fluid, a substrate liquid A, a substrate liquid B, a stop buffer, a concentrated diluent and a concentrated washing liquid. The principle of the ELISA kit for detecting adprin is solid-phase indirect competitive ELISA. In the detection method, an extracted sample, the adprin enzyme label second antibody operating fluid and the adprin antibody operating fluid are added to corresponding microholes in the ELISA plate. After incubation for a certain time, the ELISA plate is washed and is added with the substrate liquid A and the substrate liquid B, and then a color developing agent is blue in color under effect of enzymes. After the stop buffer liquid added, the color is turned into yellow from blue. An inversely proportional relationship is formed between the depth of the developed color and the content of the adprin in the standard sample or the sample. The method can be directly used for detecting residual amount of the adprin in chicken tissue.

Description

Detect enzyme linked immunological kit and the detection method thereof of Adprin

Technical field

The present invention relates to detection of veterinary drugs in food technical field, particularly detect the enzyme linked immunological kit of Adprin in Chicken Tissues.

Background technology

Currently cause the extent of injury of chicken coccidiasis very high by Eimeria coccidia, have a strong impact on poultry growth and grow and cause environmental pollution.Adprin belongs to ucleotides compound, there is a lot of research abroad, belong to a kind of novel efficient coccidiostat, it plays Anti-human globulin test by the purine metabolism of interference coccidia, its Anti-human globulin test peak phase is the agamocytogeny of coccidia, and namely after coccidium infection, 1 ~ 3 d effect is the strongest.Adprin is by being the coccidiostat of new generation that Agricultural University Of Anhui's pharmacological experiment room and Agricultural University Of Nanjing's pharmacological experiment room are researched and developed jointly, there is the advantages such as anticoccidial spectrum is wide, efficient, low toxicity, all effective to Eimeria tenella, heap-type Eimeria, Eimeria maxima and Eimeria Necatrix.In recent years, due to the continuous expansion of poultry farming scale, the frequent outburst of nationwide global-worm illness causes various anticoccidial drug to use in a large number.In addition, some raiser in order to economic interests, unreasonable use anticoccidial drug and not specifying in accordance with the off-drug period.Because Adprin mainly exists with former medicine form at chicken internal metabolism, and with former medicine form excretion, therefore cause residual in Chicken Tissues of this medicine and the pollution to environment.If human body often takes in the same residue of low dosage, the increase of drug tolerant bacteria in body can be caused and difficulty is caused to clinical treatment infectious diseases; Meanwhile, because residue is accumulated in vivo, cause various organ lesion, even canceration, this will cause serious threat to China's animal product quality safety and consumer health.The domestic relevant report still not having Adprin residue detection in chicken, therefore, is necessary to set up the detection technique that in a kind of Chicken Tissues, Adprin is residual.。

Enzyme linked immunosorbent assay is a kind of accurate, reliable, quick, special detection method, is suitable for the rapid screening of gross sample, has been widely used in food safety detection industry in recent years.The present invention is intended to set up a kind of enzyme linked immunological kit and the detection method thereof that detect Adprin.

Summary of the invention

In order to overcome chromatographic deficiency, the invention provides a kind of enzyme linked immunological kit and the detection method thereof that detect Adprin.The method is sensitive, accurate, quick, easy and simple to handle, high specificity, is applicable to the quick detection of gross sample.

The present invention detects the enzyme linked immunological kit of Adprin, comprises ELISA Plate, Adprin standard items, Adprin antibody working fluid, Adprin ELIAS secondary antibody working fluid, substrate solution A, substrate solution B, stop buffer, concentration and dilution liquid and concentrated cleaning solution.

The present invention detects the preparation of the enzyme linked immunological kit of Adprin, comprises the following steps: the preparation of the preparation of the preparation of the preparation of ELISA Plate, the preparation of Adprin standard items, Adprin antibody working fluid, the preparation of Adprin ELIAS secondary antibody working fluid, substrate solution A, the preparation of substrate solution B, stop buffer, the preparation of concentration and dilution liquid and the preparation of concentrated cleaning solution.

It is further characterized in that: described ELISA Plate is via the antigen coated preparation of Adprin, concrete steps are that Adprin haptens and the pure albumen of carrier proteins Bovine (BSA) coupling are obtained envelope antigen, with carbonate (CBS) damping fluid of 0.05 mol/L pH 9.6 as coating buffer, Adprin envelope antigen is diluted to 1:40000 ratio, 100 μ L/ holes, 37 DEG C of lucifuges hatch 2 h, take out ELISA Plate and get rid of liquid in plate, add diluted concentrated cleaning solution 300 μ L/ hole, wash plate 2 times, 30 s/ time, then 0.5% bovine serum albumin(BSA) (BSA) confining liquid is added, 150 μ L/ holes, place 1.5 h for 37 DEG C, get rid of confining liquid directly to pat dry, ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and is dried, inspect by random samples qualified after ELISA Plate is vacuum-sealed in 4 DEG C of conditions under preserve.

Adprin standard concentration is respectively 0 ng/mL, 0.1ng/mL, 0.3 ng/mL, 0.9 ng/mL, 2.7 ng/mL, 8.1 ng/mL.

Described Adprin antibody working fluid adopts Adprin artificial antigen immune mouse to obtain monoclonal antibody, is diluted to the preparation of 1:60000 ratio with antibody diluent.

Described Adprin ELIAS secondary antibody working fluid adds diluted by ELIAS secondary antibody and becomes 1:2000 ratio, described substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5 mmol/L, described substrate solution B is the ethanolic solution of tetramethyl biphenyl diamines, described stop buffer is the sulfuric acid of 2 mol/L, described concentration and dilution liquid is 10 times of concentration and dilution liquid, be the PBS of 0.1 mol/L, between pH value range 7.0-7.5, described concentrated cleaning solution is 10 times of concentrated cleaning solutions, for containing 0.5% Tween-20, the PBST of 0.1 mol/L, between pH value range 7.0-7.5.

Detect enzyme linked immunological kit and the detection method thereof of Adprin, based on the indirect competitive enzyme-linked immunosorbent reaction principle of antigen-antibody, the method comprises the following steps:

(1) pre-service testing sample is fluid sample by sample preparation to be tested, or uses Solvent Extract methods testing sample, and is redissolved in sample diluting liquid;

(2) required reagent is taken out from cold storage environment, be placed in room temperature (20 ~ 25 DEG C) and balance 30 more than min, notice that often kind of liquid reagent must shake up before using;

(3) ELISA Plate being coated with Adprin antigen is got, add standard items/sample 50 μ L/ hole in the micropore of correspondence, add Adprin ELIAS secondary antibody working fluid, 50 μ L/ holes, then Adprin antibody working fluid is added, 50 μ L/ holes, mixing of vibrating gently, reacts 30 min with in the rearmounted room temperature of cover plate membrane cover plate 25 DEG C of light protected environment;

(4) carefully open cover plate film, liquid in hole is dried, with wash operating solution 260 μ L/ hole, fully washs 4 ~ 5 times, every minor tick 10 s, pat dry (bubble be not eliminated after patting dry can be poked with original rifle head) with thieving paper;

(5) add substrate solution A 50 μ L/ hole, substrate solution B 50 μ L/ hole, mixing of vibrating gently, react 15 ~ 20 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate;

(6) add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450 nm places or dual wavelength 450/630 nm, measures every hole absorbance (please running through data in 5 min);

(7) with the logarithm of standard concentration (ppb) for horizontal ordinate, standard items percentage absorbance is ordinate, drawing standard curve, the content of Adprin in reference standard curve calculation sample.

Wherein, described testing sample following methods carries out pre-service:

A. accurately take in 5.0 ± 0.05g homogenised sample to 50 mL centrifuge tube;

B. first add organic solvent 10mL and 1g anhydrous sodium sulfate, fully to vibrate whirling motion 10min with vortex instrument;

C.5000 centrifugal 10 min of r/min;

D. get supernatant 3 mL to flow down in 50 ~ 60 DEG C of nitrogen and dry up;

E. add 1 mL normal hexane (sample that fat content is high can add 2mL normal hexane), abundant whirling motion 10 s, then add 1 mL sample diluting liquid, low speed whirling motion 10 s; 4000 r/min, centrifugal 5 min, discard upper strata normal hexane and middle layer impurity completely;

F. layer 50 μ L is taken off for analyzing.

The present invention detects the enzyme linked immunological kit of Adprin and the measuring principle of detection method thereof: antigen-specific sexual competition antibody fixing in the Adprin in sample and ELISA Plate, add ELIAS secondary antibody, with antibody response, by enzymatic chromogenic reagent, carry out the content of Adprin in judgement sample according to the depth of colour developing.If the Adprin content in sample is few, colour developing is dark; Otherwise, then develop the color shallow.Kit test method of the present invention is easy and simple to handle, detects sensitive, accurate, quick, is applicable to the quick detection of batch samples.

Embodiment

The preparation of Adprin protein conjugate:

Succinic anhydride method is adopted to obtain being with the Adprin hapten derivant of carboxyl, get 0.05 mmol and the carrier protein BSA combination by 10:1 afterwards than being blended in the carbonate buffer solution (CBS) of 0.05 mol/L pH 9.6, then 0.15 mmol carbodiimide is added, room temperature reaction 24 h is put in stirring, finally dialyse two days in the PBS damping fluid of 0.2 mol/L pH 7.6, remove unreacted haptens, the protein conjugate solution obtained is saved backup in-20 DEG C.

The preparation of Adprin antibody:

Select the purebred BALA/C mouse of healthy adult, get the immunizing antigen 50 μ g prepared with protein molecule to mix with equivalent complete Freund's adjuvant and adopt lumbar injection to carry out initial immunity, adding equivalent incomplete Freund's adjuvant every 3 weeks with same dose immunizing antigen afterwards adopts lumbar injection to carry out secondary, three immunity, after after each immune 6 days, tail vein blood measures antiserum titre to certain titre, do not add adjuvant with same dose and carry out final immunization, get spleen after 3 days and prepare Spleen cell suspensions and myeloma cell carries out Fusion of Cells, filter out required hybridoma cell line and carry out cloning, the hybridoma being in exponential phase is selected to carry out frozen, prepare for ascites, first lumbar injection 0.5 ml whiteruss is in BALB/C mouse sensitization, pneumoretroperitoneum injection 1 × 10 in 2 weeks ⁶individual hybridoma, ascites can be produced after inoculating cell 7-10 days, ascites is extracted with syringe when ascites is many as far as possible, repeatedly collect for several times, centrifugal 15 min of 4000 rpm, collect supernatant, adopt caprylic acid-ammonium purifying ascites to carry out purifying to monoclonal antibody, freeze drying saves backup in-20 DEG C after obtaining freeze-dried powder.

Preparation is coated with the ELISA Plate of Adprin envelope antigen:

Adprin haptens and the pure albumen of carrier proteins Bovine (BSA) coupling obtain by envelope antigen, with carbonate (CBS) damping fluid of 0.05 mol/L pH 9.6 as coating buffer, Adprin antigen diluent is become 1:40000 ratio, 100 μ L/ holes, place 2 h for 37 DEG C, take out ELISA Plate and get rid of liquid in plate, with the concentrated cleaning solution 300 μ L/ hole after dilution, wash plate 2 times, 30 s/ time, then add 0.5% bovine serum albumin(BSA) (BSA) to close, 150 μ L/ holes, place 1.5 h for 37 DEG C, discard confining liquid, ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and is dried, inspect by random samples qualified after by rearmounted for ELISA Plate vacuum seal 4 DEG C preserve.

Adprin standard items compound concentration is respectively 0 ng/mL, 0.1ng/mL, 0.3 ng/mL, 0.9 ng/mL, 2.7 ng/mL, 8.1 ng/mL.

The preparation of Adprin antibody working fluid: adopt Adprin artificial antigen immune mouse to obtain monoclonal antibody, be diluted to the preparation of 1:60000 ratio with antibody diluent.

Adprin ELIAS secondary antibody working fluid adds diluted by ELIAS secondary antibody and becomes 1:2000 ratio, substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5 mmol/L, substrate solution B is the ethanolic solution of tetramethyl biphenyl diamines, stop buffer is the sulfuric acid of 2 mol/L, concentration and dilution liquid is 10 times of concentration and dilution liquid, be the PBS of 0.1 mol/L, between pH value range 7.0-7.5, concentrated cleaning solution is 10 times of concentrated cleaning solutions, for containing 0.5% Tween-20, the PBST of 0.01mol/L, between pH value range 7.0-7.5.

Based on the reagent of above-mentioned preparation, the present invention comprises following material for the enzyme linked immunological kit detecting Adprin:

(1) 96 ELISA Plate × 1 piece, hole;

(2) titer × 6 bottle: (1mL/ bottle) 0 ng/mL, 0.1ng/mL, 0.3 ng/mL, 0.9 ng/mL, 2.7 ng/mL, 8.1 ng/mL;

(3) antibody working fluid 7 mL;

(4) ELIAS secondary antibody working fluid 7 mL;

(5) substrate solution A 7 mL;

(6) substrate solution B 7 mL;

(7) stop buffer 7 mL;

(8) 10 × concentration and dilution liquid 40 mL;

(9) 10 × concentrated cleaning solution 40 mL;

When kit of the present invention is for detecting Adprin residual quantity in chicken sample, implemented by following steps: sample pretreatment, with kit of the present invention carry out detecting, analysis result.

(1) sample pretreatment

A. accurately take in 5.0 ± 0.05g homogenised sample to 50 mL centrifuge tube;

B. first add organic solvent 10mL and 1g anhydrous sodium sulfate, fully to vibrate whirling motion 10min with vortex instrument;

C.5000 centrifugal 10 min of r/min;

D. get supernatant 3 mL to flow down in 50 ~ 60 DEG C of nitrogen and dry up;

E. add 1 mL normal hexane (sample that fat content is high can add 2mL normal hexane), abundant whirling motion 10 s, then add 1 mL sample diluting liquid, low speed whirling motion 10 s; 4000 r/min, centrifugal 5 min, discard upper strata normal hexane and middle layer impurity completely;

F. layer 50 μ L is taken off for analyzing.

(2) Adprin residual quantity in testing sample is detected with kit of the present invention

Get the ELISA Plate being coated with Adprin antigen, add standard items/sample 50 μ L/ hole in the micropore of correspondence; Add ELIAS secondary antibody working fluid, 50 μ L/ holes, then add Adprin antibody working fluid, 50 μ L/ holes, mixing of vibrating gently, react 30 min with in the rearmounted room temperature of cover plate membrane cover plate 25 DEG C of light protected environment; Carefully open cover plate film, dried by liquid in hole, with wash operating solution 300 μ L/ hole, fully washing 4 times, soaks 15-30 s, pats dry with thieving paper; Add substrate solution A 50 μ L/ hole, substrate solution B 50 μ L/ hole, mixing of vibrating gently, react 15 min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate; Add stop buffer 50 μ L/ hole, mixing of vibrating gently, setting microplate reader detects in 450 nm places or dual wavelength 450/630 nm, measures every hole absorbance (please running through data in 5 min); The absorbance size of contrast testing sample and standard items, Adprin residual quantity in quantitative test testing sample.

(3) analysis result

The calculating of percentage absorbance, the percentage absorbance of standard items or sample equals the absorbance of mean value (diplopore) divided by first standard (0 standard) of the absorbance of standard items or sample, then is multiplied by 100%, namely

Percentage absorbance (%)=B/B ₀× 100%

The wherein mean absorbance values of B-standard solution or sample solution, B ₀the mean absorbance values of-0 ppb standard solution.

With the logarithm of the standard concentration of Adprin (ppb) for horizontal ordinate, standard items percentage absorbance is ordinate, and drawing standard curve, obtains straight-line equation.Substituted in typical curve by the percentage absorbance of sample, read the concentration corresponding to sample from typical curve, the extension rate being multiplied by its correspondence is the actual concentrations of Adprin in sample.

Claims (8)

1. detect enzyme linked immunological kit and the detection method thereof of Adprin, comprise ELISA Plate, Adprin standard items, Adprin antibody working fluid, Adprin ELIAS secondary antibody working fluid, substrate solution A, substrate solution B, stop buffer, concentration and dilution liquid and concentrated cleaning solution.

2. detect the enzyme linked immunological kit of Adprin and detection method thereof, comprise the following steps: the preparation of the preparation of the preparation of the preparation of ELISA Plate, the preparation of Adprin standard items, Adprin antibody working fluid, the preparation of Adprin ELIAS secondary antibody working fluid, substrate solution A, the preparation of substrate solution B, stop buffer, the preparation of concentration and dilution liquid and the preparation of concentrated cleaning solution.

3. the enzyme linked immunological kit of detection Adprin according to claim 2 and detection method thereof, it is characterized in that: described ELISA Plate preparation method is for obtain Adprin envelope antigen by Adprin haptens and the pure albumen of carrier proteins Bovine (BSA) coupling, with carbonate (CBS) damping fluid of 0.05 mol/L pH 9.6 as coating buffer, envelope antigen is diluted to 1:40000 ratio, 100 μ L/ holes, hatch 2 h for 37 DEG C, take out ELISA Plate and get rid of liquid in plate, add the concentrated cleaning solution after dilution 300 μ L/ hole, wash plate 2 times, 30 s/ time, then add 0.5% bovine serum albumin(BSA) (BSA) to close, 150 μ L/ holes, place 1.5 h for 37 DEG C, discard confining liquid directly to pat dry, ELISA Plate after patting dry is placed (25 DEG C) between constant temperature and is dried, inspect by random samples qualified after ELISA Plate is vacuum-sealed in 4 DEG C of conditions under preserve.

4. the enzyme linked immunological kit of detection Adprin according to claim 2 and detection method thereof, is characterized in that: the concentration of Adprin standard items is respectively 0 ng/mL, 0.1ng/mL, 0.3 ng/mL, 0.9 ng/mL, 2.7 ng/mL, 8.1 ng/mL.

5. the enzyme linked immunological kit of detection Adprin according to claim 2 and detection method thereof, it is characterized in that: described Adprin antibody working fluid adopts Adprin artificial antigen immune mouse to obtain monoclonal antibody, is diluted to the preparation of 1:60000 ratio with antibody diluent.

6. a kind of enzyme linked immunological kit and detection method thereof detecting Adprin according to claim 2, it is characterized in that: described Adprin ELIAS secondary antibody working fluid adds diluted by ELIAS secondary antibody and becomes 1:2000 ratio, described substrate solution A is the citrate-phosphate disodium hydrogen buffer solution of the carbamide peroxide containing 0.5 mmol/L, described substrate solution B is the ethanolic solution of tetramethyl biphenyl diamines, described stop buffer is the sulfuric acid of 2 mol/L, described concentration and dilution liquid is 10 times of concentration and dilution liquid, it is the PBS of 0.1 mol/L, between pH value range 7.0-7.5, described concentrated cleaning solution is 10 times of concentrated cleaning solutions, it is for containing 0.5% Tween-20, the PBST of 0.01 mol/L, between pH value range 7.0-7.5.

7. the enzyme linked immunological kit of detection Adprin according to claim 2 and detection method thereof, based on the indirect competitive enzyme-linked immunosorbent reaction principle of antigen-antibody, the method is characterized in that: pre-service testing sample, get the ELISA Plate being coated with Adprin antigen, add standard items/sample respectively according to the order of sequence, Adprin ELIAS secondary antibody working fluid, the each 50 μ L/ holes of Adprin antibody working fluid are in the micropore of correspondence, to vibrate gently mixing, 30 min are reacted with in the rearmounted room temperature of cover plate membrane cover plate 25 DEG C of light protected environment, liquid in hole is dried, 4 ~ 5 times are fully washed with wash operating solution, every minor tick 10 s, substrate solution A 50 μ L/ hole is added after patting dry, substrate solution B 50 μ L/ hole, to vibrate gently mixing, 15 ~ 20 min are reacted with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate, add stop buffer 50 μ L/ hole, to vibrate gently mixing, setting microplate reader detects in 450 nm places or dual wavelength 450/630 nm, measure every hole absorbance (please running through data in 5 min), with the logarithm of standard concentration (ppb) for horizontal ordinate, standard items percentage absorbance is ordinate, drawing standard curve, the content of Adprin in reference standard curve calculation sample.

8. method according to claim 7, wherein, described testing sample following methods carries out pre-service: a. accurately takes in 5.0 ± 0.05g homogenised sample to 50 mL centrifuge tube;

B. first add organic solvent 10mL and 1g anhydrous sodium sulfate, fully to vibrate whirling motion 10min with vortex instrument;

C.5000 centrifugal 10 min of r/min;

D. get supernatant 3 mL to flow down in 50 ~ 60 DEG C of nitrogen and dry up;

E. add 1 mL normal hexane (sample that fat content is high can add 2mL normal hexane), abundant whirling motion 10 s, then add 1 mL sample diluting liquid, low speed whirling motion 10 s; 4000 r/min, centrifugal 5 min, discard upper strata normal hexane and middle layer impurity completely;

F. layer 50 μ L is taken off for analyzing.