CN109813919A - A kind of remaining enzyme linked immunological kit of detection Trenbolone - Google Patents

Abstract

The present invention provides a kind of remaining enzyme linked immunological kit preparation of detection Trenbolone and detection methods, it includes: that liquid is redissolved in the ELISA Plate for being coated with coating antigen, ELIAS secondary antibody, Trenbolone specific antibody working solution, Trenbolone standard solution, substrate developing solution, terminate liquid, concentrated cleaning solution and concentration.Residues detection of the enzyme linked immunological kit provided by the invention for Trenbolone in livestock and poultry, the kit have the characteristics that detection is simple and quick, testing result is accurate, testing cost is low, high sensitivity, can remain in on-site test and play a significant role in Trenbolone.

Description

A kind of remaining enzyme linked immunological kit of detection Trenbolone

Technical field

The present invention relates to enzyme-linked immune detection methods, and in particular to Trenbolone is remaining enzyme-linked in detection tissue, urine sample Immune reagent kit and its detection application.

Background technique

Trenbolone (Trenbolon, TR) chemical name trenbolone, molecular formula C₁₈H₂₂0₂, molecular weight 270, 170 DEG C of fusing point.Trenbolone is using more extensive Steroid anabolic hormones, so-called protein anabolic hormone, is that one kind can enough promote Into the growth and differentiation of cell, expand muscle, even more body hormones of the intensity and size of bone.It is by natural Such as emerald green ketone of male sex hormone reduces androgenic activity, improves semi-synthetic hormone obtained from protein assimilating activity through structure of modification Class drug.

It plays an important role in livestock and poultry control and treatment.With animal husbandry scale, intensivization development, minority production Enterprise and raiser, operator are to seek maximum profit, set state's laws regulation in ignoring, abuse or illegally disobeyed using hormone etc. Banning drugs product.As the Trenbolone stock raising of anabolic hormones additive, nutritional ingredient can be made from adipose tissue to musculature Transfer, increases lean meat.But often result in Trenbolone remained in animal derived food it is exceeded.Surpass when people have eaten residual After target animal food, accumulation the poisoning symptoms such as headache, uncomfortable in chest, palpitaition, myalgia, severe jamming can be generated in human body The natural hormonal balance of human body, leads to opposite sexization phenomenon, it is also possible to liver, kidney, cardiovascular system and reproductive system are seriously damaged, and It can make one to generate pharmacological dependence, a few peoples are also possible that serious emotion and mental illness, even result in gene mutation, induce Malignant tumour directly endangers the health and life of people.Based on the above harm, what The Ministry of Agriculture of the People's Republic of China, MOA promulgated for 2002 No. 235 file regulations must not also detect in animal derived food.In addition, year European Union proposes a type to China's animal products Residue of veterinary drug, antibiotic detect monitoring requirement, wherein explicitly pointing out, in animal derived food medicament residue, Trenbolone must not Detection.

Summary of the invention

The present invention is quasi- to provide a kind of kit specifically for Trenbolone residue detection, using indirect competitive ELISA method, And a kind of efficient, accurate, easy, meeting high-volume screening sample qualitative and quantitative analysis and reagent box preparation method are provided, it should Method is limited to 2.3 μ g/kg to tissue lowest detection, and feed lowest detection is limited to 2.5 μ g/kg, and the lowest detection of urine is limited to 2.5 μg/L。

Trenbolone residue detection kit of the present invention mainly includes following reagent: coating Trenbolone coupling is anti- Former ELISA Plate, Trenbolone drug standard solution, ELIAS secondary antibody, Trenbolone monoclonal antibody working solution, substrate solution, terminate liquid, Concentrated cleaning solution, concentration are redissolved liquid and are formed.

Kit of the present invention, based on Trenbolone haptens preparation method, this method is with Trenbolone Acetate For raw material, haptens can be obtained through succinic anhydride method two-step reaction, synthetic method craft is simple, and molecular structural formula is I institute of formula Show:

(formula I).

Trenbolone hapten synthesis route of the invention is as follows:

It weighs 5g Trenbolone Acetate raw material and is dissolved in 50-100mL ethyl alcohol, 0.5-1g hydroxylamine hydrochloride and 10mL 6mol/L is added Sodium hydroxide solution, 80 DEG C of back flow reactions, TLC method detect response situation, and 4-6h fully reacting removes ethyl alcohol, toward remaining reaction 5-10mL deionized water is added in liquid, is extracted three times with petroleum ether, each 10mL, water intaking is mutually acidified with 2mol/L hydrochloric acid, PH=6.0-7.0 is adjusted, then is extracted with ethyl acetate, until inspection does not measure ultraviolet point in extracting solution, with saturated salt solution and water difference It is dry after washing, obtain intermediate product.Intermediate product technology path is as follows:

Intermediate product prepared by previous step is dissolved in appropriate pyridine, 20-50mL succinic anhydride is added, 50 DEG C of reactions are stayed overnight, TLC detects reaction result, after reaction, adjusts pH value of solution=5.0-5.5 with HCl, then be extracted with ethyl acetate, dry.It is remaining Object is purified with silica gel column chromatography, obtains Trenbolone Acetate derivative.Be dissolved in 50mL methanol: water=1:5-10 solution is added suitable Lithium hydroxide is measured, 2-4h is reacted at room temperature, reaction solution petroleum ether is extracted three times, each 10mL, phase of fetching water slowly is added dropwise thereto There are a large amount of white solids, as Trenbolone haptens product in 2mol/L hydrochloric acid.Technology path is as follows:

In the present invention, Trenbolone Acetate molecular formula is C₂₀H₂₄O₃。

Use method of the invention that structural formula is made as the hapten compound of formula I, which remains to greatest extent The chemical structure of Trenbolone, and active group-COOH is introduced, tie point is added for antigen preparation.This chemical reaction two step method is complete At the rate of recovery 35%.

The present invention also provides a kind of preparation methods of Trenbolone antigen.

It weighs a certain amount of haptens to be dissolved in DMF solution, DEC is added and activates 30min, carrier protein is added and is dissolved in 5mL It is coupled in the phosphate buffer that pH is 7.2, prepares immunogene, dialysed with 0.01mol/L phosphate buffer at 4 DEG C 3d replaces 3 bag filters daily, removes unreacted small-molecule substance；Animal immune is used for after the completion of dialysis.Structural formula is formula II:

(formula II).

Carrier protein is bovine serum albumin, ovalbumin, white azurin.

The Trenbolone specific antibody is to obtain through Trenbolone coupled antigen as immunogen immune, and the Trenbolone is special Heterogenetic antibody can be Trenbolone monoclonal antibody or Trenbolone polyclonal antibody, wherein it is preferred that Trenbolone monoclonal antibody.

The marker enzyme of the ELIAS secondary antibody is horseradish peroxidase；Marker enzyme and antiantibody use glutaraldehyde method or acid iodide Sodium method is coupled.

To meet on-site test and great amount of samples screening, the kit further includes following reagent: Trenbolone standard items are molten Liquid, terminate liquid, cleaning solution, redissolves liquid at substrate solution.

6 bottles of the standard solution, concentration are 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.35 μ respectively g/L、4.05μg/L。

Substrate solution is horseradish peroxidase developing solution, and substrate A liquid is urea peroxide buffer, and substrate B liquid is tetramethyl Benzidine buffer.

The terminate liquid is the sulfuric acid or hydrochloride buffer of 1-2mol/L.

The cleaning solution is the phosphorus of pH=7.2 0.2-0.5mol/L of the Sodium azide of 1%-1.5% Tween-20 and 0.01% Phthalate buffer, the percentage are quality percent by volume.

It is described to redissolve the phosphate-buffered that liquid is 0.1-0.2mol/L of pH=7.2 containing 10%-15% bovine serum albumin(BSA) Liquid, the percentage are quality percent by volume.

Testing principle of the invention are as follows:

The present invention uses indirect competitive ELISA method, by the High sensitivity of the high degree of specificity of antigen-antibody reaction and enzymatic Property combine, using substrate for enzymatic activity color developing detection production concentration, pre-coated Trenbolone coupling is anti-on micropore of enzyme marker plate item Original, coupled antigen pre-coated on remaining Trenbolone and capillary strip competes anti-Trenbolone antibody in sample, and ELIAS secondary antibody is added Afterwards, it being developed the color with tmb substrate, sample absorbance value and the content of contained residue Trenbolone are negatively correlated, compared with standard curve, Multiplied by its corresponding extension rate, the residual quantity of Trenbolone in you can get it sample.

Trenbolone enzyme-linked immunologic detecting kit of the present invention has the characteristics that high sensitivity, simple and convenient, accurate, to group Vigorous dragon residue detection plays a significant role.

Detailed description of the invention

Fig. 1 Trenbolone haptens structure schema I.

Fig. 2 Trenbolone antigenic structure schema II.

Fig. 3 Trenbolone hapten synthesis reaction route.

Fig. 4 Trenbolone ELISA kit standard curve.

Specific embodiment

Embodiment one: Trenbolone kit forms preparation

One, Trenbolone hapten synthesis and identification

Weigh 5g Trenbolone Acetate, 0.5-1g hydroxylamine hydrochloride.

(1) 5g Trenbolone Acetate is dissolved in ethyl alcohol and obtains solution A；

(2) 0.5-1g hydroxylamine hydrochloride and sodium hydroxide are added in solution A, 80 DEG C are stirred to react, and extract product；

(3) appropriate pyridine, ethanedioic acid acid anhydride, 50 DEG C of reactions are added；

(4) TLC lamellae detects response situation, until it is very shallow without raw material or raw material point, terminate reaction；

(5) silica gel column purification is concentrated to get product, as haptens, obtains product 1.75g, yield 35%, reaction technology route It is as follows:

Two, the preparation of Trenbolone immunogene

It weighs 42.7mg Trenbolone haptens to be dissolved in 2mL DMF, 65mg EDC and 60mg NHS(is added and is dissolved in 2mL water) Activation 30min is carried out, 100-400mg carrier protein BSA is added to and is dissolved in 5mL aqueous solution, tune pH value of solution to 8.5-9, and It stirs at room temperature for 24 hours；With 0.01mol/L PBS in 4 DEG C of dialysis 3d, 3 bag filters are replaced, daily to remove unreacted small point Sub- substance；Packing, saves backup in -20 DEG C.

Three, the preparation of Trenbolone coating antigen

It weighs 42.7mg Trenbolone haptens to be dissolved in 2mL DMF, 65mg EDC and 60mg NHS(is added and is dissolved in 2mL water) Activation 30min is carried out, 250-520mg carrier protein BSA is added to and is dissolved in 5mL aqueous solution, tune pH value of solution to 8.5-9, and It stirs at room temperature for 24 hours；With 0.01mol/L PBS in 4 DEG C of dialysis 3d, 3 bag filters are replaced, daily to remove unreacted small point Sub- substance；Packing, saves backup in -20 DEG C.

Four, the identification of Trenbolone hapten-carrier protein conjugate

Carrier protein, Trenbolone haptens, Trenbolone hapten-carrier protein conjugate are made into 0.5mg/ with the PB of pH7.4 The solution of mL is returned to zero with the PB of 0.01mol/L pH7.4, is scanned within the scope of wavelength 200-800nm with ultraviolet specrophotometer. Obtain the absorption curve of carrier protein, Trenbolone haptens, Trenbolone hapten-carrier protein conjugate.Three as the result is shown There is different absorption curves, shows Trenbolone haptens and carrier protein couplet success, Trenbolone haptens and ox blood are pure The combination ratio of albumen is 13-20:1, and the combination ratio of Trenbolone haptens and white azurin is 15-25:1.Molecular structural formula such as formula Shown in II:

(formula II).

Five, prepared by Trenbolone monoclonal antibody

Animal immune: immunogen immune 8-10 week old Balb/c mouse is obtained by above-mentioned, immunizing dose is 50 μ g/.

Cell fusion and cloning: immune Balb/c mouse boosting cell is taken, is melted by a certain percentage with SP2/0 myeloma cell It closes, the hybridoma cell strain of stably excreting monoclonal antibody is capable of in screening

Cell cryopreservation and recovery: hybridoma is prepared into 1 × 10 with frozen stock solution⁸The cell suspension of a/mL, it is long in liquid nitrogen Phase saves；Cryopreservation tube is taken out when recovery, instant in 37 DEG C of water-baths immediately, centrifugation removal frozen stock solution moves into culture bottle culture.

Six, the preparation of ELIAS secondary antibody

Sheep anti mouse antiantibody is coupled with horseradish peroxidase (HRP) using the Over-voltage protection after optimization.After optimization HRP and Trenbolone antibody molar concentration rate are 1.5-2.5:1.

Seven, the preparation of ELISA Plate

The coating buffer that the ELISA Plate preparation process of Trenbolone coupled antigen uses is pH=9.2-9.6 concentration 0.1-0.2mol/ L carbonate buffer solution, confining liquid are pH=7.0-7.4 bovine serum albumin(BSA) containing 1%-5%, concentration 0.1-0.2mol/L Tris buffering Liquid, the percentage are quality percent by volume.

Coating antigen is coated buffer and is diluted to 10 μ g/mL, and 100 μ L, 37 DEG C of constant-temperature incubation 2h or 4 DEG C of mistakes are added in every hole Night removes aerial liquid, is washed 1 time with cleaning solution, stops 30s, pats dry, and 150 μ L confining liquids, 37 DEG C of incubation 2- are added in every hole 3h removes liquid in hole, pats dry, and is saved after dry with filter membrane vacuum sealing.

Embodiment two: Trenbolone enzyme linked immunological kit is constituted

Trenbolone Trenbolone enzyme linked immunological kit includes following each components:

(1) it is coated with the antigen ELISA Plate of Trenbolone coupling protein.

(2) ELIAS secondary antibody: sheep anti mouse antiantibody-horseradish peroxidase.

(3) Trenbolone monoclonal antibody working solution.

(4) 6 bottles of standard solution, according to gradient dilution, concentration is respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L、1.35μg/L、4.05μg/L。

(5) substrate A liquid is urea peroxide buffer, and substrate B liquid is tetramethyl benzidine buffer.

(6) terminate liquid is the sulfuric acid or hydrochloride buffer of 1-2mol/L.

(7) concentrated cleaning solution is pH=7.2 0.2-0.5mol/L of the Sodium azide of 1%-1.5% Tween-20 and 0.01% Phosphate buffer.

(8) it is slow to redissolve the phosphate that liquid is 0.1-0.2mol/L of pH=7.2 containing 10%-15% bovine serum albumin(BSA) for concentration Fliud flushing.

Embodiment three: the application of Trenbolone enzyme linked immunological kit actual measurement sample

(1) reagent configures

0.1M sodium hydroxide solution: it weighs 2.0g sodium hydroxide and the mixing of 500mL deionized water dissolving is added.

It redissolves working solution: liquid is redissolved into 2 × concentration with deionized water and is diluted by 1:1 volume ratio for extracting sample It redissolves.

Wash operating solution: 20 × concentrated cleaning solution is diluted by 1:19 volume ratio for ELISA Plate with deionized water Washing.

(2) sample pre-treatments

1. organizing (muscle, liver etc.) Sample pretreatment

8mL 0.1M sodium hydroxide is added into 50mL polystyrene centrifuge tube in tissue samples after weighing 2.0 ± 0.05g homogeneous Solution vibrates 5min, 3000g or more with oscillator, and room temperature is centrifuged 10min；1mL supernatant to 50mL polystyrene is pipetted to be centrifuged 10mL n-hexane is added in Guan Zhong, vibrates 5min, 3000g or more with oscillator, room temperature is centrifuged 10min；It is organic to pipette the upper layer 5mL It is dried up under 50-60 DEG C of water-bath nitrogen stream in phase 10mL clean dried teat glass；1mL is added and redissolves working solution, uses vortex instrument Vortex 2min dissolves dried residue, takes 50 μ L for analyzing.

2. urine sample Sample pretreatment

The limpid urine specimen of 1mL is pipetted into 50mL polystyrene centrifuge tube, 4mL 0.1M sodium hydroxide solution is added, with oscillation Device vibrates 2-5min；1mL mixed liquor is pipetted into 50mL polystyrene centrifuge tube, 10mL n-hexane is added, is vibrated with oscillator 5min, 3000g or more, room temperature are centrifuged 10min；It pipettes in 5mL upper organic phase 10mL clean dried teat glass in 50-60 DEG C It is dried up under water-bath nitrogen stream；1mL is added and redissolves working solution, dissolves dried residue with vortex instrument vortex 2min, 50 μ L is taken to be used for Analysis.

3. feed Sample pretreatment

1.0 ± 0.05g feed sample is weighed into 50mL polystyrene centrifuge tube, 1mL deionized water, 3mL 0.1M hydrogen-oxygen is added Change sodium solution, with vortex instrument vortex 1min, 10mL n-hexane is added, vibrates 10min, 3000g or more, room temperature centrifugation with oscillator 10min；It pipettes and is dried up under 50-60 DEG C of water-bath nitrogen stream in 1mL upper organic phase 10mL clean dried teat glass；It is added 1mL redissolves working solution, and with vortex instrument vortex 2min dissolution dried residue, (batch is by sample liquid and redissolves working solution by 1:9 Volume ratio dilution detection；Concentrate feed/premix is by sample liquid and redissolves working solution by 1:19 volume ratio dilution detection), take 50 μ L For analyzing.

(3) detecting step

1. plus standard items/sample and antibody working solution: adding 50 μ L of standard items/sample into corresponding micropore, antibody is then added 50 hole μ L/ of working solution, gently oscillation mixes, and reacts 30min in cover board membrane cover plate 25 DEG C of light protected environments of postposition.

2. board-washing: carefully opening cover board film, liquid in hole is dried, 250 hole μ L/ of wash operating solution is added, sufficiently washs 4-5 times, every minor tick 30s, cleaning solution in board falling hole is sprinkled, is patted dry with blotting paper.

3. plus ELIAS secondary antibody: 100 hole μ L/ of ELIAS secondary antibody is added, gently oscillation mixes, and is kept away with 25 DEG C of postposition of cover board membrane cover plate 30min is reacted in luminous environment, is taken out and is repeated step 2..

4. colour developing: 50 hole μ L/ of substrate solution A liquid is added, adds 50 hole μ L/ of substrate solution B liquid, gently oscillation mixes, with lid 15min is reacted in plate membrane cover plate 25 DEG C of light protected environments of postposition.

5. measurement: 50 hole μ L/ of terminate liquid is added, gently oscillation mixes, and sets microplate reader at 450nm, measures every hole OD Value.

(4) result judges

The percentage absorptance of the calculating of percentage absorptance, standard items or sample is equal to being averaged for the absorbance value of standard items or sample It is worth (diplopore) divided by the average value of the absorbance value of first standard items (0 standard), arrives percentage extinction multiplied by 100% Rate.Using standard items percentage absorptance as ordinate, using the logarithm of Trenbolone standard concentration as slogan banner, canonical plotting is drawn (such as Fig. 4).The percentage absorptance of sample is substituted into standard curve, concentration corresponding to sample is read from standard curve, is multiplied It is the actual concentrations of Trenbolone in sample with its corresponding extension rate.

Percentage absorptance (%)=×100%

The mean absorbance values of B-standard items or sample solution

B₀The mean absorbance values of -0ppb standard solution

Example IV: the determination of Trenbolone enzyme linked immunological kit technical parameter

(1) kit minimum detection limit and sensitivity determination

20 detections are carried out to recessive chicken, urine, feed sample, the average value of measurement result is reagent plus 3 times of standard deviations As a result the minimum detection limit of the actual sample of box obtains this method lowest detection in blank chicken sample and is limited to 2.3 μ g/L, blank Lowest detection is limited to 2.5 μ g/L in urine sample sample, and lowest detection is limited to 2.5 μ g/L in blank Feed Sample.

IC50 is frequently as evaluation competitive enzyme-linked immune kit sensitivity index.It is detected using Trenbolone standard solution, Standard curve is drawn according to actual result, kit Trenbolone standard curve is between 0.05-4.05 μ g/L as seen from the figure.Urine Middle Trenbolone sensitivity is 0.54 μ g/L.

(2) kit accuracy and precision

It takes blank chicken meat sample to be added respectively with 10 μ g/L, 20 μ g/L and 40 μ g/L Trenbolones, takes blank diaper sample point It is not added with 10 μ g/L, 20 μ g/L and 40 μ g/L Trenbolones, takes blank Feed Sample respectively with 10 μ g/L, 20 μ g/L and 40 μ g/L Trenbolone is added, and every kind of sample, each concentration each 6 parallel.3 batches of kits are extracted, every batch of kit measurement is same A sample 2 times, as a result this method to the chicken meat sample rate of recovery between 74.5%-106.2%, exist to urine sample sample recovery rate Between 81.1%-107.8%, to the Feed Sample rate of recovery between 85.1%-99.4%, within-run and between-run analysis coefficient is < 10%.

(3) kit saves experiment

Kit preservation condition is 2-8 DEG C, is measured by 15 months, OD value, the IC50 value of the zero standard product of kit, 10 μ g/ L, 20 μ g/L and 40 μ g/L add the chicken rate of recovery, and 10 μ g/L, 20 μ g/L and 40 μ g/L add the urine rate of recovery, 10 μ g/L, 20 μ G/L and 40 μ g/L adds the feed rate of recovery, within 12 months within normal range (NR).Accelerated ageing and freezing examination are done simultaneously It tests, kit is placed in 37 DEG C, -20 DEG C 9 days, measurement result also indicates that the indices of kit are normal.From above As a result obtaining Trenbolone enzyme linked immunological kit can save 12 months at 2-8 DEG C.

Claims (5)

1. a kind of remaining enzyme linked immunological kit of detection Trenbolone, ELISA Plate, group including being coated with Trenbolone coupled antigen Vigorous dragon standard items, ELIAS secondary antibody, antibody concentrated solution, substrate solution, terminate liquid, concentrated cleaning solution and the multiple solution composition of concentration.

2. a kind of remaining kit of detection Trenbolone according to right 1, Trenbolone coupled antigen is anti-by Trenbolone half Original is obtained with carrier protein couplet, and the carrier protein is bovine serum albumin, ovalbumin, white azurin；Its molecular structural formula As shown in formula II,

(formula II).

3. a kind of remaining enzyme linked immunological kit of detection Trenbolone according to right 2, Trenbolone haptens is by Trenbolone Acetic acid esters is reacted through succinic anhydride to be made, and molecular structural formula is shown in formula I,

(formula I).

4. a kind of remaining enzyme linked immunological kit of detection Trenbolone according to claim 1, it is characterised in that the group The concentration of vigorous dragon standard solution is respectively 0 μ g/L, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.35 μ g/L, 4.05 μ g/L.

5. a kind of method for detecting Trenbolone, comprising the following steps:

(1) sample pre-treatments；

(2) it is detected with enzyme linked immunological kit；

(3) Analysis of test results.