CN202693588U

CN202693588U - Detection device of florfenicol and metabolin thereof

Info

Publication number: CN202693588U
Application number: CN 201220253047
Authority: CN
Inventors: 徐飞; 王世恩; 牛兰兰; 苏丽芳
Original assignee: BEIJING WDWK BIOTECHNOLOGY Co Ltd
Current assignee: BEIJING WDWK BIOTECHNOLOGY Co Ltd
Priority date: 2012-05-31
Filing date: 2012-05-31
Publication date: 2013-01-23
Anticipated expiration: 2022-05-31

Abstract

The utility model relates to a detection device for detecting florfenicol and metabolin thereof in animal derived food. A detection kit comprises an upper layer and a lower layer, an elisa plate coated by florfenicol-ovalbumin antigens, a cover plate membrane, a zip-lock bag, a specification and a quality testing report are arranged on the upper layer, and the lower layer is provided with six bottles of florfenicol standard substances of different gradient concentrations, one bottle of high-concentration florfenicol standard substances, florfenicol antibody working solution, sample diluent, elisa II antibody working solution, concentrated cleaning solution, substrate A liquid, B liquid and stop solution. An indirect competition law is adopted, florfenicol residual of a sample to be tested and fixed antigens on the elisa plate compete for antibodies, elisa combined substances are added, color is developed through enzyme catalysis, and content of the florfenicol in the sample is calculated by using a sample absorbance value. The kit has the advantages of being simple in detection, rapid, high in sensitivity, low in detection cost, capable of meeting various detection requirements and the like.

Description

Florfenicol and metabolin pick-up unit thereof

Technical field

The invention belongs to the food safety detection field, particularly, the present invention relates to a kind of pick-up unit that detects Florfenicol in the animal derived food and metabolite residue amount thereof.

Background technology

Florfenicol is the broad-spectrum antibiotic of a class novel resisting gram-positive bacteria, Gram-negative bacteria and the Thiamphenicol drug-fast bacteria that are specifically designed to animal health market.Because its antibacterial action is obvious, and cheap, is widely used in the treatment of the various communicable diseases of all kinds of poultry, domestic animal, culture fishery and bee product.Florfenicol can pass through placental barrier, and embryotoxicity is arranged, the metabolite florfenicol amine of Florfenicol, and residual in food easily causes potential alpastic anemia.

Florfenicol (Florfenicol), claim again Florfenicol, its chemical name is that D (+)-Su-1-is to methylsulfonyl phenyl-2-dichloro acetamino-5 fluorine propyl alcohol, 3 fluorine derivatives of Thiamphenicol in the chloromycetin extensive pedigree antibiotic, to effects such as multiple gram-positive bacteria and Gram-negative bacteria and mycoplasmas obviously.The breeding toxicity test confirms that Florfenicol can pass through placental barrier, and embryotoxicity is arranged, and makes F ₁In generation,, male mouse epididymis weight obviously alleviated F ₂Little son's lactation index reduces, and survival rate reduces, thus lactation, period of pregnancy forbidding.Residual in food of the metabolite florfenicol amine of Florfenicol easily causes potential alpastic anemia.

At present the detection of Florfenicol mainly contained high performance liquid chromatography, potential method, HPLC method, polarimetry, the ultraviolet spectrophotometry, polarography etc., and for the report of Florfenicol residue detection there are no relevant monoclonal antibody (McAb) kit, monoclonal antibody is easy to production, height homogeneous and is easy to standardization, and specificity is high, highly sensitive, low residual amt also can be detected, has impayable advantage, very suitable screening analysis to medicament residue.Therefore the development of the monoclonal antibody of Florfenicol, assembling ELISA kit, develop the kit with China's independent intellectual property right, it is very necessary setting up the residual detection method of fast detecting Florfenicol and kit, this research will help to improve China to the detection technique level of animal food safety, make China synchronous with the world at the detection level of field of veterinary, thereby can ensure better people's health, advance the Green Process of agricultural byproducts.

The utility model content

The object of the present invention is to provide low, quick, easy and simple to handle, the highly sensitive Florfenicol of a kind of cost and metabolite detection kit thereof.

Technical scheme of the present invention is: comprise a Dual-layer kit box body, be equipped with on the upper strata in the box body: 96 hole ELISA Plate of coated Florfenicol-ovalbumin antigen, a cover plate film, a valve bag, a instructions, a quality inspection report, lower floor has: the recessed bottle position of the placement reagent that the fixed foam mould forms, be positioned at the reagent bottle on the recessed bottle position, it is characterized in that: 12 dismountable enzyme mark bars are housed on the ELISA Plate, each enzyme mark bar has 8 reacting holes, each enzyme mark hole is pre-coated Florfenicol-ovalbumin antigen, put into aluminium foil bag, the drying agent final vacuum of packing into; Described reagent bottle includes 6 bottles of 2mL from high concentration Florfenicol standard items, 7mL Florfenicol antibody working fluid, 30mL sample diluting liquid, the anti-working fluid of 12mL enzyme mark II, 40mL concentrated cleaning solution, 7mL substrate A liquid, 7mL substrate B liquid, the 7mL stop buffer of the Florfenicol standard items of 0 ~ 7.2ppb gradient concentration, 1 bottle of 2mL 1mg/L; The size of reagent bottle, the color of bottle cap are different, are fixed on polyfoam mould cover plate film, valve bag, instructions and quality inspection report with recessed bottle position and together are contained in the below that places ELISA Plate in the valve bag.Kit can one-time continuous be measured multiple sample, also can take plate hole apart repeated detection.

The principle that this kit detects is: the Florfenicol in the sample and with ELISA Plate on fixing antigen-specific sexual competition antibody, it is anti-to add enzyme mark II, with antibody response, by the enzymatic chromogenic reagent, come the content of Florfenicol in the judgement sample according to the depth of colour developing.If the free fluorobenzene Buddhist nun in the sample considers few, colour developing is dark, otherwise it is shallow then to develop the color.

This kit has very high sensitivity and very high specificity, and detectable sample comprises pork, chicken, beef, shrimp, the flesh of fish (peeling), and lowest detection is limited to 20ppb, and the recovery of sample is 90% ± 30%.With the cross reacting rate of florfenicol amine be 54%, Thiamphenicol is about 2.9%, chloromycetin, Chloramphenicol Succinate＜0.01%.

This kit detects easy, and required instrument is less, only needs the simple Laboratory Instruments such as microplate reader, homogenizer, hydro-extractor, and required testing cost is low.Can satisfy the self-quality control of the food enterprises, also can be used as the routine screening means of food safety supervision and testing agency, also can be used for the supplementary means of scientific research animal pharmacology and toxicologic study.

Description of drawings

Fig. 1 is upper strata of the present utility model synoptic diagram

Fig. 2 is the utility model lower floor synoptic diagram;

Fig. 3 is the vertical view after the utility model is opened the upper strata

Embodiment

Referring to accompanying drawing 1-3, but the structure that has represented the described a kind of fast detecting Florfenicol kit of present embodiment, described 96 hole ELISA Plate 2 are equipped with on described box body 1 upper strata, and described cover plate film, valve bag, instructions, quality inspection report jointly are contained in and are positioned over below the described ELISA Plate 2 in the valve bag; Has the recessed bottle position that fixed foam mould 3 forms in the described kit lower floor, have 14 recessed bottle positions: the high concentration Florfenicol standard items bottle position 4 of 2mL 1mg/L, 7mL Florfenicol antibody working fluid bottle position 5,40mL concentrated cleaning solution bottle position 6, the anti-working fluid bottle of 12mL enzyme mark II position 7,7mL substrate A liquid bottle position 8,7mL substrate B liquid bottle position 9,7mL stop buffer bottle position 10,2mL 0 μ g/L concentration Florfenicol standard items 11,2mL 0.05 μ g/L concentration Florfenicol standard items 12,2mL 0.15 μ g/L concentration Florfenicol standard items 13,2mL 0.45 μ g/L concentration Florfenicol standard items 14,2mL 1.8 μ g/L concentration Florfenicol standard items 15,2mL 7.2 μ g/L concentration Florfenicol standard items 16,30mL sample diluting liquid bottle position 17.Described recessed bottle position is used for placing the reagent bottle (14 bottles) that corresponding solution is housed.

Embodiment of the present utility model

One, the preparation of working fluid

Wash operating solution: press 1:20(1 part concentrate+19 parts of deionized waters with deionized water) dilution.

Sample diluting liquid: press 1:10(1 part concentrating sample dilution+9 parts of deionized waters with deionized water) dilution.

Tissue extract A(contains the 0.02M PB of 5% methyl alcohol and 0.5% trichloroacetic acid):

0.2M Na2HPO4: take by weighing 35.8g Na2HPO412H2O, be dissolved in 500mL water.

0.2M NaH2PO4: take by weighing 15.6g NaH2PO42H2O, be dissolved in 500mL water.

0.2M PB: get 19mL 0.2M NaH2PO4,81mL 0.2M Na2HPO4, fully mixing.

0.02M PB: get 50mL0.2M PB, add the abundant mixing of 450mL water.

Remove 25mL 0.02M PB, add successively 25mL methyl alcohol, the 2.5g trichloroacetic acid, fully mixing had both got tissue extract A.

Tissue extract B: in 500mL tissue extract A, add 22.5g NaCl, fully mixing.

Two, sample pre-treatments

Accurately take by weighing tissue sample behind 2 ± 0.1g homogeneous in the 50mL centrifuge tube; Pork, chicken, beef sample: add 10mL tissue extract A, shrimp, flesh of fish sample: add 10mL tissue extract B (being sure not the extract of different samples is added mistake); Under the room temperature (25 ± 2 ℃), high speed whirling motion 1min; More than the 4000g, centrifugal 10min; Get 100 μ L in new centrifuge tube, add the 1.5mL sample diluting liquid, fully whirling motion 30s; Getting 50 μ L detects.

Three, operation steps

1. required ELIAS strip is inserted on the ELISA Plate frame, and recorded the position of each standard items and sample, parallel laboratory test is all done in suggestion, and untapped ELIAS strip is stored in the 2-8 ℃ of environment after sealing with valve bag immediately;

2. each standard items working fluid/sample solution of 50 μ L is added respectively in corresponding standard items/sample well;

3. in each plate hole, add 50 μ L antibody working fluids;

4. build the cover plate film, the ELISA Plate of vibrating gently 10s, abundant mixing, room temperature (25 ± 2 ℃), lucifuge reaction 30min;

5. open the cover plate film;

6. outwell liquid in the plate hole, every hole adds 260 μ L wash operating solutions, soaks 15～30s;

7. repeat again previous step 3 times;

8. outwell liquid in the plate hole, ELISA Plate is inverted on the thieving paper, pat dry;

9. in each plate hole, add immediately the anti-working fluid of 100 μ L enzyme mark II;

10. build the cover plate film, the ELISA Plate of vibrating gently 10s, abundant mixing, room temperature (25 ± 2 ℃), lucifuge reaction 30min;

11. repeating step 9.5～9.8;

12. in every hole, add immediately 100 μ LA, B mixed liquor; (substrate A liquid, substrate B liquid mixed in by volume 1: 1, necessary fully mixing, mixed liquor uses in 5min, avoids using the metal splendid attire, stirs reagent);

13. build the cover plate film, the ELISA Plate of vibrating gently 10s, abundant mixing, under the room temperature (25 ± 2 ℃), lucifuge reaction 15～20min;

14. open the cover plate film, in each plate hole, add 50 μ L stop buffers, the ELISA Plate of vibrating gently 10s, fully mixing;

15. under dual wavelength 450nm, 630nm, detect absorbance with microplate reader, in 5min, read.

Four, calculate

The mean light absorbency value of each standard items (or sample) divided by zero standard (concentration is the standard items of 0ppb) absorbance, multiply by 100, can obtain the number percent of absorbance corresponding to each standard items:

In each standard items income value input semilog system, corresponding each standard items concentration can the match mark

Directrix curve sees also the quality inspection report.

Number percent substitution typical curve with the sample absorbance can draw concentration corresponding to sample, multiply by the extension rate of respective sample again, and the side gets actual drug content in the sample.

According to the pre-treating method of this instructions, each sample dilution factor is as follows:

Pork, chicken, beef, shrimp, the flesh of fish (peeling) ... 80

Five, measure points for attention

1. before kit uses, read over instructions.

2. please don't use expired kit, the reagent in the different lot number kits must not be used with.

3. before kit uses, each component in the kit is placed on the experiment table, treat that it returns to room temperature (25 ± 2 ℃) (prompting: about 2h), can use.

4. should avoid using metal species material splendid attire and stir reagent.

5. each reagent please shake up before use.

6. when detecting, should reduce ELISA Plate top Air Flow as far as possible.

7. if substrate A liquid, substrate B liquid have become blueness before use, or become immediately blue after mixing, illustrate that reagent is rotten.

8. contain 2M sulfuric acid in the stop buffer, prevent skin ambustion and corrosion clothing during use.

9. various criterion product, the used suction nozzle of sample must not be used with, otherwise can affect experimental result.

10. should avoid occurring bubble during mix reagent.

11. please immediately reagent is put back to 2～8 ℃ of preservations after using.

Six, kit parameter

This kit sensitivity 0.05ppb, standard curve range is 0.05ppb～7.2ppb;

B0 absorbance optimum value should be greater than 0.8;

Error is less than 5% in the kit absorbance plate, and error is less than 10% between plate;

The sample extraction method recovery scope 90% ± 30% that provides with this instructions;

Sample lowest detectable limit: pork, chicken, beef, shrimp, the flesh of fish (peeling) ... about 20ppb

Annotate: ppb=ng/mL or ng/g

Seven, cross reacting rate

The specificity of Florfenicol enzyme linked immunological kit determines that by carrying out the cross reaction test with corresponding material the result is as follows:

Florfenicol ... 100.0%

Florfenicol amine ... about 54%

Thiamphenicol ... about 2.9%

Chloromycetin, Chloramphenicol Succinate ...＜0.01%.

Claims

1. Florfenicol and metabolin pick-up unit thereof, it is characterized in that: a Dual-layer kit box body, be equipped with on the upper strata in the box body, and the 96 hole ELISA Plate of coated Florfenicol-ovalbumin antigen, a cover plate film, valve bag, a instructions, an a quality inspection are reported; Lower floor has: the recessed bottle position that the fixed foam mould forms, be positioned at the reagent bottle on the recessed bottle position, described reagent bottle includes 6 bottles of 2mL from high concentration Florfenicol standard items, 7mL Florfenicol antibody working fluid, 30mL sample diluting liquid, the anti-working fluid of 12mL enzyme mark II, 40mL concentrated cleaning solution, 7mL substrate A liquid, 7mL substrate B liquid, the 7mL stop buffer of the Florfenicol standard items of 0 ~ 7.2ppb gradient concentration, 1 bottle of 2mL 1mg/L.

2. Florfenicol according to claim 1 and metabolin pick-up unit thereof, the 96 hole ELISA Plate that it is characterized in that described coated Florfenicol-ovalbumin antigen: 12 dismountable enzyme mark bars are housed on the ELISA Plate, each enzyme mark bar has 8 reacting holes, each enzyme mark hole is pre-coated Florfenicol-ovalbumin antigen, put into aluminium foil bag, the drying agent final vacuum of packing into.