CN201852833U - ELISA (enzyme-linked immunosorbent assay) kit for furantoin metabolite - Google Patents

Abstract

The utility model relates to an ELISA(enzyme-linked immunosorbent assay) kit for detecting the residual quantity of furantoin metabolite in an animal-derived food product, which comprises a 96-hole enzyme-labelled plate, a second enzyme-labelled antibody, a furantoin metabolite antibody concentrated solution, standard solution in 7 concentrations, substrate colour-developing liquid, stop solution, concentrated cleaning solution, concentrated combination solution, a cover plate membrane, a self-sealed bag, a specification and a quality inspection report. By adopting an indirectly competitive ELISA method, a coupling antigen is pre-coated on a microporous strip of the enzyme-labelled plate; the furantoin metabolite contained in the sample and the coupling antigen pre-coated on the microporous strip compete for the furantoin metabolite antibody; after adding the second enzyme-labelled antibody, a TMB (etramethylbenzidine) substrate is used for developing the colour; and the residual quantity of the furantoin metabolite in the sample can be obtained by comparing the absorbance value of the sample with the standard curve and multiplying by a corresponding dilution factor. Compared with the instrument analysis technology, the ELISA kit has the advantages of being convenient to use, having high sensitivity and the like, and can play an important role during the detection of the residual quantity of the furantoin metabolite in animal-derived food products.

Description

Cistofuran metabolite ELISA detection kit

(1) technical field: the utility model relates to the kit that detects the Cistofuran metabolite residual quantity, particularly detect enzyme linked immunosorbent detection (ELISA) kit of Cistofuran metabolite residual quantity in the animal derived food, ELISA (Enzyme Linked ImmunosorbnentAssay) is the abbreviation of enzyme-linked immunosorbent assay, and it is a kind of enzyme immune technology that grows up after immunofluorescence and radioimmunoassay technique.

(2) background technology: the itrofurans medicine once was widely used because of the dynamic (dynamical) characteristic of extraordinary antibacterial action and medicine is arranged, as the somatotrophic adjuvant of bird, aquatic products and pig.But finding in the process of experimental that for a long time itrofurans medicine and metabolin all can make animal used as test that canceration and gene mutation take place, just causing this type of drug withdrawal in treatment and feed, to use Just because of this.Furantoin is disabled in nineteen ninety-five.

Because the itrofurans medicine in vivo soon can be by metabolism, the metabolic product of combination then can retain long period of time in tissue, so the product often will analyze its metabolism when analyzing this type of medicine residual after, administrative authority is that means reach the residual purpose of detection itrofurans to detect metabolic product just.Furantoin metabolic product AHD; Furaltadone metabolic product AMOZ; Furazolidone metabolite product A OZ; Nitrofurazone (nitrofurazone) metabolic product SEM.

The most popular method that is used for detecting Cistofuran metabolite is LC-UV, LC-MS and LC-MS/MS.Enzyme-linked immunoassay method combines chromatographic technique, adopts the specific antibody of AHD derivant, has very high degree of accuracy and sensitivity, and lower operative technique requires and of short duration detection time, detects characteristics such as sample size is big and well shows in detection.

(3) utility model content: problem to be solved in the utility model provides a kind of Cistofuran metabolite residual quantity detection kit of small volume and less weight, it is easy and simple to handle, detect quick, accurate, highly sensitive, cost is low, good stability, the technology content that needs is not high, can carry out the mensuration of Cistofuran metabolite residual quantity in a plurality of samples of one-time continuous, has reduced the needed time of detection sample.

The technical solution of the utility model is: a kind of Cistofuran metabolite ELISA detection kit, the ELISA Plate that comprises one 96 hole in box body and the box, a cover plate film, 15 bottles of reagent and the recessed bottle position of putting reagent, a valve bag, a instructions and a quality inspection report, it is characterized in that: ELISA Plate adopts 96 hole agent plate as solid phase carrier, the check-out console that pre-bag is made by the Cistofuran metabolite coupled antigen on the kit capillary strip, 15 bottles of reagent are respectively: 7 bottles of standard solutions, ELIAS secondary antibody, antibody concentrated solution, colour developing liquid A liquid, colour developing liquid B liquid, stop buffer, concentrated cleaning solution, concentrate redissolution liquid, totally 15 of recessed bottle positions.

ELISA Plate is made up of outer frame support and removable 12 enzymes mark reaction capillary strips of being placed on it, each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes, put into the vacuum aluminium foil bag, box body is a carton box, recessed bottle position is made by plastic foam, with the plastics dura mater is the cover plate film, cover plate film size is consistent with ELISA Plate square section size, 15 bottles of reagent are big or small with difference, the plastic bottle of different colours and vial splendid attire also are fixed in the recessed bottle position and ELISA Plate, the cover plate film, valve bag, instructions, the Quality Control report together is encapsulated in the carton box, so that carry and transport.15 bottles of reagent are respectively standard solution, 12ml ELIAS secondary antibody, 1ml antibody concentrated solution, 7ml colour developing liquid A liquid, 7ml colour developing liquid B liquid, 7ml stop buffer, 40ml concentrated cleaning solution, concentrated liquid, the 15.1mg2-nitrobenzaldehyde of redissolving of 50ml of 7 bottles of 1ml gradient concentrations.Reagent in each kit enough carries out 96 times and measures (comprising the standard analysis hole), both can carry out the detection of a plurality of samples of one-time continuous, also can take plate hole apart repeated detection.Put back in the aluminium foil bag with the valve bag sealing accuracy that like this can guarantee reagent box testing result with remaining.During detection, Cistofuran metabolite in the sample will be competed anti-Cistofuran metabolite antibody with the Cistofuran metabolite coupled antigen of pre-bag quilt on the ELISA Plate capillary strip, after adding ELIAS secondary antibody, develop the color with tmb substrate, the residual quantity of contained Cistofuran metabolite becomes negative correlation in sample absorbance and the sample, multiply by its corresponding extension rate more again with typical curve, can draw Cistofuran metabolite residual quantity in the sample, it is easy to operation.

Our experiments show that this kit has very high sensitivity, is 0.1 μ g/kg: be limited to aquatic products tissue samples detections such as 0.2 μ g/kg, shrimp, fish under tissue, honey, the milk pattern detection and be limited to 0.3 μ g/kg; The recovery of aquatic products, muscle, liver organization sample is 75% ± 15%; The recovery of honey sample is 90% ± 15%; The recovery of milk sample is 90% ± 10%.With respect to other Cistofuran metabolite detection methods, the required instrument of this kit is less, only needs microplate reader, nitrogen blow-dry device, vortex instrument, hydro-extractor, vibrating machine and micro sample adding appliance etc., and generally all there is outfit in similar laboratory, and required cost is lower.

The beneficial effects of the utility model are: can be used for the detection of Cistofuran metabolite residual quantity quick, easy, sensitive, exactly.

(4) description of drawings:

Fig. 1 is the lateral longitudinal sectional view (the long 8.55cm of being) of the utility model elisa plate;

Fig. 2 is the side drawing in side sectional elevation (the long 12.8cm of being) of the utility model elisa plate;

Fig. 3 is the vertical view of the utility model elisa plate;

Fig. 4 is the utility model cover plate membrane plane figure;

Fig. 5 is the utility model reagent bottle longitudinal profile planimetric map;

Fig. 6 is the vertical view of the utility model fixed foam mould;

Fig. 7 is the side view of the utility model box body and fixed foam mould.

Referring to accompanying drawing: enzyme mark reaction capillary strip (2) in advance coated Cistofuran metabolite coupled antigen (3) is fixed on the outer frame support (1) of ELISA Plate, and enzyme mark reaction capillary strip (2) can be with requiring dismounting; Capping enzyme mark reaction capillary strip (2) when cover plate film (4) is put water-bath or insulating box internal reaction for ELISA Plate, cover plate film size and ELISA Plate cross section in the same size; The translucent PE plastic bottle (5) of hyaline cap is used for encapsulation 40ml concentrated cleaning solution; The translucent PE plastic bottle (5) of blue cap is used for encapsulation 50ml and concentrates redissolution liquid; The white PE plastic bottle (7) of white cap (6) is used for encapsulation 7ml nitrite ion A liquid, the black PE plastic bottle (7) of red cap (6) is used for encapsulation 7ml nitrite ion B liquid, the white PE plastic bottle (7) of yellow cap (6) is used for encapsulation 7ml stop buffer, the white PE plastic bottle (8) of red cap is used for encapsulation 12ml ELIAS secondary antibody, the white PE plastic bottle (8) of green cap is used for encapsulation 7ml Cistofuran metabolite antibody working solution, and the brown glass reagent bottle (9) of white cap is used for the standard solution of encapsulation 1ml/ bottle; Foamed plastics mould (10) has 15 bottle positions, placement location is followed successively by: 40ml concentrated cleaning solution bottle position (18), 50ml concentrates redissolution liquid (11), 7ml nitrite ion A liquid bottle position (14), 7ml nitrite ion B liquid bottle position (13), 7ml stop buffer bottle position (12), 1ml Cistofuran metabolite antibody concentrated solution bottle position (16), 12ml ELIAS secondary antibody bottle position (15), the standard solution of 7 various concentration of 1ml/ bottle and; 15.1mg 2-nitrobenzaldehyde bottle position (17), box body is carton box for (19).

(5) embodiment

The pre-treatment of sample

1. dosing:

Dosing 1: derivatization reagent

In the reagent bottle that the 2-nitrobenzaldehyde is housed, add dissolve with methanol and be settled to 10ml (concentration is 10mM).

Dosing 2:C liquid 0.36M sodium nitroprusside (Na2Fe (CN) ₅NO2H ₂O) solution takes by weighing 12.5g hydration sodium nitroprusside and adds deionized water and be settled to 100ml.

D liquid (for the special use of milk sample) 1M zinc sulfate (ZnSO ₄7H ₂O) solution

Take by weighing 29.8g zinc sulfate and be settled to 100ml with deionized water.

Dosing 3:0.1M dipotassium hydrogen phosphate solution

Taking by weighing 22.8g three hypophosphite monohydrate hydrogen dipotassiums adds deionized water and is settled to 1L.

Dosing 4:1M hydrochloric acid solution

Measuring the 8.3ml concentrated hydrochloric acid adds deionized water and is settled to 100ml.

Dosing 5:1M NaOH

Taking by weighing 4.0g NaOH adds deionized water and is settled to 100ml.

Dosing 6: methanol-water solution

Measure methyl alcohol 90ml and add deionized water 10ml mixing.

Dosing 7: redissolution working fluid

With deionized water the 2 * concentrated liquid that redissolves is diluted the redissolution that (1 part 2 * the concentrated liquid+1 part deionized water that redissolves) is used to extract sample by 1: 1 volume ratio, the working fluid that redissolves can be preserved one month at 4 ℃ of environment.

Dosing 8: washing working fluid

With deionized water 20 * concentrated cleaning solution is diluted the washing that (1 part of 20 * concentrated cleaning solution+19 part deionized water) is used for ELISA Plate by 1: 19 volume ratio, the washing working fluid can be preserved one month at 4 ℃ of environment.

2, muscle, liver, aquatic products (fishes and shrimps etc.) tissue pre-treating method

With homogenizer homogeneous sample, the equal pledge of getting 1.0 ± 0.05g is to 50ml polystyrene centrifuge tube; Add 5ml methanol-water solution, with the oscillator 5min that vibrates, the above centrifugal 10min of 3000g removes whole supernatants; (this step can reduce the sample matrix interference) adds the deionized water of 4ml, with vortex instrument whirling motion dissolving, adds 0.5ml 1M hydrochloric acid solution and 100 μ l derivatization reagents, fully vibrates with oscillator; Connect (5) describing method (mark * place).Annotate: sample should be kept at shady and cool lucifuge part and stored refrigerated.

3, milk pre-treating method

Get the milk sample, more than the 3000g, 10 ℃ of centrifugal 10min absorb upper strata fat; Take out 5ml and remove fatty milk sample to 10ml glass centrifuge tube; Add 250 μ l C liquid and 250 μ l D liquid respectively; With the oscillator mixing of fully vibrating, more than the 3000g, 4-12 ℃ of (39-54) centrifugal 10min.If there is not the constant temperature hydro-extractor, then earlier sample is cooled to about 8 ℃ (46 °F), centrifugal then; Get the centrifuged supernatant 1.1ml (being equivalent to 1ml milk sample) of milk, the deionized water that adds 4ml respectively dissolves with the oscillator vibration, 0.5ml 1M hydrochloric acid and 100 μ l derivatization reagents, and 2min fully vibrates; Connect (5) describing method (mark * place).

4, honey pre-treating method

Take by weighing 1.0 ± 0.05g honey to 50ml polystyrene centrifuge tube; The deionized water that adds 4ml vibrates to honey with oscillator and all to dissolve; 0.5ml 1M hydrochloric acid solution and 100 μ l derivatization reagents fully vibrate with oscillator; Connect (5) describing method (mark * place).

5, connect the method * of face

At 37 ℃ of night incubation (approximately 16h); The ethyl acetate that adds 5ml 0.1M dipotassium hydrogen phosphate solution, 0.4ml1M sodium hydroxide solution and 5ml respectively is with oscillator thermal agitation 30s; More than the 3000g, the centrifugal 10min of room temperature (20-25 ℃/68-77); Get 2.5ml ethyl acetate to the glass test tube of 10ml drying, flow down in 50～60 ℃ of nitrogen and dry up; With the normal hexane (or normal heptane) of 1ml vortex instrument whirling motion 30s, add 1ml redissolution working fluid again, with the abundant mixing of vortex instrument whirling motion 1min; More than the 3000g, the centrifugal 10min of room temperature (20-25 ℃/68-77); Remove upper organic phase, take off layer water 50 μ l and be used for analyzing.

Two, use the operation steps of kit:

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, take out the microwell plate that needs quantity, no microwell plate is put into valve bag, be stored in 2-8 ℃.

3, the washing working fluid also need be risen again before use.

4, numbering: the corresponding micropore of sample and standard items is numbered according to the order of sequence, and it is parallel that each sample and standard items are done 2 holes, and the position at record standard hole and sample aperture place.

5, add standard items working fluid/sample solution: add standard items working fluid/sample solution 50 μ l in the micropore of correspondence, add antibody working fluid 50 μ l/ holes again, the mixing that vibrates gently is placed in 25 ℃ of lucifuge environment with cover plate membrane cover plate and reacts 30min.

6, wash plate: carefully open the cover plate film, liquid in the hole is dried, with washing working fluid 250 μ l/ holes, fully wash 4-5 time, each 10s at interval pats dry (bubble that is not eliminated after patting dry can be poked with original rifle head) with thieving paper.

7, add ELIAS secondary antibody: add ELIAS secondary antibody 100 μ l/ holes, the mixing that vibrates gently with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate, takes out and repeats to wash plate step 6.

8, colour developing: add substrate solution A liquid 50 μ l/ holes, add substrate solution B liquid 50 μ l/ holes again, the mixing that vibrates gently is with reacting 15min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate.

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

Three, the result judges

The result judges two kinds of methods, judges available the 1st kind of method roughly, quantitatively judges with the 2nd kind of method.Notice that the sample light absorption value becomes negative correlation with Cistofuran metabolite content.

1, mean light absorbency value and the standard value with sample relatively can draw its concentration range (μ g/L).For example the absorbance of sample 1 is 0.236, and the absorbance of sample 2 is 0.666, and the standard items absorbance is respectively: 0 μ g/L is 1.949; 0.1 μ g/L is 1.434; 0.3 μ g/L is .939; 0.9 μ g/L is 0.487; 2.7 μ g/L is 0.157; 8.1 μ g/L is 0.060.Then the concentration range of sample 1 is that 0.9 μ g/L-2.7 μ g/L multiply by its corresponding extension rate again and can draw the residual concentration range of Cistofuran metabolite in the sample; The concentration range of sample 2 is that 0.3 μ g/L-0.9 μ g/L multiply by its corresponding extension rate again and can draw the residual concentration range of Cistofuran metabolite in the sample.

2, quantitative test

(1) calculating of percentage absorptance, the percentage absorptance of standard items or sample equal the mean value (diplopore) of percentage absorbance of standard items or sample divided by the absorbance of first standard (0 standard), multiply by 100% again, promptly

The mean light absorbency value of B-standard solution or sample solution

The mean light absorbency value of B0-0 μ g/L standard solution

(2) drafting of typical curve and calculating

With standard items percentage absorptance is ordinate, is horizontal ordinate with the semilog of Cistofuran metabolite standard items concentration (μ g/L), the drawing standard curve map.In the percentage absorptance substitution typical curve with sample, read the pairing concentration of sample, multiply by its corresponding extension rate and be Cistofuran metabolite actual content in the sample from typical curve.

Four, measure preceding points for attention:

1, room temperature is lower than 20 ℃ or reagent and sample and does not get back to room temperature (20-25 ℃) and can cause the OD value of all standards on the low side.

2, in washing the plate process if the plate hole dry situation, it is non-linear then can typical curve to occur, the phenomenon that repeatability is bad.So should carry out next step operation immediately after washing plate and patting dry.

3, whenever adding preceding need of a kind of reagent shake up it.

4, reaction terminating liquid is a 2M sulfuric acid, avoids contacting skin.

5, do not use the kit of date of expiration; Also do not use any reagent in the kit of the term of validity, the kit that used the term of validity of mixing can cause the reduction of sensitivity; The reagent in the different lot number kits is not used in exchange.

6, condition of storage

Preserve kit in 2-8 ℃, not freezing, put no ELISA Plate microwell plate into valve bag and reseal.Therefore standard substance and colourless colour former will be avoided being directly exposed under the light to photaesthesia.

7, the rotten sign of reagent

Color development reagent has any color to show that colour former is rotten, should abandon it.When the absorbance of 0 standard (450/630nm) was worth less than 0.5 (A450nm＜0.5), expression reagent may go bad.

8, after adding substrate solution A liquid and substrate solution B liquid, the 10-15min that generally develops the color gets final product.If color is more shallow, can prolongs the reaction time to 20min (or longer), but must not surpass 30min.Otherwise, then shorten the reaction time.

9, this kit optimal reaction temperature is 25 ℃, too high or too low for temperaturely will cause detecting absorbance and sensitivity changes.

Claims (3)

1. Cistofuran metabolite ELISA detection kit, the ELISA Plate that comprises one 96 hole in box body and the box, a cover plate film, 15 bottles of reagent and the recessed bottle position of putting reagent, a valve bag, a instructions and a quality inspection report, it is characterized in that: ELISA Plate is to adopt 96 hole agent plate as solid phase carrier, the check-out console that pre-bag is made by the Cistofuran metabolite coupled antigen on the kit capillary strip, 15 bottles of reagent are respectively: 7 bottles of standard solutions, ELIAS secondary antibody, antibody concentrated solution, colour developing liquid A liquid, colour developing liquid B liquid, stop buffer, concentrated cleaning solution, concentrate redissolution liquid, totally 15 of recessed bottle positions.

2. kit according to claim 1 is characterized in that: box body is a carton box; The polystyrene ELISA Plate in 96 holes is put in the vacuum aluminium foil bag; The cover plate film is the plastics dura maters; Standard solution is with the Brown Glass Brown glass bottles and jars only of white cap, ELIAS secondary antibody is with the white PE plastic bottle of red cap, the antibody working fluid is with the white PE plastic bottle of green cap, the white PE plastic bottle of liquid A liquid with white cap develops the color, colour developing liquid B liquid is with the black PE plastic bottle of red cap, and stop buffer is with the white PE plastic bottle of yellow cap, the translucent PE plastic bottle of concentrated cleaning solution usefulness hyaline cap, concentrate the translucent PE plastic bottle of redissolution liquid with blue cap, recessed bottle position is made by plastic foam.

3. kit according to claim 1 is characterized in that: ELISA Plate is made up of outer frame support and removable 12 enzymes mark reaction capillary strips of being placed on it, and each dismantled and assembled enzyme mark reacts capillary strip 8 reacting holes; Cover plate film size is consistent with ELISA Plate square section size; 7 bottles of standard solutions, the 1ml/ bottle; 1 bottle of ELIAS secondary antibody, 12ml; 1 bottle of antibody concentrated solution, 1ml; 1 bottle of colour developing liquid A liquid, 7ml; 1 bottle of colour developing liquid B liquid, 7ml; 1 bottle of stop buffer, 7ml; 1 bottle of concentrated cleaning solution, 40ml; Concentrate 1 bottle of redissolution liquid, 50ml; 1 bottle of 2-nitrobenzaldehyde, 15.1mg.

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