CN202794173U - Enzyme linked immunosorbent assay (ELISA) kit for nitroimidazole medicament - Google Patents

Abstract

The utility model relates to an enzyme linked immunosorbent assay (ELISA) kit for detecting the residue of a nitroimidazole medicament. The kit comprises a 96-hole ELISA plate, an ELASA second antibody working solution, a nitroimidazole medicament antibody working solution, standard solutions of 7 concentrations of the nitroimidazole medicament, a substrate color development solution, a stop solution, a concentrate washing solution and a concentrate redissolving solution. Coupling antigens are pre-coated on a micropore strip of the ELISA plate by adopting an indirect competition ELISA method, the nitroimidazole medicament contained in a sample and the coupling antigens pre-coated on the micropore strip compete for nitroimidazole medicament antibodies, ELASA second antibodies are added, color is developed by using a tetramethylbenzidine (TMB) substrate, the absorbance value of the sample is compared with a standard curve and multiplied by the corresponding dilution ratio, and the residue of the nitroimidazole medicament in the sample can be obtained. Compared with an instrumental analysis technology, the kit has the characteristics of convenience in use, high sensitivity and the like and can play a great role in detection of the residue of the nitroimidazole medicament.

Description

Nitroimidazoles medicine ELISA detection kit

(1) technical field: the utility model relates to the kit that detects the nitroimidazoles medicine residual quantity, particularly detect enzyme linked immunosorbent detection (ELISA) kit of nitroimidazoles medicine residual quantity in the honey, ELISA(Enzyme Linked Immunosorbnent Assay) be the abbreviation of enzyme-linked immunosorbent assay, it is a kind of enzyme immune technology that grows up after immunofluorescence and radioimmunoassay technique.

(2) background technology: nitroimidazoles medicine is that antiprotozoan commonly used in the livestock and poultry honey infects and antimicrobial, be mainly used in preventing and treating poultry organization trichomonad and parasite, the trichomonad of ox, the hemorrhagic enteritis of pig, the parasitic infection of fish, the microsporidiosis of honeybee etc., and has a growth promoting function, but such medicine and some metabolin thereof have carcinogenic to mammal, teratogenesis, mutagenesis and genetoxic, therefore the unreasonable use of nitroimidazoles medicine causes edibility animal tissue drug residue problem to jeopardize human food security, China has forbidden metronidazole, Dimetridazole, Lip river nitre azoles uses in carnivorous food, Tinidazole is forbidden using in veterinary drug, therefore, need a kind of not only sensitivity but also accurate method mensuration nitroimidazoles medicine.

The method that detects at present nitroimidazoles medicine mainly contains: high performance liquid chromatography-ultraviolet chromatogram, high performance liquid chromatography diode display detecting device method, gas chromatography nitrogen phosphorous detector method, High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry method, vapor-phase chromatography etc., instrument detection method complex operation and expense are higher, apply being restricted.

(3) utility model content: problem to be solved in the utility model provides a kind of nitroimidazoles medicine residues 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 nitroimidazoles medicine 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 enzyme linked immunological kit of nitroimidazoles medicine residues detection, the ELISA Plate that comprises 96 holes in box body and the box, a cover plate film, 14 bottles of reagent and 15 recessed bottle positions of putting reagent, it is characterized in that: adopt 96 hole polystyrene ELISA Plate as solid phase carrier, ELISA Plate is comprised of outer frame support and removable 12 enzyme marks reaction capillary strips of being placed on it, each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes, pre-coated nitroimidazoles medicine coupled antigen is made check-out console on kit ELISA Plate capillary strip, put into the vacuum aluminium foil bag, box body is carton box, recessed bottle position is made by plastic foam, take plastic hard membrane as the cover plate film, cover plate film size and ELISA Plate square section in the same size, 14 bottles of reagent are big or small with difference, the plastic bottle splendid attire of different colours also is 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.14 bottles of reagent are respectively 7 bottles of 1ml standard solutions, 1ml ELIAS secondary antibody working fluid, 7ml nitroimidazoles medicine antibody working fluid, 7ml nitrite ion A liquid, 7ml nitrite ion B liquid, 7ml stop buffer, 40ml concentrated cleaning solution, the concentrated liquid that redissolves of 50ml.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, nitroimidazoles medicine in the sample will be competed anti-nitroimidazoles medicine antibody with pre-coated nitroimidazoles medicine coupled antigen on the ELISA Plate capillary strip, after adding ELIAS secondary antibody, develop the color with tmb substrate, the residual quantity of contained nitroimidazoles medicine becomes negative correlation in sample absorbance and the sample, multiply by its corresponding extension rate with typical curve more again, can draw nitroimidazoles medicine residual quantity in the sample, it is easy to operation.

Our experiments show that this kit has very high sensitivity: detection sensitivity is for being 0.05 μ g/kg; With respect to other nitroimidazoles medicine detection methods, the required instrument of this kit is less, only needs microplate reader, hydro-extractor, vibrating machine, vortex instrument 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 quick, easy, sensitive, exactly the detection of nitroimidazoles medicine residual quantity.

(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.

(5) embodiment

Referring to accompanying drawing: enzyme mark reaction capillary strip (2) pre-coated nitroimidazoles medicine coupled antigens (3) are 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 constant temperature oven internal reaction for ELISA Plate, cover plate film size and ELISA Plate square section in the same size; The translucent PE plastics reagent bottle (5) of hyaline cap is used for encapsulation 40ml concentrated cleaning solution; The translucent PE plastics reagent bottle (5) of blue cap is used for the concentrated liquid that redissolves of encapsulation 50ml; The white PE plastics reagent bottle (7) of white cap (6) is used for encapsulation 7ml nitrite ion A liquid, the black PE plastics reagent bottles (7) of red cap (6) are used for encapsulation 7ml nitrite ion B liquid, the white PE plastics reagent bottle (7) of yellow cap (6) is used for encapsulation 7ml stop buffer, the white PE plastics reagent bottle (8) of red cap is used for encapsulation 7ml ELIAS secondary antibody working fluid, the white PE plastics reagent bottle (8) of green cap is used for encapsulation 7ml nitroimidazoles medicine antibody working fluid, and the translucent PE plastics reagent bottle (9) of black caps is used for the standard solution of encapsulation 1ml/ bottle; Polyfoam mould (10) has 15 recessed bottle positions, placement location is followed successively by: 40ml concentrated cleaning solution bottle position (18), the concentrated liquid (11) that redissolves of 50ml, 7ml nitrite ion A liquid bottle position (14), 7ml nitrite ion B liquid bottle position (13), 7ml stop buffer bottle position (12), 7ml nitroimidazoles medicine antibody working fluid bottle position (16), 7ml ELIAS secondary antibody working fluid bottle position (15), the standard solution of 7 bottles of various concentration of 1ml/ bottle, box body is carton box for (19).

(1) use kit to detect the operation steps of nitroimidazoles medicine:

(1) takes out the microwell plate that needs quantity.

(2) 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.

(3) add standard items/sample and antibody working fluid: add standard items/sample 50 μ l in the micropore of correspondence, (annotate: the antibody working fluid places 4 ℃ of Refrigerator stores to add antibody working fluid 50 μ l/ holes, take out immediately point sample during point sample), the mixing that vibrates gently is with reacting 60min in the rearmounted 0-4 ℃ of lucifuge environment of cover plate membrane cover plate.

(4) wash plate: carefully open the cover plate film, liquid in the hole is dried, with wash operating solution 250 μ l/ holes, fully wash 4-5 time, every minor tick 10s pats dry with thieving paper.

(5) add ELIAS secondary antibody: add ELIAS secondary antibody 100 μ l/ holes, the mixing that vibrates gently is with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate.

(6) 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 the rearmounted 25 ℃ of lucifuge environment reaction 30min of cover plate membrane cover plate.

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

(2) result judges

(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%, namely again

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

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

(2) drafting of typical curve and calculating

Take standard items percentage absorptance as ordinate, take the semilog of metronidazole standard items concentration (μ g/L) as horizontal ordinate, the drawing standard curve map.In the percentage absorptance substitution typical curve with sample, read the corresponding concentration of sample from typical curve, multiply by its corresponding extension rate and be nitroimidazoles medicine practical residue limit in the sample.

Claims (3)

1. nitroimidazoles medicine ELISA detection kit, the recessed bottle position that comprises ELISA Plate, cover plate film, 14 bottles of reagent in 96 holes in box body and the box and put reagent, it is characterized in that: ELISA Plate is to adopt 96 hole agent plate as solid phase carrier, the check-out console that pre-coated nitroimidazoles medicine coupled antigen is made on the kit capillary strip, 7 bottles of standard solutions of 14 bottles of reagent difference, ELIAS secondary antibody working fluid, antibody working fluid, nitrite ion A liquid, nitrite ion B liquid, stop buffer, concentrated cleaning solution, the concentrated liquid that redissolves, totally 15 of recessed bottle positions.

2. kit according to claim 1, it is characterized in that: box body is carton box, and the polystyrene ELISA Plate in 96 holes is put in the vacuum aluminium foil bag, and the cover plate film is plastic hard membrane; Standard solution is all used the translucent PE plastic bottle of black caps, the ELIAS secondary antibody working fluid is with the white PE plastic bottle of red cap, the antibody working fluid is with the white PE plastic bottle of green cap, nitrite ion A liquid is with the white PE plastic bottle of white cap, nitrite ion 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, and concentrated cleaning solution is with the translucent PE plastic bottle of hyaline cap, concentrated redissolution liquid is made by plastic foam with the translucent PE plastic bottle of blue cap, recessed bottle position.

3. kit according to claim 1, it is characterized in that: ELISA Plate is comprised of outer frame support and removable 12 enzyme marks reaction capillary strips of being placed on it, each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes, the pre-coated nitroimidazoles medicine coupled antigen of each reacting hole; Cover plate film size and ELISA Plate square section in the same size; 7 bottles of standard solutions, the 1ml/ bottle; 1 bottle of ELIAS secondary antibody working fluid, 7ml; 1 bottle of antibody working fluid, 7ml; 1 bottle of nitrite ion A liquid, 7ml; 1 bottle of nitrite ion B liquid, 7ml; 1 bottle of stop buffer, 7ml; 1 bottle of concentrated cleaning solution, 40ml; 1 bottle of concentrated redissolution liquid, 50ml.

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