CN201535773U - Gentamicin ELISA checking reagent box - Google Patents

Abstract

The utility model relates to an enzyme immunoassay reagent box for checking gentamicin contents in animal source food. The reagent box composition comprises a 96 hole enzyme label plate, enzyme label double resistance, a gentamicin antibody working liquid, standard sample solution of gentamicin in six concentrations, substrate color development liquid, terminating liquid, concentrated washing detergent, concentrated composite solution, high concentration standard products, cover plate membrane, a self-sealing bag, an instruction book and a quality check report. Indirect competition ELISA method is adopted, coupling antigen should be wrapped in advance on an enzyme label plate micropore strip, the gentamicin contained in the sample and the coupling antigen wrapped on a micropore strip can compete for resisting the gentamicin antibody, after the enzyme label double resistance is added, TMB substrate color development liquid is used, the sample absorbance value and the standard curve are compared and then are multiplied by the corresponding dilution multiple, thereby obtaining the content of gentamicin in the samples. Compared with instrument analysis technique, the enzyme immunoassay reagent box has the characteristics of convenient use, high sensitivity and the like, and can play an important role in residue check of gentamicin in muscles and livers.

Description

Gentamicin ELISA detection kit

(1) technical field: the utility model relates to the kit that detects the gentamicin residue amount, particularly detects the enzyme linked immunological kit of gentamicin residue amount in muscle, the liver.

(2) background technology: gentamicin (Gentamicin) is an aminoglycosides antibiotics, be widely used in the treatment of Animal diseases, because it has neurotoxicity and Toxicity of Kidney, the residual meeting in animal foodstuff influence human health, and American-European countries and China all require its use of limiting the quantity of.

The chemical method that detects the gentamicin residue amount has high performance liquid chromatography at present, because complicated instrument and equipment and loaded down with trivial details process are not suitable for on-site supervision and great amount of samples examination.

(3) utility model content: problem to be solved in the utility model provides a kind of gentamicin residue amount 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 gentamicin residue amount 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 that detects gentamicin residue amount in the animal derived food, the ELISA Plate that comprises one 96 hole in box body and the box, a cover plate film, 14 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: adopt 96 hole polystyrene agent plate as solid phase carrier, 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, pre-bag is made check-out console by the gentamicin coupled antigen on kit ELISA Plate capillary strip, put into the vacuum aluminium foil bag, box body is a carton box, with the plastics dura mater is the cover plate film, to put into the vacuum aluminium foil bag, standard solution with 6 bottles of 1ml gradient concentrations, 1ml high concentration standard solution, the 12ml ELIAS secondary antibody, 7ml gentamicin antibody working fluid, 7ml colour developing liquid A liquid, 7ml colour developing liquid B liquid, the 7ml stop buffer, the 40ml concentrated cleaning solution, 50ml concentrates and redissolves liquid reagent with different big or small, the plastic bottle of different colours and vial splendid attire also are fixed in the polyfoam mould 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.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, gentamicin in the sample will be competed anti-gentamicin antibody with the gentamicin 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 gentamicin becomes negative correlation in sample absorbance and the sample, multiply by its corresponding extension rate more again with typical curve, can draw gentamicin residue amount in the sample, it is easy to operation.

Our experiments show that this kit has very high sensitivity: the lowest detection that the lowest detection of muscle sample is limited to 4 μ g/kg, liver samples is limited to 4 μ g/kg; The recovery of muscle sample is 90% ± 10%; The recovery of liver samples is 90% ± 10%; With respect to other gentamicin detection methods, the required instrument of this kit is less, only needs microplate reader, constant temperature oven, vortex instrument, hydro-extractor and micro sample adding appliance, 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 gentamicin residue amount 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 gentamicin 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; The translucent plastic reagent bottle (5) of hyaline cap is used for encapsulation 40ml concentrated cleaning solution; The translucent plastic reagent bottle (5) of blue cap is used for encapsulation 50ml and concentrates redissolution liquid; The white plastic reagent bottle (7) of white cap (6) is used for encapsulation 7ml nitrite ion A liquid, the black plastic reagent bottle (7) of red cap (6) is used for encapsulation 7ml nitrite ion B liquid, the white plastic reagent bottle (7) of yellow cap (6) is used for encapsulation 7ml stop buffer, the white plastic reagent bottle (8) of red cap is used for encapsulation 12ml ELIAS secondary antibody, the white plastic reagent bottle (8) of green cap is used for encapsulation 7ml gentamicin antibody working solution, and the brown glass reagent bottle (9) of white cap is used for standard solution and the 1ml high concentration 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), 7ml gentamicin antibody working solution bottle position (16), 12ml ELIAS secondary antibody bottle position (15), the standard solution of 6 kinds of various concentration of 1ml/ bottle and 1 bottle of 1ml high concentration standard solution bottle position (17), box body is carton box for (19).

Embodiment

Embodiment of the present utility model:

One, the pre-treatment of sample

1. dosing:

Dosing 1:0.1M PBS solution (PH=10-11)

Taking by weighing 13.4g disodium hydrogen phosphate dodecahydrate, 20g sodium chloride, 0.32g NaOH, 0.5g two hypophosphite monohydrate potassium dihydrogens, 0.5g potassium chloride, to add deionized water dissolving fixed molten to 500ml.

Dosing 2: 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.

Dosing 3: redissolution working fluid

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

2. muscle, the liver samples treatment step that adds with the gentamicin standard items:

Take by weighing 2.0 ± 0.05g and go the homogeneous sample to 50ml polystyrene centrifuge tube, add 6ml 0.1M PBS (PH=10～11) damping fluid 6ml, mixing 10min; Place 60 ℃ of water-bath environment 60min, taking-up is cooled to room temperature and shakes up; More than the 3000g, the centrifugal 10min of room temperature; Pipette supernatant, add to redissolve working fluid, dilute (50 μ l supernatants+450 μ l redissolve working fluid) with 1: 9 volume ratio; Getting 50 μ l is used for analyzing.

Two, use the operation steps of kit:

1, required reagent is taken out from cold storage environment, put room temperature (20-25 ℃) more than the balance 30min, note to shake up before every kind of liquid reagent uses.

2, take out grillage and need the micropore of quantity, no micropore is put into valve bag, be stored in 2-8 ℃.

3, cleansing solution also needs to rise again before use.

4, numbering: the corresponding micropore of sample solution and standard items working fluid is numbered according to the order of sequence, and it is parallel that each sample solution and standard items working fluid 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 micropore separately, add antibody working fluid 50 μ l/ holes then, the mixing that vibrates gently with cover plate membrane cover plate, reacts 30min in 37 ℃ of environment.

6, take out microwell plate, throw away liquid in the hole, wash plate 4-5 time by 250 μ l/ holes, soaked 10 seconds at every turn, pat dry (bubble that is not eliminated after patting dry can puncture with clean rifle head) with thieving paper with cleansing solution.

7, add ELIAS secondary antibody working fluid 100 μ l/ holes, the mixing that vibrates gently places 37 ℃ of environment reaction 30min with cover plate membrane cover plate, takes out repeating step 7.

8, colour developing: every hole adds substrate solution A liquid 50 μ l earlier, adds B liquid 50 μ l again, the mixing that vibrates gently, lucifuge colour developing 15min in 37 ℃ of environment.

9, measure: every hole adds stop buffer 50 μ l, the mixing that vibrates is gently set microplate reader in 450nm place (suggestion runs through data with dual wavelength 450/630nm detection in 5min), measure every hole OD (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 the gentamicin residue amount.

1, mean light absorbency value and the standard items absorbance with sample relatively can draw its concentration range (μ g/L).The absorbance of supposing sample 1 is 0.512, and the absorbance of sample 2 is 0.889, and the titer absorbance is respectively: 0 μ g/L is 1.900; 0.1 μ g/L is 1.450; 0.3 μ g/L is 1.000; 0.9 μ g/L is 0.600; 2.7 μ g/L is 0.300; 8.1 μ g/L is 0.150.Then the concentration range of sample 1 is 0.9 μ g/L-2.7 μ g/L multiply by its corresponding extension rate; The concentration range of sample 2 is that 0.3 μ g/L-0.9 μ g/L multiply by its corresponding extension rate.

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 gentamicin 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 gentamicin practical residue limit 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, can be accompanied by then that typical curve to occur non-linear, the phenomenon that repeatability is bad.So should carry out next step operation immediately after washing plate and patting dry.

3, reagent mix is wanted evenly, otherwise can the bad phenomenon of duplicating property.

4, reaction terminating liquid is a 2mol/L sulfuric acid, avoids contacting skin.

5, condition of storage

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

6, do not use the kit of date of expiration, diluted or mixed up the variation that use can cause sensitivity, OD value.The reagent in the different lot number kits is not used in exchange.

7, the rotten sign of reagent

Colour developing liquid is rotten if there is any color to show, should abandon it.The absorbance of 0 standard is during less than 0.5 (A450nm＜0.5), and expression reagent may go bad.

8, add that general colour developing 15min get final product behind A, the B liquid, more shallow as if color, developing time can be prolonged, but 30min can not be surpassed.

9, this kit optimal reaction temperature is 37 ℃, too high or too low for temperaturely will cause detecting absorbance and sensitivity changes.

Claims (3)

1. enzyme linked immunological kit that detects gentamicin residue amount in the animal derived food, the ELISA Plate that comprises one 96 hole in box body and the box, a cover plate film, 14 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 gentamicin coupled antigen on the kit capillary strip, 6 bottles of standard solutions of 14 bottles of reagent difference, 1 bottle of high concentration standard solution, ELIAS secondary antibody, the antibody working fluid, 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 aluminide-coating bag; The cover plate film is the plastics dura maters; Standard solution and high concentration standard solution are all used 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, the black PE plastic bottle of liquid B liquid with red cap develops the color, stop buffer is with the white PE plastic bottle of yellow cap, the concentrated cleaning solution translucent PE plastic bottle of 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, it 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, each dismantled and assembled enzyme mark reaction capillary strip has 8 reacting holes, and each reacting hole wraps in advance by the gentamicin coupled antigen; Cover plate film size is consistent with ELISA Plate square section size; 6 bottles of standard solutions, the 1ml/ bottle, concentration is respectively 0 μ g/L, 0.1 μ g/L, 0.3 μ g/L, 0.9 μ g/L, 2.7 μ g/L, 8.1 μ g/L; 1 bottle of high concentration standard solution, 1ml; 1 bottle of ELIAS secondary antibody, 12ml; 1 bottle of antibody working fluid, 7ml; 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.

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