CN201130184Y - Kit for testing tsiklomitsin ELISA - Google Patents

Abstract

The utility model relates to an ELISA detection kit used to detect the tetracycline residue in animal derived food, comprising an enzyme label plate equipped with 96 holes, an enzyme labeled second antibody, a tetracycline antibody working fluid, a tetracycline standardized product solution having six concentration, a substrate coloured solution, a stopping solution, a concentrated washing solution, a concentrated complex solution, standard product solution with high concentration, a cover plate film, a self-sealing bag, an instruction and a quality inspection report. The utility model is characterized in that, indirect competition ELISA method is adopted; the micropore strip of the enzyme label plate is coated by coupling antigens in advance; the tetracycline left in the sample and the coupling antigens coated on the micropore strip in advance compete for the antibody of tetracycline; after the enzyme labeled second antibody is added, TMB is used to display the color of substrate; the sample absorbance value having a negative correlation with the content of tetracycline is compared with the standard curve, and corresponding dilution ratio is multiplied, then the residue of tetracycline in the sample can be calculated. Compared with instrument analysis technology, the ELISA detection kit has the advantages of easy operation, low cost, and high sensitivity.

Description

Tetracycline ELISA detection kit

(1) technical field:

The utility model relates to the detection kit of animal derived food Chinese traditional medicine residual quantity, particularly detects the kit of tetracycline residual quantity in the animal derived food.

(2) background technology:

The total amount that (goes out to close on March 18th, 1992) tentatively to be defined in the telracycline family compound in muscle and the milk in European Union's the 675/92nd command must not surpass 100 μ g/L, at kidney, must not distinguish in liver and the egg and surpass 600 μ g/L, 300 μ g/L and 200 μ g/L.

At present, the residual detection of telracycline family antibiotic is carried out with high performance liquid chromatography, and the shortcoming of HPLC method is that the preparation sample will expend the plenty of time, and needs expensive experimental chamber equipment.

(3) utility model content: problem to be solved in the utility model provides a kind of tetracycline residue 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 tetracycline Determination on content in a plurality of samples of one-time continuous, has reduced the needed time of test samples.

The technical solution of the utility model is: adopt 96 hole polystyrene agent plate as solid phase carrier, put into the vacuum aluminide-coating bag, pre-bag is made check-out console by the tetracycline coupled antigen on kit ELISA Plate capillary strip, with the plastics dura mater is the cover plate film, with the 40ml concentrated cleaning solution, 50ml concentrates redissolution liquid, the 12ml ELIAS secondary antibody, 7ml tetracycline antibody working fluid, 7ml colour developing liquid A liquid, 7ml colour developing liquid B liquid, the standard solution of 7ml stop buffer and 6 bottles of 1ml gradient concentrations and 1 bottle of 1ml high concentration standard solution, reagent is with different big or small, the plastic bottle of different colours and vial splendid attire also are fixed in the polyfoam mould and agent plate, the cover plate film together is encapsulated in the 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.During detection, residue in the sample (tetracycline) will with the coupling tetracycline antigenic competition tetracycline resistance antibody of pre-bag quilt on the ELISA Plate capillary strip, after adding ELIAS secondary antibody, develop the color with tmb substrate, the content of contained residue (tetracycline) becomes negative correlation in sample light absorption value and the sample, multiply by its corresponding extension rate more again with typical curve, can draw the residual quantity of tetracycline 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 tissue sample is limited to 5 μ g/kg, honey is limited to 4 μ g/kg; The recovery of tissue sample is 80% ± 15%, the recovery of honey is 80% ± 15%; Be 125%, be 110%, be 70%, be 20% with the cross reacting rate of miaow promise tetracycline with the cross reacting rate of terramycin with the cross reacting rate of aureomycin with the cross reacting rate of pyrrolidine first tetracycline.With respect to other tetracycline method for detecting residue, the required instrument of this kit is less, only needs microplate reader, even matter device, vibrating machine and micro sample adding appliance, and generally all there is outfit in similar laboratory, and required cost is lower.

The novel beneficial effect of this experiment is: can be used for the residual detection of animal derived food tetracycline quick, easy, sensitive, exactly.

(4) description of drawings:

Fig. 1 is the side drawing in side sectional elevation (long is 12.8cm) of this experiment novel enzyme yoke plate;

Fig. 2 is the vertical view of this experiment novel enzyme yoke plate;

Fig. 3 is the lateral longitudinal sectional view (long is 8.55cm) of this experiment novel enzyme yoke plate;

Fig. 4 is this experiment blind flange membrane plane figure;

Fig. 5 is this experiment novel agent bottle longitudinal profile planimetric map;

Fig. 6 tests the vertical view of novel fixed foam mould for this.

Referring to accompanying drawing: enzyme mark reaction capillary strip (2) in advance coated tetracycline coupled antigen (3) is fixed in the enzyme mark On the outer frame support (1) of plate, enzyme mark reaction capillary strip (2) can be with requiring dismounting; Cover plate film (4) is used Capping enzyme mark reaction capillary strip (2) when ELISA Plate is put water-bath or insulating box internal reaction; Hyaline cap translucent Plastics reagent bottle (5) is used for encapsulation 40ml concentrated cleaning solution; The translucent plastic reagent bottle (5) of blue cap Be used for encapsulation 50ml and concentrate 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) are 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 of green cap Reagent bottle (8) is used for encapsulation 7ml tetracycline antibody working solution, the white plastic reagent bottle (8) of red cap Be used for encapsulation 12ml ELIAS secondary antibody, the brown glass reagent bottle (9) of white cap is used for the mark of encapsulation 1ml/ bottle Accurate product solution and 1ml high concentration standard solution; Foamed plastics mould (10) has 15 bottle positions, places the position Put and be 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 tetracycline Antibody working solution bottle position (16), 12ml ELIAS secondary antibody bottle position (15), the standard of 6 kinds of various concentration of 1ml/ bottle Product solution and 1 bottle of 1ml high concentration standard solution bottle position (17).

The embodiment that this experiment is novel:

One, the pre-treatment of sample

1. dosing:

Dosing 1: 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 for sample by 1: 1 volume ratio

Dosing 2:0.02M phosphate buffer

Taking by weighing 2.58g disodium hydrogen phosphate dodecahydrate and 0.435g two hypophosphite monohydrate sodium dihydrogens adds deionized water dissolving and is settled to 500ml

Dosing 3:Mcllvain-buffer

Take by weighing Citric acid monohydrate 12.9g, two hypophosphite monohydrate sodium dihydrogen 10.9g, disodium EDTA (EDTA-Sodium salt) 37.2g adds deionized water dissolving and is settled to 1L, transfers pH to 3.8 with phosphoric acid

Dosing 4: eluent

Contain 20mM Oxalic acid methanol solution (=1.8g/l)

Taking by weighing 1.8g Oxalic acid adds dissolve with methanol and is settled to 1L

Dosing 9: washing working fluid

With deionized water 20 * concentrated cleaning solution is diluted (or by requirement dilution) (1 part of 20 * concentrated cleaning solution+19 part deionized water) by 1: 19 volume ratio and be used for the washing of ELISA Plate

2. honey sample pre-treatment:

Take by weighing 1 ± 0.05g honey to in the bottle of gong flap (80ml), dilute (1 part of phosphate buffer+49 part deionized water) with deionized water by 1: 49 volume ratio with the 0.02M phosphate buffer; With ultrasound wave homogeneous 5 minutes; The strong mixing with vortex instrument whirling motion 2min; Be mixed before the application of sample (putting upside down vibration); Getting 50 μ l analyzes.

3. honey sample pre-treatment:

Accurately take by weighing 4 ± 0.05g honey sample to 50ml polystyrene centrifuge tube, add 0.5ml 1MNaOH and leave standstill 20min after with oscillator vibration mixing; Add 0.5ml 1M HCl, with the oscillator mixing (pH should about 3) that vibrates if please do not transfer to accurately with hydrochloric acid or NaOH in this scope, add 7ml acidifying acetonitrile (about pH4.0) again, with the oscillator 10min that fully vibrates, more than the 3000g, the centrifugal 10min of room temperature; Get in the clean glass test tube of supernatant 3ml to 10ml, flow down in 50～60 ℃ of water-bath nitrogen and dry up; Add 1ml redissolution working fluid dissolving dried residue; Getting 50 μ l is used for analyzing.

4. meat tissue sample pre-treatments

With homogenizer homogeneous structure sample; Take by weighing the equal pledge of 5 ± 0.05g to 50ml polystyrene centrifuge tube, add 25ml Mcllvain-buffer with the oscillator 30min that vibrates, more than the 3000g, 10 ℃ of centrifugal 10min collect supernatant to 50ml polystyrene centrifuge tube; Sediment repeats to extract once, and twice extract is united two into one, and filters with folded filter paper, collects filtrate; Get 5ml filtrate and use RIDA C18 post purifying according to the following steps; With 4ml methyl alcohol (100%) washing pillar, with 3ml deionized water wash pillar; Get 5ml filtrate and advance post, with 3ml deionized water wash pillar; The liquid that careful extrusion is all, with 2ml eluent elution samples, flow velocity is 15 droplets/minute; The sample liquid of wash-out is diluted (50 μ l sample liquid add 450 μ l redissolution working fluid mixing) with the redissolution working fluid by 1: 9 volume ratio; Getting 50 μ l analyzes.

Two, use the operation steps of kit:

1, from 4 ℃ of cold storage environment, takes out required reagent, put room temperature (20-25 ℃) more than the balance 30min, note to shake up before every kind of liquid reagent uses.

2, take out framework and need the micropore of quantity, no micropore 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/sample: add standard items/sample 50 μ l/ holes in micropore separately, add antibody working fluid 50 μ l/ holes again, the mixing that vibrates gently is with reacting 60min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate.

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

7, 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.Taking-up repeats to wash plate step 6.

8, colour developing: add substrate solution A liquid 50 μ l/ holes, add B liquid 50 μ l/ holes again, the mixing that vibrates gently is with lucifuge reaction 20-30min (seeing points for attention 8) 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, 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 its contained tetracycline.

1, mean light absorbency value and the standard value with sample relatively can draw its concentration range (μ g/L).The absorbance of supposing sample 1 is 0.210, and the absorbance of sample 2 is 0.820, and the titer absorbance is respectively: 0 μ g/L is 1.810; 0.05 μ g/L is 1.520; 0.15 μ g/L is 1.130; 0.45 μ g/L is 0.560; 1.35 μ g/L is 0.289; 4.05 μ g/L is 0.111.Then the concentration range of sample 1 is that 1.35 μ g/L-4.05 μ g/L multiply by its corresponding extension rate again and can draw the residual concentration range of tetracycline in the sample; The concentration range of sample 2 is that 0.15 μ g/L-0.45 μ g/L multiply by its corresponding extension rate again and can draw the residual concentration range of tetracycline in the sample.

2, quantitative test

(1) calculating of percentage absorptance, the percentage absorptance that the percentage absorptance of standard items or sample equals standard items or sample equals 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

Percentage absorbance (%)=B/B ₀* 100%

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

With standard items percentage absorptance is ordinate, is horizontal ordinate with the semilog of tetracycline 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 the practical residue limit that its corresponding extension rate is tetracycline in the sample from typical curve.

If utilize kit specialty analysis software to calculate, accurate, the express-analysis of a large amount of samples of being more convenient for.

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; Putting no ELISA Plate capillary strip into valve bag reseals; 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.The absorbance of 0 standard (450/630nm) value is less than 0.5 (A _450nm＜0.5) time, expression reagent may go bad.

8, after adding substrate solution A and substrate solution B liquid, general developing time is that 30min gets final product.If color is more shallow, can prolongs the reaction time to 35min (or longer), but must not surpass 40min.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. ELISA detection kit that detects tetracycline 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 tetracycline coupled antigen on the kit capillary strip, 14 bottles of reagent are respectively 6 bottles of standard solutions, 1 bottle of high concentration standard solution, ELIAS secondary antibody, tetracycline 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 transparent plastic dura mater; 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, tetracycline 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 concentrates the translucent PE plastic bottle of redissolution liquid with blue cap with the white PE plastic bottle of yellow cap, concentrated cleaning solution with the translucent PE plastic bottle of hyaline cap; Recessed bottle position is made by plastic foam.

3. according to the described kit of 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 tetracycline 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, 0.05 μ g/L, 0.15 μ g/L, 0.45 μ g/L, 1.35 μ g/L, 4.05 μ g/L; 1 bottle of high concentration standard solution, 1ml; 1 bottle of ELIAS secondary antibody, 12ml; 1 bottle of tetracycline 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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