| Customization: | Available |
|---|---|
| Classification: | Food Diagnostic |
| Assay Time: | 1.5 H |
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7. Solutions
Solution 1: 0.5M PB
Dissolve 11.0g Na2HPO4.12H2O, 34.2g NaH2PO4 with deionized water and dilute to 500ml.
Solution 2: 0.2M HCl
Dissolve 8.3ml of concentrated HCl with deionized water, dilute to 500ml.
Solution 3: 1M NaOH
Dissolve 4.0g of NaOH with deionized water and dilute to 100ml.
Solution 4: Extraction solution
Dilute the 3×concentrated extraction solution with deionized water in the volume ratio of 1:2, which will be used for sample extraction. This solution can be conserved for a month at 4ºC.
Solution5: Wash buffer
Dilute the 20×concentrated wash solution with deionized water in the volume ratio of 1:19, which will be used to rinse the plate. This buffer can be conserved at 4ºC for 1 month.
8. Sample Preparations
8.1 Notice and precautions before operation
(a) Please use one-off tips in the process of experiment, and change the tips when absorbing different reagent.
(b) Make sure that all experimental instruments are clean.
(c) Prepared tissue sample should be tested within 2h.
(d) Test liver sample should be freshly collected.
8.2 Urine
----Take 50μl of the clear urine sample directly for assay.
Note: if the sample is not clear, please centrifuge:10min/ 20-25ºC/ 3000g, till the sample is transparent or filter with filter paper. Keep unused samples in freeze.
8.3 Tissue
----Homogenize the test sample;
----Take 2.0±0.05g of homogenate into a 50ml polystyrene centrifuge tube;
----Add 3ml of 0.5M PB (solution 1), 1ml of 0.2M HCl (solution 2), vortex to dissolve thoroughly;
----Centrifuge for separation: 5min / ambient temperature / 3000g;
For liver sample: Take 1ml supernate (Do not take the impurity), add approximate 80μl of 1M NaOH (solution 3), mix thoroughly (pH should be approx. 6.5);
For muscle sample: Take 1ml of supernate (Do not take the impurity), add approximate 50μl of 1M NaOH (solution 3), mix thoroughly (pH should be approx. 6.5);
----Take 50μl of the prepared solution for assay.
Notice: the sample cannot be stored after centrifuge, which should be tested immediately.
8.4 Feed
----Homogenize a certain amount of the feed.
----Take 2.0±0.05g feed homogenate into a 50ml polystyrene tube;
----Add 10ml of methanol, vortex for 1min;
----Centrifuge for separation: 5min / ambient temperature / 3000g;
----Take 1ml of supernate organic phase into a 10ml glass tube, dry with 50-60ºC water bath under nitrogen gas stream;
----Add 1ml of n-hexane, vortex for 1min, then add 1ml of extraction solution(solution 4), vortex for 30s.
----Centrifuge for separation: 5min / ambient temperature / 3000g
-------Remove the supernatant organic phase, take 200ml of the substrate aqueous phase and dilute with 200ml of extraction solution (solution 4), mix completely, take 50ml per well for assay.
9. Assay process
9.1 Notice before assay
9.1.1 Make sure all reagents and microwells are all at room temperature (20-25ºC).
9.1.2 Return all the rest reagents to 2-8ºC immediately after used.
9.1.3 Washing the microwells correctly is an important step in the process of assay; it is the vital factor to the reproducibility of the ELISA analysis.
9.1.4 Avoid the light and cover the microwells during incubation.
9.2 Assay Steps
9.2.1 Take all reagents out at room temperature (20-25ºC) for more than 30min, shake gently before use.
9.2.2 Get the microwells needed out and return the rest into the zip-lock bag at 2-8ºC immediately.
9.2.3 The diluted wash solution should be brought to room temperature before use.
9.2.4 Number: Numbered every microwell positions and all standards and samples should be run in duplicate. Record the standards and samples positions.
9.2.5 Dilution of concentrated enzyme conjugate: Take enough concentrated enzyme conjugate (Kit insert), and dilute it with enzyme conjugate diluent (Kit insert) in the volume ratio of 1:10. (e.g. 0.5ml of concentrated enzyme conjugate + 5ml of enzyme conjugate diluent). Mix thoroughly before use.
Note: The diluted enzyme conjugate solution can be stored for 1 month at 2-8ºC.
9.2.6 Add standard solution/sample and diluted enzyme conjugate: Add 50µl of standard solution(Kit insert) or prepared sample to corresponding wells, and then add 100µl of the diluted enzyme conjugate to each well, shake gently and cover the plate with cover, incubate at 25ºC for 40min;
9.2.7 Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with diluted wash buffer (solution 5) at interval of 10s for 4-5 times. Absorb the residual water with absorbent paper (the rest air bubble can be eliminated with unused tip).
9.2.8 Coloration: Add 50µl of solution A(Kit insert) and 50µl of solution B(Kit insert) to each well. Mix gently by rocking the plate manually and incubate for 15min at 25ºC with cover.
9.2.9 Measure: Add 50µl of stop solution (Kit insert) to each well. Mix gently by rocking the plate manually and measure the absorbance at 450nm (It's suggested measure with the dual-wavelength of 450/630nm. Read the result within 5min after addition of stop solution.)
10. Results
10.1 Percentage absorbance
The mean values of the absorbance values obtained for the standards and the samples are divided by the absorbance value of the first standard (zero standard) and multiplied by 100%. The zero standard is thus made equal to 100% and the absorbance values are quoted in percentages.
=
B --absorbance standard (or sample)
B0 --absorbance zero standard
10.2 Standard Curve
----To draw a standard curve: Take the absorbance value of standards as y-axis, semi logarithmic of the concentration of the β-agonist standards solution (ppb) as x-axis.
----The β-agonist concentration of each sample (ppb), which can be read from the calibration curve, is multiplied by the corresponding dilution factor of each sample followed, and the actual concentration of sample is obtained.
Notice: Analysis software can be provided on request.
Dilution factor
Urine: 1
Tissue: 2
Feed: 10
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.2 ppb
Detection limit
Salbutamol
Urine …........................….0.4ppb
Tissue ...................…......….…0.6ppb
Feed...............................…2ppb
Clenbuterol
Urine …........................….0.4ppb
Tissue ...................…......….…0.5ppb
Feed...............................…2ppb
Accuracy
Urine..........….............….....100±20%
Tissue....…...................…..…90±20%
Feed.........................….....90±20%
Precision:
CV of the ELISA kit is less than 10%.
12. Notice
12.1 The mean values of the absorbance values obtained for the standards and the samples will be reduced if the reagents and samples have not been regulated to room temperature (20-25ºC).
12.2 Do not allow microwells to dry between steps to avoid unsuccessful reproducibility and operate the next step immediately after tap the microwells holder.
12.3 Shake each reagent gently before use.
12.4 Keep your skin away from the stop solution for it is the 0.5M H2SO4 solution.
12.5 Don't use the kits out of date. Don't exchange the reagents of different batches; otherwise it will drop the sensitivity.
12.6 Keep the ELISA kits at 2-8ºC, do not freeze. Seal rest microwell plates, Avoid straight sunlight during all incubations. Covering the microtiter plates is recommended.
12.7 Substrate solution should be abandoned if it turns colors. The reagents may be deteriorated if the absorbance value (450/630nm) of the zero standard is less than 0.5 (A450nm<0.5).
12.8 The coloration reaction needs 15min after the addition of solution A and solution B. You can prolong the incubation time to 20min or more if the color is too light to be determined. Never exceed 30min. On the contrary, shorten the incubation time properly.
12.9 The optimal reaction temperature is 25ºC. Higher or lower temperature will lead to the changes of sensitivity and absorbance values.
13. Storage
Storage condition: 2-8ºC.
Storage period: 12 months.
Stuff
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Stock Market
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GMP (Good Manufacturing Practice) factory and SPF animal house. The cleanliness of producing department can reach above level 105.
Instruments
HPLC, GC, LC-MS/MS for test result calibration, which are expected to provide better quality control of our test products.
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