| Customization: | Available |
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| Classification: | Food Diagnostic |
| Test Sample: | Honey, Milk Bee, Egg, Milk |
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7. Reagents Preparation
Solution 1: 0.2M Phosphas buffer (pH=6)
Weigh 4.4g of Na2HPO4*12H2O and 13.68g of NaH2PO4*2H2O, dissolve with deionized water and dilute to 500ml;
Solution 2: Wash solution
Dilute the concentrated wash solution with deionized water in the volume ratio of 1:19(e.g. 5ml of 20×concentrated wash solution + 95ml of deionized water), which will be used to rinse the plates. The diluted wash solution can be conserved for a month at 4ºC.
Solution 3: milk diluent
Take 10ml of extraction solution, add 2ml of methanol, mix completely for assay.
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) Treated samples can be stored at 2-8oC for 24h in dark.
8.2 Honey
------Weigh 2.0±0.05g of honey into a 50ml polystyrene centrifuge tube;
------Add 2ml of 0.2M Phosphas buffer solution (solution 1);
------Add 8ml of ethyl acetate, shake for 5min with shaker till all samples dissolved, centrifuge for separation: 5min / 3000g / ambient temperature;
------Transfer 4ml of supernatant organic phase to a 10ml clean glass tube, dry under nitrogen gas with 50-60ºC water bath;
------Add 1ml of n-hexane, vortex for 30s, add 0.5ml of extraction solution (kit component), vortex for 30s, centrifuge for separation: 5min / 3000g / ambient temperature;
------Remove the supernatant organic phase, and take 50μl of the substrate solution for assay.
Dilution factor: 0.5
8.3 Milk
------Take 100μl milk sample into a 2ml polystyrene centrifuge tube, add 500μl milk diluent( see solution 3), vortex for 30s to mix it completely;
------Take 50μl for assay.
Dilution factor: 6
9. Assay process
9.1 Notice before assay
9.1.1 Make sure all reagents and microwells are all at room temperature (20-25ºC). Notice: The antibody solution should be stored at 4ºC, which will be used immediately after taking out.
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 Please avoid direct sunlight during the incubation, which means the plate should be covered with the plate cover provided in the kit.
9.2 Assay Steps
9.2.1 Take all reagents out at room temperature (20-25ºC) for more than 30min. Shake gently before use. Notice: The antibody solution should be stored at 4ºC, which will be used immediately after taking out.
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: Number every microwell position and all standards and samples should be run in duplicate. Record the standards and samples positions.
9.2.5 Add standard /sample and antibody: Add 50µl of standard solution(kit component) or prepared sample to corresponding wells. Add 50µl of antibody solution(kit component, The antibody solution should be stored at 4ºC, which will be used immediately after taking out), mix gently by shaking the plate manually and incubate for 60min at 4ºC with cover (or in dark place).
9.2.6 Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with 250µl of diluted wash solution (solution 2) 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).
9.2.7 Add enzyme conjugate: Add 100µl of enzyme conjugate(kit component) to each well, mix gently by shaking the plate manually and incubate for 30min at 25ºC with cover. Take out and wash the plate again following the Wash Step 9.2.6.
9.2.8 Coloration: Add 50µl of solution A(kit component) and 50µl of solution B(kit component) to each well. Mix gently by shaking the plate manually and incubate for 15 min at 25ºC with cover(see 12.8).
9.2.9 Measure: Add 50µl of stop solution(kit component) to each well. Mix gently by shaking 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 from the standards and the samples are divided by the absorbance value of the first standard (zero standard) and multiplied by 100%.
=
B --the mean absorbance value of each standards or each samples
B0 --absorbance value of zero standard
10.2 Standard Curve
---To draw a standard curve, the absorbance value of standards as y-axis, semilogarithmic of the concentration of the standards (ppb) as x-axis.
---The nitroimidazoles 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.
Please notice:
Special software has been developed for all data reduction, which can be provided on request.
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.05ppb
Detection limit
Honey..................…......…0.1ppb
Milk..............................0.4ppb
Accuracy
Honey...........................…100±15%
Milk...........................…100±20%
Precision
C.V. 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). The antibody solution should be stored at 4ºC, which will be used immediately after taking out. If the antibody solution is return to room temperature before assay, the OD values will be higher, and the result of the assay will not be right.
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, or else 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 turn bad if the absorbance value (450/630nm) of the zero standard is less than 0.5(A450nm<0.5).
12.8 The coloration reaction need 20-30min after the addition of solution A and solution B, but you can prolong the incubation time ranges to 35min or more if the color is too light to be determined, never exceed 40min, on the contrary, shorten the incubation time properly.
12.9 After adding standard, antibody solution, the incubation temperature is 0-4ºC. While after adding enzyme conjugate, substrate A and B, the incubation temperature will be 25ºC. Please make sure the temperature is correct during all steps. Higher or lower temperature will lead to experiment failure.
13. Storage
Storage condition: 2-8ºC.
Storage period: 12months
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