| Customization: | Available |
|---|---|
| Classification: | Food Diagnostic |
| Test Sample: | Fish, Shrimp, Honey |
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7. Reagents Preparation
Solution 1: 1% trichloroacetic acid solution
Weigh 2.0g of trichloroacetic acid(Cl3CCOOH), dilute with deionized water to 200ml.
Solution 2: 0.2M NaOH
Weigh 4.0g sodium hydroxid(NaOH), dissolve with deionized water to 500ml.
Solution 3: Wash solution
Dilute the 20×concentrated wash solution(kit provided) with deionized water in the volume ration of 1:19(e.g. 10ml of 20×concentrated wash solution+ 190ml of deionized water), which will be used to wash the plates. This diluted solution can be conserved for 1 month at 4ºC.
8. Sample Preparations
8.1 Notice and precautions for the users before operation
(a) Please use one-off tips in the process of experiment, and change the tips when absorb different reagent.
(b) Make sure that all experimental instruments are clean.
(c) Treated samples should be tested immediately.
8.2 Honey
Weigh 1.0±0.05g of honey sample into a 10ml polystyrene centrifuge tube;
Add 2ml of 1% trichloroacetic acid solution(solution 1), shake to dissolve completely.
Centrifuge for separation: 3000g / 5min / ambient temperature.
Transfer 100ml of supernate into a 2ml polystyrene centrifuge tube, add 25ml of 0.2M NaOH(solution 2), then add 375ml of extraction solution(Kit component), mix completely.
Take 50ml of the prepared solution for assay.
Dilution factor: 10
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 repetitiveness 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, homogenize 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 rewarmed to be at 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 Dilute the concentrated enzyme conjugate: Dilute the concentrated enzyme conjugate(Kit component) with enzyme conjugate diluent(Kit component) in the volume ratio of 1:10(e.g. 0.4ml of concentrated enzyme conjugate + 4ml of enzyme conjugate diluent)
Notice: The diluted enzyme conjugate can not be preserved, use immediately.
9.2.6 Add standard solution/sample and diluted enzyme conjugate: Add 50ml of standard sample solution or prepared sample to corresponding wells. Add 50µl of diluted enzyme conjugate(see 9.2.5). Mix gently by shaking the plate manually and incubate for 60min at 25ºC with cover.
9.2.7 Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with 250µl of diluted wash solution (solution 3) at interval of 10s for 4 to 5 times. Absorb the residual water with absorbent paper (the rest air bubble can be eliminated with unused tip).
9.2.8 Coloration: Add 50ml of solution A(kit component) and solution B(kit component) to each well respectively. Mix gently by shaking the plate manually and incubate for 15min at 25ºC with cover(see 12.8).
9.2.9 Measure: Add 50ml of the stop solution(kit component) to each well. Mix gently by shaking the plate manually and measure the absorbance at 450nm against an air blank. (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 (%) = -- ×100%
B0
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 Neomycin standards solution (ppb) as x-axis.
----The tetracyclines concentration of each sample (ppb), which can be read from the calibration curve, is multiplied by the corresponding dilution multiple of each sample followed, and the actual concentration of sample is obtained.
Please notice:
Special software has been developed for all data analysis, which can be provided on request.
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.2ppb
Detection limit
Tetracycline.........…........…....….…2.0ppb
Aureomycin.................................…1.2ppb
Terramycin...............................…....3.0ppb
Doxycycline............................2.0ppb
Accuracy
Honey...............................90±15%
Precision
Variation coefficient 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 repetitiveness and operate the next step immediately after tap the microwells holder.
12.3. Homogenize each reagent before using.
12.4. Keep your skin away from the stop solution for it is the 2M H2SO4 solution.
12.5 Don't use the kits out of date. Don't exchange the reagents of different batches, for it will drop the sensitivity.
12.6 Storage condition:
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 Indications for the reagents going bad:
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 15min after the addition of solution A and solution B; But you can prolong the incubation time to 20min if the color is too light to be determined., 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.
Certificate
ISO9001: 2008 & ISO13485: 2003;
UKAS (United Kingdom Accreditation Service);
CNAS (China National Accreditation Service for Conformity Assessment);
CQC (China Quauty Certification Center);
CE (Communate Europpene).
Awards
High-Tech Enterprise;
Key High-Tech Enterprise of China Torch Program;
Beijing Engineering Research Center;
Industrialization Demonstration Project of China Torch Program;
Beijing Science and Technology Award;
Tianjin Science and Technology Award;
Shandong Science and Technology Award;
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