Contents of Kit
1. Microtiter plate with 96 wells coated with antigen
2. Standard solutions (6×1ml/bottle): 0ppb, 0.05ppb, 0.15ppb, 0.45ppb, 1.35ppb, 4.05ppb
3. Spiking standard solution: (1ml/bottle), 1ppm
4. Concentrated Enzyme conjugate: 1ml, red cap
5. Enzyme conjugate diluent: 10ml, green cap
6. Substrate solution A: 7ml, white cap
7. Substrate solution B: 7ml, red cap
8. Stop solution: 7ml, yellow cap
9. 20× wash solution: 40ml, transparent cap
10. 2× extraction solution: 50ml , blue cap
Animal Tissue (muscle, liver, fish and shrimp) and Feed.
Solution 1: 1% trichloroacetic acid solution
Dissolve 1.0g of trichloroacetic acid with deionized water and to 100ml.
Solution 2: Extraction solution
Dilute the 2Xconcentrate extraction solution with deionized water in the volume ratio of 1:1 (or according to the requirement), which will be used for diluting the concentrated antibody solution and sample extraction. The diluted extraction solution can be conserved for 1 month at 4°C.
Solution 3: Wash solution
Dilute the 20Xwash solution with deionized water in the volume ratio of 1:19 (or according to the requirement), which will be used to rinse the plate. The diluted wash solution can be conserved for 1 month at 4°C.
1 Notice before assay
1.1 Make sure all reagents and microwells are all at room temperature (20-25°C).
1.2 Return all the rest reagents to 2-8°C immediately after used.
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.
1.4 Avoid the light and cover the microwells during incubation.
2 Assay Steps
2.1 Take all reagents out at room temperature (20-25°C) for more than 30min, shake gently before use.
2.2 Get the microwells needed out and return the rest into the zip-lock bag at 2-8°C immediately.
2.3 All reagents should be rewarmed before use.
2.4 Number: Number every microwell position and all standards and samples should be run in duplicate. Record the standards and samples positions.
2. 5 Dilute the concentrated enzyme conjugate : Dilute the concentrated enzyme conjugate(Kit provided) with the enzyme conjugate diluent(Kit provided) in the volume ratio 1:10(e.g. 0.5ml of concentrated enzyme conjugate + 5ml of enzyme conjugate diluent), mix thoroughly, ready for use. Note:▲▲▲ The diluted enzyme conjugate can not be preserved, use immediately.
2.6 Add standard solution/sample and diluted enzyme conjugate: Add 50µl of standard solution(Kit provided) or prepared sample to corresponding wells, add 50µl of diluted enzyme conjugate solution(see 9.2.5) to each well, and then mix gently by shaking the plate manually and incubate for 30min at 25°C with cover.
2.7 Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with 250µl diluted wash solution (solution 3) at interval of 10s for 4-5 times. Absorb the residual water with absorbent paper.
2.8 Coloration: Add 50µl of solution A(Kit provided) and 50µl of solution B(Kit provided) to each well. Mix gently by shaking the plate manually and incubate for 15 min at 25°C with cover.
2.9 Measure: Add 50µl the stop solution(Kit provided) 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. We can also measure by sight without stop solution in short of the ELISA reader)
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.
Absorbance (%) = B/B0 x 100%
B ——absorbance standard (or sample)
B0 ——absorbance zero standard
Reconstitution And Storage
Florfenicol is a derivative of thiamphenicol, which is broadly applied in animal industry for controlling and treating diseases for it has a relative low cost and strong inhibition against a number of gram bacterium and mycoplasma. The residue of this drug will lead to potential aplastic anemia, so the MRL of which is now restricted to 0.1ppm in food by CAC and many other countries.
This kit is a new product for drug residue detection based on ELISA technology, which can considerably minimize operation errors and work intensity compared with instrumental analysis.