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Product Details:
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Specifications: | 96 Wells/kit | Shelf Life: | 12 Months When Store At 2~8℃ |
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Sample Performance: | Honey, Tissue, Egg, Milk, Feed, Urine | Sensitivity: | 0.1ppb |
Recovery Rate: | 90 ±30% | Incubation Temperature: | 25℃ |
Incubation Time: | 30min~15min | ||
High Light: | ELISA test kit,flor elisa assay kit,Florfenicol diagnostic kit |
Florfenicol ELISA Test Kit
Catalog No. LSY-10008
1. Principle
This test kit is based on the competitive enzyme immunoassay for the detection of Florfenicol in samples. The coupling antigens are pre-coated on the micro-well stripes. The Florfenicol in the sample and the conjugate antigens pre-coated on the micro-well stripes compete for the anti-Florfenicol antibodies. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value has a negative correlation with the Florfenicol concentration in the sample. This value is compared to the standard curve and the Florfenicol concentration is subsequently obtained.
2. Technical specifications
Sensitivity: 0.1ppb
Incubator temperature: 25℃
Incubator time: 30min~15min
Detection limit:
Honey, tissue, egg........................................................................... 0.1ppb
Milk, feed, Urine(Method 1).............................................................. 0.2ppb
Urine(Method 2)................................................................................. 1ppb
Recovery rate:
Tissue, honey, milk, egg, feed, urine ............................................ 90 ±30%
Cross-reaction rate:
Florfenicol........................................................................................ 100%
Florfenicol Amine................................................................................ 11%
Thiamphenicol............................................................................... < 0.1%
TheChloramphenicol............................................................................ < 0.1%
3. Components
1 | Micro-well strips | 12 strips with 8 removable wells each | |
2 | 6× standard solution (1mL each) | 0ppb | 0.1ppb |
0.2ppb | 0.4ppb | ||
0.8ppb | 1.6ppb | ||
3 | Enzyme conjugate | 7ml | red cap |
4 | Antibody working solution | 7ml | blue cap |
5 | Substrate A solution | 7ml | white cap |
6 | Substrate B solution | 7ml | black cap |
7 | Stop solution | 7ml | yellow cap |
8 | 20× concentrated washing buffer | 40ml | white cap |
9 | 2×concentrated redissolving solution | 50ml | transparent cap |
4. Materials required but not provided
5. Sample pre-treatment
Instructions
The following points must be dealt with before the pre-treatment of any kind of sample:
Solution preparation before sample pre-treatment:
Solution I Sample redissolving solution:
Dilute the 2× concentrated redissolving solution with deionized water at 1:1 (1 part 2× concentrated redissolving solution +1 part deionized water).
Solution Ⅱ Sample extracting solution:
Mix Acetonitrile and Ethyl acetate at 1:2 evenly (1 part Acetonitrile + 2 parts Ethyl acetate )
Samples preparation
a) Tissue (Chicken/liver, pork/liver, shrimp, fish etc.)
Fold of dilution of the sample: 1
b) Honey
1 Put 2±0.05 g honey sample into centrifugal tube, dissolved with 4 mL deionized water. Add 4 mL ethyl acetate, shake for 2 min. Centrifuge at above 4000 r/min at room temperature for 10 min.
2 Transfer 2ml supernatant into another centrifuge tube, blow to dry with nitrogen at 50-60 ℃. Add 1 mL of the sample redissolving solution to dissolve dry residue, mix for 30 seconds.
3 Take 50 µL for analysis.
Fold of dilution of the sample: 1
c) Milk
1. Take 1mL of homogenized milk sample into a 5ml centrifuge tube, add 2ml Sample extracting solution, shake for 1min, centrifuge at above 4000 r/min at room temperature for 10 min.
2. Take 1 mL of the supernatant, blow to dry with nitrogen at 50-60 ℃.
3. Add 1 mL N-hexane to dissolve the dry residues, then add 1 mL of the sample redissolving solution, mix for 30 seconds; centrifuge at above 4000 r/min at room temperature for 5 min. Remove up-layer organic phase.
4. Take 50 µL of the down-layer for analysis.
Fold of dilution of the sample: 2
d) Feed
Fold of dilution of the sample: 2
e) Egg
Fold of dilution of the sample: 1
(If the 3ml supernatant cannot be obtained during processing, the solution can be taken after repeated centrifugation, or 5ml can be taken out after adding 10ml of ethyl acetate, and the dilution can be maintained.)
f) Urine method 1
1)Weight 1 ± 0.05g (or 1ml)of sample into a 50ml centrifuge tube, add 6 mL of ethyl acetate, shake properly for 1 min, centrifuge at above 4000 r/min at room temperature for 10 min.
2)Take 3 mL of the supernatant, blow to dry with nitrogen at 50-60 ℃.
3)Add 1 mL N-hexane to dissolve the dry residues, then add in 1 mL of the sample redissolving solution, mix for 30 seconds; centrifuge at above 4000 r/min at room temperature for 10 min. Remove up-layer organic phase.
4)Take 50 µL of the down-layer for analysis.
Fold of dilution of the sample: 2
g) Urine method 2
1) Take 100μl of thawed clear urine, add 900μl sample redissolving solution,Vortex it.
2) Take 50 µL of above mixed liquid for analysis.
Fold of dilution of the sample: 10
6. ELISA procedures
6.1 Instructions
6.2 Test implementation
1) Take out all the necessary reagents from 2~8 ℃ environment, bring them to the room temperature (20-25 ℃) for at least 30 min, note that each liquid reagent must be shaken evenly before use.
2) Take the required micro-well strips and plate frames. Re-sealed the unused microplate, store at 2-8℃, not frozen.
3) Solution preparation: dissolve 40ml of the 20×concentrated washing buffer with deionized water at 1:19 (1 part of 20×concentrated washing buffer + 19 parts of deinonized water ) or just to the required volume for use.
4) Numbering: number the micro-wells according to samples and standard solution; each testing sample and standard solution should be performed in duplicate; record their positions.
5) Add 50 µL of the sample or standard solution to separate duplicate wells, add 50 µL of the enzyme conjugate, then add 50 µL of the antibody working solution into each well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, and incubate at 25 ℃ for 30 min.
6) Wash the microplate with the washing buffer at 250 µL/well for 4-5 times. Each time soak the well with the washing buffer for 15-30s and then flap to dry (if there are the bubbles after flapping, cut them with the clean tips).
7) Coloration: add 50 µL of the substrate A solution and then 50 µL of the substrate B solution into each well. Mix gently by shaking the plate manually, and incubate at 25 ℃ for 15 min at dark for coloration.
8) Determination: add 50 µL of the stop solution into each well, Mix gently by shaking the plate manually. Set the wavelength of the microplate reader at 450 nm to determine the OD value (we recommend to read the OD value at the dual-wavelength 450/630 nm within 5 min).
7. Result judgment
There are two methods to judge the results; the first one is the rough judgment, while the second is the quantitative determination. Note that the OD value of the sample has a negative correlation with the content of Florfenicol.
7.1 Qualitative determination
The concentration range (ng/mL) can be obtained from comparing the average OD value of the sample with that of the standard solution. Assuming that the OD value of the sampleⅠ is 0.3, and that of the sampleⅡ is 1.0, the OD value of standard solutions is: 2.243 for 0ppb, 1.816 for 0.1ppb, 1.415 for 0.2ppb, 0.74 for 0.4ppb, 0.313 for 0.8ppb, 0.155 for 1.6ppb, accordingly the concentration range of the sampleⅠ is 0.8 to 1.6, and that of the sampleⅡ is 0.2 to 0.4ppb.
7.2 Quantitative determination
The mean values of the absorbance values obtained for the average OD value (B) of the sample and the standard solution divided by the OD value (B0) of the first standard solution (0 standard) and subsequently multiplied by 100%, that is,
Percentage of absorbance value = | B | ×100% |
B0 |
B—the average (double wells) OD value of the sample or the standard solution
B0—the average OD value of the 0 ng/mL standard solution
Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithm values of the Florfenicol standard solutions (ng/mL) as Y- and X-axis, respectively. Read the corresponding concentration of the sample from the standard curve by incorporating its absorption percentage into the standard curve. The resulting value is subsequently multiplied by the corresponding dilution fold, thus finally obtaining the Florfenicol concentration in the sample.
Using the professional analyzing software of this kit will be more convenient for the accurate and rapid analysis of a large amount of samples. (Please contact us for this software).
8. Precautions
9. Storage and expiry date
Storage: store at 2-8 ℃, not frozen.
Expiry date: 12 months; date of production is on box.
Shenzhen Lvshiyuan Biotechnology Co., Ltd
D Building, National Biological Industrial Park of Marinelife, No.2 Binhai Road, Dapeng, Shenzhen, 518120 China
Tel. 86-755-28438788 Fax 86-755-28938800
Email: info@lsybt.com
www.lsybt.com