Shenzhen Lvshiyuan Biotechnology Co.,Ltd |
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TheChloramphenicol ELISA test Kit
Catalog No. LSY-10007
1. Principle
This test kit is based on the indirect competitive enzyme immunoassay for the detection of theChloramphenicol in the sample. The coupling antigen is pre-coated on the micro-well stripes. TheChloramphenicol in the sample and the coupling antigen pre-coated on the micro-well stripes compete for the anti-theChloramphenicol antibody. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value of the sample has a negative correlation with theChloramphenicol in it. This value is compared to the standard curve and theChloramphenicol concentration is subsequently obtained.
2. Technical specifications
Sensitivity: 15 ppt
Incubation Temperature: 25℃
Incubation Time: 30min~15min
Detection limit:
Tissue/Aquatic(method 1).................................................................. 7.5ppt
Tissue/Aquatic(method 2), egg, honey ............................................... 15ppt
Milk, milk powder .............................................................................. 50ppt
Feed .............................................................................................. 200ppt
Recovery rate
Tissue,Aquatic, egg........................................................................ 95±25%
Feed.............................................................................................. 90±30%
Milk, milk powder, honey............................................................... 100±30%
Cross-reaction rate:
TheChloramphenicol................................................................................ 100%
Thiamphenicol................................................................................. < 0.1%
Florfeniol......................................................................................... < 0.1%
3. Components
1 | Micro-well strips | 12 strips with 8 removable wells each | |
2 | 7× standard solution (1mL each) | 0ppt | 15ppt |
45ppt | 135ppt | ||
405ppt | 1215ppt 10ppb | ||
3 | Enzyme conjugate | 7ml | red cap |
4 | Antibody working solution | 7ml | blue cap |
5 | Substrate A | 7ml | white cap |
6 | Substrate B | 7ml | black cap |
7 | Stop solution | 7ml | yellow cap |
8 | 20× concentrated washing buffer | 15ml | white cap |
9 | 2×concentrated redissolving solution | 50ml | transparent cap |
10 | Sample extracting buffer | 50ml | yellow 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:
1) Only the disposable tips can be used for the experiments and the tips must be changed when used for absorbing different reagents;
2) Before the experiment, each experimental equipment must be checked to be clean and should be re-cleaned if necessary, in order to avoid the contamination which interferes with the experimental results.
Solution preparation before sample pre-treatment
5.1 Tissue (Chicken, duck, pork, fish, shrimp, beef, lamb) Method 1
Fold of dilution of the sample: 0.5
(If cannot take 4ml supernatant during sample preparation, repeat centrifuge then to take solution, or add 9ml ethyl acetate then take 6ml supernatant blow to dry, the dilution factor is same)
5.2 Tissue (Chicken, duck, pork, fish, shrimp, beef, lamb) Method 2
Fold of dilution of the sample: 1
(If cannot take 3ml supernatant during sample preparation, repeat centrifuge then to take solution, or add 10ml ethyl acetate then take 5ml supernatant blow to dry, the dilution factor is same)
5.3 Other tissue samples with high fat content (kidney, liver, intestine, skin, heart, pork belly, etc.)
Fold of dilution of the sample: 1
5.4 Egg
Fold of dilution of the sample: 1
(If cannot take 3ml supernatant during sample preparation, repeat centrifuge then to take solution, or add 10ml ethyl acetate then take 5ml supernatant blow to dry, the dilution factor is same)
5.5 Feed
1. Take 1±0.05g of the homogenized sample into a 50ml centrifuge tube, firstly add 9 mL deionized water, then add 5mL ethyl acetate, shake properly for 3 min.
2. Centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.
3. Take 5mL of the middle-layer water phase, transfer into another clean 50ml centrifuge tube, add 10mL ethyl acetate,shake properly for 10S.
4. Centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.
5. Take 5mL of the up-layer liquid into another clean centrifuge tube, blow to dry by nitrogen or air at 50℃.
6. Dissolve the dry residues in 2 mL N-hexane, add 1 mL of the sample redissolving solution, mix for 30 seconds; centrifuge at above 4000 r/min at room temperature (20-25℃) for 10 min, remove the up-layer organic phase.
4. Take 50 µL down-layer liquid for analysis.
Fold of dilution of the sample: 4
5.6 Honey
Fold of dilution of the sample: 0.5
5.7 Raw milk, pasteurized milk, sterilized milk, modified milk, reconstituted milk, fermented milk, milk beverages
1. Take 2±0.05ml fresh sample into a 50ml centrifuge tube, add 0.1mL Sample extracting buffer, then add 6mL ethyl acetate, shake properly for 1 min, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min.
2. Take 3mL of the up-layer liquid into a new centrifuge tube, blow to dry by nitrogen in 50-60℃.
3. Dissolve the dry residues in 1mL N-hexane, then add 1mL of the sample redissolving solution, mix for 30 seconds, centrifuge at above 4000 r/min at room temperature (20-25℃) for 10 min, discard up-layer organic phase;
4. Take 50 µL down-layer liquid for analysis.
Fold of dilution of the sample: 1
5.8 Milk powder
1. Take 2±0.05g fresh milk powder sample into a 50ml centrifuge tube, add 5mL deionized water, then add 0.5mL Sample extracting buffer, shake properly for 1 min;
2. Add 3g NaCl solid, then add 10mL ethyl acetate, shake properly for 1 min, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min;
3. Take 5mL of the up-layer liquid into a new centrifuge tube, blow to dry by nitrogen in 50-60℃.
4. Dissolve the dry residues in 1 mL N-hexane, then add 1mL of the sample redissolving solution, mix for 30 seconds, centrifuge at above 4000 r/min at room temperature (20-25℃) for 5 min, discard up-layer organic phase;
Fold of dilution of the sample: 1
6. ELISA procedures
6.1 Instructions
6.2 Operation procedures
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 theChloramphenicol.
7.1 Qualitative determination
The concentration range (ng/mL) can be obtained from the comparison 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, while those of the standard solutions are as the followings: 2.243 for 0ppt, 1.816 for 15ppt, 1.415for 45ppt, 0.74 for 135ppt, 0.313 for 405ppt and 0.155 for 1215ppt, accordingly the concentration range of the sampleⅠis 405 to 1215ppt, and that of the sampleⅡ is 45 to 135ppt.
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 OD value of the sample or the standard solution
B0—the average OD value of the 0ng/mL standard solution
Draw the standard curve with the absorption percentages of the standard solution and the semilogarithm values of theChloramphenicol standard solution (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 theChloramphenicol 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 the box.
Note: If the Vacuum package of microplate has leakage, it is still valid to use, do not affect the test result, be relax to use.
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
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