Elisa Test Kit for Florfenicol

• Environmental Protection: Yes
• Certification: REACH
• Color: White
• Classification: Food Diagnostic
• Function: Food Testing
• Appearance: Liquid
• Model NO.: KA00301H
• Transport Package: Foam Box with Ice Bag
• Trademark: kwinbon
• Origin: China
• HS Code: 38229000
• Production Capacity: 50000PCS/Year

Product Description

Product Description

 

Competitive Enzyme Immunoassay Kit for
Quantitative Analysis of Florfenicol

1. Background
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.
2. Test Principle
This kit is based on indirect-competitive ELISA technology. The microtiter wells are coated with coupling antigen. Florfenicol residue in the sample competes with the antigen coated on the microtiter plate for the antibody. After the addition of enzyme labeled anti-antibody, TMB substrate is used to show the color. Absorbance of the sample is negatively related to florfenicol residue in it, after comparing with the Standard Curve, multiplied by the dilution factor, florfenicol residue quantity in the sample can be calculated.
3. Applications
This kit can be used in quantitative and qualitative analysis of Florfenicol residue in animal tissue (muscle, liver, fish and shrimp), milk and feed.
4. Cross-reactions
Florfenicol...........................…100%
Florfenicol amine.......................…0.04%
Chloramphenicol.........…..............<0.1%
Thiamphenicol.........................…2.2%
Thiamphenicol amine....................…<0.1%
5. Materials Required
5.1 Equipments:
Microtiter plate spectrophotometer (450nm/630nm)
Rotary evaporator or nitrogen drying instruments
Homogenizer
Shaker
Vortex mixer
Centrifuge
Analytical balance (inductance: 0.01g)
Graduated pipette: 10ml
Rubber pipette bulb
Volumetric flask: 500ml
Glass bottle:15ml
Polystyrene Centrifuge tubes:2ml, 50ml
Micropipettes: 20ml-200ml, 200ml-1000ml
250ml-multipipette
5.2 Reagents:
Ethyl acetate (AR)
N-hexane (AR)
Trichloroacetic acid(AR)
Deionized water
6. Kit Components

1. Microtiter plate with 96 wells coated with antigen
2. Standard solutions(6×1ml/bottle)

0ppb, 0.5ppb,1.5ppb, 4.5ppb,13.5ppb,40.5ppb   

1. Spiking standard solution:(1ml/bottle) 1ppm
2. Enzyme conjugate 12ml..............…red cap
3. Antibody solution  7ml................green cap
4. Substrate solution A  7ml..........…...white cap
5. Substrate solution B  7ml...….........…red cap
6. Stop solution    7ml.......….......yellow cap
7. 20Xconcentrated wash solution 40mltransparent cap
8. 2Xconcentrated extraction solution  50ml…blue cap

7. Reagents Preparation:
Solution 1: 0.8% trichloroacetic acid solution(for milk sample)
Weigh 1.6g trichloroacetic acid and add 200ml deionized water to mix completely.
Solution 2: Extraction solution
Dilute the 2Xconcentrated 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 4ºC.
Solution 3: Wash solution
Dilute the 20Xconcentrated wash 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 4ºC.
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 absorb different reagent.
(b) Make sure that all experimental tools are clean.
(c) Keep untreated samples in freeze.
(d) Treated samples can be conserved for 24h in dark at 2-8ºC, dry samples (before dissolved with n-hexane) is recommended.
(e) Remove part of the supernatant organic phase if the treated sample become emulsified, and keep in 60ºC water bath for 5-10min to eliminate the emulsion, then repeat the centrifuge step.
8.2 Tissue (chicken, chicken liver, pork, pig liver, fish and shrimp):
Homogenize the tissue sample with homogenizer;
Weigh 3.0±0.05g homogenized sample into a 50ml polystyrene centrifuge tube, then add 6ml ethyl acetate, shake for 10min with shaker, then centrifuge for 10min at room temperature (20-25ºC), at least 3000g;
Transfer 4ml of the supernatant organic phase(equal to 2g sample) into a 10ml clean glass test tube, dry with 50-60ºC water bath nitrogen gas stream;
Add 1ml n-hexane, mix for 30s with vortex mixer, then add 1ml extraction solution(solution 1), vortex for 1min, then centrifuge for 15min at room temperature (20-25ºC) ,at least 3000g;
Remove the supernatant organic phase, take 50ml of the substrate phase for assay;
Notice: add 1ml n-hexane to dissolve the dry leftover when detecting animal liver, then add 1ml extraction solution, shake for 10s, centrifuge and take the substrate phase for assay; Increase the volume of n-hexane to 2-3ml when detecting samples high in fat.
8.3 Feed sample:
Weigh 2.0±0.05g of the homogenate into a 50ml polystyrene centrifuge tube, add 8ml ethyl acetate. Shake for 5min with shaker, then centrifuge at room temperature(20-25ºC) for 5min, at least 3000g;
Transfer 1ml of the supernatant organic phase into a 10ml clean glass tube, dry with 50-60ºC water bath nitrogen gas stream;
Add 1ml n-hexnae, vortex for 1min, then add 1ml extraction solution (solution 2), vortex for 1min, then centrifuge at room temperature (20-25ºC) for 5min, at least 3000g;
Remove the supernatant organic phase, take 100ml of the substrate phase and dilute with 900ml extraction solution (solution 2), mix completely, take 50ml per well for assay.
8.3 Raw milk
Homogenize the milk sample with homogenizer;
Take 1ml milk sample into a 2ml polystyrene centrifuge tube, add 1ml 0.8% trichloroacetic acid solution(solution 1), shake with shaker for 5min to mix completely;
Centrifuge for 5min at room temperature (20-25ºC), at least 3000g;
Transfer 100μl of the supernatant into 2 ml polystyrene centrifuge tube, add 400μl extraction solution(solution 2), mix completely;
Take 50ml per well for assay.
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 reproducibility 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, shake gently 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 All reagents should be rewarmed 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 solution/sample: Add 50µl of standard solution or prepared sample to corresponding wells, add 50µl antibody solution to each well, and then mix gently by shaking the plate manually and incubate for 30min at 25ºC with cover.
9.2.6 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 3-4 times. Absorb the residual water with absorbent paper.
9.2. 7 Add enzyme conjugate : add 100ml enzyme conjugate to each well, shake gently, then incubate for 30min at 25ºC with cover, repeat the Wash step after taking out.
9.2.8 Coloration: Add 50µl solution A and 50µl solution B 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 the stop solution 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)
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 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 florfenicol standards solution (ppb) as x-axis.
--- The florfenicol 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:
For data reduction of the ELISA kits, special software has been developed, which can be provided on request.
Dilution factor of samples:
Tissue samples (Muscle, liver, fish and shrimp): 0.5
Feed sample: 40
Milk: 10
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.5ppb
Tissue samples (muscle, liver, fish and shrimp)…...0.25ppb
Feed sample........................….20ppb
Milk...........................….......5ppb
Accuracy:
Chicken/chicken liver......................95±15%
Pork/pig liver.......................... 95±15%
Fish and shrimp.....................… 90±15%
Feed...........................…..100±30%
Milk..............................100±30%
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 Shake each reagent gently before use.
12.4. Keep your skin away from the stop solution for it is 2M H₂SO₄.
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 for the standard sample and the colorless chromogenic reagent are sensitive to light.
12.7 Substrate solution should be abandoned if it turns colors. The reagents may 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 needs 10-15min after adding Solution A and Solution B. And you can prolong the incubation time ranges to 20min or more if the color is too light to be determined, never exceed 30min, 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 condition and storage period
     Storage condition: 2-8ºC.
Storage period: 12 months.
 

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