Clopidol Elisa Test Kit for Tissue

• Environmental Protection: Yes
• Certification: REACH
• Color: White
• Classification: Food Diagnostic
• Function: Food Testing
• Appearance: Liquid
• Model NO.: KA09201H
• 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 Clopidol

 

1. Background

Clopidol belongs to pyridines compounds. Because the clopidol has the broad spectrum insect resistance, it becomes the most widely used coccidiostat in China and it is the banned drugs to the company which export meat in China.
This kit is a new product based on ELISA. It needs 1.5 hours in one operation. It can considerably minimize operation error and work intensity. 
2.  Test Principle
This ELISA kit is designed to detect clopidol based on the principle of "indirect-competitive" enzyme immunoassay. The microtiter wells are coated with coupling antigen. Colpidol in the sample competes with the coating antigen for binding to the limited number of antibody added. After the addition of a ready-to-use TMB substrate the signal is measured in an ELISA reader. The absorption is inversely proportional to the clopidol concentration in the sample.
3.  Applications
This kit can be used in quantitative and qualitative analysis of clopidol residue in tissue (pork, pork liver, chicken, chicken liver), milk, egg.
4.  Cross-reactions
Clopidol..................….....100%
5. Materials Required 
5.1 Equipments 
----Microtiter plate spectrophotometer (450nm/630nm) 
----Rotary evaporator / Nitrogen drying instrument
----Homogenizer
----Shaker
----Vortex mixer
----Centrifuge
----Analytical balance (inductance: 0.01g)
----Graduated pipette: 10ml
----Rubber pipette bulb
----Volumetric flask: 100ml, 500ml
----Glass test tube: 10ml
----Polystyrene Centrifuge tube: 2ml, 50ml
----Micropipettes: 20μl-200μl, 100μl-1000μl
250μl- multipipette 
5.2 Reagents 
----Acetonitrile (AR)
----n-hexane (AR)

----Hydrochloric acid (HCl, AR)

----NaCl (AR)
----Trichloroacetic acid
----Deionized water
6.  Kit Components

1. Microtiter plate coated with antigen, 96wells
2. Standard solutions (5×1ml/bottle)

0ppb,0.5ppb,1.5ppb,4.5ppb,18ppb

1. Spiking standard solution: (1ml/bottle) ...…1ppm
2. Enzyme conjugate  12ml..........….red cap
3. Antibody solution    7ml...........................green cap
4. Solution A  7ml...........….......white cap
5. Solution B  7ml.........…......…...…red cap
6. Stop solution  7ml.........…....…yellow cap
7. 20×Concentrated wash solution   40ml

.....................….…transparent cap

1. 2×Concentrated extraction solution 50ml......blue cap

7.  Reagents Preparation
Solution 1: 0.1M HCl solution
Mix 4.15ml concentrated HCl solution and 500ml deionized water up.
Solution 2: 10% NaCl solution
Mix 10.0g NaCl and 100ml deionized water up.
Solution 3: 2% trichloroacetic acid solution
Mix 2.0g trichloroacetic acid and 100ml deionized water up.
Solution 4: Extraction solution:
Dilute the 2×concentrated extraction solution with deionized water in the volume ratio of 1:1(e.g. 10ml of 2×concentrated extraction solution + 10ml of deionized water), which will be used for dissolving the extracted sample. The diluted extraction solution can be conserved for 1 month at 4ºC.
Solution 5: Wash solution:
Dilute the 20×concentrated wash solution with deionized water in the volume ratio of 1:19(e.g. 5ml of 2×concentrated wash solution + 95ml of deionized water), which will be used for washing the microtiter plate. The 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)The untreated sample should be stored in frozen.
(d)The treated sample should be used immediately.
8.2 Tissue (pork, pork liver, chicken, chicken liver), egg
----Homogenize the sample.
----Take 1.0±0.05g homogenized sample into a 50ml polystyrene centrifuge tube, add 1ml of 0.1M HCl solution (solution 1) and shake for 2min. Then add 7ml of acetonitrile and shake for 3min. Centrifuge for separation: 3000g / 5min/ ambient temperature.
----Take 1ml super organic phase into 10ml clean dry glass tube. Dry in 50-60°C water bath under nitrogen flow (air).
----Add 1ml n-hexane and vortex for 30s. Add 1ml extraction solution (solution 4) and vortex for 1min. Centrifuge for separation: 3000g / 5min/ ambient temperature.
----Remove the upper organic phase and take 50ul of the lower phase for assay.
8.3 Raw milk
----Take 1ml of milk sample into 2ml polystyrene centrifuge tube. Add 1ml of 2% trichloroacetic acid solution (solution 3) and vortex for 1min. Centrifuge for separation: 7200g / 3min/ ambient temperature.
----Take 200ul of upper clear liquid and add 800ul of extraction solution (solution 4) and vortex for 30s.
----Take 50ul for assay.
8.4 Finish milk
----Take 1ml of milk sample into 2ml polystyrene centrifuge tube. Add 200ul of 10% NaCl solution (solution 2) and 800ul of 2% trichloroacetic acid solution (solution 3).
----Vortex for 1min. Centrifuge for separation: 7200g / 3min/ ambient temperature.
----Take 200ul of upper clear liquid and add 800ul of extraction solution (solution 4) and vortex for 30s.
----Take 50ul for assay.

9 Assay Steps
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 The concentrated wash solution and concentrated extraction solution 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 and antibody solution: Add 50µl of standard solution (kit insert) or prepared sample to corresponding wells. And add 50ul of antibody solution to each well. Shake gently. 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 of wash solution (solution 5) at interval of 10s for 4-5 times. Absorb the residual water with absorbent paper (eliminate the air bubble with unused tips).
9.2.7 Add the enzyme conjugate: Add 100ul of the enzyme conjugate per well, Mix gently by shaking the plate manually and incubate for 30min at 25ºC with cover. Take out and repeat the step 9.2.6.
9.2.8 Coloration: Add 50µl of solution A (kit insert) and 50µl of solution B(kit insert) 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 of the stop solution (kit insert) to each well. Mix gently by shaking the plate manually and measure the absorbance at 450nm (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 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 clopidol standards solution (ppb) as x-axis.
The clopidol 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 analysis of the ELISA kits, special software has been developed, which can be ordered on request.

Diluents ratio:
Tissue, egg...............…8
Milk......................10
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.5ppb
Detection limit
Tissue, egg.....................….4ppb
Milk........................…...…5ppb
Accuracy:
Tissue, egg, milk...............…100±20%
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 The stop solution is high concentrated sulfuric acid.
Don't touch it.
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 be turn bad if the absorbance value (450/630nm) of the zero standard is less than 0.5(OD450nm<0.5).
12.8 The coloration reaction needs 15min after adding Solution A and Solution B. And you can prolong the incubation time to 20min if the color is too light to be determined. Never exceed 25min, 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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