Tiamulin Ellisa Test Kit for Tissue

• Environmental Protection: Yes
• Certification: REACH
• Color: White
• Classification: Food Diagnostic
• Function: Food Testing
• Appearance: Liquid
• Model NO.: KA06101H
• Transport Package: Foam Box with Ice Bag
• Trademark: kwinbon
• Origin: China
• HS Code: 38229000
• Production Capacity: 50000PCS/Year

Product Description

 

Competitive enzyme immunoassay Kit for
Quantitative Analysis of Tiamulin

1. Background
Tiamulin is a pleuromutilin antibiotic drug that is used in veterinary medicine particularly for pigs and poultry. Strict MRL has been established due to the potential side effect in human.
HPLC or LC-MS is the common approach to detect tiamulin, and it is limited for the high expense and cost in sample pre-treatment. ELISA is rapid and accurate, which can be used for tiamulin residue detection muscle samples.
2. Test Principle
This kit is based on indirect-competitive ELISA technology. The microtiter wells are coated with coupling antigen. Tiamulin 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 the tiamulin reside in it, after comparing with the Standard Curve, multiplied by the dilution factor, tiamulin residue quantity in the sample can be calculated.
3. Applications
This kit is intended for quantitative analysis of tiamulin residue in animal tissue (pork and chicken, etc).
4. Cross-reactions
Tiamulin ...........................…100%
Lincomycin….....................….....< 0.1%
Spectinomycin...................….....< 0.1%
Apramycin.....................….....< 0.1%
5. Materials Required
5.1 Equipment
----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, 1L
----Glass test tube: 10ml
----Polystyrene Centrifuge tube: 2ml, 10ml, 50ml
----Glass Centrifuge tube: 10ml
----Micropipettes: 20μl-200μl, 200μl-1000μl
250ul-multipipette
5.2 Reagents
----Ethyl acetate (AR)
----Dimethylformamide (DMF,  AR)
----Deionized water
6. Kit Components

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

0ppb,3ppb,9ppb,27ppb,81ppb,243ppb

1. Spiking standard solution : 1ppm (1ml/bottle)
2. Enzyme conjugate  7ml...….........….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. 20×concentrated wash solution  40ml

.........................transparent cap

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

7. Reagents Preparation
Solution 1: Extraction solution
Dilute 2×concentrated extraction solution with deionized water in the volume ratio of 1:1, which will be used for sample extraction. This solution can be stored for 1 month at 4ºC.
Solution 2: Sample diluent
Mix extraction solution with DMF in the volume ratio of 22:3. Mix completely for use.
Solution 3: Standard solution
Dilute standard solution concentrate with extraction solution (solution 1) in the volume ratio of 1:9 (50ul standard solution concentrate + 450ul extraction solution). This diluted standard solution can be stored for 2h at 20-25ºC.
Solution 4: Wash solution
Dilute the 20xconcentrated wash solution with deionized water in the volume ratio of 1:19, which will be used to rinse the plate. The diluted wash solution can be stored for a 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.
8.1 animal tissues (pork, chicken, etc)
----Weigh 2.0±0.05g homogenized tissue sample into a 50ml polystyrene centrifuge tube, and add 8ml of ethyl acetate, shake fiercely for 10min, and then centrifuge for 5min, at 20-25ºC, at 3000g.
----Transfer 1ml of the supernatant transparent liquid to a 10ml clean glass tube, dry with 50-60ºC nitrogen gas stream.
----Dissolve the dry leftover with 1ml of sample diluent (solution 2), vortex for 1min(the sample may be turbid, it will not influence the result).
----Take 50μl of the prepared solution for assay.

Dilution Factor..................4

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 use.
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 Number: Number every microwell position and all standards and samples should be run in duplicate. Record the standards and samples positions.
9.2.4 Add standard solution/ sample / enzyme conjugate / antibody: Add 50µl of standard solution(solution 3) / sample to corresponding wells, add 50µl of enzyme conjugate(kit component), 50µl of antibody(kit component), shake gently to mix completely. And then incubate at 37ºC for 30min with cover.
9.2.5 Wash: Remove the cover gently and pour the liquid out of the wells and rinse the microwells with 250µl of diluted wash solution (solution 4) at interval of 10s for 4-5 times. Absorb the residual water with absorbent paper.
9.2.6 Coloration: Add 50µl of solution A(kit component) and 50µl of solution B(kit component) to each well. Mix gently by shaking the plate manually and incubate for 15min at 37ºC with cover (see 12.8).
9.2.9 Measure: Add 50µl of the stop solution(kit component) to each well. Mix gently by shaking the plate manually and measure the absorbance at 450nm (It's suggested measure with dual-wavelength 450/630nm. Read the result within 5min after adding 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 tiamulin standards solution (ppb) as x-axis.
The tiamulin 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.
Notice:
Special software has been developed for all data interpretation, which can be provided on request.
11. Sensitivity, accuracy and precision
Test Sensitivity: 0.3ppb
Detection limit:
Tissue.........…...….......................................2ppb
Accuracy:
Tissue....................................…80±15%
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 the 0.5M H₂SO₄ solution.
12.5 Don't use the kits out of date. Don't exchange the reagents of different batches, for it will drop the sensitivity.
12.6 Keep the ELISA kits at 2-8ºC, do not freeze. Seal rest microwell plates Avoid straight sunlight during all incubations. Covering the microtiter plates is recommended for the standard sample and 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 (A450nm<0.5).
12.8 The coloration reaction needs 15min after adding Solution A and Solution B. And you can prolong the incubation time ranges to 20min or longer 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 37ºC. Higher or lower temperature will lead to the changes of sensitivity and absorbance values.
13. Storage
Storage condition: 2-8ºC.
Storage period:  12months