Tiamulin Elisa Kit DEIA036

Tiamulin ELISA Kit, Quantitative
Cat.No: DEIA036
Lot. No. (See product label)
Size
96T
Intended Use
This kit is intended for quantitative analysis of tiamulin residue in animal tissue (pork and chicken, etc).
General Description
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.
Principle Of The Test
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.
Reagents And Materials Provided
1. Microtiter plate with 96 wells coated with antigen

2. Standard solutions concentrate(6 bottles×1ml/bottle): 0ppb, 3ppb, 9ppb, 27ppb, 81ppb, 243ppb
3. Spiking standard solution: 1ppm (1ml/bottle)
4. Enzyme conjugate 7ml, red cap
5. Antibody solution 7ml, green cap
6. Substrate solution A 7ml, white cap
7. Substrate solution B 7ml, red cap
8. Stop solution 7ml, yellow cap
9. 20×concentrated wash solution 40ml, transparent cap
10. 2×extraction solution 50mlblue cap
Materials Required But Not Supplied
1. Microtiter plate spectrophotometer (450nm/630nm)
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45-16 Ramsey Road Shirley, NY 11967, USA
Tel: 1-631-624-4882 Fax: 1-631-938-8221
E-mail: info@creative-diagnostics.com
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2. Rotary Evaporator/Nitrogen drying instrument
3. Homogenizer
4. Shaker
5. Vortex mixer
6. Centrifuge
7. Analytical balance (inductance: 0.01g)
8. Graduated pipette: 10ml
9. Rubber pipette bulb
10. Volumetric flask: 100ml, 500ml, 1L
11. Glass test tube : 10ml
12. Polystyrene Centrifuge tube: 2ml, 10ml, 50ml
13. Glass Centrifuge tube : 10ml
14. Micropipettes: 20μl-200μl, 200μl-1000μl 250ul-multipipette
15. Ethyl acetate (AR)
16. Dimethylformamide (DMF, AR)
17. Deionized water
Storage
Storage condition: 2-8°C.

Storage period: 12 months
Specimen Collection And Preparation
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.
Animal tissues (pork, chicken, etc)
a) 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 20-25°C, at 3000g.
b) Transfer 1ml of the supernatant transparent liquid to a 10ml clean glass tube, dry with 50-60°C nitrogen gas stream.
c) 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).
d) Take 50μl of the prepared solution for assay.
Reagent 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 4°C.
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 20-25°C.
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 4°C.

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Assay Procedure
1. Notice before assay

1) Make sure all reagents and microwells are all at room temperature (20-25°C).
2) Return all the rest reagents to 2-8°C immediately after use.
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.
4) Avoid the light and cover the microwells during incubation.
2. Assay Steps
1) Take all reagents out at room temperature (20-25°C) for more than 30min, shake gently before use.
2) Get the microwells needed out and return the rest into the zip-lock bag at 2-8°C immediately.
3) Number: Number every microwell position and all standards and samples should be run in duplicate. Record the standards
and samples positions.
4) Add standard solution/sample/enzyme conjugate/antibody: Add 50μL of standard solution(solution 3)/sample to corresponding
wells, and 50μL of enzyme conjugate(kit component), 50μL of antibody(kit component), shake gently to mix completely. And
then incubate at 37°C for 30min with cover.
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 3-4 times. Absorb the residual water with absorbent paper.
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 37°C with cover.
7) 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)
Interpretation Of Results
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
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.

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3
Performance Characteristics
Cross-reactions
Tiamulin: 100%
Lincomycin: < 0.1%
Spectinomycin: < 0.1%
Apramycin: < 0.1%
Precision
Variation coefficient of the ELISA kit is less than 10%.
Detection Limit
Detection limit: 2ppb

Accuracy: 80±15%
Sensitivity
0.3ppb
Specificity
Cross-reactions
Tiamulin: 100%
Lincomycin: < 0.1%
Spectinomycin: < 0.1%
Apramycin: < 0.1%
Precautions
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 (20-25°C).
2. Do not allow microwells to dry between steps to avoid unsuccessful repetitiveness and operate the next step immediately after
tap the microwells holder.
3 Shake each reagent gently before use.
4 Keep your skin away from the stop solution for it is the 0.5M H2SO4 solution.
5. Don't use the kits out of date. Don't exchange the reagents of different batches, for it will drop the sensitivity.
6. Keep the ELISA kits at 2-8°C, 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.
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).
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.

Tel: 1-631-624-4882 Fax: 1-631-938-8221
4
9. The optimal reaction temperature is 37°C. Higher or lower temperature will lead to the changes of sensitivity and absorbance
values.

Tel: 1-631-624-4882 Fax: 1-631-938-8221

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Tiamulin Elisa Kit DEIA036

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Tiamulin ELISA Kit, Quantitative

Principle Of The Test

Reagents And Materials Provided

1. Microtiter plate with 96 wells coated with antigen

Materials Required But Not Supplied

1. Microtiter plate spectrophotometer (450nm/630nm)

Creative Diagnostics. All rights reserved

Storage condition: 2-8°C.

Specimen Collection And Preparation

Notice and precautions before operation

Solution 1: Extraction solution

Creative Diagnostics. All rights reserved

1. Notice before assay

B——absorbance standard (or sample)

Creative Diagnostics. All rights reserved

Variation coefficient of the ELISA kit is less than 10%.

Detection limit: 2ppb

Creative Diagnostics. All rights reserved

Creative Diagnostics. All rights reserved

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