Tiamulin ELISA Test Kit

Catalog No. LSY-10076

1. Principle

This test kit is based on the indirect competitive enzyme immunoassay for the detection of Tiamulin in samples. The coupling antigens are pre-coated on the micro-well stripes. The Tiamulin in the sample and the conjugate antigens pre-coated on the micro-well stripes compete for the anti- Tiamulin antibodies. After the addition of the enzyme conjugate, the TMB substrate is added for coloration. The optical density (OD) value has a negative correlation with the Tiamulin concentration in the sample. This value is compared to the standard curve and the Tiamulin concentration is subsequently obtained.

 

2. Technical specifications

Sensitivity:  0.03ppb

Incubator temperature:  25℃

Incubator time:  30min～15min

Detection limit:

Tissue, egg   1ppb

Feed 2ppb

Recovery rate:

Tissue, egg, feed  90 ±30%

Cross-reaction rate:

Tiamulin 100%

 

3. Components

1	Micro-well strips	12 strips with 8 removable wells each
2	5× standard solution (1mL each)	0ppb	0.03ppb
		0.09ppb	0.27ppb
		0.81ppb
3	High concentration standard	1ml	100ppb
3	Enzyme conjugate	7ml	red cap
4	Antibody working solution	7ml	blue cap
5	Substrate A solution	7ml	white cap
6	Substrate B solution	7ml	black cap
7	Stop solution	7ml	yellow cap
8	20× concentrated washing buffer	40ml	white cap
9	2×concentrated redissolving solution	50ml	transparent cap

 

4. Materials required but not provided

1)     Equipment: microplate reader, homogenizer, printer, nitrogen-drying device, oscillator, centrifuge, measuring pipets, balance( a reciprocal sensibility of 0.01 g), incubator.

2)     Micropipette: single-channel 20-200µL and 100-1000 µL, and multi-channel 30～300 μl;

3)     Reagents: Anhydrous methanol, Anhydrous ethanol

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 utensil must be clean and should be re-cleaned if necessary, in order to avoid the contamination that interferes with the experimental results.

Solution preparation before sample pre-treatment:

Solution I  Sample redissolving solution:

Dilute the 2× concentrated redissolving solution with deionized water at 1:1 (1 part 2× concentrated redissolving solution +1 part deionized water).

Samples preparation

a) Tissue (Chicken, pork, duck)

1）  Weight 1 ± 0.05 g of the homogenized sample into a 10ml centrifuge tube, add 2 mL of Anhydrous methanol, shake properly for 3 min, centrifuge at above 4000 r/min at room temperature for 5 min.

2）  Take 100 µl of the supernatant, add 900µl Sample redissolving solution, shake to mix it evenly.

3）  Take 50 µL liquid for analysis.

Fold of dilution of the sample: 30

 

b) Feed

1)    Weight 1 ± 0.05 g of the crushed feed sample into a 10ml or 50ml centrifuge tube, add 5 mL of Anhydrous methanol, shake properly for 3 min, centrifuge at above 4000 r/min at room temperature for 5 min.

2)    Take 100 µl of the supernatant, add 900µl Sample redissolving solution, shake to mix it evenly.

3)    Take 50 µL liquid for analysis.

Fold of dilution of the sample: 50

 

c) Egg

1)    Weight 1 ± 0.05 g of the egg sample into a 10ml centrifuge tube, add 2 mL of Anhydrous ethanol, shake properly for 3 min, centrifuge at above 4000 r/min at room temperature for 5 min.

2)    Take 100 µl of the supernatant, add 900µl Sample redissolving solution, shake to mix it evenly.

3)    Take 50 µL liquid for analysis.

Fold of dilution of the sample: 30

 

6. ELISA procedures

6.1 Instructions

1       Bring all reagents and micro-well strips to the room temperature before use (20-25 ℃).

2       Return all reagents to 2-8 ℃ immediately after use.

3       The reproducibility of the ELISA analysis, to a large degree, depends on the consistency of plate washing. The correct operation of plate washing is the key point in the ELISA. procedures.

4       For the incubation at constant temperatures, all the samples and reagents must avoid light exposure, and each microplate should be sealed by the cover membrane.

6.2 Test implementation

1) Take out all the necessary reagents from 2~8 ℃ environment, bring them to the room temperature (20-25 ℃) for at least 30 min, note that each liquid reagent must be shaken evenly before use.

2) Take the required micro-well strips and plate frames. Re-sealed the unused microplate, store at 2-8℃, not frozen.

3) Solution preparation: dissolve 40ml of the 20×concentrated washing buffer with deionized water at 1:19 (1 part of 20×concentrated washing buffer + 19 parts of deinonized water ) or just to the required volume for use.

4) Numbering: number the micro-wells according to samples and standard solution; each testing sample and standard solution should be performed in duplicate; record their positions.

5)  Add 50 µL of the sample or standard solution to separate duplicate wells, add 50 µL of the enzyme conjugate, then add 50 µL of the antibody working solution into each well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, and incubate at 25 ℃ for 30 min.

6) Wash the microplate with the washing buffer at 250 µL/well for 4-5 times. Each time            soak the well with the washing buffer for 15-30s and then flap to dry (if there are the bubbles after flapping, cut them with the clean tips).

7) Coloration: add 50 µL of the substrate A solution and then 50 µL of the substrate B solution into each well. Mix gently by shaking the plate manually, and incubate at 25 ℃ for 15 min at dark for coloration.

8) Determination: add 50 µL of the stop solution into each well, Mix gently by shaking the plate manually. Set the wavelength of the microplate reader at 450 nm to determine the OD value (we recommend to read the OD value at the dual-wavelength 450/630 nm within 5 min).

 

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 Tiamulin.

7.1 Qualitative determination

The concentration range (ng/mL) can be obtained from comparing 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 0.7, the OD value of standard solutions is: 2.043 for 0ppb, 1.716 for 0.03ppb, 1.015 for 0.09ppb, 0.44 for 0.27ppb, 0.064 for 0.81ppb, accordingly the concentration range of the sampleⅠ is 0.27 to 0.81, and that of the sampleⅡ is 0.09 to 0.27ppb.

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 (double wells) OD value of the sample or the standard solution

B₀—the average OD value of the 0 ng/mL standard solution

Draw the standard curve with the absorption percentages of the standard solutions and the semilogarithm values of the Tiamulin standard solutions (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 the Tiamulin 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

1     The room temperature below 25 ℃ or the temperature of the reagents and the samples being not returned to the room temperature (20-25 ℃) will lead to a lower standard OD value.

2     Dryness of the microplate in the washing process will be accompanied by the situations including the non-linear standard curves and the undesirable reproducibility; So continue to next step immediately after washing.

3     Mix evenly, otherwise there will be the undesirable reproducibility;

4     The stop solution is the 2 M sulfuric acid solution, avoid contacting with the skin.

5     Do not use the kit exceeding its expiry date. The use of diluted or adulterated reagents from the kits will lead to the changes in the sensitivity and the detecting OD values. Do not exchange the reagents from the kits of different lot numbers to use.

6     Put the unused microplate into an auto-sealing bag to re-seal it. The standard substance and the colorless color former is light sensitive, and thus they cannot be directly exposed to the light.

7     Discard the coloration solution with any color that indicates the degeneration of this solution. The detecting value of the 0 standard solution of less than 0.5 indicates its degeneration.

8     The optimum reaction temperature is 25 ℃, and too high or too low temperatures will result in the changes in the detecting sensitivity and OD values.

 

9. Storage and expiry date

Storage:  store at 2-8 ℃, not frozen.

Expiry date: 12 months; date of production is on box.

Tip: If there is any air leakage in the vacuum packaging bag of the enzyme-linked immunosorbent assay (ELISA) plate, the ELISA plate is still valid and does not affect the experimental results. Please feel free to use it.

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

www.lsybt.com