Trenbolone ELISA Test Kit 

Catalog No. LSY-10068

1. Principle

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

 

2. Technical specifications

Sensitivity: 0.03ppb

Incubation Temperature:  25℃

Incubation Time:  30min～15min 

Detection limit:

Tissue  1.5ppb

Milk,urine  0.6ppb

Milk powder  1.2ppb

Recovery rate

Tissue, milk, urine, milk powder 70%~120%

Cross-reaction rate:

Trenbolone  100%

 

3. Components

1	Micro-well strips	12 strips with 8 removable  wells each
2	6× standard solution (1mL each)	0ppb	0.03ppb
		0.06ppb	0.12ppb
		0.24ppb	0.48ppb
3	Spiking standard solution	1ml	100ppb
4	Enzyme conjugate	7ml	red cap
5	Antibody working solution	7ml	blue cap
6	Substrate A	7ml	white cap
7	SubstrateB	7ml	black cap
8	Stop solution	7ml	yellow cap
9	10×Sample diluent	50ml	transparent cap
10	20× concentrated washing buffer	15ml	white cap

 

4. Materials required but not provided

1) Equipments:microplate reader, homogenizer, vortex, centrifuge, measuring pipets, and balance( a sensibility reciprocal of 0.01 g), incubator, printer.

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

3) Reagents: Methanol, Sodium hydroxide (NaCl).

 

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

Solution preparationbefore sample pre-treatment 

1) 50% Methanol solution: 1 part of Methanol + 1 part of deionized water (100ml anhydrous methanol + 100ml deionized water), mix it evenly.

2) 0.01M NaOH solution: Weigh 0.04g solid sodium hydroxide (NaCl) and add deionized water to make the volume to 100ml.

3) Sample diluent: 1 part of 10× Sample diluent + 9 parts of deionized water, mix it evenly.

 

5.1 Tissue sample preparation

1) Take 1± 0.05 g of the homogenized tissue sample into 10mL/15mL/50mL centrifuge tube;

2) Add 4mL 50% Methanol solution, vortex for 3min; centrifuge at above 4000 r/min at room temperature (20 - 25 ℃) for 5 minutes;

3) Take 100 µl up-layer clear liquid, add 900 µl Sample diluent,mix it evenly;

4) Take 50uL above liquid for analysis.

Fold of dilution of the sample:40  

 

5.2 Milk and Urine sample preparation

1) Take 1ml of the milk or urine sample into 10mL/15mL/50mL plastic centrifuge tube;

2) Add 4mL 0.01M NaOH solution, vortex for 3min;

3) Centrifuge at above 4000 r/min at room temperature (20 - 25 ℃) for 5 minutes;

4) Take 200 µl up-layer clear liquid, add 600 µl Sample diluent,mix it evenly;

5) Take 50uL above liquid for analysis.

Fold of dilution of the sample:20  

 

5.3 Milk powder sample preparation

1) Take 1g of the milk powder sample into 10mL/15mL/50mL plastic centrifuge tube;

2) Add 4mL 0.01M NaOH solution, vortex for 3min;

3) Centrifuge at above 4000 r/min at room temperature (20 - 25 ℃) for 5 minutes;

4) Take 100 µl up-layer clear liquid, add 900 µl Sample diluent,mix it evenly;

4) Take 50uL above liquid for analysis.

Fold of dilution of the sample:40  

 

6. ELISA procedures

6.1Instructions 

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

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 ELISA the 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. 

6.2Operation procedures

1. Take out all the necessary reagents from the kit and place at the room temperature (20-25 ℃) for at least 30 min. Note that each reagent must be shaken to mix 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: dilute 15mL of the concentrated washing buffer (20 × concentrated) with the deionized water at 1:19 (1 part of 20X concentrated washing buffer + 19 parts of deionized water), or prepare as quantity needed.

4. Numbering: number the micro-wells according to samples and standard solution; each 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; then add 50 µL enzyme conjugate into each well, at last add 50 µL of antibody working solution into each well. Mix gently by shaking the plate manually, seal the microplate with the cover membrane, andincubate at25 ℃ for 30 min.

6. Pour the liquid, wash the microplate with the diluted washing buffer at 250 µL/well for 4-5 times. Each time soak the well with the washing buffer for 15-30 sec, flap to dry with absorbent paper (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, andincubate at 25 ℃ for 15minatdark 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 microplate reader at 450nm to determine the OD value. (recommend to read the OD value at the dual-wavelength 450/630nm 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 Trenbolone.

7.1 Qualitative determination

The concentration range (ng/mL) can be obtained from the comparison 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 1.0, while those of the standard solutions are as the followings: 2.243 for 0ppb, 1.816 for 0.03ppb, 1.415for 0.06ppb, 0.74 for 0.12ppb, 0.313 for 0.24ppb and 0.155 for 0.48ppb, accordingly the concentration range of the sampleⅠis 0.24ppb～0.48ppb, and that of the sampleⅡ is 0.06ppb～0.12ppb.

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,

 

B—the average OD value of the sample or the standard solution

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

Draw the standard curve with the absorption percentages of the standard solution and the semilogarithm values of the Trenbolone standard solution (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 Trenbolone 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.

3. Mix every reagent and reaction mixture evenly and wash the microplate thoroughly, otherwise there will be the undesirable reproducibility.

4. The stop solution is the 0.5 M hydrochloric acid, avoid contacting with the skin;

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

6. 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.

7. Discard the colouration solution with any color that indicates the degeneration of this solution. The OD value of the standard solution 1 (0 ppb) 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 andexpiry date

Storage: store at 2-8 ℃, not frozen.

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

Note: If the Vacuum package of microplate has leakage, it is still valid to use, do not affect the test result, be relax to use.