Microsoft word - tbs-21141.doc
Salbutamol Fast Detection ELISA Kit
Enzyme-Linked Immunosorbent Assay Kit for Detection of
Salbutamol is a potent beta-2 agonist that can stimulate beta-2 receptors in mammals,
which in turn leads to fat loss by allowing the body to release and burn more stored fat. It
has been illegally used for decades in the veterinary world to increase the lean yield of
Salbutamol can cause a multitude of adverse effects upon entering human body, such as
dizziness, nausea, diarrhea, agitations, muscle cramps, hypertension, and even acute
myocardial infarction (heart attack). Therefore, it has been banned in many countries to be
Salbutamol ELISA Kit can quickly
, and accurately
the presence of Salbutamol in animal feed, and urine and tissue samples of animals,
providing a vital tool to prevent consumption of food tainted with this toxic chemical.
Salbutamol ELISA Kit utilizes competitive ELISA for the quantitative and
qualitative analysis of Salbutamol in animal feed and tissue/urine samples of farm
animals. The limit of detection (LOD) of Salbutamol in ELISA Kit is 0.05ng/ml (0.05ppb).
To receive complete safety information on this product, contact Tribioscience, Inc. and
request Material Safety Data Sheets. Stop solution is 1N sulfuric acid. Handle with care.
Salbutamol ELISA Kit is a competitive enzyme-labeled immunoassay. Each well of
the 96-well microtiter plate has been pre-coated with an anti-Salbutamol antibody. During
the assay, Salbutamol standard solution or samples are added to test wells, followed by
adding horse radish peroxidase (HRP)-Salbutamol conjugate, which will compete with
Salbutamol in standard or sample for binding to antibody during the 30-minute incubation.
After plate wash, a clear HRP substrate is added to the wells leading to a colored product
only in the presence of HRP, and optical density is inversely related to Salbutamol
concentrations in the samples. The accurate concentration of Salbutamol can then be
determined by interpolation using the standard curve constructed in the same run.
Reagents and Materials Provided
The reagents included in the kit are sufficient for performing 96 measurements (including
standards and samples). Reagents and materials in each kit include:
a) 1 microtiter plate containing 12 test strips of 8 wells sealed in an aluminized pouch
b) 6 vial each containing 0.5 mL of Salbutamol standard with 0, 0.1, 0.3, 0.9, 2.7, 8.1
c) 1 vials containing 0.1 mL Salbutamol-HRP conjugate (100×).
d) 1 bottle containing 10 mL sample diluents (5 x).
e) 1 bottle containing 10 mL assay buffer (1 x).
f) 1 bottle containing 15 mL microtiter plate wash solution (20×).
g) 1 bottle containing 11 mL substrate (1×).
h) 1 bottle containing 11 mL stop solution (1×).
Materials required but not provided
a) Microplate reader with 450 nm filter.
b) Pipet capable of dispensing 20-200 µl
Equilibrate kit components at room temperature (20-25oC) for at least 30 min prior to
running the test, and thoroughly mix all liquid components before use.
Use test strips as needed on the frame, and store unused strips in the resealable bag
Number standards and samples according to positions on microtiter plate. All
standards and samples need duplicate measurement for accuracy.
Samples need to be processed as followings before ELISA assay:
3. Add 18 mL distilled water and 2 mL 1N HCl
5. Centrifuge at 2000 rpm for 20 min at room temperature
6. Take supernatant and adjust pH to 7-8 using 1N NaOH
7. Centrifuge at 2000 rpm for 20 min at room temperature
8. Use supernatant directly for ELISA assay
9. Sample processed in this method has dilution factor of 10
2. Add 2ml ethyl acetate and mix by vortex thoroughly
3. Centrifuge at 4000 rpm for 10 min at room temperature
5. Evaporate to dryness in a nitrogen evaporator
9. Centrifuge at 4000 rpm for 10 min at room temperature
10. Discard upper layer, and transfer 50 µl bottom aqueous phase to ELISA plate
11. Sample processed in this method has dilution factor of 2
1. Centrifuge at 4000 rpm for 5 min at room temperature
2. Take supernatant and dilute 2 fold with sample diluents for ELISA assay
3. Sample processed in this method has dilution factor of 2
1. Prepare Wash Solution by diluting 1 part of Wash Solution Concentrate (20x) with
19 parts of distilled water to obtain Wash Solution (1x)
2. Prepare HRP working solution by diluting 1 part of HRP conjugate (100x) with 99
parts of Assay Buffer to obtain HRP working solution (1x)
3. Add Salbutamol standard sample, or unknown sample, 50 µl/well in duplicate
4. Add HRP working solution 50 µl/well, gently shake plate by hand for 1 min, and
5. Wash plate 4 times with wash solution (1x), 200 µl/well wash buffer each time
6. Add 100 µl/well TMB-Ultra solution, and incubate plate at room temperature for 15
Add 100 µl/well stopping solution, read OD450nm
Two approaches can be used to obtain Salbutamol concentration results from the assay.
Semi-quantitative results can be obtained with the first approach while quantitative results
can be calculated with the second. Please note the negative correlation between
absorbance reading (OD450nm) and Salbutamol concentration in the sample.
1. Semi-quantitative detection of Salbutamol
A simple comparison of average sample absorbance to absorption of standards will give
the range of Salbutamol concentration (ng/mL or ppb) in the samples. For example,
Sample 1 has an average absorbance of 0.6, and Sample 2 of 1.2, and OD450nm of standard solutions are as follows: 2.300 for 0 ng/mL; 2.000 for 0.1 ng/mL; 1.650 for 0.3
ng/mL; 0.950 for 0.9 ng/mL; 0.400 for 2.7 ng/mL; 0.165 for 8.1 ng/mL. It is immediately
known that Salbutamol concentration of Sample 1 is between 0.9 ng/mL and 2.7 ng/mL,
while Sample 2 contains 0.3-0.9 ng/mL Salbutamol.
2. Quantitative calculation of Salbutamol concentration
a) Calculate B/B0 Dividing average absorbance of each standard and sample (B) by absorbance of first
standard (the standard with 0 ng/mL Salbutamol concentration, B0) to obtain percentage absorbance.
Percentage absorbance (%) = B/B0 x 100% B — average absorbance of a standard or sample
B0 — average absorbance of 0 ng/mL standard b) A standard curve is obtained by graphing the percentage absorbance of standards (Y
axis) versus their corresponding concentration (X axis) on semi-log graph paper (which
should be a linear relationship), and sample concentration can be read from this standard
curve. Alternatively, Salbutamol concentration in the samples can be calculated with
regression equation correlating percentage absorbance to Salbutamol concentration.
Graphing software can also be used for quick analyses of large number of samples.
Sensitivity (defined as the average of absorbance from 6 zero-standards minus 3 times of
Assay kit should be stored at 2-8oC and avoid freezing conditions; unused test
strips should be sealed in reclosable bag; colorless substrate is sensitive to light so
prolonged exposure to light needs to be avoided.
Reagents should be brought to room temperature (20-25oC) prior to use. A room
temperature of lower than 20oC or failure to equilibrate reagents or samples to room
temperature could result in low OD readings for all samples. All unused portion of
reagents should be put back into 2-8oC storage immediately after use.
Adhere to assay protocol on reaction temperature and time, and use pipet to add
components whenever possible. Results are solely based on OD450nm readings from
Reagents need to be thoroughly mixed to improve reproducibility.
During all incubation steps, avoid light and seal plate with sealer.
If wells are dried out during plate wash steps, linearity of standard curve will be
negatively affected and reproducibility will be poor. Therefore, substrate addition
should be carried out immediately after tapping the plate dry (following the last wash).
The stop solution is 1N sulfuric acid. Avoid contact with skin or clothing.
Immediately clean up any spills and wash area with copious amounts of water. If
contact should occur, immediately flush with copious amounts of water.
Do not use reagents beyond expiration date. Dilution or adulteration of reagents
or samples not called for in the procedure may result in adverse changes in sensitivity
and OD reading. Do not substitute reagents from kits with different lot numbers.
Obvious color in substrate suggests expiration and it should be discarded. When
absorbance of zero-standard is lower than 0.6, the reagents may have expired.
Storage and Expiration Date
Storage: All components of the kit should be stored at 2-8oC.
Expiration Date: This kit expires 12 months after manufacturing date.
For ordering or technical assistance regarding this kit, or for additional information about
Tribioscience products, please email info@Tribioscience.com or call (650) 917-9269.
General Limited Warranty
Tribioscience, Inc. warrants its manufactured products against defects in materials and
workmanship when used in accordance with the applicable instructions for a period not to
extend beyond a product’s printed expiration date. Tribioscience makes no other warranty,
expressed or implied. There is no warranty of merchantability or fitness for a particular
purpose. The warranty provided herein and the data, specifications and descriptions of
Tribioscience products appearing in published catalogues and product literature may not
be altered except by express written agreement signed by an officer of Tribioscience.
Representations, oral or written, which are inconsistent with this warranty or such
publications are not authorized and, if given, should not be relied upon.
In the event of a breach of the foregoing warranty, Tribioscience Inc.’s sole obligation shall
be to repair or replace, at its option, any product or part thereof that proves defective in
materials or workmanship within the warranty period, provided the customer notifies
Tribioscience promptly of any such defect. The exclusive remedy provided herein shall not
be deemed to have failed of its essential purpose so long as Tribioscience is willing and
able to repair or replace any nonconforming Tribioscience product or part. Tribioscience
shall not be liable for consequential, incidental, special or any other indirect damages
resulting from economic loss or property damage sustained by a customer from the use of
its products. However, in some states the purchaser may have rights under state law in
addition to those provided by this warranty.
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