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TriboTM Salbutamol Fast Detection ELISA Kit Enzyme-Linked Immunosorbent Assay Kit for Detection of General Description

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 The TriboTM Salbutamol ELISA Kit can quickly, sensitively, and accurately determine
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.
Intended Use

The TriboTM 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). Safety Instructions

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.

Assay Principle

TriboTM 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 Assay Procedure
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
Calculating Results

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. Performance Data

Sensitivity (defined as the average of absorbance from 6 zero-standards minus 3 times of Precautions

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. Technical Assistance

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.

Source: http://www.tribioscience.com/files/TBS21141.pdf