The Abraxis Tetracycline Plate Kit is a competitive ELISA for the quantitative analysis of Tetracycline in Honey. ASSAY PRINCIPLES The Abraxis Tetracycline plate kit is a competitive enzyme-labeled immunoassay. Tetracycline is extracted from honey by shaking with extraction solution. The Tetracycline sample extract and calibrators are pipetted into the test wells followed by Tetracycline antibody into the test wells to initiate the reaction. During the 30 minute incubation period, Tetracycline from the sample and Tetracycline protein conjugate compete for binding to Tetracycline antibody. Following this 30 minute incubation, the contents of the well are removed and the wells are washed to remove any unbound Tetracycline and free Tetracycline antibody. After wash, the Goat anti-Rabbit HRP conjugate is added to each well and the plate is incubated for 30 min. After a second wash with wash solution, a clear substrate is then added to the wells and any bound enzyme conjugate causes the conversion to a blue color. Following a 30 minute incubation, the reaction is stopped and amount of color in each well is read. The color of unknown samples is compared to the color of the calibrators and the Tetracycline concentration of the samples is derived. SPECIFICITY: The Abraxis TETRACYCLINE Plate Kit can not differentiate between the various Tetracyclines, but detects their presence to differing degrees. The following table shows the relative values for the % cross reactivity versus Tetracycline. All concentrations are in parts per billion (ppb). Compound Tetracycline Rolitetracycline Chlorotetracycline-Hcl Demeclocycline-Hcl Oxytetracycline Minocycline Doxycycline Hyclate DETECTION LIMIT: Honey: 2 ppb REAGENTS AND MATERIALS PROVIDED The kit in its original packaging can be used until the end of the month indicated on the box label when stored at 2 – 8ºC. •
Plate containing 12 test strips of 8 wells each vacuum-packed in aluminized pouch with indicating
6 vials each containing 2 mL of Tetracycline calibrators corresponding to 0, 0.4, 1.2, 3.6, 10.8, and 32.4 µg/L (ppb) of Tetracycline. Calibrators are provided 10X concentrated and should be diluted prior to running the assay.
1 vial containing 22 mL Goat anti-Rabbit HRP Enzyme Conjugate.
1 vial containing 12 mL of Rabbit anti-Tetracycline antibody.
1 vial containing 12 mL of Stop Solution. (Caution! 1N HCl. Handle with care.)
1 vial containing 100 mL of 5X concentrated Wash Buffer. Solution needs to be diluted with deionized water (100 mL of 5X Wash buffer and 400 mL of deionized water)
1. Each reagent is optimized for use in the Abraxis Tetracycline Plate Kit. Do not substitute reagents from
any other manufacturer into the test kit. Do not combine reagents from other Abraxis Tetracycline Plate Kits with different Lot numbers.
2. Dilution or adulteration of reagents or samples not called for in the procedure may result in inaccurate
3. Do not use reagents after expiration date. 4. Reagents should be brought to room temperature, 20 – 28ºC (62 – 82ºF) prior to use. Avoid prolonged
(> 24 hours) storage at room temperature.
5. Tetracycline is antibiotics and should be treated with care. 6. The Stop Solution is 1N hydrochloric acid. Avoid contact with skin and mucous membranes.
Immediately clean up any spills and wash area with copious amounts of water. If contact should occur, immediately flush with copious amounts of water.
MATERIALS REQUIRED BUT NOT PROVIDED
Laboratory quality distilled or deionized water.
Glassware for sample extraction and extract collection.
Pipet with disposable tips capable of dispensing 50 µL.
Multi-channel pipet; 8 channel capable of dispensing 50 and 100 µL.
Paper towels or equivalent absorbent material.
Microwell plate or strip reader with 450nm filter.
20 mM PBS, pH 5.0. [2.76 g NaH2PO4-H2O (F.W. 137.99) + 8.5 g NaCl, filling with 1 liter of laboratory grade water]
10% Methanol/20 mM PBS is prepared as follows: 100 mL of methanol and 90 mL of 20 mM PBS.
ASSAY CALIBRATORS PREPARATION The calibrators were provided in the kit are 10X concentrates. Before each assay, they should be diluted into 10% MeOH/20 mM PBS. For example, mix 100 ul of 0.4 ppb with 900 ul of 10% MeOH/20 mM PBS in a small glass vial or glass tube to obtain 0.04 ppb working calibrator. All the 10X calibrators from 0 ppb to 32.4 ppb should be diluted the same way before assay. HONEY SAMPLE PREPARATION
1. Weigh 1 gram of honey in a screw cap glass bottle (60-80 ml size)
2. Add 49 ml of 20 mM PBS (1ml sample + 49 ml buffer, 1:50 dilution)
3. Put the sample bottle in an ultrasonic water bath for 5 min.
5. Before transferring 100 ul for the assay, invert the sample bottle several times to mix.
TEST PROCEDURE (Note: Running calibrators and samples in duplicate will improve assay precision and accuracy.) 1. Allow reagents and sample extracts to reach room temperature prior to running the test. Prepare the
wash solution by mixing 100 ml of 5X wash buffer concentrate with 400 ml of lab grade water.
2. Place the appropriate number of test wells and into a microwell holder. Be sure to re-seal unused wells
3. Using a pipet with disposable tips, add 100 μL of calibrators and samples to the appropriate test
wells. Be sure to use a clean pipet tip for each.
100 µL of Antibody Solution into each test well.
5. Shake the plate gently for 30 seconds and incubate the test wells for 30 minutes. 7. Dump the contents of the wells into an appropriate waste container. Add 250 uL of diluted (1X) wash
buffer and dump. Repeat 3X for a total of four washes.
8. Following the last wash tap the inverted wells onto absorbent paper to remove the last of the wash
9. Add 200 ul of Goat anti-Rabbit HRP conjugate to each well. 10. Incubate for 30 min.
11. Repeat Step 7 and 8 12. Dispense 100 µL of Substrate into each well. 13. Incubate the wells for 30 minutes. 14. Dispense 100 µL of Stop Solution into each test well. 15. Read and record the absorbance of the wells at 450nm using a strip or plate reader. RESULTS INTERPRETATION
1. Semi-quantitative results can be derived by simple comparison of the sample absorbances to the
absorbance of the calibrator wells: Sample containing less color than a calibrator well will have a concentration of Tetracycline greater than the concentration of the calibrator. Samples containing more color than a calibrator well have a concentration less than the concentration of the calibrator.
2. Quantitative interpretation requires graphing the absorbances of the calibrators (X axis) versus the log
of the calibrator concentration (Y axis) using software programs such as 4-parameters or alternatively logit-log. Samples with absorbances greater than the lowest calibrator or less than the highest calibrator must be reported as < 0.04 ppb or >3.24 ppb, respectively.
3. To obtain the final concentration of the sample in honey, the concentration obtained in the assay needs
to be multiplied by 50 to account for dilution factors in the extraction procedure.
Alternatively, Abraxis can supply a spreadsheet template which can be used for data reduction. Please contact Abraxis for further details.
GENERAL LIMITED WARRANTY
Abraxis LLC. (“Abraxis”) warrants the products manufactured by it 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. ABRAXIS 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 Abraxis products appearing in published catalogues and product literature may not be altered except by express written agreement signed by an officer of Abraxis. 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, Abraxis’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 Abraxis promptly of any such defect. The exclusive remedy provided herein shall not be deemed to have failed of its essential purpose so long as Abraxis is willing and able to repair or replace any nonconforming Abraxis product or part. Abraxis 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. ABRAXIS LLC www.abraxiskits.com
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