PROCEDURE FOR THE DETERMINATION OF
Three vials of Positive Calibrator (0.75 ml)
After incubation, dispose the solution in the
ATENOLOL IN SAMPLE
containing 1.0, 7.5 and 60 ng/ml of atenolol.
wells by inverting and shaking. Wash microtiter wells
with wash buffer to remove the non-bound
3.4 One bottle containing 10.5 ml of Atenolol-
conjugate. Washing may be done manually as
Alkaline Phosphatase conjugate (ATL-ALP).
follows: use squeeze bottle to fill wells gently with wash buffer, dumping the wells between each wash
by inverting and shaking. After the third wash, tamp
One bottle (#5)
containing 10.5 ml of p-
holder with washed strips onto a piece of absorbent
antagonist used in the treatment of angina and
Nitrophenyl Phosphate (pNPP) substrate. Ready to
hypertension. Atenolol is poorly protein bound.
Plasma half-life is 6-8 hours but can rise to very long
Add 100 µl
of pNPP substrate (#5)
to each well
hours in severe renal impairment. There is no liver
One bottle (#6)
containing 15 ml of Wash Buffer
and incubate at room temperature for 20 min
metabolism, 90 % of absorbed drug is excreted
(10xPBS-Tween). Dilute 10 fold with distilled or
avoid contamination, place the needed amount of
substrate into a test tube and dispense only from the tube itself.
2. Principle of Atenolol Determination
One bottle (#7)
containing 5.5 ml Stop Solution,
Add 50 µl
of Stop Solution (#7)
to each well and
The enzyme immunoassay for Atenolol is based on
tap the strip holder for proper mixing .
the competition between the Atenolol in the sample
and the Atenolol-alkaline phosphatase conjugate, for binding to rabbit antibody directed against Atenolol,
4. Optional Equipment and Material Required
Read absorbance at 405 nm
using an ELISA
coated onto microwells. The sample containing the
Atenolol, and the Atenolol-alkaline phosphatase
Pipettors capable of delivering 25 µl, 50 µl and
conjugate, when added to the microtiter wells,
compete for binding to a limiting number of antibody sites. After incubation, each well is rinsed in order to
4.2 Microtiter plate reader (wavelength 405 nm).
6. Preparation of Standard Curve
enzymatic activity is then measured by the addition of
Plate washer or squeezable wash bottle.
a chromogenic substrate. If no or small amount of
Atenolol is present in the sample more enzyme
labeled Atenolol will bind the antibody on the solid
concentration of atenolol including 0 ng/ml (OD0).
surface. On the other hand, if large or significant
(b) % of Inhibition = 100 ( ODs / OD0 ) x100
amount of Atenolol is present in urine sample, less
enzyme labeled Atenolol will bind to the antibody,
Plot values of % of Inhibition, step 6.1
producing less color signal. Therefore, the intensity of
against their corresponding concentrations on Log10
the color developed is inversely proportional to the
5. Assay Procedure
concentration of Atenolol in the sample. The
concentration is calculated on the basis of a standard
Let the components of the kit equilibrate to room
Calculate atenolol concentration in the sample by
interpolation and multiply by the sample’s dilution factor to obtain the actual quantity of atenolol.
Carefully add 25 µl
of standard or samples to the
bottom of each well. Slightly tap the side of the strip
Reagents are for in vitro research use only. All
reagents of the kit are stable, if stored at 2-80 C, until
7. General Remarks
the expiration date stated on the kit.
Avoid touching the well with pipette tip and add 100 µl
of ATL-ALP conjugate (#3)
to each well.
Do not mix reagents from different lots.
96-well microtiter plate (#S)
. Twelve strips of 8
Slightly tap the side of the strip holder to properly mix
detachable wells, coated with Anti-Atenolol antibody.
If concentrations of atenolol in the samples are high, dilute sample such that points fall in the middle
One vial of 0.9 ml Negative Calibrator containing
Incubate at room temperature for 40 minutes
7.3 Do not return unused reagents back into their
Samples tested should have a pH of 7.0 ( 1.0). Excessive alkaline or acidic conditions may affect the
The stop solution contains NaOH. Avoid contact with skin or eyes. If exposed, flush with water.
EIA FOR ATENOLOL
Dispose of all materials, containers and devices
Enzyme Immunoassay for the Determination of
in the appropriate receptacle after use.
Catalog Number: ATL-K1 (96 wells)
8. Simplified Assay Procedure
Add sample or standard (25 l).
Add Enzyme conjugate (100 l). 40 min at RT.
Add pNPP (100 l), wait for 20 min. at RT.
Add stop solution (50 l) and read at 405 nm.
9. Atenolol Inhibition Curve
US Biocontract Inc.
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