3.3 Three vials of Positive Calibrator (0.75 ml) 5.4 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 3 times 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 1. Introduction
Alkaline Phosphatase conjugate (ATL-ALP). follows: use squeeze bottle to fill wells gently with wash buffer, dumping the wells between each wash Atennolol, 4-(2-hydroxy-3-isopropyl-aminopropoxy)- by inverting and shaking. After the third wash, tamp 3.5 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 5.5 Add 100 µl of pNPP substrate (#5) to each well
hours in severe renal impairment. There is no liver 3.6 One bottle (#6) containing 15 ml of Wash Buffer
and incubate at room temperature for 20 min. To
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
3.6 One bottle (#7) containing 5.5 ml Stop Solution,
5.6 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
5.7 Read absorbance at 405 nm using an ELISA
coated onto microwells. The sample containing the Atenolol, and the Atenolol-alkaline phosphatase 4.1 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 4.3 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, 6.2 Plot values of % of Inhibition, step 6.1 (b), 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 6.3 Calculate atenolol concentration in the sample by interpolation and multiply by the sample’s dilution factor to obtain the actual quantity of atenolol. 3. Reagents
5.1 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. 5.2 Avoid touching the well with pipette tip and add
100 µl of ATL-ALP conjugate (#3) to each well.
7.1 Do not mix reagents from different lots. 3.1 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. 7.2 If concentrations of atenolol in the samples are high, dilute sample such that points fall in the middle 3.2 One vial of 0.9 ml Negative Calibrator containing 5.3 Incubate at room temperature for 40 minutes.
7.3 Do not return unused reagents back into their 7.4 Samples tested should have a pH of 7.0 ( 1.0). Excessive alkaline or acidic conditions may affect the 7.5 The stop solution contains NaOH. Avoid contact with skin or eyes. If exposed, flush with water. EIA FOR ATENOLOL
7.6 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
8.1 Add sample or standard (25 l). 8.2 Add Enzyme conjugate (100 l). 40 min at RT. 8.4 Add pNPP (100 l), wait for 20 min. at RT. 8.5 Add stop solution (50 l) and read at 405 nm. 9. Atenolol Inhibition Curve
US Biocontract Inc.



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