Determination of Atrazine by Immunoassay

Experiment #3

Determination of Atrazine by Immunoassay

Purpose

The purpose of this experiment is to determine the concentration of atrazine in a water sample by immunoassay methods, and to investigate the sensitivity and selectivity of the method used.

Theoretical Background

The intense need for analytical methods that are rapid, sensitive and field portable has led in recent years to the development of immunochemical methods for many analytes. Advantages of such methods include very high sensitivity (parts per trillion in many cases), good (although sometimes not excellent) selectivity for the target analyte, rapid turnaround, low cost, and the ability to carry out the entire procedure in the field with a minimum of equipment.

Triazine herbicides (such as atrazine) are widely used in road maintenance, soil sterilization, and agriculture to control broadleaf weeds. The U.S. EPA Safe Drinking Water Act sets an allowable limit of 3 ppb atrazine for drinking water.

In this experiment, you will carry out magnetic particle based solid-phase enzyme linked immunosorbent assay (ELISA) tests, with atrazine as the antigen (analyte). The Abraxis 100T rapid magnetic atrazine ELISA kit employs super-paramagnetic particles that are fabricated out of silanized metal oxide. Polyclonal antiserum against atrazine is covalently coupled to the particles. The size of 1 m is optimal because the particles will remain suspended in the sample solution for an hour, and yet can be pulled to the bottom in <2 min. Advantages of magnetic particles include high surface area,fast reaction and great precision.

Methods

Experimental

Part 1. Determination of the concentration of atrazine in a water sample

1. Label 12 polyethylene test tubes as follows. Dry them thoroughly.

Tube Number / Contents
1,2 / Diluent/Zero Standard, 0 ppb
3,4 / Standard, 0.1 ppb
5,6 / Standard, 1.0 ppb
7,8 / Standard, 5.0 ppb
9 / Control (3.0 ppb for interference detection)
10 / Sample 1
11 / Sample 2
12 / Sample 3

2. Add 250 L of the appropriate standard, control or sample to each tube.
3. Add 250 L of the atrazine enzyme conjugate to each tube.
4. Mix the bottle of atrazine antibody coupled magnetic particles thoroughly and add 500 L of it to each tube.
5. Vortex for 1-2 seconds.
6. Incubate for 15 minutes at room temperature.
7. Separate in the magnetic separator for 2 minutes.
8. With the tubes remaining in the magnetic separator, decant all tubes at the same time with a quick flip of the whole assembly towards the sink. Blot the open end of each tube gently on paper towels in a consistent manner.
9. Add 1 mL of washing solution to each tube, and allow them to remain in the magnetic separator for 2 minutes.
10. Decant and blot as above.
11. Repeat steps 9 and 10 once more.
12. Remove the tubes from the magnetic separator and add 500 L of the color solution to each.
13. Vortex for 1-2 seconds.
14. Incubate for 20 minutes at room temperature. Note that this is the most time-sensitive step.
15. Add 500 L of stopping solution to each tube.
16. Read results at 450 nm within 15 minutes of adding stopping solution. Use 1 mL of stopping solution as a blank to zero the spectrometer.

Calculations

1. Calculate mean absorbance value for each of the standard concentrations.
2. Calculate the %B/Bo for each standard by dividing the mean absorbance value for the standard by the mean absorbance value of the zero standard.
3. Plot %B/Bo versus atrazine concentrationon the special graph paper provided.
4. Regress the curve, and determine the concentration of the unknown from the regression and the mean absorbance value of the samples.

5. Estimate the detection limit, which is defined as the concentration at B/Bo = 85%. [T. Giersch, Journal of Agriculture and Food Chemistry, 1993, 41, 1006-1011.]

Part 2. Determination of interferences

For this part of the experiment, you will design the experiments yourself to answer the question of whether or not compounds similar (or not) to atrazine interfere with its analysis. You may find, for instance, that a compound similar to atrazine enhances the analytical signal, leading to anomalously high results, due to some interaction with the antibodies. Other compounds may depress the signal. You are provided with a few compounds similar to atrazine (i.e. other triazinessuch assimazine, aldrin, ametryn, endrin, prometryn, propazine and tertutyryn), as well as some typical degradation products. You may use other compounds as well which might typically be found in water samples along with atrazine (i.e. you need to do some reading!). Make solutions of two or three of these along with a known concentration of atrazine, analyze as above, and compare to the results in the absence of interfering compounds.