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Chapter 19: Q23P (page 488)

Here is an immunoassay to measure explosives such as trinitrotoluene (TNT) in organic solvent extracts of soil. The assay employs a flow cytometer, which counts small particles (such as living cells) flowing through a narrow tube past a detector. The cytometer in this experiment irradiates the particles with a green

laser and measures fluorescence from each particle as it flows past the detector.

1. Antibodies that bind TNT are chemically attached to 5mmdiameter latex beads.

2. The beads are incubated with a fluorescent derivative of TNT to saturate the antibodies, and excess TNT derivative is removed. The beads are resuspended in aqueous detergent.

3. 5mlof the suspension are added to 100mlof sample or standard. TNT in the sample or standard displaces some derivatized TNT from bound antibodies. The higher the concentration of TNT, the more derivatized TNT is displaced.

4. An aliquot is injected into the flow cytometer, which measures fluorescence of individual beads as they pass the detector. The figure shows median fluorescence intensity 6 standard deviation. TNT can be quantified in the ppb to ppm range.

Draw pictures showing the state of the beads in steps 1, 2, and 3and explain how this method works.

Short Answer

Expert verified

When bead passes in front of the detector, it excites, and the fluorescence is measured. According to the graph, the more TNT in the standards, the less fluorescence related to the beads.

Step by step solution

01

Binding of TNT antibodies to the latex beads


Antibodies that bind TNT are attached to the latex beads.

02

Saturation of antibody with fluorescent labeled TNT

The antibody is saturated with fluorescent labelled TNT, the excess TNT is removed.

03

Displacement of fluorescence derivatized TNT from bound antibodies

TNT in the standard or sample displaces some fluorescence derivatized TNT from bound antibodies

04

Injection of aliquot

Then an aliquot is injected into the flow cytometer. When bead passes in front of the detector, it excites, and the fluorescence is measured. According to the graph, the more TNT in the standards, the less fluorescence related to the beads.

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