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Immunoassay of Theophylline by fluorescence polarisation

4 shows excitation and emission spectra of bound, tagged theophylline and antibody taken with a bandpass of 7.2 nm on the excitation, and 3.6 nm on the emission spectrometers. The excitation maximum occurs at 485 nm, while the emission maximum occurs at 513 nm. The tagged drug shows virtually no polarization when not bound to the antibody, as shown by the dashed curve in Figure 5. Upon binding, a polarization of about 0.27 is observed (solid curve). This demonstrates that the complexed and uncomplexed theophylline can be distinguished readily by polarization without the need for chemical separation.


To generate a calibration curve, the spectrometers were set at the excitation and emission maxima, and the polarization was directly calculated. Eleven readings with a 5-second integration time were averaged at each of six concentrations (0, 2.5, 5, 10, 20, and 40 g/mL of theophylline in buffer) to produce the calibration curve (see Figure 6).


Finally, a trial sample was produced which contained the following concentrations of eleven different drugs:
gentamicin at 5 g/mL
theophylline at 17.8 g/mL
tobramycin at 6.5 g/mL
quinidine at 2.6 g/mL
phenytoin at 12 g/mL
digoxin at 2.2 ng/mL
salicylate at 220 g/mL
acetaminophen at 217 g/mL
lithium at 1.2 meq/L
meclofenamic at 6.0 g/mL
phenobarbital at 51.4 g/mL


Three determinations of different aliquots of this sample produced an average polarization of 0.122 0.002, which corresponds to a theophylline concentration of 18.0 0.3 g/mL on the calibration curve (see Figure 6). This is agrees with the actual concentration listed above.

In a normal fluorescence experiment, interference would be anticipated form gentamicin, qu
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