Navigation Links
Immunoassay of Theophylline by fluorescence polarisation

Fluorescence immunoassay is applied to the detection of theophylline, a drug used for myocardial stimulation. Interference from other sample components is limited by use of polarization measurements, which discriminate between bound and unbound fluorophores. The detection system includes a T-format sampling module mounting two emission spectrometers and programmable polarizers. A calibration curve is generated and the technique demonstrated by running test samples in serum.

Immunoassay methods are based on competitive-binding reactions between labeled and unlabeled ligand for receptor sites on a specific antibody.1 Measurement of some physical property of the label permits the construction of a calibration curve of that property versus concentration of the unlabeled ligand. The selectivity of the antibodies for a specific antigen allows the analysis of the ligand (analyte) of interest in the presence of similar substances.

To date, the most popular immunoassay technique has been radioimmunoassay (RIA) because of its high sensitivity. There are a number of problems, however, associated with RIA.2 A license is required to handle radioactive material, waste disposal is complicated, and reagents have short shelf-lives. Consequently, other techniques have been developed to supplant RIA. One of the most promising, generally applicable approaches, is fluorescence immunoassay (FIA). With FIA, the label is a small fluorescent molecule (the probe), which is bound covalently to the analyte. The intensity of fluorescence produced by this probe is related to the ligand concentration, to produce the calibration curve.

Ideally, a fluorescent label should satisfy certain criteria:
The labeled ligand should be water-soluble to match physiological conditions.
The label should have at least one reactive functional group, such as isothiocyanate, acid chloride, or diazonium salt, to ease its conjugation to the ligand.
The label should not alter the specificity of the ligand for its antibody by shielding the reactive sites for antibody recognition.
The shelf-life and stability of the complex should be adequate for practical laboratory use.

The tag should have a strong absorption at the excitation wavelength of the experiment.
The quantum yield of the labeled species should be large.
The absorption and emission bands should be in the visible range to avoid interference from native proteins.
The separation between excitation and emission peaks should be large.

A further requirement of any immunoassay method is the possibility of distinguishing between free and bound labeled ligand, or the two species must be separated somehow. Here, FIA has a distinct advantage over RIA: by monitoring fluorescence polarization, the separation step is circumvented.

In fluorescence polarization, the excitation light is passed through a polarizing prism, and the emitted light is analyzed with another polarizer that is alternately oriented parallel and perpendicular to the excitation polarizer. The amount of polarization retained by the molecule depends upon its Brownian rotation between absorption and emission. A bound ligand will retain much of its polarization because the large molecular volume of the antibody gives the complex a long rotational relaxation time. The free ligand, on the other hand, has a much smaller volume, so the rotational relaxation time is shorter than its fluorescence lifetime. The emitted radiation, therefore, has a polarization that is totally randomized.

The degree of polarization is defined as

where Pr is defined as I||/ I, with I|| the intensity of emitted light polarized in the same direction as the excitation beam, and I the intensity of emitted light polarized perpendicular to the excitation beam. In practice, I|| = IVV, where V is the polarization vector normal to the optical plane, and the double subscript refers to the orientation of excitation and emission polarizers (see Figure 1). I should be equivalent to IHH, IHV, and IVH, where H is the polarization vector lying in the optical plane. Because of instrument artifacts, however, to IHH, IHV, and IVH are not equivalent. Measuring all four intensities is therefore necessary. The artifacts are then excluded by calculating PP:

The required number of measurements may be reduced to two by observing the emission simultaneously through two polarizers, using two detectors (Figure 2). In this T-format, one emission polarizer is fixed in the H position, the other in the V position, while the excitation polarizer is rotated between the V and H positions. With the SPEX Third Polarization Unit, the emission wavelength of one detector is selected by the emission monochromator, while the other detector has its wavelength selected by a filter.

Though convenient and economical, the filter method is insufficient when highly scattering samples are being analyzed, or scanning the emission spectrum is necessary, or when two wavelengths must be monitored simultaneously. In such cases, the T-format Sample Module must be employed, for it mounts two emission monochromators at 90 to the excitation beam.

Finally, polarization measurements can be performed completely under external control by adding an Autopolarization Assembly. This is a stepper-motor-driven polarizer mount that may be positioned in the excitation beam to rotate the polarizer between the H and V orientations under computer control. The host computer records the ratios IVV/IVH and IHV/IHH, then calculates either polarization or anisotropy, depending on the operators the antibody, as shown by the dashed curve in Fig-choice of ACQUISITION MODE.

The concentration of theophylline in serum or plasma is monitored by fluorescence polarization immunoassay. Theophylline (Figure 3) is a drug administered for myocardial stimulation, increased coronary blood flow, and smooth-muscle relaxation. It can be a bronchodilator in the control of asthma, for example. The observed fluorescence polarization of a fluorescein-labeled theophylline tracer is inversely related to the drug concentration. The drug level in a given sample therefore can be determined by comparison with a calibration curve.

A pre-treatment solution, the theophylline standard in a pH-7.2 buffer, the theophylline-fluorescein tracer, and antiserum were mixed together and incubated for three minutes before determining the fluorescence polarization. The theophylline samples were from General Diagnostics; Abbot Laboratories supplied the standards and other reagents.

Polarization measurements were performed on a SPEX FLUOROLOG spectrofluorometer consisting of three single-grating spectrometers, the T-format Sampling Module, a T-format Polarization Kit, the Autopolarization Assembly, and two Hamamatsu R928P detectors. A 450-W xenon lamp supplied the excitation.

Results and Discussion
Figure 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, quinidine, salicylate, and acetaminophen. In this experiment, however, the antibodys specificity for a particular drug, combined with polarization measurements, eliminates interference from other fluorophores as well as unbound tags.

This example demonstrates that fluorescence immunoassay has, like radioimmunoassay, the specificity and sensitivity to perform at biologically relevant concentrations. The advantage of FIA, however, is that no separation step is required.

1 Visor, G.C.; Schulman, S.G. J. Pharm. Sci. 1981, 70, 469.
2 Smith, D.S.; Hassan, M.; Nargessi, R.D. In Modern Fluorescence Spectroscopy; Wehry, E.L., Ed.; vol. 3, 93, Plenum Press: New York, 1981.

back to top


Page: All 1 2 3 4 5 6

Related biology technology :

1. Enzyme Immunoassay for Studying Intracellular Levels of cAMP
2. Bio-Plex Cytokine Immunoassays and ELISA: Comparison of Two Methodologies in Testing Samples From Asthmatic and Healthy Children, Rev A
3. Principles of Curve Fitting for Multiplex Sandwich Immunoassays, Rev B
4. Mix-and-Read, Bead- and Cell-Based Immunoassays Using the FMAT 8100 HTS System
5. Coated Plate Immunoassay on the MicroBeta - eg. TSH - IRMA
6. Monitoring fluorescence resonance energy transfer (FRET) between GFP fusions in lysates of the yeast Saccharomyces cerevisiae using the Varian Cary Eclipse
Post Your Comments:
TAG: Immunoassay Theophylline fluorescence polarisation

(Date:10/7/2015)... --  VWR (NASDAQ: VWR ), a ... and solutions, announced today that it has acquired ... that performs high volume purification of selected high ... laboratory and production chemical manufacturing portfolio to better ... Manuel Brocke-Benz , President and CEO of ...
(Date:10/7/2015)... ... October 07, 2015 , ... Optivia ... to develop a dynamic, multi-scale, physiologically based pharmacokinetic (dmPBPK) modeling platform for predicting ... combine their respective expertise in in vitro transporter assay technology and modeling methods ...
(Date:10/6/2015)... Texas , Oct. 6, 2015  The ... announce that natural ingredients manufacturer Teawolf has adopted ... ABC,s Adopt-an-Herb Program . HerbMedPro ™ ... and clinical research on guayusa, a caffeine-containing member ... and comprehensive database available on ABC,s website that ...
(Date:10/6/2015)... , Oct. 6, 2015 Actinium Pharmaceuticals, Inc. ... yesterday,s session. Trading volume was light at 217,180 shares. ... $1.74 per share on September 29th. ... show investors of Actinium Pharmaceuticals, Inc. information about the ... our report at the link below. ...
Breaking Biology Technology:VWR Acquires Purification Technologies 2VWR Acquires Purification Technologies 3VWR Acquires Purification Technologies 4VWR Acquires Purification Technologies 5Optivia Biotechnology and The Sugiyama Laboratory/RIKEN Sign Collaborative Agreement 2Optivia Biotechnology and The Sugiyama Laboratory/RIKEN Sign Collaborative Agreement 3Teawolf "Adopts" Guayusa under Agreement with American Botanical Council 2Teawolf "Adopts" Guayusa under Agreement with American Botanical Council 3Teawolf "Adopts" Guayusa under Agreement with American Botanical Council 4Teawolf "Adopts" Guayusa under Agreement with American Botanical Council 5Teawolf "Adopts" Guayusa under Agreement with American Botanical Council 6Teawolf "Adopts" Guayusa under Agreement with American Botanical Council 7Teawolf "Adopts" Guayusa under Agreement with American Botanical Council 8Teawolf "Adopts" Guayusa under Agreement with American Botanical Council 9Teawolf "Adopts" Guayusa under Agreement with American Botanical Council 10Teawolf "Adopts" Guayusa under Agreement with American Botanical Council 11Teawolf "Adopts" Guayusa under Agreement with American Botanical Council 12Small Cap Street - Actinium Pharmaceuticals Due Diligence Report 2Small Cap Street - Actinium Pharmaceuticals Due Diligence Report 3Small Cap Street - Actinium Pharmaceuticals Due Diligence Report 4Small Cap Street - Actinium Pharmaceuticals Due Diligence Report 5Small Cap Street - Actinium Pharmaceuticals Due Diligence Report 6
... Pharmaceutical Companies Select Drug Model ... VIEW, Calif., April 2, 2008 ,Pharsight Corporation (Nasdaq: ... consulting, and regulatory services for optimizing clinical drug,development, ... have become customers for its Drug Model Explorer ...
... are focusing on the development of novel biomarkers ... for cancer, reports Genetic Engineering and Biotechnology News ... much stronger and targeted emphasis on diagnosis will ... April 1 issue of GEN ( ). ...
... 2008 The biotech industry came,through a very turbulent ... a price-weighted index tracking 20 of,biotech,s "blue chip" companies, ... and the Nasdaq, whose value dropped 14%., "Despite ... the Burrill,Biotech Select Index recorded a credible gain of ...
Cached Biology Technology:Pharsight Signs Two New DMX(R) License Customers 2Pharsight Signs Two New DMX(R) License Customers 3Biotech Avoids Fall Out From Tough Quarter for Capital Markets 2Biotech Avoids Fall Out From Tough Quarter for Capital Markets 3Biotech Avoids Fall Out From Tough Quarter for Capital Markets 4
(Date:9/9/2015)... British Columbia , Sept. 9, 2015 /PRNewswire/ ... numerous organizational and solution-based milestones, furthering the company,s ... perils of online fraud. NuData Security,s ... in enhancing the company,s growth cycle. The product ... learning to determine good user behavior from fraudulent ...
(Date:9/2/2015)... YORK , Sept. 2, 2015 About ... concern for enterprises, banks, government organizations, telecommunication and hospitals ... assets. Keys, identity badges, and numeric keypads are used ... the structure of palm and fingers of an individual, ... Hand geometry scanners with infrared light and reflectors can ...
(Date:9/1/2015)... , Sept. 1, 2015  Automation and ... during airport passenger screening. This is the first ... screening process will become unintrusive. An assortment of ... on voluntary background investigations will transform the passenger ... & Sullivan, Global Airport Passenger Screening Market ...
Breaking Biology News(10 mins):NuData Security Achieves Significant Milestones, Furthers Commitment to Providing Unparalleled Online Fraud Detection 2Global Hand Geometry Biometrics Market 2015-2019 2Global Hand Geometry Biometrics Market 2015-2019 3The Future of Airport Security: Passenger Screening Transitioning Into an Invisible Process 2The Future of Airport Security: Passenger Screening Transitioning Into an Invisible Process 3The Future of Airport Security: Passenger Screening Transitioning Into an Invisible Process 4
... WINSTON-SALEM, N.C., July 18, 2013 -- Scientists at Wake Forest ... the cumulative effect of impacts to the head incurred by ... (RWE), can capture players, exposure to the risk of concussion ... frequency and magnitude of all impacts, said senior author of ...
... now open for the Mushrooms and Health Summit held at ... 2013. The meeting is of interest to scientists, writers and ... examination of the global research that links mushrooms to today,s ... event , Learn from the world,s top scientists, researchers ...
... have identified biochemical changes in people taking antidepressants ... occur in a neurotransmitter pathway that is connected to ... that controls the sleep cycle, suggesting an added link ... published on July 17, 2013, in the journal ...
Cached Biology News:Scientists develop new way to measure cumulative effect of head hits in football 2Scientists develop new way to measure cumulative effect of head hits in football 3Biochemical mapping helps explain who will respond to antidepressants 2Biochemical mapping helps explain who will respond to antidepressants 3
Total Proteins from over 50 different human Alzheimer, Diabetic, Liver Cirrhosis, and Lupus diseased tissues are available....
UGT1A6 (D-20)...
... pump features T Squareds patented high efficiency ... and efficiency. With the most compact, lightweight ... ideal for use in portable air and ... life, and high efficiency. , Includes DC ...
Biology Products: