Fluorescent polarization immunoassay

Fig. 7. Fluorescence polarization (FP). (a) The formation of the large FITC—protein A—IgG complex which leads to a net increase in plane polarized light transmitted from the solution. Molecular weights of the protein A-FITC, IgG, and complex are ca 43,000, 150,000, and 343,000, respectively, (b) Detection of IgG by fluorescence polarization immunoassay using A, a laboratory fluorimeter where (O) represents AP = change in polarization, and B, a portable detection unit where (D) is —fiV = change in voltage (27). The field detector proved to be more sensitive than the fluorimeter.

Guo XQ, Castellano FN, Li L, Lakowicz JR (1998) Use of a long lifetime Re(I) complex in fluorescence polarization immunoassays of high-molecular weight analytes. Anal Chem 70 632-637... 

This is used for synthesis of porphobilinogen (Eq. 10.24).25 Porphobilinogen is the key building block in the biosynthesis of pigments of life such as porphyrins, heme, and vitamin B12. Interesting application of porphobiliogen to synthesis of immunocomponents for the measurement of lead (Pb) by fluorescence polarization immunoassay has been reported.26... 

A competitive fluorescence-polarization immunoassay method was described for the monitoring of 12 drugs including valproic acid [18]. Samples (serum or plasma) were deproteinated. Fluorescence from the fluorescein-labeled analyte used as tracer was excited at 488 nm and polarization of light emitted at 531 nm was measured. The calibration was stable for 4 weeks and the coefficient of variation was below 10%. A single measurement took 8-10 min. 

Five anticonvulsants including valproic acid were determined by the Abbott TD x fluorescence polarization immunoassay automatic analyzer. Recoveries were 94.8-106% and the coefficients of variations were 1.0-9.7% [23], Fluorescence polarization immunoassay and enzyme immunoassay were compared for the determination of free valproic acid in serum [24], Good correlation (R = 0.9992) was obtained between the two assays. Higgins [25] reported on the determination of valproic acid in serum by enzyme immunoassay with use of EMIT reagents and the Abbot ABA-200 analyzer. Responses were rectilinear up to 150 mg/L. 

A.Y. Kolosova, J.H. Park, S.A. Eremin, S.J. Kang and D.H. Chung, Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of the organophosphorus pesticide parathion-methyl. J. Agric. Food Chem. 51, 1107-1114 (2003). 

Rao et al.20 demonstrated a fluorescence polarization immunoassay for evaluating serum concentrations of tricyclic antidepressants (amitriptyline, imipramine, clomipramine, and doxepin) with respect to nonresponse, compliance, therapeutic window, and influences of age, sex, substance abuse, and toxicity. Abbott Laboratories TDx/TDxFLx Toxicology Tricyclic Assay FPIA (fluorescence polarization immunoassay) was used. This assay of 50 /uL samples contained tricyclic antidepressant antibodies raised in rabbits and fluorescein-labeled tricyclic antidepressant as a tracer. The assay was calibrated with imipramine in the range of 75 to 1000 fig/L (268 to 3571 nmol/L). Intra-assay and inter-assay coefficients of variation for internal quality control samples from the manufacturer were 4.2 and 4.7%, respectively. The limits of detection were 72,71,64, and 72 nmol/L for amitriptyline, imipramine, clomipramine, and doxepin, respectively. This high-throughput immunoassay was easy to use although amitriptyline, dosulepine, desipramine, and nortriptyline showed cross-reactivities ranging from 74 to 100%. 

Rao, M.L. et al. 1994. Monitoring tricyclic antidepressant concentrations in serum by fluorescence polarization immunoassay compared with gas chromatography and HPLC. Clin Chem. 40 929. 

Fluorescence microscopy, 16 483 Fluorescence polarization (FP), 14 149-150 Fluorescence polarization immunoassay (FPIA), 12 97... 

Figure 14.5. Representation of fluorescence polarization immunoassay measurements, (a) Unbound tracer (b) bound tracer.

W. B. Dandliker, R. J. Kelly, J. Dandliker, J. Farquhar, and J. Levin, Fluorescence polarization immunoassay. Theory and experimental method, Immunochemistry 10, 219-227 (1973). 

P. Urios and N. Cittanova, Adaptation of fluorescence polarization immunoassay to the assay of macromolecules, Anal. Biochem. 185, 308-312(1990). 

S. H. Grossman. Fluorescence polarization immunoassay applied to macromolecules Creatine kinase-BB, /. Clin. Immunoassay 7, 96-100(1984). 

M. Tsuruoka, E. Tamiya, and I. Karube, Fluorescence polarization immunoassay employing immobilized antibody, Biosensors and Bioelectronics 6, 501-505 (1991). 

Figure 6.1. Fluorescence polarization immunoassay for theophylline. (A) Effect of theophylline rabbit antiserum ( ) and normal rabbit serum (A) on the fluorescence polarization of the theophylline-umbelliferone conjugate. (B) Fluorescence polarization of the theophylline-umbelliferone conjugate in the presence of varying concentrations of theophylline. (Reprinted from Ref. 1, with permission from Academic Press.)...

Jolley, M.E., Fluorescence polarization immunoassay for the determination of therapeutic drug levels in human plasma, ]. Anal. Toxicol., 5, 236,1981. 

MEGX is readily detected by HPLC and fluorescence polarization immunoassay techniques [14,21,25,40,41]. The test is simple, normally requiring a onetime blood sampling, and informative because it depends on the capacity of the hepatic enzymes to metabolize lidocaine. While the analysis of lidocaine metabolites is rapid, this method has not been adapted for continuous hepatic function monitoring, which may be possible with the radiolabeled analogues such as Tc-Sn-lidocaine iminodiacetic acid [42]. 

Figure 2.3 Relationship between the concentration of teicoplanin in the serum of patients as determined using high-performance liquid chromatography (HPLC) versus fluorescence polarization immunoassay (FPIA) (r = 0.974). (From McCann et al., 2002.)...

McCann S.J., O. White, and B. Keevil (2002). Assay of teicoplanin in serum Comparison of high-performance liquid chromatography and fluorescence polarization immunoassay. Journal of Antimicrobial Chemotherapy 50 107-110. 

The fluorescence polarization immunoassay is used for routine, automated immunoassay of small molecules, such as drugs. It depends on the principle that a fluorophore attached to a macromolecule such as an antibody is not free to rotate in solution. If polarized light is used to stimulate the fluorophore to fluoresce, emission from the bound fluorophore (attached to the antibody, which is bound to a surface) will continue to be polarized, but polarization will be lost from free fluorophore (111). 

Because of the requirement to use radioactive substances, RIAs are frequently being replaced by other immunologic assays, such as ELISA and fluorescence polarization immunoassays (FPIA) (Niemann et al. 1985). These have similar degrees of sensitivity. FPIAs are highly quantitative, as are RIAs, and ELISAs can be designed to be quantitative. 

Niemann, A., Oellerich, M., Schumann, G., Sybrecht, G.W. (1985). Determination of theophylline in saliva, using fluorescence polarization immunoassay (FPIA). J. Clin. Chem. Clin. Biochem., 23(11), 725-732. 

Benzodiazepines are an important group of drugs with tranquilizing properties. Available immunochemical methods include radioimmunoassays (164, 165), a radioreceptor assay (166), and nonseparation immunoassays such as the widely used enzyme-monitored immunotest (EMIT) and fluorescent polarization immunoassays (167, 168). Such assays generally require sophisticated apparatus and dedicated laboratories. However, a relatively simple enzyme-linked immunosorbent assay was recently described for screening benzodiazepines in urine (169). 

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