Fluorescence Polarization Immunoassays

The versatility of luminescence goes beyond intensity-, wavelength- and kinetic-based measurements. Fluorescence polarization (or anisotropy) is an additional parameter still largely unexplored for optical sensing yet widely used in Biochemistry to study the interaction of proteins, the microfluidity of cell membranes and in fluorescence immunoassays. Although only a few optosensors based on luminescence polarization measurements can be found in the literature, elegant devices have recently been reported to measure chemical parameters such as pFI or O2 even with the bare eye41. 

In addition to fluorescence intensity and polarization, fluorescence spectroscopy also includes measurement of the lifetime of the excited state. Recent improvements in the design of fluorescence instrumentation for measuring fluorescence lifetime have permitted additional applications of fluorescence techniques to immunoassays. Fluorescence lifetime measurement can be performed by either phase-resolved or time-resolved fluorescence spectroscopy. 

Methods for the Detection of Antigens/Antibodies Equilibrium and kinetic inhibition assays based upon fluorescence polarization, 70, 3 fluorescence excitation transfer immunoassay (FETI), 70, 28 indirect quenching fluoroimmunoassay, 70, 60 the homogeneous substrate-labeled fluorescent immunoassay, 70, 79 fluorescence immunoassays using plane surface solid phases (FIAPS), 70, 87. 

Two rather interesting variations on fluorescence immunoassay that do not require the fluorescence spectra of free and bound labeled materials to have different spectral positions have fairly recently become rather popular in clinical analysis. These are fluorescence polarization immunoassay and time-resolved fluorescence immunoassay. 

Enzyme activities may also be measured in urine, cerebrospinal fluid, bone marrow cells or fluid, amniotic cells or fluid, red blood cells, leukocytes, and tissue cells. Cytochemical localization is possible in leukocytes and biopsy specimens (e.g., from liver and muscle). Under ideal conditions, both the concentration of the enzyme and its activity would be measured. Radioimmunoassay (RIA) and its alternative modes such as fluorescence immunoassay (FIA), fluorescence polarization immunoassay (FPIA), and chemiluminescence immunoassay (CLIA) (discussed later), can be used to measure enzyme concentration as well as other clinically important parameters. 

Immunoassay or methods based on fluorescence measurements (fluorescence polarization, fluorescence intensity)... 

B Rollman, et al. Dibekacin assay in serum by automated fluorescence polarization immunoassay (Abbot TDX)—Comparison with high performance liquid chromatography, substrate labeled fluorescent immunoassay and radioimmunoassay. J Pharm Biomed Anal 4 53, 1986. 

Heterogenous fluorescence immunoassays can be carried out with the aid of the same separation procedures used in radioimmunoassay. The more expedient homogenous fluorescence immunoassays require quenching, enhancement, polarization, or shifting of the fluorescence of the label upon binding of the labeled analyte to its antibody. Occasionally, a second antibody, directed at the anti analyte antibody, will be used in a double-antibody method to precipitate the bound labeled and unlabeled analyte or to alter the optical properties of the label in such a way as to make the analysis more sensitive. 

In addition to wavelength and time-resolved fluorescence techniques, polarization fluorescence can yield important information about an analyte." This is especially true when differentiating between chiral compounds. Combining, for example, a fluorescently tagged antibody immunoassay with polarization detection allows for very sensitive detection limits of chiral enantiomers. Laser-induced fluorescence polarization (LIFP) has been used to detect concentrations as low as 0.9 nM of an antibody-boimd cyclosporine A (an immunosuppressive drug) in human blood. A conventional single channel fluorescence detector can be easily modified to perform such measurements, simply by adding the appropriate polarization filters. 

Immunoanalytical methods occupy an important position in bioanalytical chemistry. Chemical derivatization plays a fundamental role in this technique. Small analyte molecules (haptenes) are covalently bound to proteins to raise the antibody, which is the basis for their highly selective and sensitive assay. Various labeled derivatives of the analyte are then prepared for competitive binding on the antibody (radiolabeling, labeling for enzyme immunoassay, fluorescence immunoassay, fluorescence polarization immunoassay, and luminescence immunoassay). These derivatization reactions are carried out by the... 

Depending on the manner of labeling, various kinds of immunoassays are used in the analysis of steroid hormones RIA, enzyme immunoassay, fluorescence immunoassay, fluorescence polarization immunoassay, and luminescence 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... 

---