Fluorescence detection immunoassays

Although vitamins can be determined, both qualitatively and quantitatively, by MS, routine analysis is usually best conducted by other means (e.g., HPLC with ultraviolet (UV) or fluorescence detection, immunoassay methods, or microbiological methods). Analytically, MS does have an important role as a reference technique, especially when used in isotope... 

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.

Hybrid probe—immunoassays are expected to find a specific niche in clinical analysis, especially as a means to adapt probe assays to existing immunoanaly2ers which are locked into a specific enzyme or fluorescence detection technology. Commercialization of the first of these assays is expected by the year 2000. 

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). 

X. Zhao and S.A. Shippy, Competitive immunoassay for microliter protein samples with magnetic beads and near-infrared fluorescence detection. Anal. Chem. 76, 1871-1976 (2004). 

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%. 

An interesting practical application is the detection of the citrate anion in soft drinks, as shown in Box 10.3. The strategy is quite different from that of the preceding examples because the anion receptor is not linked to a fluorophore. The latter simply acts in competition with the citrate anion in a fashion that resembles fluorescence-based immunoassays. 

In addition to these research applications of fluorescence, there is a continuing use of fluorescence detection to replace analytical methods based on radioactivity, as can be judged from the recent books and conferences on fluorescence sensing methods. (7 n) These emerging applications of fluorescence can be seen by the growth and introduction of improved methods for immunoassays, enzyme-linked immunoassays... 

The detection of flu viruses via a fluorescent sandwich immunoassay was reported by Bucher.(10) However, the method sensitivity was too low for direct detection of the virus. A novel sandwich immunoassay was described by Ogcr((lff7 for the detection of Botulinum Toxin A. Antibodies specific for Clostridium botulinum were covalently attached to the surface of a tapered fiber. After the capture of the antigen, a sandwich was formed with a rhodamine-labeled anti-toxin IgG, and the evanescent wave was measured. The assay was highly specific with detection limits near 5 ppb. 

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]. 

NM Schultz, RT Kennedy. Rapid immunoassays using capillary electrophoresis with fluorescence detection. Anal Chem 65 3161-3165, 1993. 

L Steinmann, J Caslavska, W Thormann. Feasibility study of a drug immunoassay based on micellar electrokinetic capillary chromatography with laser-induced fluorescence detection—determination of theophyllin in serum. Electrophoresis 16 1912-1916, 1995. 

Q Wang, G Luon, J Ou, WSB Yeung. Noncompetitive immunoassays using protein G affinity capillary chromatography and capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr A 848 139-148, 1999. 

Czajka, J. and Batt, C.A. (1996) A solid phase fluorescent capillary immunoassay for the detection of Escherichia coli 0157 H7 in ground beef and apple cider. Journal of Applied Bacteriology 81(6), 601-7. 

Like in RILAs, an advantage of fluorescence detection is the possibility of developing homogeneous FILAs using direct or indirect (competitive or displacement) approaches. The fluorescence polarization immunoassay (PFIA) and their homolog fluorescence polarization immuno-like assay (PFILA) are two of the most widely used procedures in homogeneous fluoroassays. Both are based on the principle that fluorescence polarization gives a direct measure of the bound/free ratio of the labeled analyte (tracer) without the need for their separation [23, 28]. 

Mohamadi et al. [96] reported online preconcentration of human serum albumin (HSA) and its immunocomplex with a monoclonal antibody on-chip coupled to isotachophoresis. The sample injection, preconcentration, and separation were carried out continuously and controlled by a sequential voltage switching program. Preconcentration was carried out with on-chip nondenaturing gel electrophoresis in methylcellulose solution. Furthermore, the authors applied this method for immunoassay of HSA. The separation of HSA and its immunocomplex was achieved in 25 seconds in 1 cm of the microchannel with induced fluorescence detection at 7.5 pM. 

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