Fluorescence and Luminescence Immunoassay

Fluorescent or luminescent substances are known to be excellent markers for antigen-antibody reactions. The fluorescent and luminescent substances with low molecular weight can be coupled easily to antigen or antibody 

Enzymatic Hydrolysis of Antigen-Fluorescent Dye Conjugate Immunoassay 

In this assay, marker-labeled antigen itself acts as substrate. Kohen et al. (K9) developed a homogeneous immunoassay for steroid using a steroid-fluorescent dye conjugate that yields fluorescent products upon hydrolysis with enzyme. The steroid-fluorescent dye conjugate is inactive as a substrate when bound to the antibody to steroid [F-Ag Ab]. But when unlabeled steroid [Ag] is added, it binds competitively to the antibody [Ab Ag],. and the free form of steroid-fluorescent dye conjugate [F-Ag], which is 

Antibody-Enhanced Hydrolysis of Steroid-Fluorescent Dye Conjugate Immunoassay 

Antigen --Q Antibody—Excitation---wav Emission- ftWl Brownian movement —J ) 

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ILA23 A Radioimmunoassays and Enzyme, Fluorescence, and Luminescence Immunoassays... 

CLSI. 2004b. ILA23-A. Radioimmunoassay and enzyme, fluorescence and luminescence immunoassays. Approved guideline. 

LA21-A2. 2008. Clinical evaluation of immunoassays, 2nd ed. Approved guidehne. 1LA-23A. 2004. Immunoassay systems Radioimmunoassays and enzyme, fluorescence and luminescence immunoassays, 1st ed. Approved guideline. 

Other immunoassays are based on the same antibody-antigen binding reaction but use a different labeling system for detection. Instead of an enzyme label, there are radioactive isotopes, and fluorescent and luminescent labels. Some important immunoassays are defined below ... 

Due to the high specificity and sensitivity of immunoassays, there are bioanalytical methods for the measurement of an analyte of interest, with little or without preconcentration or purification of the samples. The principle behind immunoassays is based on an interaction between an antibody and a corresponding antigen, and the detection of the specific interaction using radiolabels (247), enzyme, fluorescent and luminescent compounds (178, 179,181,183), electroactive markers (177,180, 228, 248), or nanomaterials (249-251). 

Enzyme immunoassays that require physical separation of free and bound T4 are generally based on principles analogous to those of conventional RIAs except that enzyme activity rather than radioactivity is measured. Most enzyme immunoassays use labeled T4 as antigen. An assortment of photometric, fluorescent, and luminescent substrates have been used to monitor the enzyme activity of the antibody-bound fraction. 

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. 

Other variants of immunoassay methods used in the analysis of steroid hormones are the fluorescence polarization immunoassay, where the label is fluorescein, coupled to the steroid, and luminescence immunoassay, where the steroid is bound to amino-alkylethylluminol. The basis of the luminescence assay is the luminescence generated by the action of hydrogen peroxide/peroxidase, which is measured by a luminometer (LIA, CELIA). 

The first quantitative immunoassays were based on the use of radioactive reporter molecules, mainly and " C. These radioimmunoassays (RIAs) have gradually been replaced by nomadioactive reporter molecules or labels, e.g., enzymes, fluorescent, and luminescent molecules and combinations of these. The nonradio-active labels facilitated development of automated immunoassays, and they also contributed to the design of assays with substantially reduced detection limits. However, nomadioactive assay have an advantage only in immunometric sandwich assays in inhibition assays, the use on nonradioactive labels has not provided any improvement in assay sensitivity as compared to RIA [1]. The development in immunoassay technology has been reviewed by Ekins [2]. 

Aslan K, Holley P, Geddes CD (2006) Microwave-accelerated metal-enhanced fluorescence (MAMEF) with silver colloids in 96-well plates Application to ultra fast and sensitive immunoassays, high throughput screening and dmg discovery. J Immunol Methods 312 137-147 Matveeva E, Gryczynski Z, Malicka J et al (2004) Metal-enhanced fluorescence immunoassays using total internal reflection and silver-coated surfaces. Anal Biochem 334 303-311 Blue R, Kent N, Polerecky L (2005) Platform for enhanced detection efficiency in luminescent-based sensors. Electron Lett 41 682-684... 

There is increasing interest in the use of nonisotopic labels in immunoassay. The main advantages of such labels in labeled antibody techniques are the expected longer shelf life of the reagent and the ability to use a detection apparatus that may for one reason or another prove to be more convenient—e.g., for automation or cheapness. Some alternatives that are being explored are bacteriophages, spin labels, enzymes, fluorescent compounds, and luminescent compounds. ... 

Most immunoassays currently employed in the biomedical field are either radioimmunoassays, enzyme immunoassays, or luminescence immunoassays (including fluorescence immunoassays [FIA] and chemiluminescence immunoassays). Although radioimmunoassay is currently the most sensitive of these (10 -10 M concentrations are often detectable), due to the problems inherent to dealing with radioactive materials, such as licensing, radiation hazard, short shelf-life of expensive radioisotopes, the expense of the counting equipment, and the tedium associated with heterogeneous immunoassay, it has fallen, in popularity, behind the non-isotopic methods of analysis. 

See also Chemiluminescence Overview. Chiroptical Analysis. Derivatizatlon of Analytes. Enzymes Enzyme-Based Assays. Fluorescence Overview Ciinicai and Drug Appiications. Gas Chromatography Coiumn Technoiogy Mass Spectrometry. Immunoassays Overview. Immunoassays, Applications Clinical. Immunoassays, Techniques Enzyme Immunoassays Luminescence Immunoassays. Infrared Spectroscopy Overview. Liquid Chromatography Column Technology Normal... 

See also. Chemiluminescence Oven/iew. Derivatiza-tion of Analytes. Electrophoresis Oven/iew. Enzymes Oven/iew Immobiiized Enzymes Enzyme-Based Eiec-trodes Enzymes in Physioiogicai Sampies Industriai Products and Processes Enzyme-Based Assays. Fluorescence Clinical and Drug Applications. Immunoassays Overview Production of Antibodies. Immunoassays, Applications Clinical Food Forensic. Immunoassays, Techniques Radioimmunoassays Enzyme Immunoassays Luminescence Immunoassays. Mass Spectrometry Polymerase Chain Reaction Products. Nucleic Acids Chromatographic and Electrophoretic Methods Electrochemical Methods. Polymerase Chain Reaction. 

In quantitative immunoassays, e.g., enzyme-linked immunosorbent assay and radioimmunoassay, a known amount of labeled vitamin is mixed with sample extract in which the vitamin content should be determined. Methods of labeling include radioisotopes (e.g., cobalamine), fluorescence, or luminescence markers (e.g., folate). The mixture is subjected to binding agent, equally forming complexes with both labeled and unlabeled vitamin. This complex is then isolated, and the amount of labeled vitamin present is measured. Sample vitamin concentration can be deduced from the ratio of labeled vitamin added to labeled vitamin measured after isolation. Advantages of immunoassays are short analysis time, and the possibility of automating them on clinical analyzers. 

Historically, the immunoassay variant—the enzyme-linked immunosorbent assay (ELISA)—is a format where the enzyme label is followed optically as a change of absorbance, fluorescence or luminescence. The electrochemical immunoassays employ an electrode to measure the electroactive product released from a biocatalytic reaction of the label enzyme in this case, the immunoassay and electrode reaction occur at different surfaces. These concepts finally inspired various types of immunosensors where the immunorecognition event proceeds directly at the electrode surface. Thus, the electrochemical immunosensor is obtained when the immunorecognition element (antibody, antigen, hapten) becomes immobilised on the surface of the electrode as a transducer. The assays can be realised in the following formats ... 

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