Fluorescent antibody techniques

An even more indirect method which does not require the use of a fluorescence microscope is to use enzyme linked antibodies, e.g. the peroxidase anti-peroxidase (PAP) method of Stemberger et al. (1970). Here, after the antigen has been reacted with a rabbit antibody preparation this is conjugated to sheep anti-rabbit IgG which in turn is conjugated to a complex of rabbit anti-horse-radish peroxidase and horseradish peroxidase. This then reacts with diaminobenzidine and hydrogen peroxide when a brown colour indicates the presence of the antigen. 

If the second antibody is complexed with biotin it will react with streptavidin. The steptavidin may be labelled with a fluorescent dye or an enzyme such as horseradish peroxidase or alkaline phosphatase which can be detected in situ. Kits to perform such assays are available from Amersham or Bio-Rad Laboratories (Appendix 3). 

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Kawamura Jr., A. (ed.) (1977) Fluorescent Antibody Techniques and Their Application. University of Tokyo Press, Baltimore, Maryland. 

Moller, G. (1961). Demonstration of mouse isoantigens at the cellular level by the fluorescent antibody technique. J. Exp. Med. 114,415-434. 

Forerunners of nonisotopic immunoassay had already appeared before radioimmunoassay was developed. For example, nephelometry is based on precipitation, which is known as the classical immune reaction, and the ideas of particle immunoassay and viroimmunoassay seem to have developed from the hemagglutination test. The principles of enzyme and fluorescence immunoassay had already been used as enzyme and fluorescence antibody techniques in histochemical analysis. In 1971, two groups reported use of an enzyme immunoassay (E5, V2). Leute et al. reported spin immunoassay, which has spurred recent development of nonisotopic immunoassays (L5). 

The compounds of the second group will be of primary concern in the following lines. The most important advantage of the application of these fluorescent dyes (fluorophores) in comparison with the ordinary ones (chromophores) is a considerable increase in the nsitivity of protein detection. The introduction of fluorescent dyes in order to increase the sensitivity of the dye techniques is ascribed to Creech and Jones who treated proteins with benzantryl isocyanate to form the corresponding carbamido conju tes. Approximately at the same time, Coons et al. started to apply the fluorescence antibody technique. Since that time this technique is generally... 

Cellular component or artifact-based technologies GL chromatographic fatty acid profiles MALDl-TOF mass spectrometry Fluorescence antibody techniques Enzyme-linked immunosorbent assay Limulus ameboc5de lysate-endotoxin assay... 

The viral infection elicits an immunological reaction, but in spite of measurable circulating antibodies, infection persists and virus particles can be detected by the fluorescent antibody technique. 

As a result of this active protein synthesis, the total protein mass is increased, and new proteins appear. The development of new proteins has been established by immunological methods and by determination of enzyme activity. During the development of the chick embryo lens, seven antigenic proteins appear. The antigen reactive groups of myosin appear in the heartforming area of the chick embryo. Fluorescent antibody techniques have demonstrated that myosin is diffusely distributed in the early embryo it is later restricted to the heart and muscle-developing areas [16]. 

The infusion of calcitonin to rats induces increased calcium deposition in the bone and hypocalcemia. Thus, the hormone has properties antagonistic to those of parathormone. Fluorescent antibody techniques have demonstrated that the hormone is secreted by the C cells of the thyroid gland. A light cell hyperplasia outside the follicular walls of the thyroid has been observed in mice with hereditary hypocalcemia. 

Probably the most important applications of fluorescence microscopy are to the study of living cells and tissues, to protein tracing by the Coons fluorescent antibody technique and to the study of nucleic acids by in situ hybridization. 

EIA was originally developed as a histological technique to localize specific ceUular sites using the specificity of an immunological reaction (23). The resulting fluorescent antibodies can be detected in animal tissues at levels as low as 1 /tg/mL of body fluid. Eluorophore-labeled antibodies have also been used widely for flow cytometry appHcations using fluorescein antibodies to cell surface markers to detect and quantify specific cells (24). 

Fluorescent labels, by contrast, can provide tremendous sensitivity due to their property of discrete emission of light upon excitation. Proteins, nucleic acids, and other molecules can be labeled with fluorescent probes to provide highly receptive reagents for numerous in vitro assay procedures. For instance, fluorescently tagged antibodies can be used to probe cells and tissues for the presence of particular antigens, and then detected through the use of fluorescence microscopy techniques. Since each probe has its own fluorescence emission character, more... 

Buchwalow IB, Minin EA, Bocker W (2005) A multicolor fluorescence immunostaining technique for simultaneous antigen targeting. Acta Histochemica 107 143 148 Harlow E, Lane D (1999) Using Antibodies A Laboratory Manual. Cold Spring Harbor Labora... 

The fluorescence polarization technique is a very powerful tool for studying the fluidity and orientational order of organized assemblies (see Chapter 8) aqueous micelles, reverse micelles and microemulsions, lipid bilayers, synthetic non-ionic vesicles, liquid crystals. This technique is also very useful for probing the segmental mobility of polymers and antibody molecules. Information on the orientation of chains in solid polymers can also be obtained. 

Wessels, B. W., and Rogus, R. D. (1984) Radionuclide selection and model absorbed dose calculations for radiolabeled tumor associated antibodies. Med. Phys. 11, 638—645. Wessendorf, M. W. (1990) Characterization and use of multi-color fluorescence microscopic techniques. Handbook of Chemical Neuroanatomy, Vol. 8, Chapter 1. 

Antibodies coated onto MTP wells capture kinase or phosphatase substrate phosphorylation state is detected by anti-phosphopeptide antibody coupled to detector dye can be read by time-resolved fluorescence (DELFIA) technique... 

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