Fluorescent antibodies

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). 

Fujiwara, K., Pollard, T.D. (1976). Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow and mitotic spindle of human cells. J. Cell Biol. 71, 848-875. 

Mikhailov, I. F. and Stanislavskii, E. S. (1963). Staining isolated bacterial structures with fluorescent antibodies. Science 40, 74-9. 

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

CDC Case Definition Laboratory criteria for diagnosis is (1) a positive direct fluorescent antibody test (preferably performed on central nervous system tissue) or (2) isolation of rabies virus (in cell culture or in a laboratory animal). 

Rapid antigen and point-of-care tests, direct fluorescence antibody test, and the reverse-transcription polymerase chain reaction assay may be used for rapid detection of virus. 

Laboratory findings include leukocytosis with predominance of mature and immature granulocytes in 50% to 75% of patients. Because L. pneumophila stains poorly with commonly used stains, routine microscopic examination of sputum is of little diagnostic value. Fluorescent antibody testing can be performed to diagnose Legionnaires disease. 

Because T. pallidum is difficult to culture in vitro, diagnosis is based primarily on dark-held or direct fluorescent antibody microscopic examination of serous material from a suspected syphilitic lesion or on results from serologic testing. 

Tests that allow rapid identification of chlamydial antigens in genital secretions are the direct fluorescent antibody test, the enzyme immunoassay (requires just 30 minutes for results), the DNA hybridization probe and nucleic acid amplification tests. 

Quenching can actually be used to create analytical methods. An example is that of fluorescent antibody assay. Here, a fluor is bound to an antibody but the binding constant is less than that for the antibody-antigen pair for which the antibody was generated. Binding quenches the fluorescence of the fluor. When a solution containing the native antigen is mixed with the fluor complex, the fluor is released and can fluoresce normally. There are numerous examples of this approach. 

Albert H. Coons (Fig. 1.2) was the first who attached a fluorescent dye (fluorescein isocyanate) to an antibody and used this antibody to localize its respective antigen in a tissue section. The concept of putting a visible label on an antibody molecule appeared both bold and original. His initial results were described in two brief papers in the early 1940s (Coons et al. 1941,1942), but the research was halted while he joined the army and spent the next 4 years in the South Pacific. His later studies (Coons and Kaplan 1950) contributed immensely to the use of the fluorescent antibody method in a wide variety of experimental settings. In our time, the use of antibodies to detect and localize individual or multiple antigens in situ has developed into a powerful research tool in almost every field of biomedical research (http //books.nap.edu/html/biomems/acoons.pdf). 

Whereas multicolor immunoenzyme staining is applicable only for separately located antigens (see Chap. 7), multicolor fluorescence immmunostaining makes it possible to colocalize antigens not only in the same cell but also in the same cellular compartment. Simultaneous immunolocalization of antigens using fluorescent antibodies can be fulfilled both by the direct (see Sect. 4.1) and indirect (see Sect. 4.2) methods. With the direct method, primary antibodies are labeled with fluorescent dyes, while with the indirect method, primary antibodies are applied as unlabeled antibodies and the visualization is performed with secondary antibodies that are labeled with fluorescent dyes. 

Coons AH, Creech H, Jones R (1941) Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med 47 200 202 Coons AH, Creech H, Jones R, Berliner E (1942) The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J Immunol 45 159 170 Dabbs DJ (ed) (2006) Diagnostic immunohistochemistry. Churchill Livingstone, Philadelphia Dairkee SH, Mayall BH, Smith HS, Hackett AJ (1987) Monoclonal marker that predicts early recurrence of breast cancer. Lancet 1 (8531) 514... 

Weller TH, Coons AH. 1954. Fluorescent antibody studies with agents of varicella and herpes zoster propagated in vitro. Proc Soc Exp Biol Med 86 789-794. 

DCs were generated (BmDC) from thigh bone marrow of naive C57BL/6 mice according to established procedures. In a spleen cell mixture of the same mice breed, DCs were identified using a fluorescent antibody. 

Add the appropriate amount of secondary fluorescent antibody diluted in PBSG with 10% bovine serum albumin (see Note 2). 

Fluorescent Antibodies Enzyme-linked immunosorbant assays... 

It is now clear that fluorescent antibody to malaria persists for years after the patent malaria infestation has long disappeared, and malaria antibodies were still detectable in some West Africans after they had been in Britain for up to seven years (K5, V4). 

In areas where malaria is hyper- or holoendemic, newborn infants had high titers of malarial antibody which decreased during the first 6 months of life and then showed a gradual rise throughout childhood until adult levels were attained. The pattern of the malarial fluorescent antibody titer in children in a malaria endemic area, reflected the development of the serum IgG more closely than that of any other immunoglobulins, and the early fall in the titer of the malaria fluorescent antibody coincided with that of the loss of maternal IgG from the children s circulation, implying that malarial antibody is quite capable of traversing the human placenta (M13) (Table 6, Fig. 6). 

Fig. 6. Pattern of development of IgG, IgM, IgA (mg/ml) and malarial antibodies (reciprocal of dilution units) in Nigerians, plotted on semilog paper. MFAT, malarial fluorescent antibody titer (M6). By courtesy of the publisher of Tropical and Geographical Medicine.

Study No. Agar gel diffu- Serum immunoglobulins (mg/ml) Malarial fluorescent antibody titer ... 

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