Immunocytochemical Detection Systems
Lars-Inge Larsson in Immunocytochemistry: Theory and Practice, 2020
The beginnings of direct and indirect immunofluorescence were reviewed in the Introduction. Today, a number of different procedures and labels are available for producing fluorescent antibodies. These include fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylaminofluorescein (DTAF), Texas red, bimane, and several others.33,217,243 The two most-used reagents are FITC and TRITC. Both reagents are now commercially available as highly reactive isothiocyanates, so earlier dangerous methods involving phosgene to prepare them do not have to be used in the research laboratory.243 The isothiocyanates react directly with primary amino groups under suitable conditions of pH and salt concentration. Labeling, therefore, is conveniently carried out by mixing the desired isothiocyanate and protein in a suitable buffer, incubating them, and then separating unincorporated fluorochrome from protein. This separation is most conveniently achieved by gel filtration, although different solid-phase scavengers for unreacted fluorochrome (e.g., lysine-Sepharose®) also may be used. Details for labeling IgG with FITC and TRITC are given in the Appendix.
Diagnostic applications of immunology
Gabriel Virella in Medical Immunology, 2019
The principles of quantitative immunofluorescence are similar to those described for indirect fluorescence assays: antigen is bound to a solid phase, exposed to a serum sample containing specific antibody, unbound immunoglobulins are rinsed off, and a fluorescein-labeled antibody is added to reveal specific antibody. A fluorometer is used to measure the amount of fluorescence emitted by the second antibody. Since the amount of fluorescent antibody added to the system is fixed, the amount that remains bound is directly proportional to the concentration of antibody present in the sample. Thus, a quantitative correlation can be drawn between the intensity of fluorescence and the concentration of antibody. These quantitative tests have been adapted to microbiological assays and can identify IgG and IgM antibodies.
Pathology and Epidemiology
John T. Kemshead in Pediatric Tumors: Immunological and Molecular Markers, 2020
In the earlier days of the application of immunohistological methods to tumor diagnosis, the method used to demonstrate the site of antigen localization was immunofluorescence using the action of ultraviolet light on antibodies conjugated with fluorescein or other fluorogen. Indeed, immunofluorescence is still the method of choice in some areas of study because of its high degree of sensitivity and ability to locate small quantities of antigen. It does suffer from disadvantages in the short storage life of the sections and the inconvenience of requiring a separate facility for ultraviolet light microscopy in addition to the white light microscopy available in most laboratories.
Therapeutic effects of thymoquinone or capsaicin on acrylamide-induced reproductive toxicity in rats mediated by their effect on oxidative stress, inflammation, and tight junction integrity
Published in Drug and Chemical Toxicology, 2022
Ekram Nemr Abd Al Haleem, Walaa Yousef Soliman Hasan, Hossam Mohamed Mohamed Arafa
Immunofluorescence is a technique for detecting antigens and antibodies in which the antibodies are labeled with fluorescent dyes, and the resulting antigen-antibody complex is visualized using a fluorescent microscope. The indirect immunofluorescence assay was used in this research (Odell and Cook 2013). Occludin Antibody (E-5) was obtained from Santa Cruz Biotechnology, which is a mouse monoclonal IgG2b (kappa light chain) (Oregon, USA). Using a Leica fluorescence microscope (Model: Leica DM 5500B, Leica Microsystems, Wetzlar, Germany), tissue sections were examined and imaged in blue, green, and red channels. The fluorometric analysis of the results was performed using Image-J software on a minimum of five fields from each rat testicular segment (minimum of two rats from each group) (NIH, USA).
Enhanced detection of ATTR amyloid using a nanofibril-based assay
Published in Amyloid, 2021
M. Mahafuzur Rahman, Benjamin Schmuck, Henrik Hansson, Torleif Härd, Gunilla T. Westermark, Mats Sandgren
Since our concept yielded promising results in the microplate format, we next examined whether Ab-bNF can be used to improve the sensitivity of an immunofluorescence assay. As with the microplate assay, we first compared the binding pattern of TTR-bNF and TTR primary antibody (1899) to TTR amyloid-containing tissue from Case 1. The indirect immunofluorescence labelling was carried out with 0.25 µg, 0.5 µg, and 1 µg primary antibody per tissue section, followed by the addition of a fluorophore-conjugated secondary antibody. Strong fluorescence, indicating TTR amyloid presence, was observed in sections incubated with 0.25 µg primary antibody, while the signal in sections incubated with 0.5 µg and 1 µg primary antibody appeared weaker (Figure 3(a–c)). The lower fluorescence signal observed in tissue sections incubated with higher antibody concentrations is likely due to steric hindrance [30,31].
A review on current diagnostic techniques for COVID-19
Published in Expert Review of Molecular Diagnostics, 2021
Islam El Jaddaoui, Malika Allali, Sanae Raoui, Sofia Sehli, Nihal Habib, Bouchra Chaouni, Najib Al Idrissi, Najwa Benslima, Wissal Maher, Houda Benrahma, Noureddine Hamamouch, Kamal El Bissati, Sahar El Kasmi, Salsabil Hamdi, Youssef Bakri, Chakib Nejjari, Saaïd Amzazi, Hassan Ghazal
As with antibody testing, the principle of antigen detection relies on the mobilization of the sample along the test strip by capillary action [190]. These assays are not limited to a particular format as the target antigen can be captured by fluorescent-labeled or colloidal gold antibodies. In the case of colloidal gold-based immunoassays, the antigen is highlighted by visible lines appearing on the test strip [192]. The first line, which comprises the capture antibodies specific to a specific region of SARS-CoV-2 antigen, is commonly the test line while the second line, with dedicated antibodies, is the control line intended to validate the results [190]. In the case of fluorescence immunoassays, the antigens are detected through fluorescence using an immunofluorescence analyzer [192].
Related Knowledge Centers
- Antibody
- Biomolecule
- Dye
- Fluorescence
- Fluorophore
- Immunostaining
- Microscopy
- Epitope
- Antigen
- Fluorescence Microscope