Primary Reactions 2.3 IMMUNOFLOURESCENT Flashcards
________ employs the visual detection of fluorescent dyes coupled (conjugated) to antibodies which react with the antigen present using Fluorescent Microscopy
Fluorescent Antibody Assay
An analytical technique to identify and characterize minute amount of a substance by excitation of a substance with a beam of ultraviolet/visible light and measure the characteristic wavelength of fluorescent light emitted.
IMMUNOFLUORESCENT ANTIBODY TECHNIQUE
_____ (1941) introduced the immunofluorescence technique for the first time. He used _______, a blue fluorescing compound, coupled to pneumococcal antiserum to detect bacterial antigens in tissue sections
Albert COONS
Beta-anthracene
_______ is a histochemical or cytochemical technique for the detection and localization of antigens.
Specific antibody is conjugated with fluorescent compounds resulting in a sensitive tracer of unaltered immunologic reactivity
Immunofluorescence
➢Virtually, any antigen can be detected in ______ or in live cell suspension by immunofluorescence.
➢ Allows the competent user to identify visually the site of the _____ reaction, thereby rendering significant specificity.
➢ Is a combination of great ____ and ______with the use of histologic techniques.
fixed tissue sections
antigenantibody
sensitivity & specificity
______is the emission of light of one color, i.e., wavelength, while a substance is irradiated with light of a different color. e.g., FITC absorbs light (UV light) or short blue light at a wavelength of 490-495 nm and emits its characteristic green color (visible part of the electromagnetic spectrum) at 517 nm.
Fluorescence
_______- a chemical that can absorb electromagnetic energy of relatively short wavelength, and almost instantaneously emit light at a longer frequency (lower energy level).
Fluor or Fluorochrome
Also known as fluorophore, a chemical compound able to re-emit light upon excitation.
● are dyes that fluoresce when exposed to specific wavelength, often used as indicator molecules on antibodies and antigens.
Fluor or Fluorochrome
When fluorochromes ____ light of a particular wavelength, they are instantly excited and then emit light of a longer wavelength as they ____ to their unexcited state.
absorb
return
The ______ is fluorescent light or simply fluorescence, which is the emission of light of one color, i.e., wavelength, while a _____ is irradiated with light of a different color.
emitted light
substance
The intensity of the light is ________ to the amount of Fluorochrome present in the sample.
directly proportional
Most popular Fluorochromes used:
- Fluorescein isothiocyanate (FITC); emits ______
- Lissamine Rhodamine B
- 1-dimethylaminonaphthalene-5-sulfonyl chloride (DANSYL)
- Tetramethylrhodamine isothiocyanate – emits a ______
green light
red color
These fluorescent compounds have different characteristic absorption and emission spectra; absorb light (UV light) or short blue light ____; and emit light on the visible part of the electromagnetic spectrum ______.
● Readily bind covalently to proteins at an _____ pH, primarily through the epsilon amino residues of lysine and terminal amino groups.
● They covalently join to free amino groups of proteins (immunoglobulins) without affecting the antigen binding capacity of the antibody as in during ______or labelling
(200-400nm)
(400-700 nm)
alkaline
tagging
To view fluorescence, a Fluorescent Microscope is used. The Fluorescent microscope was invented in the early part of the 20th century by _________
August Kohler, Carl Reichert and Henrich Lehmann, among others.
The basic task of the fluorescence microscope is to let excitation light radiate the specimen and sort out the much weaker emitted light from the image. The radiation collides with the atoms in your specimen and electrons are excited to a higher energy level. When they relax to a lower level, they ______.
emit light
The actual brightness of fluorescence observed by the eye depends on:
a. The _____ at which the dye converts incident light (absorbed light) into fluorescent light.
b. The _______ of the dye in the tissue specimen.
c. The ______ of the exciting (absorbed) radiation
efficiency
concentration
intensity
Parts and function of a Fluorescent Microscope:
- _________ – produces an intense beam
- _________ - limits the heat transfer from the light source.
- _______ – transmits only the desired wavelength
- _________ - designed to efficiently reflect excitation wavelengths
- _______ - provides a dark background against which the fluorescent objects glow
- _________- Removes any remaining ultraviolet light which could damage the viewer’s eyes, or blue & violet light which could reduce the image’s
Mercury vapor arc lamp Heat Filter or Infrared Filter Exciter Filter Dichroic mirrors Darkfield Condenser Barrier Filter (Lens)
Fluorescence may be detected with
- Spectrofluorometers
- Filter Fluorometer
- Flow Cytometer
- Fluorescence Microscope
Fluorescence on slide – may be observed using ________ Fluorescence in solution - the following may be used:
Fluorescence Microscope
a. Spectrofluorometer
b. Filter Fluorometer
c. Flow cytometer
For immunoassays, fluorescence intensity can be converted to _______ by using a standard curve generated under the same conditions as that used for the patient sample
protein concentration
Factors affecting Fluorescence Measurements:
- _______________
- Exciting light can collide with non-fluorochrome molecules in the sample, and, cause the light to be scattered. The scattered light is of the same wavelength as the exciting light, and so contribute to the background noise /reading. - ___________
- Refers to the decrease in fluorescent intensity. - May occur when the fluorochrome interacts with molecules in the solution. Or, some of the radiant energy absorbed by the fluorochrome is transferred to the molecules rather than being emitted as fluorescence. - ________ are used to measure fluorescence and can contribute to the background noise as some materials in the cuvettes can fluoresce.
- _______________. - It is important therefore that calibration curve and the samples be measured at the same temperature
Light scattering in the solution
Quenching of the solvent
Cuvettes
Fluorescence intensity decreases with increasing temperature
General Steps involved in Immunofluorescence Technique -
- Preparation of immune antiserum or purified gamma globulin
- Conjugation with Fluorescent dye
- Staining procedure
Specific antibody is conjugated with fluorescent compounds. The conjugated antiserum is added to cells or tissues and becomes fixed to antigens, thereby forming a stable immune complex.
Non-antibody proteins are removed by washing and when the resultant preparation is observed in a Fluorescence microscope against a dark background, antigens bound specifically to fluorescent antibody can be detected by virtue of the bright color of the antibody.
Principle
Qualitative Fluorescent Antibody Technique:
Direct Immunofluorescence Test / Single Layer Technique
Indirect Immunofluorescence Test/ Double Layer Technique
Inhibition Technique
Complement Staining Technique
Biotin-Avidin System
