Primary Reactions 2.3 IMMUNOFLOURESCENT

Question 1

________ employs the visual detection of fluorescent dyes coupled (conjugated) to antibodies which react with the antigen present using Fluorescent Microscopy

Answer 1

Fluorescent Antibody Assay

Question 2

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.

Answer 2

IMMUNOFLUORESCENT ANTIBODY TECHNIQUE

Question 3

_____ (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

Answer 3

Albert COONS

Beta-anthracene

Question 4

_______ 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

Answer 4

Immunofluorescence

Question 5

➢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.

Answer 5

fixed tissue sections
antigenantibody
sensitivity & specificity

Question 6

______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.

Answer 6

Fluorescence

Question 7

_______- a chemical that can absorb electromagnetic energy of relatively short wavelength, and almost instantaneously emit light at a longer frequency (lower energy level).

Answer 7

Fluor or Fluorochrome

Question 8

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.

Answer 8

Fluor or Fluorochrome

Question 9

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.

Answer 9

absorb

return

Question 10

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.

Answer 10

emitted light

substance

Question 11

The intensity of the light is ________ to the amount of Fluorochrome present in the sample.

Answer 11

directly proportional

Question 12

Most popular Fluorochromes used:

1. Fluorescein isothiocyanate (FITC); emits ______
2. Lissamine Rhodamine B
3. 1-dimethylaminonaphthalene-5-sulfonyl chloride (DANSYL)
4. Tetramethylrhodamine isothiocyanate – emits a ______

Answer 12

green light

red color

Question 13

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

Answer 13

(200-400nm)
(400-700 nm)

alkaline
tagging

Question 14

To view fluorescence, a Fluorescent Microscope is used. The Fluorescent microscope was invented in the early part of the 20th century by _________

Answer 14

August Kohler, Carl Reichert and Henrich Lehmann, among others.

Question 15

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 ______.

Answer 15

emit light

Question 16

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

Answer 16

efficiency
concentration
intensity

Question 17

Parts and function of a Fluorescent Microscope:

1. _________ – produces an intense beam
2. _________ - limits the heat transfer from the light source.
3. _______ – transmits only the desired wavelength
4. _________ - designed to efficiently reflect excitation wavelengths
5. _______ - provides a dark background against which the fluorescent objects glow
6. _________- Removes any remaining ultraviolet light which could damage the viewer’s eyes, or blue & violet light which could reduce the image’s

Answer 17

Mercury vapor arc lamp
Heat Filter or Infrared Filter
Exciter Filter
Dichroic mirrors
Darkfield Condenser
Barrier Filter (Lens)

Question 18

Fluorescence may be detected with

Answer 18

1. Spectrofluorometers
2. Filter Fluorometer
3. Flow Cytometer
4. Fluorescence Microscope

Question 19

Fluorescence on slide – may be observed using ________ Fluorescence in solution - the following may be used:

Answer 19

Fluorescence Microscope

a. Spectrofluorometer
b. Filter Fluorometer
c. Flow cytometer

Question 20

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

Answer 20

protein concentration

Question 21

Factors affecting Fluorescence Measurements:

1. _______________
   - 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.
2. ___________
   - 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.
3. ________ are used to measure fluorescence and can contribute to the background noise as some materials in the cuvettes can fluoresce.
4. _______________. - It is important therefore that calibration curve and the samples be measured at the same temperature

Answer 21

Light scattering in the solution
Quenching of the solvent
Cuvettes
Fluorescence intensity decreases with increasing temperature

Question 22

General Steps involved in Immunofluorescence Technique -

Answer 22

1. Preparation of immune antiserum or purified gamma globulin
2. Conjugation with Fluorescent dye
3. Staining procedure

Question 23

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.

Answer 23

Principle

Question 24

Qualitative Fluorescent Antibody Technique:

Answer 24

Direct Immunofluorescence Test / Single Layer Technique
Indirect Immunofluorescence Test/ Double Layer Technique
Inhibition Technique
Complement Staining Technique
Biotin-Avidin System

Question 25

Purpose: To identify unknown antigen by using fluoresce in labeled antibody.

Principle: Conjugated antiserum is added directly to the tissue section or viable cell suspension. Subsequent washing in physiologic buffer saline removes excess uncombined labeled antibody. Mixture is then examined under the Fluorescent microscope. Performed to identify foreign substances (antigens) in tissues. e.g.,
bacteria
Particulate viruses & their soluble antigens Protozoa
fungal antigens

Answer 25

Direct Immunofluorescence Test / Single Layer Technique

Question 26

Purpose: detects antibody in unknown sera or unlabeled sera.
➢ Basically an adaptation of the antiglobulin reaction (Coomb’s Test) or Double antibody Technique.
➢ Advantage: Eliminates the need to purify and individually conjugate each serum sample.

Principle: Unlabeled antibody is exposed to antigen, followed by the addition of labeled antiglobulin sera directed against the globulin of the species used in the initial exposure. In this case,fluorescence indicates that the reaction between the antigen, the unknown antibody, and the labelled antiglobulin has taken place.

➢ To detect a wide variety of human antigen-antibody reactions, fluorescinated rabbit or goat antihuman globulin is used.

Answer 26

Indirect Immunofluorescence Test/ Double Layer Technique

Question 27

Principle: Unlabeled antibody is added to antigen. Antigen becomes saturated with unlabeled specific antibody. Labeled antibody is then added. No fluorescence is seen and the test remains non-reactive because no antigen sites remain to react with the labeled antibody

➢ Antigen becomes ______ when treated with unlabeled antibody. Therefore, upon subsequent exposure to specific labeled antibody, no fluorescence can be detected.

Answer 27

Inhibition Technique

saturated

Question 28

➢ Similar to the Indirect Technique, except that the labeled/conjugated antibody is directed against the complement components, often of Guinea pig origin.

➢ Advantage: A single conjugate (anti-pig complement) may be used to test the serum from any species.

➢ Fluorescence indicates that a reaction has occurred between the unknown antibody and the antigen, resulting in the fixation of complement to the complex, and that a subsequent reaction has occurred between the guinea pig complement and the anti-guinea pig complement

Answer 28

Complement Staining Technique

Question 29

a basic glycoprotein derived from egg albumin with a MW of 68,000.
- It shows a remarkably high affinity (1015K cal/mol.) for the vitamin Biotin.

Answer 29

Avidin

Question 30

_____ can be easily coupled covalently to a protein (antibody) and then reacted with fluorochrome-coupled Avidin.

Answer 30

Biotin

Question 31

Principle: After reaction of antigen with labeled antibody, the Biotin-labeled second antibody is added. Since many molecules of Biotin can be coupled to an antibody, the subsequent addition of fluorochrome-labeled Avidin results in a firm bond with exceedingly _______

Advantage: Lack of _______ of fluorochrome-coupled Avidin to various substrates and general use of avidin conjugates in binding to Biotin-labeled antibodies regardless of their derivation.

Answer 31

Biotin-Avidin System

bright fluorescence.
non-specific binding

Question 32

Quantitative fluorescent Antibody Technique –

_________measures the fluorescence spectra of microscopic samples or microscopic area of a larger object.
Basic types: ___

Answer 32

Microfluorometer

a) the fully integrated microfluorometer ; and
b) the fluorescence spectrometer unit.

Question 33

_______ can also be configured to measure the transmission and reflectance spectra of microscopic sample areas in addition to fluorescence. With special software, they are capable of colorimetry as well.

Use: Measures _______

Answer 33

Microfluorometers

IgG, IgA, IgM C3, C4, ANA, anti-DNA antibodies