Immunofluorescence assay

Question 1

What is the purpose of IFA?

Answer 1

visualization of a specific protein or antigen in cells or tissue sections by binding a specific antibody chemically conjugated with a fluorescent dye such as fluorescein isothiocyanate

Question 2

What are the two major types of immunofluorescence staining methods, and how to they work?

Answer 2

• direct immunofluorescence - the primary antibody is labelled with a fluorescent dye
• indirect immunofluorescence - a secondary reagent is used to recognize a primary antibody

Question 3

What can be secondary reagents in indirect IFA?

Answer 3

antiglobulin or protein A/G body labelled fluorochrome

Question 4

Why do we use immunofluorescence staining?

Answer 4

Diagnosis of:
- viral diseases
- identify and enumerate lymphocyte populations
- autoimmune disorders (indirect IFA)

Question 5

What is the principle of quantitative immunofluorescence assays?

Answer 5

Antigen bound to a solid phase, is exposed to a serum sample containing specifric antibody.
Unbound immunoglobulins are rinsed off, and a fluorescein-conjugated antibody is added to reveal the antibody that reacted specifically with the immobilised antigen.

Question 6

What happens in the direct method?

Answer 6

Cells are stained with antimembrane antibody (anti-mAg) that is labelled with fluorochrome.

Question 7

What happens in the indirect method?

Answer 7

Cells are first incubated with unlabelled anti-mAg antibody (primary) and then stained with a fluorochrome labelled secondary reagent that binds to the primary antibody

Question 8

IFA is seen using what kind of microscope?

Answer 8

fluorescent microscope

Question 9

Indirect IFA is most commonly used for:

Answer 9

autoimmune disorders

Question 10

Direct IFA is used for:

Answer 10

determination of antigen in a cell or tissue by direct labelling with antibodies labelled with FITC

Question 11

Flow cytometry is used for:

Answer 11

analysis of the cells based on their differential ability to bind fluorescens labelled antibody

Question 12

Size of cells in flow cytometer is detected by:

Answer 12

forward scatter

Question 13

Sensitized erythrocytes are:

Answer 13

erythrocytes labelled antibody (amboceptor)

Question 14

Name the steps in immunofluorescens test:

Answer 14

1. Fixation with methanol, formaldehyde or paraformaldehyde/glutaraldehyde
2. Staining

Question 15

What is double immunofluorescens?

Answer 15

Single labelling. Antibodies from different animals can be mixed and incubated as a cocktail.

Question 16

What is a common clinical use of flow cytometer?

Answer 16

determination of what kind and number of white blood cells in each population of a patients blood sample

Question 17

What can one obtain from flow cytometer?

Answer 17

• How many cells express the target antigen.
• The distribution of cells in a sample population according to antigen densities as determined by fluorescens intensity.
• The size of cells

Question 18

Flow cytometer is one of the essential tools for classifying what?

Answer 18

Leukaemia