PRIMARY SEROLOGICAL TEST IMMUNOFLUORESCENT TEST

Introduction Immunofluorescence is a serological test where the labeling of antibodies or antigens is done with fluorescent dyes ( fluorochromes). Fluorochromes are dyes which have the ability to absorb the short wavelength UV radiation and emit light of longer wavelength fluorescence ( visible green light). Examples : FITC, Rhodamine, Acridine orange

Immunofluorescence - Most commonly used fluorescent dyes are: Fluorescein and Rhodamine, but other highly fluorescent substances (Phycoerythrin and Phycobiliproteins) have also come into use. - Fluorescein: absorbs blue light and emits an intense yellow- green fluorescence - Rhodamine: absorbs yellow-green range and emits a deep red fluorescence - Phycoerythrin: efficient absorbers of light and brilliant emitters of red fluorescence

Assumptions that are important for 1 serological tests One of reactants (Ag,Ab) must be rendered insoluble (fixed ) on solid phase such as slide or microtiter well or glass bead. The other reactant is conjugated to a labeling material( enzyme, fluorochrome, radioactive substance) without loss of activity.

There are two ways of doing IF staining – Direct immunofluorescence – Indirect immunofluorescence Direct immunofluorescence Use: Direct detection of Pathogens or their Ag’s in tissues or in pathological samples Ag is fixed on the slide Fluorescein labeled Ab’s are layered over it Slide is washed to remove unattached Ab’s Examined under UV light in an fluorescent microscope The site where the Ab attaches to its specific Ag will show apple green fluorescence

Direct immunofluorescence

Indirect immunofluorescence: Indirect test is a double-layer technique The unlabelled antibody is applied directly to the tissue substrate Treated with a fluorochrome-conjugated anti-immunoglobulin serum

Advantage over direct IF – Because several fluorescent anti- immunoglobulins can bind to each antibody present in the first layer, the fluorescence is brighter than the direct test. – It is also more time-efficient since it is only one signal labeled reagent, the anti- immunoglobulin, is prepared during the lengthy conjugation process

Indirect Elisa for detection of ANA Principle ( Diagram) Solid phase :slide Ag Nucleated tissues Patient serum Solid phase :slide Ag Anti human IgG conjugated to FITC Incubation period followed by washing Non specific AB FITC

Indirect IFA for detection of ANA in patient sample Serum sample must be diluted 1:40 to dilute non specific Ab due to aging or autoimmune diseases such as RA. Nucleated tissues(Ag) are immobilized on slide Diluted patient serum is added(25ul),the slide is put in a humid chamber. After addition of patient serum,there is an incubation period followed by washing(3-4).

Indirect IFA for detection of ANA in patient sample Conjugate is added. After addition of conjugate, there is an incubation period followed by washing. Read using Fluorescent microscope. UV light is produced by Fluorescent microscope special lamp.This microscope must have barrier filter to protect observer’s eye from UV light.

Indirect IFA for detection of ANA in patient sample Washing is an important step to remove unbound ( excess/ non specific)Ag or Ab Insufficient washing results in false +ve results Excessive washing results in false –ve results Patient serum must be diluted 1:40 before performing test(why)

Indirect IFA for detection of ANA in patient sample(+ve/x40)

Indirect IFA for detection of ANA in patient sample (+ve/x20)

Indirect IFA for detection of ANA in patient sample (-ve/x40)

Indirect IFA for detection of ANA in patient sample (-ve)

False +ve and False -ve results False +ve Cross reacting Ab Presence of Rheumatoid factor or other autoimmune disease Use of non specific Ag Improper washing Aging False –ve Improper Immune system Blocking Ab(compete for Ag binding site of Ab) Prozone phenomenon Excessive washing