Title: Indirect Immunofluorescence Method

Aim: To perform an indirect immunofluorescence method on a sample obtained

Principle: Indirect immunofluorescence Method

Immunofluorescence is a technique allowing the 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 (FITC). There are two major types of

immunofluorescence staining methods: 1) direct immunofluorescence staining in which the

primary antibody is labeled with fluorescence dye, and 2) indirect immunofluorescence staining

in which a secondary antibody labeled with fluorochrome is used to recognize a primary

antibody. Immunofluorescence staining can be performed on cells fixed on slides and tissue

sections. Immunofluorescence stained samples are examined under a fluorescence microscope or

confocal microscope. Indirect immunofluorescence is employed to detect antibodies in patient

serum. The antigen on smear are made to react with specific unlabeled antibody (raised in

mouse) and washed. The unbound antibody gets washed off. The presence of specific mouse

antibody bound to the antigen on smear is detected by adding another antibody. The second

antibody is labeled anti-gamma globulin (rabbit antibody against mouse antibody) antibodies.

This antibody binds to Fc portion of first antibody and persists despite washing. The presence of

the second antibody is detecting by observing under fluorescent microscope. It is often used to

detect autoantibodies. Commonly used in the detection of anti-nuclear antibodies (ANA) found

in the serum of patients with SLE.

Method: see lab handout

Results:

Types of A B Parainfluenzae Parainfluenzae Parainfluenzae

Influenzae 1 2 3
Virus
Results Negative Negative Negative Positive Negative

TABLE SHOWING IMMUNOFLUORESCENCE RESULTS FOR THE PATIENT

Discussion: Immunofluorescence is a technique allowing the 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 (FITC). The fluorescent antibody technique is

based upon the capacity of antibodies to bind certain fluorescent staining without any alteration

in their immunological properties. A known antigen is placed on a slide; the patient's serum is

added, then rinsed away. A fluorescein-labeled antiglobulin is added, then rinsed away. The

presence of fluorescence over the antigen indicates the presence of antibodies to this antigen in

the patient. After the incubation period in a moist chamber a rinse in PBS to remove unbound

serum proteins, sections are treated with a FITC-conjugated antiIgG or other antibody conjugates

of defined specificity. Following which another rinse with in PBS to remove the unbound

conjugate is done then slides are mounted and examined under a fluorescence microscope. The

influenza virus that was found to be present in the patient was Para influenzae 2 and this was

obtained by reading the slides which indicates the presence of the virus where the fluorescein –

labeled antiglobulin bounded to the antigen and when viewed under a fluorescence microscope

an apple green fluorescent was seen on the slide where the virus was present. The limits to IF

include specificity of the antibodies, specimen preparation, autofluorescence, and performance of

the microscope and user. Specificity of the antibodies depends on the purity of the antigen used

for immunization and can often be satisfied with either affinity-purified polyclonal antibodies or

by monoclonal antibodies.