Lab 10: Anti-Nuclear Antibodies - Indirect Immunofluorescence

Principle of ANA

Autoimmune disease will produce antibodies to various nucleoprotein’s and DNA materials found within the nucleus. A well with tissues test cells is incubated with patient serum. If the patient has antibodies to nuclear antigens, they will bind to various nuclear components in the test cells. After incubation, the well is washed and & fluorescent labeled anti-human antibody is added and incubated. The fluorescent antibody will bind to any patient antibody that has attached to the nuclear material of the test cells. The unbound fluorescent antibody is washed off and the well is observed under a fluorescent microscope. Different patterns of fluorescence are suggestive of different autoimmune diseases.

what material in the nucleus can bind human antibody in ANA?

double stranded or native DNA (dsDNA), single stranded DNA (ssDNA), nuclear membrane, histones, etc

what do the fluorescent patterns observed under the microscope indicate ?

indicates which of the nuclear materials have patient antibodies bound to them — suggestive to different diseases

are ANA patterns diagnostic ?

NO

Homogenous ANA pattern

entire nucleus is smoothly stained (dsDNA)

rim or peripheral ANA pattern

outer rim of the nucleus is stained = most likely lupus

speckled ANA patter

speckles in nucles no staining of nucleoli (Sm protein)

Speckled ANA pattern is found in which health conditions?

lupus & other connective tissue diseases