Research
HAPS Executive
Locked Bag 1
Hunter Region Mail Centre
NSW 2310, Australia
Phone: (02) 4921 4000
Fax: (02) 4921 4400
International Enquiries
Phone: +61 2 4921 4000
Fax: +61 2 4921 4400
Auto-Antibody Testing Methods
(Download 
Auto-Antibody Testing Methods.pdf)
Indirect immunofluorescence uses fluorescein-conjugated antibodies directed against human immunoglobulin to detect antinuclear antibodies which bind to the nuclear antigens in a monolayer of human epithelial (HEp 2) cells on a glass slide. The bound fluorescin conjugated antibodies emit light that is detected by a fluorescent microscope.
Immunoblotting uses enzyme-labelled antibodies directed against human immunoglobulin to detect antinuclear antibodies which bind to nuclear antigens displayed in characteristic positions on the agarose gel after they have been electrophoretically separated. The antibodies are detected by reaction of the enzyme labelled antibody with a colour producing enzyme substrate. The positive colour changes in the bands are read visually.
The ELISA uses enzyme-labelled antibodies directed against human immunoglobulin to detect surface antinuclear antibodies which have bound to isolated nuclear antigens fixed to a solid phase, such as a microtitre plate well. The antibodies are detected by reaction of the enzyme labelled antibody with a colour producing enzyme substrate, which is read by a spectrophotometer.
The modified Farr radioimmunoassay is based on the precipitation of specific antinuclear antibodies which have bound to radio-labelled DNA antigen preparations. The amount of radioactivity of the immunoprecipitate is proportional to the antibody titre. The radioactive label is the gamma emitting iodine 125 (125I), which is measured on a gamma counter.
Counter-Immunoelectrophoresis, used to detect extractable nuclear antigens, refers to the formation of precipitation lines when patient antinuclear antibodies move toward and bind to nuclear antigens in an electric field. Known positive antibodies are assayed adjacent to the patient sera. When the precipitation lines cross-over the patient is negative for the antibody. When the lines join and do not cross, the patient is positive for the antibody. The precipitation lines are interpreted visually.
Written by: Dr Glenn Reeves, Immunology, HAPS
Written: 6 October 1998
Reviewed: August 2001