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Immunology—Autoimmunity 687

1990 Burlingame and Rubin described one of the most expression systems, as most individuals have antibodies
informative analyses of autoantibody reactivity as deter- to bacterial components and contamination of recombi-
mined by ELISA. These investigators used subnucleo- nant protein with bacterial antigens can lead to significant
some structures as substrates in ELISA to demonstrate that “false-positive” reactions.
complexes of DNA and histones (e.g., DNA complexed to
a dimer of histone 2A and 2B) are better antigens than the
individual components (e.g., histone 2B). These studies C. Immunoblotting
introduced an additional dimension to the concept that au-
1. History
toantibodies recognize conformational antigenic determi-
nants by suggesting that the quarternary macromolecular The term immunoblotting essentially describes the
structure may be the target of an autoantibody response. transfer of antigenic material from one phase to another
Molecular cloning of autoantigens and their expression and the use of immunological reagents (i.e., antibody)
andpurificationasrecombinantproteinshavesignificantly to detect the transferred protein. The technique has also
increasedtheusefulnessofELISAinidentifyingthespeci- been called protein blotting and Western blotting, an at-
ficity of autoantibodies in autoimmunity. tempt to distinguish the method from the related tech-
niques of Southern and Northern blotting, which allow
identification of DNA and RNA, respectively. The term
2. Principle immunoblotting is more applicable to the technique, par-
In a typical screening assay to detect an antigen-specific ticularly when used to characterize the antigenic speci-
(auto)antibody, the antigen is adsorbed onto a solid sup- ficity of autoantibodies. An early and elegant application
port, usually a polystyrene microtiter plate. A source of the procedure was described by Towbin and collegues in
of soluble antibody (e.g., serum) is allowed to react 1979. They electrophoretically transferred proteins, sepa-
with antigen, unbound material is washed away, and rated by polyacrylamide gel electrophoresis, onto a sheet
then an enzyme-conjugated antiimmunoglobulin is added. of nitrocellulose (Fig. 3) and then detected the transferred
Unbound (excess) antibody conjugate is removed by protein using specific antibody that had been either radio-
washing, and the amount of antigen-specific antibody de- labeled or conjugated with a fluorochrome or an enzyme.
termined by the addition of an enzyme substrate and mea- This method exploited the ability of polyacrylamide gel
surement of the enzyme-generated signal. The signal can electrophoresis to separate a mixture of proteins on the
be in the form of a color change as observed when alkaline basis of their molecular weights and allowed that level of
phosphatase hydrolyzes p-nitrophenylphosphate to pro- resolution to be transferred to nitrocellulose, where spe-
duce the yellow p-nitrophenolate. This color change can cificantibodycouldbeusedtoidentifyindividualproteins.
be measured using a spectrophotometer at 400 nm. Other This method was of immediate applicability to autoim-
means of generating signal include enzyme-generated flu- munity, as contemporary immunological techniques only
orescence and luminescence. allowed sera to be identified as having the same antigenic
specificity, and did not allow identification of the specific
antigen at the molecular level. Within a matter of a few
3. Method years immunoblotting provided not only the molecular
The majority of ELISA procedures that seek to measure weight of many autoantigens, but also clues to the macro-
the presence of autoantibodies employ direct adsorption of molecular complexity of multicomponent antigens such
autoantigen onto a solid support such as a microtiter plate. as the snRNP particles which contain the Sm and nRNP
The most important aspect of these assays is the purity of antigens. As with immunofluorescence, immunoblotting
the antigen. If impure antigen is used, it may be difficult to continues to be one of the major techniques used to char-
define accurately the antigenic specificity of the autoan- acterize both autoantigens and autoantibodies.
tibody. For example, if the antigenic extract contains the
nuclear snRNP particles, then both anti-Sm and anti nRNP 2. Principle
autoantibodies will be detected. As these autoantibodies
can define different clinical syndromes, any confusion in Since the early descriptions of the method, immunoblot-
their detection is undesirable. On the other hand, the use of ting has undergone considerable modification and many
nucleosomes, rather than purified histone, is preferred for different techniques are in use today (see the Bibliogra-
detection of antichromatin autoantibodies. Recent devel- phy for additional reading). The description that follows
opments in ELISA include the use of purified recombinant is essentially the principle of electrophoretic transfer to
autoantigens. Again, these antigenic preparations must be nitrocellulose from polyacrylamide gels, the technique
carefully characterized, particularly those from bacterial pioneered by Towbin, Staehelin, and Gordon in 1979. The