Immunofixation electrophoresis (IFE)

TEST
Immunofixation electrophoresis (IFE)
METHOD
TEST EVALUATION
Rhoda Noall, M.D.
The specimen is electrophoresed on a gel system of choice. Subsequently, specific
antiserum (or antigen) is applied to the area of interest on the gel. Antigenantibody
precipitates are formed, the gel is washed, and then stained for final
reading. Alternatively, the direct precipitate can be read if it is sufficiently clear.
Turn around time is the same day or the next day.
CLINICAL APPLICATIONS
Immunofixation electrophoresis is a powerful tool for studying antigens or antibodies
with slightly different electrophoretic mobilities. In particular, the method is good
for the evaluation of protein polymorphisms and gammopathies. Because the
method minimizes diffusion, sharp separations can be obtained among molecular
species, as compared to standard Immunoelectrophoresis (IEP). Alternate,
comparable techniques which have been used to study polymorphisms are two
dimensional immuno-electrophoresis (Laurell "rockets") or isoelectric focusing.
(^ However, these methods are technically more difficult and cumbersome than IFE
(3).
IFE has been used in the clinical laboratory to evaluate monoclonal/biclonal
gammopathies (2,4,5,7) and ai-antitrypsin variants (2,3,10). This method can
resolve biclonal immunoglobulins or "microspike" gammopathies better than
conventional IEP. In addition, abnormal light chain proliferations associated with
IgM or IgA monoclonal spikes are evaluated more easily with IFE than IEP. In
general, IFE is superior to IEP in demonstrating monoclonal bands through
background, polyclonal immunoglobulin, or other protein (such as urine with massive
proteinuria). Unlike IEP, a monoclonal band does not appear morphologically
"abnormal" in shape, but rather is identified as a denser line of precipitate compared
to normal samples (see diagram). Commercial systems are available from Helena
Laboratories and other suppliers. Those laboratories using IFE regard it as an
adjunct method rather than as a replacement for standard immunoelectrophoresis
(5,7).
Clinical or basic research groups have used the technique of IFE in a variety of
investigative settings. Oligoclonal banding in CSF and the heterogeneity of antibody
production in an immune response have been examined by several groups (2,4-7,11).
Metabolic, tissue, and genetic variants of numerous proteins have also been studied,
including complement fractions, Gc globulins, ceruloplasmin, ai,-antitrypsin,
a2-macroglobulin, and transferrin (1,2,3,5,10). The technique has been adjusted to
study both viral antigens and anti-viral antibodies in body fluids and in tissue
extracts (6). Given sufficient clinical interest and material, it would be possible to
apply these research methods to questions about selected patients in the setting of
the clinical lab.

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TECHNICAL AND CLINICAL LIMITATIONS
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Immunofixation electrophoresis is technically uncomplicated and rapid, especially
when compared to alternate methodology such as isoelectric focusing. The
preparations are dependent on the electrophoretic method, the quality of the
antisera, and the staining method. Specimens may be sera, other body fluids, or
tissue extracts. The gels can be read directly for qualitative evaluation, or if
electrophoresis and staining methodology are sufficiently clean, densitometry can be
used (3). Gel systems have included agarose, cellulose-acetate, and starch gel
electrophoresis. After the antigen-antibody precipitates are formed, any stain or
visualization method can be used. Published variations include Coomasie blue,
nigrosin, amido black, FITC-conjugated antibodies, horse radish peroxidaseconjugated
antibodies, and autoradiography for maximum sensitivity. Direct or
indirect antibody techniques have been employed for different sensitivities (see
chart). Although the range of optimum antibody-antigen concentration is narrower
in IFE than IEP, prozone phenomena are easily detected as a zone of central
clearing (7). IFE is superior to other techniques such as isoelectric focusing or
rocket electrophoresis in technical ease, turn-around time, amount of sample
required, and numbers of samples which can be run with ease. For example, using
IFE, a larger number of samples can be screened for a j-antitrypsin polymorphisms
in less time and using less experienced personnel than using rocket electrophoresis
(3). However, in some worker's laboratories, immunofixation electrophoresis is less
reproducible and requires more skill than IEP. Finally, although this technique has
not found the widespread use of IEP, in the correct setting of sufficient appropriate
material IFE can be a useful adjunct method in protein evaluation.
MOLECULE
monoclonal lg
monoclonal lg
albumin
albumin
albumin
STAIN
direct Ab-conjugated to HPO
indirect Ab-conjugated to FITC
0.1% nigrosine (direct stain,
no antibody applied to gel)
0.1% nigrosine after rabbit
anti-human antibody
indirect Ab-conjugated to HPO
or alkaline phosphatase
DETECTED
CONCENTRATION
2.6 ng/yl
>2.6 ng/yl
500 ng/yl
125 ng/wl
31 ng/ul
REF
5
5
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Diagram (from Ref. 7)
Biclonal gammopathy
SPEP $» to
anti-X
anti-K
a n t i - M fi t f t
%
electrophoreses are all in register
REFERENCES
I
Polyclonal gammopathy
SPEP m a •» Jg»^
a n t i - X W
a n t i - K *
a n t i - M f c
anti-A *»
anti-G $
all electrophoreses in register, anti
Page 3
bodies applied to "boxed" area of SPEP
1. Alper CA, Johnson AM: Immunofixation electrophoresis: A technique for the
study of protein polymorphism. Vox Sang 17:445-452, 1969.
2. Ritchie RF, Smith R: Immunofixation. I. General principles and application
to agarose gel electrophoresis. Clin Chem 22(4):497-499, 1976.
3. Ritchie RF, Smith R: Immunofixation. n. Application to typing of
a;rantitrypsin at acid pH. Clin Chem 22(10):1735-1737, 1976.
4. Ritchie RF, Smith R: Immunofixation. HI. Application to the study of
monoclonal proteins. Clin Chem 22(10):1982-1985, 1976.
5. Cawley LP, Minard BJ, Tourtellotte WW, Ma BI, Chelle C: Immunofixation
electrophoretic techniques applied to identification of proteins in serum and
cerebrospinal fluid. Clin Chem 22(8):1262-1268, 1976.
6. Nordal HJ, Vandvik B, Norrby E: Demonstration of electrophoretically
restricted virus-specific antibodies in serum and cerebrospinal fluid by imprint
electroimmunofixation. Scand J Immunol 7:381-388, 1978.
7. Sun T, Lien YL, Degnan T: Study of gammopathies with immunofixation
electrophoresis. Am J Clin Pathol 72:5-11, 1979.
8. Voller A, Bartlett A, Bidwell D (ed). Immunoassays for the 80[s. University
Park Press, Baltimore, 1981, pp. 55-56.

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9. Chang C-H, Inglis NR: Convenient immunofixation electrophoresis on
cellulose acetate membrane. Clin Chim Acta 65:91-97, 1975.
10. Johnson AM: Genetic typing of a j- antitrypsin by immunofixation
electrophoresis. Identification of subtypes of PiM. J Lab Clin Med 87(1):152-
163, 1976.
11. George PM, Lorier M, Donaldson I: An evaluation of cerebrospinal fluid
oligoclonal banding confirmed by immunofixation on agarose gel. J Neurol,
Neurosurg, Psychiat 46(6):500-504, 1983.