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rev:February 5, 1997
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The following book on the IgG Subclass is made available by the permisssion
of CLB Reagents (Netherlands). It is for your personal use and may not be
reproduced without the permission of CLB Reagents. This material is for
information use only and should not be construed as medical advice. We are
not responsible for any misprints or error or ommissions. Certain charts
,graphs and table have been ommitted or changed in style (not content)
to fit this web site. Please use this information as part of your discussion
with your medical professional.
5.3 Radio Immuno Assay (RIA)
In the RIA,IgG subclasses are quantified as immune complexes after binding of radioactively labelled specific antibody.In case of a 'direct' technique, a radioactively labelled anti-IgG subclass-specific antibody is used. In an 'indirect' technique, an anti-IgG subclass-specific antibody directed against the first antibody. Since working with radioactively labelled reagents requires special precautions and is relatively costly,radio immunoassays have been largely replaced by enzyme-linked immunoassays.
5.4 Enzyme-linked Immunosorbent Assay (ELISA)
In this technique, enzyme-labelled antibodies are used for detection. In
the 'sandwich'-type of enzyme immunoassay, the IgG subclass of interest is
captured by anti-human IgG subclass-specific antibody and quantified by an
enzyme-labelled anti-IgG antibody. Because of its high sensitivity and
specificity, this assay allows accurate measurement of very low levels of
IgG subclasses. In figure 9, a schematic outline of the ELISA technique is
shown. The sensitive ELISA comprises many incubation-and washing steps. Because
of the need for high dilutions when measuring IgG sublasses in sera,ELISA
assays may be less reproducible in comparison with RID and nephelometry.
For measurement of IgG and its subclasses in large numbers of samples,ELISA
is increasingly being replaced by nephelometry. ELISA may be advocated for
measuring IgG subclasses in other body fluids than serum/plasma, e.g.saliva,
cerebrospinal fluid and broncho-alveolar lavage fluid.
Brief outline of the method (figure 9):
An ELISA is generally performed in wells of microplates.
-Wells of the plates are coated with unlabelled monoclonal antihuman IgG
subclass-specific antibody and washed (figure 9A);
-Test samples,standard-and control sera are introduced in the respective wells and incubated; The IgG subclass to be determined will bind to the solid phase and non-bound IgG is removed by washing (figure 9B);
-Enzyme-labelled anti-human IgG antibodies are added to each well and non-bound conjugate is removed by washing (figure 9C);
-Plates are incubated with substrate solution;
-After incubation,the coloured reaction product is measured photometrically (figure 9D);
-The concentration of the IgG subclasses in the test samples is calculated relative to the values of the calibration curve.
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