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5.5 Immuno-affinity chromatography

In immuno-affinity chromatography the IgG subclasses are determined by monoclonal anti-IgG subclass-specific antibodies, bound to a solid phase in an open column. The captured IgG subclass molecules are labelled with a polyclonal, fluorescently-labelled anti-IgG reagent. The amount of bound conjugate is proportional to the amount of captured IgG subclass. The conjugate is eluted from the column and subsequently measured in a fluorescence reader. The IgG subclass concentrations of the test samples are calculated using the calibration curves.

5.6 Agglutination techniques, Direct Antiglobulin Test (DAT)

Haemolytic disease in adults and new-borns may be due to the presence of anti-erythrocyte (auto-) antibodies. Such antibodies, bound to the red cell membrane in vivo, can be detected in the direct antiglobulin test (DAT). In case of a positive DAT due to IgG antibodies it is important to determine the IgG isotype of the antibodies, because sequestration of sensitised red cells is dependent upon the IgG subclass of the antibody. This is caused by differences between IgG subclasses in their ability to activate complement and to bind to Fcg receptors on phagocytic cells (table II). In general, the haemolytic effect of the IgG subclasses ranges from low to high in the following order: IgG4<1gG2<IgG1<IgG3.

Brief outline of the method:

In V-shaped wells of a microplate, serial dilutions of anti-IgG subclass-specific antisera are introduced. Next, sensitised red cells are added. After overnight incubation at 4 C, the agglutination is read macroscopically. Agglutination may also be detected by running the red cell mixture over a gel column.

5.7 Comparison of the most frequently used assays

Comparison of the RID-, nephelometry and ELISA assays for determination of human IgG subclass levels



Intra assay

Inter assay


Assay time

Work load






long (>48 hours)




very small

very small


very short (few minutes)







Short (few hours)


5.8 Standardization and reference values

Officially recognised as an International Laboratory for Biological Standards since the 1970's, CLB is well aware of the value of reliable standardisation. Thus, CLB's IgG subclass standard sera are accurately calibrated against the IgG subclass levels in the WHO-reference serum 67/97, recommended for human immunoglobulins. Measurements of the IgG subclass content of this preparation has been performed by a number of different research groups resulting in somewhat divergent results. The IgG subclass levels in the WHO-reference serum 67/97 were assessed at 5.0 g/l for IgG1, 2.6 g/l for IgG2, 0.4 g/I for IgG3 and 0.5 g/I for IgG4 by Klein and colleagues (115). These values for the IgG subclass levels have been suggested by the International Union of Immunological Societies (IUIS) as the "correct" future standard (116).

However, a high specificity of the anti-IgG subclass antisera and accurate standardisation are not sufficient to ensure a high-quality assay for human IgG subclasses, if no reliable reference values are available. Therefore, CLB has conducted extensive studies by measuring IgG subclass levels in more than 1000 serum samples from health individuals. Likewise, more than 5000 serum samples from patients have been evaluated for their IgG subclass concentrations. Approximately 28% of the latter sera had one or more decreased IgG subclass levels. Based upon the results of these studies, together with extensive literature data, a set of reference values was derived for IgG subclass levels in sera of healthy individuals (see section 3.1 table III ).

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