IgG subclass antisera for agglutination techniques
The reagents for agglutination are designed for detection of immunoglobulins bound to human erythrocytes. The antisera are adsorbed to human AB0 cells and have a very high specificity. No cross-reactivity is detectable with human serum proteins other than the intended IgG subclass.
Haemolytic disease in adults and newborns may be caused by 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 on the IgG subclass of the antibody. This is caused by differences between IgG subclasses in their ability to activate complement and to bind to Fc receptors on phagocytic cells.
In general, the haemolytic effect of the IgG subclasses ranges from low to high as follows:
IgG4 < IgG2 < IgG1 < IgG3.
Erythrocytes are incubated with specific anti-human IgG subclass reagents. If red cells are sensitised (have IgG antibodies bound to their surface), the anti-human IgG subclass antiserum causes the cells to agglutinate.
Sanquin has developed a kit of monospecific IgG subclass antisera, that can be used in a microtitre plate agglutination technique. In V-shaped wells of a microtitre plate serial dilutions of anti-IgG subclass specific antisera are introduced. Next, (sensitised) erythrocytes are added. After overnight incubation at 2-8°C, the microtitre plates are placed at an angle of 60°. The agglutination is read macroscopically.