[Isotyping of human C4 complement using differences in the functional activity of C4A and C4B isotypes]

Area of Science:

• Immunology
• Biochemistry

Background:

• The human complement system plays a crucial role in innate and adaptive immunity.
• Component C4 (C4) is a key protein in the classical and lectin pathways of complement activation.
• C4 exists as two main functional isotypes, C4A and C4B, with distinct functional activities.

Purpose of the Study:

• To develop and validate ELISA methods for quantifying the functional activity of human C4 isotypes A and B.
• To establish a method for determining the ratio of C4A and C4B functional activities.
• To enable the detection of inherited deficiencies or absence of C4 isotypes.

Main Methods:

• Development of ELISA assays for quantitative measurement of total C4 and functional C4 isotypes.
• Activation of the classical complement pathway using immobilized activators (IgG3 for C4A, Shigella sonnei LPS for C4B).
• Utilized rabbit polyclonal monospecific antibodies against human C4 and horseradish peroxidase conjugates.

Main Results:

• Successful quantitative assay for C4 functional activity based on isotype-specific binding to target activators.
• Demonstrated differential binding of C4A to protein (IgG3) and C4B to carbohydrate (LPS).
• The ratio of determined activities effectively indicates the presence, absence, or deficiency of individual C4 isotypes.

Conclusions:

• Developed sensitive and specific ELISA methods for assessing C4 isotype functional activity.
• The developed methods allow for the reliable detection of inherited C4 isotype deficiencies.
• This approach provides a valuable tool for immunological and genetic studies involving the complement system.