Immunoaffinity purification of human complement component C9 using monoclonal antibodies.

Area of Science:

• Immunology
• Complement System Biology

Background:

• The complement system is crucial for innate immunity.
• Human complement component 9 (C9) is the terminal protein in the complement cascade, polymerizing to form the membrane attack complex (MAC).
• Understanding C9's structure and function is vital for immunomodulation and therapeutic development.

Purpose of the Study:

• To produce monoclonal antibodies against human C9.
• To characterize the antigenic sites on C9 recognized by these antibodies.
• To utilize an antibody for the purification of C9 from human plasma.

Main Methods:

• Production and characterization of five monoclonal antibodies against human C9.
• Affinity determination of antibodies using binding assays.
• Immobilization of a low-affinity antibody onto Sepharose 4B for affinity chromatography.
• Purification of C9 from human plasma using the immobilized antibody.

Main Results:

• Five monoclonal antibodies to human C9 were successfully generated.
• Antibody affinities ranged from 0.1–0.3 x 10^9 M^-1 to 1.6–3.1 x 10^9 M^-1.
• Four distinct antigenic sites on C9 were identified.
• A 1200-fold purification of C9 from human plasma was achieved with a 30–40% yield using immobilized antibody.
• The purified C9 retained full cytolytic activity.

Conclusions:

• Monoclonal antibodies are effective tools for studying complement proteins like C9.
• Affinity chromatography using specific antibodies can efficiently purify functional C9.
• The purified C9 is suitable for further investigations into its role in the complement cascade and cytolytic mechanisms.