UNIT 13.1 Classical Pathway Evaluation

  1. Patricia C. Giclas

Published Online: 1 MAY 2001

DOI: 10.1002/0471142735.im1301s09

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Giclas, P. C. 2001. Classical Pathway Evaluation. Current Protocols in Immunology. 9:13.1:13.1.1–13.1.26.

Author Information

  1. National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: MAR 1994


This unit describes several assay methods that can be used to determine the functional status of the classical pathway of complement and to quantitate its component proteins. The classical pathway includes C1qrs, C2, C4, C3, C5, C6, C7, C8, and C9, listed in the order in which they interact. Two CH50 assay procedures are presented that test total classical pathway function; one is carried out in test tubes, whereas the alternate protocol describes a variation that is carried out in 96-well microtiter plates. Three support protocols describe preparing serum and erythrocyte-antibody complexes (EA) for use in CH50 assays,and titrating of hemolysin for use in EA preparation. As an alternative to functional assays, radial immunodiffusion (RID) methods are also presented. These can be used to measure the concentrations of most circulating complement proteins and to evaluate the functional status of C1-esterase inhibitor. A fourth support protocol provides a method to determine antibody concentrations for RID assays.