Immunoaffinity Methods for Purification of Complement Components and Regulators

Immunoaffinity protocols offer a rapid, efficient, and simple way of obtaining a protein of interest. Almost all of the complement components, receptors, and regulators have been successfully isolated by immunoaffinity protocols. The use of these methods is limited only by the availability of suitable antibodies in sufficient quantities (1 –6 ). Immunoaffinity methods are compatible with most detergents used in the solubilization of membrane proteins and are thus well suited to the purification of membrane regulators of complement and complement receptors (3 ,7 ,8 ). Proteins isolated by immunoaffinity methods, particularly on monoclonal antibody solid phases, are usually of high purity and require either no downstream processing or simple “polishing” steps, such as gel filtration to remove aggregates. Elution of the bound protein from the antibody-solid phase is commonly achieved by subjecting the column to extremes of pH to disrupt antibody-antigen interaction. Some complement proteins are labile at these pH extremes and methods must be modified accordingly (Table 1 ). Here, I will describe the steps followed in establishing an immunoaffinity protocol for a complement protein. These differ little from those used for any other target protein. Example protocols will be provided for a serum complement protein and a membrane bound complement regulator.

Componen	Problem	Solution
C l	Dissociates in absence of	Avoid chelating agents in serum/plasma,
	divalent cations	use VBS instead of PBS, avoid chaotropes,
		include protease inhibitor NPGB (1 mM).
C2/fB	Very prone to	Ensure all steps are performed at 4�C,
	proteolysis/denaturation	include protease inhibitors in plasma and
		buffers.
C3/C4	Very abundant in plasma,	Reduce the volume of plasma used-20 mL
	column saturates	plasma should yield >10 mg C3!
fD	Very low concentrations	Consider alternative sources-urine from
	in plasma	patients with renal pathology?
Properdin	Tendency to form	Minimize time of procedure keep at 4�C
	aggregates	include protease inhibitors.
C8	Noncovalently associated	Avoid high salt wash (250 mM max), use
	subunits (αγ/β)dissociate	solid phase which binds both subunits
	in high salt	(mixed MAb).
C9	Labile at pH extremes	Elute in chaotropes (e.g., 2 M MgCl2 ) or
		minimize time of exposure by neutralizing
		immediately.