Immunoaffinity Methods for Purification of Complement Components and Regulators

  • B. Paul Morgan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 150)

Abstract

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, 2, 3, 4, 5, 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.
Table 1

Modifications of Basic Protocol for Specific Components

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.

Keywords

EDTA Epoxy Glycine Butanol Ghost 

References

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Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • B. Paul Morgan
    • 1
  1. 1.Department of Medical BiochemistryUniversity of Wales College of MedicineCardiff

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