Isolation and Purification of C3 from Human Plasma

Lynda D. O'Rear1, Gordon D. Ross1

1 University of Louisville, Louisville, Kentucky
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 13.3
DOI:  10.1002/0471142735.im1303s14
Online Posting Date:  May, 2001
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Abstract

The alternative pathway of complement shares its terminal components (C3 and C5 through 9) with the classical pathway, but has several unique components, including factors D, B, and P (properdin). This unit presents methods for assaying total alternative pathway activity and the activity of factors B and D. Radial immunodiffusion (RID) can also be used to measure factor D, B, and P concentrations.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of C3 from Human Plasma
  • Support Protocol 1: Preparation and Testing of Lysine‐Agarose
  • Support Protocol 2: Preparation of DEAE‐Sephacel Column
  • Support Protocol 3: Double‐Immunodiffusion Assay for Complement Factors
  • Support Protocol 4: Hemolytic Assay for C5
  • Support Protocol 5: Preparative FPLC Chromatography of C3 Fraction
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Isolation of C3 from Human Plasma

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Four blood donors with the same ABO blood‐group type
  • recipeProtease inhibitor stock solution (see recipe)
  • recipePhenylmethylsulfonyl fluoride (PMSF) stock solution (see recipe)
  • Lysine‐agarose equilibrated with LA buffer and tested for activity (first protocol 2support protocol)
  • recipeLA buffer (see recipe)
  • ε‐amino‐n‐caproic acid (EACA)
  • recipeDEAE starting buffer (see recipe)
  • Polyethylene glycol (PEG) 4000 (4000 to 6000 MW; Union Carbide)
  • recipeLow‐ionic‐strength diluent (see recipe)
  • recipeDEAE starting buffer with 0.3 M sodium chloride (dissolve solid sodium chloride in buffer)
  • recipeS‐300 starting buffer (see recipe)
  • Sephacryl 300 HR filtration medium (Pharmacia Biotech)
  • Blood collection bags coated with acid citrate/dextrose (ACD) anticoagulant
  • 1‐liter or 500‐ml and 250‐ml centrifuge bottles
  • Sorvall RC‐3B centrifuge with H6000A rotors or equivalent
  • 3‐liter Buchner funnel with sintered‐glass filter
  • 4‐liter side‐arm flask
  • Pellicon concentration apparatus (Millipore)
  • Pellicon cassette with 30,000‐MW cutoff (Millipore)
  • Conductivity meter
  • Equilibrated 5 × 95–cm DEAE‐Sephacel column (second protocol 3support protocol)
  • Peristaltic pump (e.g., Pharmacia Biotech P‐1 pump)
  • UV monitor/recorder (e.g., Pharmacia Biotech UV‐1 monitor)
  • Two 7‐liter carboys with bottom spouts
  • Tygon tubing of appropriate diameter for carboy spouts
  • Pinch‐clamp for Tygon tubing
  • Sorvall RC‐24 centrifuge with GSA and SS‐34 rotors or equivalent
  • Preparative anion‐exchange FPLC column (e.g., Pharmacia Biotech Mono Q column)
  • Ultrafiltration concentrator cell and YM‐30 membrane (Amicon)
  • Additional reagents and equipment for dialysis ( appendix 3A) and SDS‐polyacrylamide gel electrophoresis (unit 8.4)
NOTE: Plasma must be kept at 4°C at all times; therefore, all steps should be performed in a cold room and all reagents should be ice cold unless otherwise indicated.

Support Protocol 1: Preparation and Testing of Lysine‐Agarose

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Sepharose CL‐4B gel filtration medium (Pharmacia Biotech)
  • recipe10 M and 1 M sodium hydroxide
  • Cyanogen bromide (CNBr)
  • 0.1 M bicarbonate buffer (pH 8.9), ice cold
  • L‐lysine monohydrochloride
  • recipe1 M monoethanolamine, pH 8.0 (see recipe)
  • recipeLA buffer (see recipe)
  • Fresh plasma ( protocol 1basic protocol)
  • 0.2 M ε‐amino‐n‐caproic acid (EACA)
  • recipePhosphate‐buffered saline (PBS; appendix 2A)
  • recipePBS containing 10 mM sodium azide
  • 3‐liter glass Buchner funnel with sintered‐glass filter
  • 4‐liter side‐arm flask
CAUTION: This procedure uses cyanogen bromide to couple L‐lysine to Sepharose. During the procedure, deadly hydrogen cyanide gas is generated. Accordingly, great care must be used to prevent cyanide exposure. The entire procedure should be carried out under a fume hood that is known to be functioning properly and gloves must be worn at all times.

Support Protocol 2: Preparation of DEAE‐Sephacel Column

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • 1 M sodium chloride
  • DEAE‐Sephacel ion exchange medium (Pharmacia Biotech)
  • recipeDEAE equilibration buffer (see recipe)
  • recipeDEAE starting buffer (see recipe)
  • 3‐liter Buchner funnel with sintered‐glass filter
  • Conductivity meter
  • 5 × 100–cm glass column
  • UV monitor/recorder (e.g. Pharmacia Biotech UV‐1 monitor)

Support Protocol 3: Double‐Immunodiffusion Assay for Complement Factors

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Agarose (powdered electrophoresis‐grade)
  • 43.55 mM sodium barbital, pH 8.6
  • Antigen‐containing samples to be tested
  • Antisera directed against the factor(s) to be assayed
  • recipePhosphate‐buffered saline (PBS; appendix 2A)
  • 0.1% (w/v) Coomassie brilliant blue in 30% (v/v) methanol/10% (v/v) acetic acid
  • 30% (v/v) methanol/10% (v/v) acetic acid
  • 20‐ml glass test tubes
  • Template (unit 2.3)
  • Coplin jars
  • Lens paper

Support Protocol 4: Hemolytic Assay for C5

  Additional Materials
    For recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Rabbit IgM anti‐sheep erythrocyte antibody (Diamedix)
  • recipeGVBE buffer (see recipe), room temperature and ice cold
  • Sheep erythrocytes at 2 × 109 cells/ml in recipeGVBE buffer
  • recipeGVB++ buffer (see recipe), room temperature and ice cold
  • C5‐containing samples ( protocol 1basic protocol)
  • C5 standard purified from human serum (Quidel)
  • C5‐depleted human serum (Quidel)
  • Sorvall H6000A rotor (or equivalent)
  • 96‐well V‐bottom microtiter plates
  • 25‐µl pipet droppers (PGC Scientifics)
  • 25‐µl manual microdiluters (PGC Scientifics)
  • Refrigerated centrifuge with microplate carriers (e.g., Sorvall RT6000B with H1000B rotor)
  • Minishaker (PGC Scientifics)
  • Test‐reading mirror (PGC Scientifics)

Support Protocol 5: Preparative FPLC Chromatography of C3 Fraction

  Additional MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Mono Q HR 16/10 preparative FPLC column (Pharmacia Biotech)
  • recipeMono Q starting buffer (see recipe)
  • recipeMono Q starting buffer with 0.24 M sodium chloride (dissolve solid sodium chloride in buffer)
  • Activated Thiol Sepharose 4B (ATS) coupling gel medium (Pharmacia Biotech)
  • Small column (2 × 20–cm) with fritted glass filter
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Figures

Videos

Literature Cited

Literature Cited
   Basta, M. and Hammer, C.H. 1991. A rapid FPLC method for purification of the third component of human and guinea pig complement. J. Immunol. Methods 142:39‐44.
   Deutsch, D.G. and Mertz, E.T. 1970. Plasminogen: Purification from human plasma by affinity chromatography. Science 170:1095‐1096.
   Hammer, C.H., Wirtz, G.H., Renfer, L., Gresham, H.D., and Tack, B.F. 1981. Large scale isolation of functionally active components of the human complement system. J. Biol. Chem. 256:3995‐4006.
   Hsiung, L.‐M., Barclay, A.N., Brandon, M.R., Sim, E., and Porter, R.R. 1982. Purification of human C3b inactivator by monoclonal‐antibody affinity chromatography. Biochem. J. 203:293‐298.
   Ripoche, J., Erdei, A., Gilbert, D., Al Salihi, A., Sim, R.B., and Fontaine, M. 1988. Two populations of complement factor H differ in their ability to bind to cell surfaces. Biochem. J. 253:475‐480.
   Tack, B.F. and Prahl, J.W. 1976. The third component of human complement: Purification from plasma and physicochemical characterization. Biochemistry 15:4513‐4521.
Key References
   Bokisch, V.A., Müller‐Eberhard, H.J. and Cochrane, C.G. 1969. Isolation of a fragment (C3a) of the third component of human complement containing anaphylatoxin and chemotactic activity and description of an anaphylatoxin inactivator of human serum. J. Exp. Med. 129:1109‐1130.
  The first paper that attempted to isolate and characterize structurally the important fragments of C3: C3a, C3b, C3c, and C3d.
   Hammer, C.H., Wirtz, G.H., Renfer, L., Gresham, H.D., and Tack, B.F. 1981. See above.
  Described methods for the large scale isolation of C components from human plasma; the major basis for the methods currently in use.
   Janatova, J. 1988. C3, C5 components and C3a, C4a, and C5a fragments of the complement system. Methods Enzymol. 162:579‐625.
  A more recent review that summarized some alternative methods to isolate C3 and C5, as well as their activation peptides that function as anaphylatoxins.
   Tack, B.F. and Prahl, J.W. 1976. See above.
  Described major advances in the methods used to isolate C3, most importantly, the control of proteolytic inactivation of C3 with protease inhibitors; a starting point for subsequent improvements in C3 isolation techniques.
   Tack, B.F., Janatova, J., Thomas, M.L., Harrison, R.A., and Hammer, C.H. 1981. The third, fourth, and fifth components of human complement: Isolation and biochemical properties. Methods Enzymol. 80:64‐101.
  A review that summarized isolation procedures and the state of knowledge at that time of structure and function of the related C components C3, C4, and C5 (subsequent advances included cDNA and gene sequence analysis of this C protein family).
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