Classical Pathway Evaluation

Patricia C. Giclas1

1 National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 13.1
DOI:  10.1002/0471142735.im1301s09
Online Posting Date:  May, 2001
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Abstract

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.

     
 
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Table of Contents

  • Basic Protocol 1: CH50 Assay for Total Classical Pathway Hemolytic Activity
  • Alternate Protocol 1: Microtiter CH50 Assay
  • Support Protocol 1: Collection and Storage of Samples for Complement Function Assays
  • Support Protocol 2: Preparation of Standardized Erythrocyte‐Antibody Complex (EA) Suspensions
  • Support Protocol 3: Titration of Hemolysin
  • Basic Protocol 2: Measurement of Complement Component Levels by Radial Immunodiffusion (RID)
  • Basic Protocol 3: Measurement of C1 Esterase Inhibitor (C1‐INH) Function by Indirect RID
  • Support Protocol 4: Titering Antibody for RID
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: CH50 Assay for Total Classical Pathway Hemolytic Activity

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Serum sample (first protocol 3support protocol)
  • Control sera: calibrated standard serum and/or normal pooled serum (NPS; see Critical Parameters)
  • recipeGVB++ buffer (see recipe), ice cold
  • 2 × 108 EA/ml suspension (second protocol 4support protocol)
  • 0.15 M NaCl, ice cold
  • Disposable 12 × 75–mm borosilicate glass culture tubes
  • Centrifuge with carriers for 12 × 75–mm and 50‐ml tubes
  • Probability × 3‐log‐cycle paper (Keuffel & Esser) or 2‐cycle log‐log paper
NOTE: All reagents, sera, and cells are to be kept on ice throughout this protocol unless otherwise noted.

Alternate Protocol 1: Microtiter CH50 Assay

  Additional MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • U‐ or V‐bottom 96‐well microtiter plate with lid
  • Flat‐bottom 96‐well microtiter plate
  • Centrifuge with carriers for microtiter plates
  • Microtiter plate reader with 540‐nm filter
NOTE: All reagents, sera, and cells are to be kept on ice throughout this protocol unless otherwise noted.

Support Protocol 1: Collection and Storage of Samples for Complement Function Assays

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Sheep whole blood in Alsevers solution (e.g., from Colorado Serum)
  • recipeGVBE buffer (see recipe)
  • recipeGVB++ buffer (see recipe)
  • Titrated hemolysin (third protocol 5support protocol)
  • 50‐ml conical‐bottom plastic tubes with screw caps
NOTE: All reagents, sera, and cells are to be kept on ice throughout this protocol unless otherwise noted.

Support Protocol 2: Preparation of Standardized Erythrocyte‐Antibody Complex (EA) Suspensions

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • Packed sheep red blood cells (step or of second protocol 4support protocol)
  • Normal human or guinea pig serum (as complement source)
  • recipeGVB++ buffer (see recipe)
  • recipeHemolysin (see recipe)
  • 56°C water bath
  • Disposable 12 × 75–cm borosilicate glass culture tubes
  • Semilog graph paper

Support Protocol 3: Titration of Hemolysin

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2A; for suppliers, see suppliers appendix .
  • 2% (w/v) SeaKem ME agarose ( FMC Bioproducts) in modified Mancini buffer, dissolved and allowed to harden (store at 4°C)
  • recipeModified Mancini buffer (see recipe), 60°C
  • Titered polyclonal antiserum specific for the complement component to be assayed (third protocol 5support protocol)
  • 0.15 M NaCl
  • recipeSerum test sample (see recipe)
  • Control sera: normal pooled serum (NPS) and calibrated standard serum or purified antigen of known concentration (see Critical Parameters)
  • 0.2% (w/v) Coomassie brilliant blue in recipedestaining solutionor recipeCrowles double stain (see reciperecipe)
  • recipeDestaining solution (see recipe)
  • Disposable 16 × 100–mm glass test tubes
  • GelBond film (FMC BioProducts), 60‐ or 100‐mm petri dishes with lids, or microscope slides
  • Leveling table
  • Humidified chamber (e.g., box with damp paper towel or sponge)
  • 3‐mm stainless steel hole punch (e.g., 3‐mm skin biopsy punch)
  • Pasteur pipet attached to a suction device (e.g., vacuum‐trap apparatus)
  • Whatman No. 1 filter paper
  • 6 × 8–in. glass or similar smooth flat plate
  • Loupe with inset millimeter scale or equivalent measuring device

Basic Protocol 2: Measurement of Complement Component Levels by Radial Immunodiffusion (RID)

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • recipeSerum test sample (see recipe)
  • Positive control: normal pooled serum (NPS) or serum from an individual with normal C1‐INH function (store in 0.25‐ml aliquots at −70°C and discard unused portion after thawing; see Critical Parameters)
  • recipeNegative control: serum with abnormal C1‐INH function (see recipe and Critical Parameters)
  • recipeHeat‐aggregated IgG (HAGG; see recipe)
  • 0.15 M NaCl
  • Agarose gels (second protocol 6basic protocol)
  • Goat anti‐human C1r antiserum (IgG fraction, Incstar), titered (third protocol 5support protocol)
  • 0.25 M EDTA
  • 2‐mm hole punch
  • Pasteur pipet attached to a suction device (e.g., vacuum‐trap apparatus)
  • Humidified chamber (e.g., box with damp paper towel or sponge), 4°C
  • Loupe with inset millimeter scale or equivalent measuring device
NOTE: All sera are to be kept on ice throughout this protocol unless otherwise noted.

Basic Protocol 3: Measurement of C1 Esterase Inhibitor (C1‐INH) Function by Indirect RID

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • 2% (w/v) SeaKem ME agarose (FMC Bioproducts) in modified Mancini buffer, dissolved and allowed to harden (store at 4°C)
  • recipeModified Mancini buffer (see recipe), prewarmed to 60°C
  • Polyclonal goat antisera specific for the individual complement component(s) to be tested ( Quidel, Incstar, Kent Labs, or Binding Site)
  • Controls: normal pooled serum (NPS) and calibrated serum or purified antigen of known concentration (see Critical Parameters)
  • GelBond film (FMC Bioproducts)
  • Leveling table
  • 60°C and boiling water bath
  • Disposable 16 × 100–mm glass test tubes
  • Pipets, prewarmed to 60°C
  • 3‐mm stainless steel hole punch (e.g., 3‐mm skin biopsy punch)
  • Humidified chamber (e.g., box with damp paper towel or sponge), 4°C
  • Loupe with inset millimeter scale or equivalent measuring device
  • Additional reagents and equipment for agarose gel electrophoresis (unit 10.4)
NOTE: All reagents, sera, and cells are to be kept on ice throughout this protocol unless otherwise noted.
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Figures

Videos

Literature Cited

Literature Cited
   Barta, V. and Barta, O. 1993. Testing of hemolytic complement and its components. In Veterinary Clinical Immunology Laboratory (O. Barta, ed.) pp. C6.1‐C6.40. Bar‐Lab, Virginia.
   Caldwell, J.R., Ruddy, S., and Austen, K.F. 1972. Assay of complement components C1, C4, C2, C3 and C9 in whole rat serum. Int. Arch. Allergy Appl. Immunol. 43:887‐897.
   Check, I., Piper, M., and Papadea, C. 1992. Immunoglobulin quantitation. In Manual of Clinical Laboratory Immunology, 4th ed., (N.R. Rose, E.C. DeMacario, J.L. Fahey, H. Friedman, and G.M. Penn, eds.) pp. 74‐80. American Society for Microbiology, Washington, DC.
   Crowle, A.J. 1961. Immunodiffusion. Academic Press, San Diego.
   Cullmann, W. and Opferkuch, W. 1986. Deficiencies in regulator proteins. 1. C1 Inhibitor. In Hereditary and Acquired Complement Deficiencies in Animals and Man (K. Rother and U. Rother, eds.). Prog. Allergy 39:311‐334.
   Donaldson, V.H. and Evans, R.R. 1963. A biochemical abnormality in C′1 esterase inhibitor. Am. J. Med. 35:37‐44.
   Gelfand, J.A., Boss, G.R., Conley, C.L., Rheinhart, R., and Frank, M.M. 1979. Acquired C′1 esterase inhibitor deficiency and angioedema: A review. Medicine 58:321‐327.
   Giclas, P.C., Keeling, P.J., and Henson, P.M. 1981. Isolation and characterization of the third and fifth components of rabbit complement. Mol. Immunol 18:113‐123.
   Goldman, M.B. and Goldman, J.N. 1976. Relationship of functional levels of early components of complement to the H‐2 complex of mice. J. Immunol. 117:1584‐1588.
   Harbeck, R.J. and Giclas, P.C. 1991. Section I: Assessment of Complement Components and their Function. In Diagnostic Immunology Laboratory Manual (R.J. Harbeck and P.C. Giclas, eds.) pp.21‐78. Raven Press, New York.
   Harrison, R.A. and Lachmann, P.J. 1986. Complement technology. In Handbook of Experimental Immunology, Vol. 1 (D.M. Weir, ed.) pp. 39.1‐39.49. Blackwell Scientific, Oxford.
   Lowry, O.H., Rosenbrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265‐276.
   Mancini, G., Carbonara, A.O., and Heremans, F.J. 1965. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235‐254.
   Mayer, M.M. 1961. Complement v. complement fixation. In Experimental Immunochemistry, 2nd ed (E.A. Kabat and M.M. Mayer, eds.) pp. 133‐239. Thomas, Springfield, Ill.
   Opferkuch, W., Rother, K., and Schultz, D.R. eds. 1978. Clinical Aspects of the Complement System. PSG Publishing, Mass.
   Rapp, H.J. and Borsos, T. 1970. Molecular Basis of Complement Action Appleton‐Century‐Crofts, New York.
   Ruddy, S. 1988. Hereditary angioedema: Undersuspected, underdiagnosed. Hosp. Pract. (Aug. 15):91‐106.
   Sim, R.B. (ed.) 1993. Activators and Inhibitors of Complement. Kluwer Academic Publishers, Boston.
   Smith, J.A. 1987. Colorimetric methods. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 10.1.1‐10.1.3. Greene Publishing and John Wiley & Sons, New York.
   Weeke, B. 1973. Rocket immunoelectrophoresis. In A Manual of Quantitative Immuno‐Electrophoresis (Scand. J. Immunol 2, Suppl. 1) pp. 37‐46.
   Whaley, K. 1985. Measurement of complement. In Methods in Complement for Clinical Immunologists (K. Whaley, ed.) pp. 77‐144. Churchill Livingston, Edinburgh.
   Ziccardi, R.J. and Cooper, N.R. 1979. Development of an immunochemical test to assess C1 inactivator function in human serum and its use for the diagnosis of hereditary angioedema. Clin. Immunol. Immunopathol. 15:465‐471.
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