Alternative 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 13.2
DOI:  10.1002/0471142735.im1302s09
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: AH50 Assay for Total Alternative Pathway Hemolytic Activity
  • Basic Protocol 2: Factor B or D Hemolytic Assay (FBHA or FDHA)
  • Support Protocol 1: Preparation of Standardized Erab
  • Support Protocol 2: Preparation of Factor D–Depleted Serum
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: AH50 Assay for Total Alternative Pathway Hemolytic Activity

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2A ; for suppliers, see appendix 55.
  • Test serum sample (see )
  • Control sera: calibrated standard serum and/or normal pooled serum (NPS) or serum from an individual donor with normal AH 50 (see )
  • recipeGVB/MgEGTA buffer (see recipe)
  • 2 × 108 cell/ml E rab suspension (first protocol 3support protocol)
  • 0.15 M NaCl, ice cold
  • Disposable 12 × 75–mm borosilicate glass culture tubes
  • Probability × 3‐log‐cycle paper or 2‐cycle log‐log paper
NOTE: All reagents, cells, and sera are to be kept on ice throughout this protocol unless otherwise noted.

Basic Protocol 2: Factor B or D Hemolytic Assay (FBHA or FDHA)

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2A ; for suppliers, see appendix 55.
  • Test serum sample (see )
  • Control sera: calibrated standard serum and/or normal pooled serum (NPS) or serum from an individual donor with normal AH 50 (see )
  • recipeGVB/MgEGTA buffer (see recipe), ice cold
  • recipeFactor B–depleted serum (see recipe) or factor D–depleted serum (second protocol 4support protocol)
  • 2 × 108 cell/ml E rab suspension (first protocol 3support protocol)
  • 0.15 M NaCl, ice cold
  • Disposable 12 × 75–mm borosilicate glass culture tubes
NOTE: All reagents, cells, and sera are to be kept on ice throughout this protocol unless otherwise noted.

Support Protocol 1: Preparation of Standardized Erab

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2A ; for suppliers, see appendix 55.
  • recipeRabbit blood in Alsevers solution (see recipe)
  • recipeGVB/MgEGTA buffer (see recipe), ice‐cold and 37°C
  • 50‐ml conical‐bottom plastic centrifuge tube
  • Disposable 12 × 75–mm borosilicate glass culture tubes
NOTE: All reagents, cells, and sera are to be kept on ice throughout this protocol unless otherwise noted.

Support Protocol 2: Preparation of Factor D–Depleted Serum

  MaterialsFor recipes, see in this unit (or cross‐referenced unit); for common buffers and stock solutions, see appendix 2A; for suppliers, see appendix 55.
  • BioRex 70 chromatography resin (Bio‐Rad)
  • recipeVBS buffer (see recipe)
  • ACD‐plasma from a local blood bank or donor blood collected in ACD (acid‐citrate‐dextrose)
  • 1 M HCl ( appendix 2A)
  • 0.5 M EDTA ( appendix 2A)
  • recipeVBS/MgEGTA buffer (see recipe)
  • Normal human serum or NPS, diluted 1/20 in recipeGVB/MgEGTA
  • 2 × 108 cells/ml E rab suspension (first protocol 3support protocol)
  • recipeGVB/MgEGTA buffer (see recipe)
  • 0.15 M NaCl, ice cold
  • 100‐ to 150‐ml chromatography column
  • Additional reagents and equipments for column chromatography and dialysis ( appendix 3A)
NOTE: All reagents, cells, and sera are to be kept on ice throughout this protocol unless otherwise noted.
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Figures

Videos

Literature Cited

Literature Cited
   Harbeck, R.J. and Giclas, P.C. 1991. Diagnostic Immunology Laboratory Manual. Raven Press, New York.
   Lesavre, P.H., Hugli, T.E., Esser, A.F., and Muller‐Eberhard, H.J. 1979. The alternative pathway C3/C5 convertase: Chemical basis of factor B activation. J. Immunol 123:529‐534.
   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.
   Pangburn, M.K. 1981. The alternative pathway. In Immunobiology of the Complement System (G. Ross, ed.) pp. 45‐62. Academic Press, San Diego.
   Pangburn, M.K. 1988. Alternative pathway of complement. Methods Enzymol. 162:648‐649.
   Platts‐Mills, T.A.E. and Ishizaka, K. 1974. Activation of the alternative pathway of human complement by rabbit cells. J. Immunol. 113:348‐358.
   Reid, K.B.M. and Porter, R.R. 1981. The proteolytic activation systems of complement. Annu. Rev. Biochem. 50:433‐464.
   Reid, K.B.M., Johnson, D.M.A., Gagnon, J., and Prohaska, R. 1988. Preparation of human factor D of the alternative pathway of complement. Methods Enzymol. 80:134‐143.
   Whaley, 1985. Methods in Complement for Clinical Immunologists. Churchill Livingstone, Edinburgh.
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