Assays for the Mannan‐Binding Lectin Pathway

Mihaela Gadjeva1, Steffen Thiel1, Jens C. Jensenius1

1 University of Aarhus, Aarhus
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 13.6
DOI:  10.1002/0471142735.im1306s58
Online Posting Date:  February, 2004
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Abstract

This unit contains protocols that can be used to measure mannan‐binding lectin (MBL) levels and MBL pathway activity in human plasma or serum. Using a modification of the conventional ELISA, the detection reagent used in the methods described (e.g., an antibody or streptavidin) is labeled with Eu3+ instead of an enzyme. The sensitizing reagent used in these assays employ the use of anti‐MBL antibody or mannan. The unit also includes an MBL/MASP complex activity assay to evaluate MBL activity in serum or plasma samples, which allows for the measurement of the specific activity of the MBL pathway.

Keywords: complement; lectin pathway activity; mannan binding lectin; mannose binding lectin (MBL)

     
 
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Table of Contents

  • Basic Protocol 1: Quantification of MBL as an Antigen
  • Alternate Protocol 1: Quantification of MBL as a Lectin
  • Basic Protocol 2: MBL/MASP Complex Activity Assay
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Quantification of MBL as an Antigen

  Materials
  • Coating antibody: anti‐MBL antibody of choice (e.g., monoclonal 131‐1, Immunolex)
  • Biotinylation kit (e.g., Pierce)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 5 µg/ml anti‐MBL antibody
  • TBS ( appendix 2A) containing 0.05% (v/v) Tween 20 (store at room temperature)
  • Serum (unit 13.1) or plasma (unit 13.3) samples for analysis (store in aliquots at −80°C until use)
  • MBL binding buffer for antigen assay (see recipe)
  • Detection antibody: monoclonal anti‐MBL‐antibody of choice (e.g., 131‐1, Immunolex)
  • Streptavidin‐Eu3+ (Wallac/Perkin‐Elmer)
  • TBS ( appendix 2A) containing 0.05% (v/v) Tween 20 and 25 µM EDTA
  • Enhancement solution (see recipe)
  • 96‐well FluoroNunc microtiter plates (Nunc)
  • Moist chamber (see, e.g., unit 3.17)
  • Time‐resolved fluorometer: e.g., Wallac 1232 DELFIA fluorometer (Wallac Oy/Perkin‐Elmer)
  • Additional reagents and equipment for biotinylation of antibodies (unit 7.12 or unit) and ELISA (unit 2.1)

Alternate Protocol 1: Quantification of MBL as a Lectin

  • Coating buffer (see recipe) containing 10 µg/ml mannan (Sigma)
  • Blocking buffer (see recipe)
  • TBS containing 0.05% (v/v) Tween 20 and 5 mM CaCl 2
  • MBL binding buffer for lectin assay (see recipe)

Basic Protocol 2: MBL/MASP Complex Activity Assay

  Materials
  • Anti‐human C4 antibodies (DAKO) or monclonal anti‐human C4 antibodies 162.1 and 162.2 (State Serum Institute, Denmark)
  • Biotinylation kit (e.g., Pierce)
  • Coating buffer (see recipe) containing 10 µg/ml mannan (Sigma)
  • Blocking buffer (see recipe)
  • TBS ( appendix 2A) containing 0.05% (v/v) Tween 20, with and without 5 mM CaCl 2 (store at room temperature)
  • Serum (unit 13.1) or plasma (unit 13.3) samples for analysis (store in aliquots at −80°C until use)
  • MBL binding buffer for MBL/MASP complex activity assay (see recipe)
  • 2 µg/ml human complement C4 (Quidel) in C4 dilution buffer (see recipe for buffer)
  • Streptavidin‐Eu3+ (Wallac/Perkin‐Elmer)
  • TBS ( appendix 2A) containing 0.05% (v/v) Tween 20 and 25 µM EDTA
  • Enhancement solution (see recipe)
  • 96‐well FluoroNunc microtiter plates (Nunc)
  • Moist chamber (see, e.g., unit 3.17)
  • Time‐resolved fluorometer: e.g., Wallac 1232 DELFIA fluorometer (Wallac Oy/Perkin‐Elmer)
  • Additional reagents and equipment for biotinylation of antibodies (unit 7.12 or unit 10.19) and ELISA (unit 2.1)
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Figures

Videos

Literature Cited

Literature Cited
   Dodds, A.W. 1993. Small‐scale preparation of complement components C3 and C4. Methods Enzymol. 223:46‐61.
   Ikeda, K., Sannoh, T., Kawasaki, N., Kawasaki, T., and Yamashina, I. 1987. Serum lectin with known structure activates complement through the classical pathway. J. Biol. Chem. 262:7451‐7454.
   Kuipers, S., Aerts, P.C., Sjoholm, A.G., Harmsen, T., and van Dijk, H. 2002. A hemolytic assay for the estimation of functional mannose‐binding lectin levels in human serum. J. Immunol. Methods 268:149‐157.
   Petersen, S.V., Thiel, S., Jensen, L., Steffensen, R., and Jensenius, J.C. 2001. An assay for the mannan‐binding lectin pathway of complement activation. J. Immunol. Methods 257:107‐116.
   Super, M., Levinsky, R.J., and Turner, M.W. 1990. The level of mannan‐binding protein regulates the binding of complement‐derived opsonins to mannan and zymosan at low serum concentrations. Clin. Exp. Immunol. 79:144‐150.
   Thiel, S., Moller‐Kristensen, M., Jensen, L., and Jensenius, J.C. 2002. Assays for the functional activity of the mannan‐binding lectin pathway of complement activation. Immunobiology 205:446‐454.
   Turner, M.W., Johnson, M., Booth, C., Klein, N., Rolland, J., and Davies, J. 2003. Assays for human mannose‐binding lectin. J. Immunol. Methods 276:147‐149.
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