51Cr Release Assay of Antibody‐Dependent Cell‐Mediated Cytotoxicity (ADCC)

David L. Nelson1, Carole C. Kurman1, Deborah E. Serbousek1

1 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.27
DOI:  10.1002/0471142735.im0727s08
Online Posting Date:  May, 2001
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Abstract

Antibody‐dependent cell‐mediated cytotoxicity (ADCC) is an immunologic cytotoxic effector mechanism that is dependent on the cooperative interaction of humoral and cellular effector elements. This unit describes an assay of ADCC activity that can be used as a test for immunocompetence in effector cells or to test the activity of a monoclonal antibody to mediate ADCC. In this form of cytotoxicity, effector cells with receptors for the Fc portion of immunoglobulin produce target cell lysis by attachment to the Fc portion of antibodies that are bound to target cells via their antigen‐combining sites. Therefore, an ADCC assay involves three essential components: labeled target cells, antibodies with specificity for target‐cell surface antigens, and effector‐cell populations. The basic protocol describes a method of measuring ADCC effector activity in lymphoid cells (peripheral blood mononuclear cells, or PBMC) that employs 51Cr‐labeled target cells. The three components are mixed in microtiter‐plate wells and lysis of the target cells is detected by measuring the release of radioactivity into the cell supernatant. Support protocols describe procedures for preparing anti‐target cell antiserum and 51Cr‐labeled target cells.

     
 
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Table of Contents

  • Support Protocol 1: Preparation of Anti–Target Cell Antiserum
  • Support Protocol 2: Preparation of 51Cr‐Labeled Target Cells
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Complete RPMI‐10 medium ( appendix 2A) with 25 mM HEPES
  • Effector cells: Ficoll‐Hypaque‐gradient‐separated preparation of peripheral blood mononuclear cells (PBMC; unit 7.1) suspended in complete RPMI‐10 medium at 1 × 107 cells/ml
  • Antibody‐coated and non‐antibody‐coated 51Cr‐labeled target‐cell (e.g., Chang‐cell) suspensions in complete RPMI‐10 medium (1 × 105 cells/ml; second protocol 3support protocol)
  • 5% (v/v) Triton X‐100
  • Single and multichannel pipettors
  • 6‐ml centrifuge tubes (e.g., Falcon)
  • 96‐well round‐bottom microtiter plates with covers (Costar)
  • Sorvall RC‐3B centrifuge with holder for plates or equivalent
  • Humidified 37°C, 5% CO 2 incubator
  • 15‐ml centrifuge tubes (e.g., Sarstedt)
  • Weighing boat or other apparatus for pipetting solutions using multichannel pipettor
  • Tubes for γ counting: e.g., snap‐cap BioVials ( Beckman)
  • Multiwick supernatant harvesting system (e.g., Skatron; optional)
  • Mechanism for counting γ emission (e.g., γ scintillation counter)
  • Additional reagents and equipment for counting cells using a hemacytometer and for trypan blue exclusion testing of cell viability ( appendix 3B)

Support Protocol 1: Preparation of Anti–Target Cell Antiserum

  Materials
  • Target cells: Chang cells (ATCC# CCL13) or other suitable cells
  • (see )
  • Freunds complete and incomplete adjuvants (Difco)
  • Additional reagents and equipment for subcutaneous and intramuscular injection (unit 1.6), bleeding (unit 1.7), and immunization and preparation of serum from blood (unit 2.4)

Support Protocol 2: Preparation of 51Cr‐Labeled Target Cells

  Materials
  • Target cells: Chang cells (ATCC# CCL13) or other suitable cells (See )
  • Rabbit anti–target cell antiserum (previous protocol 2support protocol)
  • 1 mCi/ml Na 251 CrO 4 (250 to 500 mCi/mg; Amersham)
  • Sorvall RC‐3B centrifuge with tube holder or equivalent
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Figures

Videos

Literature Cited

Literature Cited
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