Titering Antibodies

Carleton C. Stewart1, Sigrid J. Stewart1

1 Roswell Park Cancer Institute, Buffalo, New York
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 4.1
DOI:  10.1002/0471142956.cy0401s14
Online Posting Date:  May, 2001
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Abstract

Flow cytometry using fluorochrome‐conjugated antibodies has emerged as a major approach to automated cellular identification. One of the most important issues in immunophenotyping is using the correct amount of antibody. This unit presents techniques for ascertaining the optimal titer for individual, dual, and multiple antibodies used for simultaneous phenotyping, stressing the importance of quality control in making batches of antibody for routine use. Keywords: flow cytometry; monoclonal antibodies; antibody titration; immunophenotyping.

     
 
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Table of Contents

  • Basic Protocol 1: Titering Directly Conjugated Antibodies to Extracellular Antigens
  • Alternate Protocol 1: Titering Biotinylated or Hapten‐Conjugated Antibodies
  • Alternate Protocol 2: Titering of Indirect Antibody to Extracellular Antigen
  • Basic Protocol 2: Titering Directly Conjugated Antibodies to Intracellular Antigens
  • Alternate Protocol 3: Titering Biotinylated, Hapten‐Conjugated, or Secondary Antibodies to Intracellular Antigens
  • Basic Protocol 3: Epitope Titering
  • Basic Protocol 4: Verifying Performance of Antibody Combinations
  • Basic Protocol 5: Batch Production of Antibody Combinations
  • Commentary
  • Key Reference
  • Figures
     
 
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Materials

Basic Protocol 1: Titering Directly Conjugated Antibodies to Extracellular Antigens

  Materials
  • Stock solution of specific, fluorochrome‐conjugated antibody to be titered
  • Target cells for antibody to be titered
  • PBS without calcium or magnesium (e.g., appendix 2A or Life Technologies)
  • Normal mouse IgG (e.g., Caltag)
  • 2% ultrapure formaldehyde in PBS (prepared from 10% EM‐grade solution; Polysciences)
  • Ammonium chloride lysing solution ( appendix 2A), prepared fresh
  • Isotype control myeloma proteins (fluorochrome conjugated)
  • 12 × 75–mm polypropylene test tubes (Falcon)
  • Sorvall centrifuge and H1000B rotor, or equivalent
  • Flow cytometer

Alternate Protocol 1: Titering Biotinylated or Hapten‐Conjugated Antibodies

  • Biotinylated or hapten‐conjugated primary antibody
  • Labeled strepavidin, or anti‐hapten, secondary antibody with conjugated fluorochrome

Alternate Protocol 2: Titering of Indirect Antibody to Extracellular Antigen

  • Antibodies or antisera against extracellular antigen
  • Normal goat IgG against primary antibody species, fluorochrome conjugated (Caltag)

Basic Protocol 2: Titering Directly Conjugated Antibodies to Intracellular Antigens

  • Target cells that do and do not express the desired epitope

Alternate Protocol 3: Titering Biotinylated, Hapten‐Conjugated, or Secondary Antibodies to Intracellular Antigens

  • Avidin or anti‐hapten antibody, or second antibody with conjugated fluorochrome

Basic Protocol 3: Epitope Titering

  • Specific antibodies with appropriate fluorochromes
  • Isotype control myeloma proteins with appropriate fluorochromes

Basic Protocol 4: Verifying Performance of Antibody Combinations

  • Previous antibody combination batch
  • Antibody combination batch
  • Isotype control combination batch
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Figures

Videos

Literature Cited

Key Reference
   Stewart, C.C. and Stewart, S.J. 1994. Cell preparation for the identification of leukocytes. Methods Cell Biol. 41:39‐60.
  General reference on immunophenotyping.
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