Measurement of Polyclonal Immunoglobulin Synthesis Using the Reverse Plaque Technique

Giovanna Tosato1, Sandra E. Pike1, Michael Blaese2

1 Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, 2 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.13
DOI:  10.1002/0471142735.im0713s00
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


This unit describes the reverse hemolytic plaque assay, an effective method for measuring the number of immunoglobulin (Ig)‐secreting cells present in a cell population at any particular time. Cell populations that can be assayed using the technique include peripheral blood mononuclear cells or cells from tissues such as the tonsils. The basic protocol is divided into three stages. First, protein A‐sensitized sheep red blood cells (SRBC), guinea pig complement, and anti‐Ig antibody are prepared. Test samples are then combined and incubated with the SRBC, complement, and antibody in appropriate chambers. Finally, the resulting plaques are scored. A support protocol describes the preparation of plaquing chambers.

PDF or HTML at Wiley Online Library

Table of Contents

  • Support Protocol 1: Preparation of Plaquing Chamber
  • Reagents and Solutions
  • Commentary
PDF or HTML at Wiley Online Library


Basic Protocol 1:

  • 1:2 SRBC/ Alsevers solution (unit 7.2)
  • Normal saline (GIBCO/BRL)
  • Protein A from Staphylococcus aureus (Pharmacia)
  • Chromium chloride
  • Balanced salt solution (GIBCO/BRL)
  • Phosphate‐buffered saline (PBS; appendix 2A), cold
  • Lyophilized guinea pig complement in diluent (GIBCO/BRL)
  • Rabbit anti‐Ig antibody, polyclonal or class‐specific (heat‐inactivated 30 min, 56°C; Cappel)
  • recipeWash medium
  • Paraffin wax
  • White petroleum jelly
  • 50‐ and 15‐ml conical tubes
  • Beckman centrifuge with JS‐4.2 rotor (or equivalent)
  • 30°C water bath
  • 4°C ice bath
  • 96‐well round‐bottom microtiter plate (Costar #3596)
  • Plaquing chamber ( protocol 2support protocol)
  • Dissecting microscope or semiautomated plaque counter ( Optomax)

Support Protocol 1: Preparation of Plaquing Chamber

  Additional Materials
  • 70% ethanol
  • 3 1/4 × 4–in. glass lantern slides
  • 1/4‐in.‐wide, double‐sided scotch tape (4.5 mm thick)
  • 3 × 1–in. microscope slides (1.17‐ to 1.27‐mm thick)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Eby, W.C., Chong, C.A., Dray, S., and Molinars, G.A. 1975. Enumerating immunoglobulin‐secreting cells among peripheral human lymphocytes. A hemolytic plaque assay for a B cell function. J. Immunol. 115:1700‐1703
   Forsgren, A. and Sjoquist, J. 1966. Protein A from Staphylococcus aureus. I. Pseudo‐immune reactions with human‐γ globulin. J. Immunol. 97:822‐828
   Ginsburg, W.W., Finkelman, F.D., and Lipsky, P.E. 1978. Circulating and mitogen‐induced immunoglobulin secreting cells in human peripheral blood: Evaluation by a modified reverse hemolytic plaque assay. J. Immunol. 120:33‐39.
   Gold, E.R. and Fudemberg, H.H. 1967. Chromium chloride: A coupling reagent for positive hemagglutination reactions. J. Immunol. 99:859‐866
   Jerne, N.K., Henry, C., Nordin, A.A., Fuji, H., Koros, A.M.C., and Lefkovits, I. 1974. Plaque forming cells: Methodology and theory. Transplant. Rev. 18:130‐191
   Sjoquist, J. and Stalenheim, G. 1969. Protein A from Staphylococcus aureus. IX. Complement fixing activity of protein A‐IgG complexes. J. Immunol. 103:467‐472
   Stanworth, D.R. and Turner, M.W. 1978 Immunoglobulins. In Handbook of Experimental Immunology, Vol. 1 Immunochemistry. (D.M. Weir, eds.) pp. 6.1‐6.90. Blackwell, Oxford
Key Reference
   Jerne, N.K., 1974. See above
  Contains detailed description of hemolytic hxplaquehy assays.
PDF or HTML at Wiley Online Library