Measurement of In Vitro Specific‐Antibody Synthesis

Robert Yarchoan1

1 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.15
DOI:  10.1002/0471142735.im0715s17
Online Posting Date:  May, 2001
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In this unit, a protocol is provided in which antigen‐specific responses by human lymphocytes are elicited and then quantitated by the enzyme‐linked immunosorbent assay (ELISA). Initially, Ficoll‐Hypaque‐isolated mononuclear cells are treated to remove cytophilic antibody. The antibody response being studied is then elicited by the appropriate stimulating antigen (in this case, influenza virus, although other antigens can also be used) or mitogen. Finally, after a 12‐day incubation, the supernatant is harvested for assay.

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Table of Contents

  • Commentary
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Basic Protocol 1:

  • Stimulating antigen or mitogen—live (or dialyzed rmalin‐inactivated) influenza virus or other antigen such as tetanus toxoid
  • Phosphate buffered saline (PBS; appendix 2A)
  • Ficoll‐Hypaque‐gradient separated PBMC (unit 7.1)
  • Complete RPMI‐5 medium ( appendix 2A)
  • 100% fetal calf serum (FCS; heat‐inactivated 30 min, 56°C)
  • Complete RPMI‐10 medium ( appendix 22)
  • 0.4‐µm filter (Nalgene)
  • Sorvall centrifuge equipped with H‐2000 rotor (or equivalent)
  • 15‐ml centrifuge tubes
  • 24‐well flat‐bottom microtiter plate (Costar)
  • Sorvall RC‐3B plate centrifuge (or equivalent)
  • Additional reagents and equipment for dialysis ( appendix 3H), cell counting ( appendix 3A), and trypan blue exclusion ( appendix 3B), B and T cell enrichment (unit 7.1; optional), B cell precursor antibody assay (unit 3.5; optional), and antigen‐specific ELISA (unit 7.11)
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Literature Cited

Literature Cited
   Callard, R.E. 1979. Specific in vitro antibody response to influenza virus by human blood lymphocytes. Nature (Lond.) 282:734‐736.
   Dosch, H.M., Percey, M.E., and Gelfand, E.W. 1977. Functional differentiation of B lymphocytes in congenital agammaglobulinemia. I. Generation of hemolytic plaque‐forming cells. J. Immunol. 119:1959‐1964.
   Dulbecco, R. 1980. The nature of viruses. In Virology (R. Dulbecco and H.S. Ginsberg, eds.) pp. 873‐875. Harper & Row, New York.
   Lane, H.C., Volkman, D.J., Whalen, G., and Fauci, A.S. 1981. In vitro antigen‐induced, antibody‐specific antibody production in man. Specific and polyclonal components, kinetics, and cellular requirements. J. Exp. Med. 154:1043‐1057.
   Stevens, R.H. and Saxon, A. 1978. Immunoregulation in humans. Control of antitetanus toxoid antibody production after booster immunization. J. Clin. Invest. 62:1154‐1160.
   Tosato, G., Magrath, I.T., Koski, I.R., Dooley, N.J., and Blaese, R.M. 1980. B cell differentiation and immunoregulatory T cell function in human cord blood lymphocytes. J. Clin. Invest. 66:383‐390.
   Waldmann, T.A., Broder, S., Blaese, R.M., Durn, M., Blackman, M., and Strober, W. 1974. Role of suppressor T cells in the pathogenesis of common variable hypogammaglobulinemia. Lancet 2:609‐613.
   Yarchoan, R. and Nelson, D.L. 1984. Specificity of in vitro anti‐influenza virus antibody production by human lymphocytes: Analysis of original antigenic sin by limiting dilution cultures. J. Immunol. 132:928‐935.
   Yarchoan, R., Murphy, B.R., Strober, W., Schneider, H.S., and Nelson, D.L. 1981. Specific anti‐influenza virus antibody production in vitro by human peripheral blood mononuclear cells. J. Immunol. 127:2588‐2593.
Key Reference
   Yarchoan et al., 1981. See above.
  Provides an overview of the technique described here and the results that can be attained from a group of normal donors. Also describes the use of limiting dilution cultures to measure the precursor frequency of B cells producing anti‐influenza antibody in vitro.
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