Induction and Measurement of In Vivo Antibody Responses

Renata J.M. Engler1, Carole C. Kurman2, David L. Nelson2

1 Walter Reed Army Institute of Research, Washington, D.C., 2 National Cancer Institute, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.16
DOI:  10.1002/0471142735.im07016s00
Online Posting Date:  May, 2001
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Abstract

The capacity of the human immune system to mount an antibody response following in vivo immunization with a protein or polysaccharide antigen is a revealing indication of the overall integrity of both the B and T cell arms of the immune system. As such, in vivo immunization followed by measurement of the antibody response is an appropriate test of immune function in the various acquired and congenital immunodeficiencies and in a host of other conditions affecting the immune system. This unit describes procedures for in vivo immunization and for the measurement of the subsequent immune response using an ELISA technique.

     
 
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Table of Contents

  • Basic Protocol 1: Induction of In Vivo Antibody Responses to Protein/Polysaccharide Antigens
  • Basic Protocol 2: Measurement of In Vivo Antibody Response Using ELISA
  • Support Protocol 1: Coupling of Polysaccharide to Protein
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Induction of In Vivo Antibody Responses to Protein/Polysaccharide Antigens

  Materials
  • Toxoid mixture: 25 Lf U/ml diphtheria toxoid plus 10 Lf U/ml tetanus toxoid (see )
  • recipePolyvalent pneumococcal vaccine (see )
  • Sterile hypodermic syringes and alcohol swabs
  • Additional reagents and equipment for drawing blood samples ( appendix 3F)

Basic Protocol 2: Measurement of In Vivo Antibody Response Using ELISA

  Materials
  • Carbonate buffer, pH 9.6 ( appendix 2A) or PBS ( appendix 2A)
  • recipeAntigen: diphtheria toxoid, tetanus toxoid, or pneumococcal polysaccharide types I or II
  • 0.05% Tween 20 (v/v) in PBS (Tween/PBS; store ≤1 month at 4°C)
  • recipeReference and test serum samples
  • 1% fetal calf serum (heat‐inactivated 1 hr, 56°C) in Tween/PBS (FCS/Tween/PBS)
  • recipeAlkaline phosphatase–conjugated heterologous antibody specific for human immunoglobulins
  • recipeSubstrate solution, freshly prepared (∼6 ml per plate)
  • 3 N NaOH
  • 96‐well flat‐bottom microtiter plates with lids (Costar #3596)
  • Semiautomatic or automatic microtiter plate washer (Dynatech)
  • ELISA reader with appropriate software
NOTE: See following protocols for descriptions of and suppliers for antigens, antibodies, and serum samples.

Support Protocol 1: Coupling of Polysaccharide to Protein

  Additional Materials
  • recipeIndicator solution
  • Cyanuric chloride crystals
  • 0.1% poly‐L‐lysine (molecular weight 30,000 to 70,000; Sigma)
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Figures

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Literature Cited

Literature Cited
   Burke, C.P., Smith, K.A., Stocking, R.I., Ferm, M., and McIntire, O.R. 1977. Anti‐keyhole limpet hemocyanin antibody in normal unsensitized individuals. J. Aller. Clin. Immunol. 59:309‐313.
   Channing‐Rogers, R.P. 1986. Data processing of immunoassay results. In Manual of Clinical Laboratory Immunology., 3rd ed. (N.R. Rose, H. Friedman, and J.L. Fahey, eds.) pp. 82‐87. American Society for Microbiology, Washington, D.C.
   Gaspari, M.M., Brennan, P.T., Solomon, S.M., and Elson, C.O. 1988. A method of staining, processing, and analyzing human intestinal secretions for antibody content. J. Immunol. Methods 110:85‐91.
   Gray, B.M., 1979. ELISA methodology for polysaccharide antigens: Protein coupling of polysaccharides for adsorption to plastic tubes. J. Immunol. 28:187‐192.
   Hammarström, L. and Smith, C.I.E. 1986. Immunoglobulin diversity and its functional significance. In Immunoglobulins in Health and Disease. (M.A.H. French, ed.) pp. 31‐53. MTP Press, Lancaster and The Hague.
   Murphy, B.R., Nelson, D.L., Wright, P.R., Tierney, E.L., Phelan, M.A., and Chanock, R.M. 1982. Secretory and systemic immunologic response in children infected with live attenuated influenza A virus vaccines. Infect. Immun 36:1102‐1110.
   Rodbard, D. and McClean, S. 1977. Automated computer analysis for enzyme‐multiplied immunological techniques. Clin. Chem. 23:112‐115.
   Schubert, J.H. and Cornell, R.G. 1958. Determination of diphtheria and tetanus antitoxin by the hemagglutination test in comparison with tests in vivo. J. Lab. Clin. Med. 52:737‐743.
   Schiffman, G., Douglas, R.M., Bonner, M.J., Robbins, M., and Austrian, R. 1980. A radioimmunoassay for immunologic phenomena in pneumococcal diseaseand for the antibody response to pneumococcal vaccines. J. Immunol. Methods 33:133‐144.
   Voller, A. and Bidwell, D. 1986. Enzyme‐linked immunosorbent assay. In Manual of Clinical Laboratory Immunology, 3rd ed. (N.R. Rose, H. Friedman and J.L. Fahey, eds.) pp. 99‐109. American Society for Microbiology, Washington, D.C.
   Wisdom, G.B. 1976. Enzyme‐Immunoassay. Clin. Chem. 22:1243‐1255.
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