In Vitro Antibody Production

James J. Mond1, Mark Brunswick1

1 Uniformed Services University of the Health Sciences, Bethesda, Maryland
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.13
DOI:  10.1002/0471142727.mb1113s50
Online Posting Date:  May, 2001
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Abstract

This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. A generalized system for inducing in vitro antibody production is presented along with a procedure for quantifying the number of antibody‐producing cells by plaque‐forming cell (PFC) assays: the Cunningham‐Szenberg technique and the Jerne‐Nordin technique. The assay can be modified as described to measure all classes of antibodies or to enumerate total immunoglobulin‐secreting B cells. A protocol for preparing the resting B cells by Percoll gradient centrifugation is also described.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Induction of Antigen‐Specific and Polyclonal Antibody Production
  • Plaque‐Forming Cell Assays
  • Alternate Protocol 1: Measurement of Isotype‐Specific Antibody and Polyclonal Antibody Responses
  • Support Protocol 1: Preparation of Cunningham‐Szenberg Chambers
  • Support Protocol 2: Preparation of Glass Slides for the Jerne‐Nordin Assay
  • Preparation of Modified Indicator SRBC for PFC Assays
  • Support Protocol 3: Isolation of B Cells by Percoll Gradient Centrifugation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Induction of Antigen‐Specific and Polyclonal Antibody Production

  Materials
  • recipeAntigens (see recipe): SRBC, TNP‐BA, TNP‐Ficoll, TNP‐dextran, TNP‐ovalbumin (TNP‐OVA), or TNP‐LPS
  • Recombinant lymphokines (for use with purified B cells): rIL‐2 (Cetus or Schering) and/or rIL‐1 (Hoffman La Roche)
  • recipeComplete RPMI‐10 medium (see recipe) or recipecomplete DMEM‐10 medium (see recipe)
  • B lymphocytes purified from mice ( protocol 9) either nonimmunized or immunized 3 to 5 weeks previously with specific hapten‐carrier conjugate (unit 11.12 and Table 11.13.1)
  • T cells primed to antigen or carrier (Kruisbeek and Shevach, ; Table 11.13.1)
  • 96‐well flat‐bottom microtiter plates (Costar)
  • Refrigerated low‐speed centrifuge (e.g., IEC 7R with 216 rotor)
  • Sterile gauze

Basic Protocol 2:

  Materials
  • recipeSupplemented complete RPMI‐10 (see recipe) or recipeDMEM‐10 medium (see recipe)
  • 7.5% (v/v) SRBC or 15% (v/v) TNP‐SRBC (see protocol 7) in supplemented complete medium
  • Source of complement: 50% (v/v) recipeguinea pig serum (Life Technologies; also see recipe) in supplemented complete medium
  • Wax (tissue preparation grade; Fisher)
  • 96‐well round‐bottom microtiter plate (Linbro # 36‐311‐05)
  • Cunningham‐Szenberg chambers (see Fig. and protocol 5)
  • 120‐cm2 glass petri dish
  • Dissecting microscope
NOTE: All reagents should be room temperature before addition to chambers, because cold reagents result in bubble formation during incubation.

Basic Protocol 3:

  Materials
  • 1% agarose (SeaPlaque; FMC Bioproducts)
  • 2× basal Eagle medium (Life Technologies)
  • 10% (v/v) SRBC or 20% (v/v) TNP‐SRBC (see protocol 7) in supplemented complete medium
  • Phosphate‐buffered saline (PBS; appendix 22), 4°C
  • Source of lyophilized guinea pig serum (Life Technologies)
  • 5‐ml pipets and 5‐ml glass test tubes, prewarmed
  • 42°C water bath
  • Precoated agarose slides (see protocol 6)
  • Tray for holding slides (Fig. ; not commercially available)

Alternate Protocol 1: Measurement of Isotype‐Specific Antibody and Polyclonal Antibody Responses

  Materials
  • 70% ethanol
  • Microscope slides
  • 1/4‐in. double‐sided tape (3M)

Support Protocol 1: Preparation of Cunningham‐Szenberg Chambers

  Materials
  • 1% agarose (SeaPlaque; FMC Bioproducts)
  • Microscope slides
  • 2 × 2–in. sterile gauze pad (Johnson & Johnson)

Support Protocol 2: Preparation of Glass Slides for the Jerne‐Nordin Assay

  Additional Materials
  • recipeModified barbital buffer (MBB; see recipe)
  • 2,4,6‐trinitrobenzenesulfonic acid, sodium salt (TNBS; Eastman Kodak)
  • recipeCacodylate buffer (see recipe)
  • 120 mg glycylglycine (gly‐gly; Sigma #10022) in 15 ml MBB (see recipe for MBB)
  • 15‐ml graduated conical tube (Costar)

Support Protocol 3:

  Materials
  • Saline (0.15 M NaCl; Life Technologies)
  • 0.5 mg/ml protein A (Pharmacia Biotech) in saline (store frozen in saline at 1 mg/ml)
  • recipe1× chromic chloride solution (see recipe)
  • 15‐ml conical test tube (Costar)

Support Protocol 4:

  Additional Materials
  • Hank's balanced salt solution (HBSS; appendix 22), ice cold
  • Percoll (store at 4°C; Pharmacia Biotech #17‐0891‐01)
  • recipePercoll mix solution (see recipe)
  • 15‐ and 50‐ml polypropylene centrifuge tubes
  • Sorvall H‐1000B rotor (or equivalent)
  • Additional reagents and equipment for preparation of depleted B cell suspensions (Hathcock, ; Mage, ), making cell suspensions (Kruisbeek, 1993), and counting cells ( appendix 3F)
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Figures

Videos

Literature Cited

Literature Cited
   Cunningham, A.J. and Szenberg, A. 1968. Further improvements in the plaque technique for detecting single antibody forming cells. Immunology 14:599‐600.
   Eisen, H., Belman, S., and Carsten, M. 1953. The reaction of 2,4‐dinitrobenzene sulfonic acid with free amino groups of proteins. J. Am. Chem. Soc. 75:4583‐4591.
   Gronowicz, E., Coutinho, A., and Melchers, F. 1976. A plaque assay for all cells secreting Ig of a given type or class. Eur. J. Immunol. 6:588‐590.
   Hathcock, K. 1991a. T cell enrichment by cytotoxic elimination of B cells and accessory T cells. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.), pp. 3.3.1‐3.3.5. John Wiley & Sons, New York.
   Hathcock, K. 1991b. T cell depletion by cytotoxic elimination. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.), pp. 3.4.1‐3.4.3. John Wiley & Sons, New York.
   Inman, J. K., 1975. Thymus‐independent antigens: The preparation of covalent, hapten‐Ficoll conjugates. J. Immunol. 114:704‐709.
   Jerne, N.K. and Nordin, A.A. 1963. Plaque formation in agar by single antibody producing cells. Science 140:405‐408.
   Kruisbeek, A.M. and Shevach, E. 1991. Proliferative assays for T cell function. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.), pp. 3.12.1‐3.12.14. John Wiley & Sons, New York.
   Lycke, N.Y. and Coico, R. 1996. Measurement of immunoglobulin synthesis using the ELISPOT assay. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober eds.), pp. 7.14.1‐7.14.9. John Wiley & Sons, New York.
   Mage, M.G. 1993. Fractionation of T cells and B cells. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober eds.), pp. 3.5.1‐3.5.6. John Wiley & Sons, New York.
   McCarthy, M.M. and Dutton, R.W. 1975a. The humoral response of mouse spleen cells to two types of sheep erythrocytes. I. Genetic control of the response to H and L SRBC. J. Immunol. 115:1316‐1321.
   McCarthy, M.M. and Dutton, R.W. 1975b. The humoral response of mouse spleen cells to two types of sheep erythrocytes. II. Evidence for gene expression in the B lymphocyte. J. Immunol. 115:1322‐1329.
   Mishell, R.I. and Dutton, R.W. 1966. Immunization of normal mouse spleen cell preparations in vitro. Science 153:1004‐1006.
   Mishell, R.I. and Dutton, R.W. 1967. Immunization of dissociated spleen cell cultures from mice. J. Exp. Med. 126:423‐442.
   Mitchison, N.A. 1971. The carrier effect in the secondary response to hapten‐protein conjugates. II. Cellular cooperation. Eur. J. Immunol. 1:18‐27.
   Mond, J.J., Scher, I., Mozier, D.E., Blacsy, M., and Paul, W.E. 1978. T independent responses in B cell defective CBA/N mice to Brucella abortus and to trinitrophenyl (TNP) conjugates of Brucella abortus. Eur. J. Immunol. 8:459‐463..
   Mond, J.J., Farrar, W.E., Paul, W.E., Fuller‐Farrar, J., Schaeffer, M., and Howard, M., 1983. T cell dependence and factor reconstitution of in vitro antibody responses to TNP‐B. abortus and restoration of depleted responses with chromatographed fractions of a T cell derived factor. J. Immunol. 131:633‐637.
   Rittenberg, M.B. and Pratt, C. 1969. Anti‐trinitrophenyl (TNP) plaque assay. Primary response of BALB/c mice to soluble and particulate immunogen. Proc. Soc. Exp. Biol. Med. 132:575
   Shiigi, S.M. and Mishell, R.I. 1975. Sera and the in vitro induction of immune responses. I. Bacterial contamination and the generation of good fetal bovine sera. J. Immunol. 115:741‐744.
   Silverman, M.S. and LaVia, M.F. 1976. Letters to the editor, J. Immunol. 117:2270‐2279.
   Thompson, C‐B., Scher, I., Schaeffer, M., Lindsten, T., Finkelman, F.D., and Mond, J.J. 1984. Size dependent B lymphocyte subpopulations: Relationship of cell volume to surface phenotype, cell cycle, proliferative response and requirement for antibody production to TNP‐Ficoll and TNP‐BA. J. Immunol. 133:2333‐2342.
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