Immunization of Mice

Tomás Maira‐Litrán1

1 Division of Infectious Diseases, Department of Medicine Brigham and Women's Hospital/Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.4
DOI:  10.1002/cpmb.30
Online Posting Date:  January, 2017
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Abstract

The selection of an appropriate immunization strategy depends largely on the properties of an antigen, including its nature, purity, solubility, and availability. This unit describes the critical steps in the production of monoclonal and polyclonal antibodies against soluble molecules (such as proteins, peptides, polysaccharides, oligosaccharides, or hapten‐conjugate vaccines), complex antigens (such as whole pathogens or outer membrane vesicles), and antigens embedded in or eluted from gel matrix following electrophoresis. © 2017 by John Wiley & Sons, Inc.

Keywords: adjuvant; antigen; monoclonal antibody

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Production of Immune Spleen Cells: Immunization with Soluble Antigens
  • Basic Protocol 2: Immunization with Complex Antigens (Membranes, Whole Cells, and Microorganisms)
  • Basic Protocol 3: Immunization with Antigen Embedded in Polyacrylamide Gel Following Electrophoresis
  • Alternate Protocol 1: Immunization with Antigens after Electroelution from Polyacrylamide Gel
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Production of Immune Spleen Cells: Immunization with Soluble Antigens

  Materials
  • Mice (BALB/c is the most widely used strain)
  • Phosphate‐buffered saline (PBS), pH 7.2 to 7.4
  • Antigen
  • Complete Freund's adjuvant (Sigma or Colorado Serum Co.)
  • Incomplete Freund's adjuvant (Sigma or Colorado Serum Co.)
  • Sterile scalpel blades
  • Microtainer serum separator tubes (BD Biosciences, cat. no. 365967)
  • Heat lamp
  • 1‐ to 3‐ml Luer‐Lok syringes (BD Biosciences)
  • Female‐to‐female Luer‐Lok connector (BD Biosciences, cat. no. 513110)
  • 20‐G and 22‐G needles
  • Webglue (Patterson Veterinary, cat. no. 07‐856‐6128)
  • Microcentrifuge
  • Additional reagents and equipment for enzyme‐linked immunosorbent assay (ELISA; unit 11.2; Hornbeck et al., ) and fusion of myeloma cells with immune spleen cells (unit 11.7; Fuller et al., )

Basic Protocol 2: Immunization with Complex Antigens (Membranes, Whole Cells, and Microorganisms)

  Materials
  • Antigen mixture
  • Preparative SDS polyacrylamide gel
  • 0.2 M imidazole‐0.1% (w/v) SDS
  • 0.2 M ZnSO 4
  • 0.2 M glycine, 25 mM Tris (pH 8.3)
  • Vertical SDS‐PAGE apparatus and power pack
  • Battery‐operated pestle grinder system (Fisher Scientific)
  • Additional reagents and equipment for one‐dimensional SDS gel electrophoresis of proteins (unit 10.2; Gallagher, )

Basic Protocol 3: Immunization with Antigen Embedded in Polyacrylamide Gel Following Electrophoresis

  Materials (also see protocol 3)
  • D‐Tube Dialyzer Mini, e.g., 6 to 8 kDa cutoff (EMD Millipore, cat. no. 71504) or 12 to 14 kDa cutoff (EMD Millipore, cat. no. 71505)Choose a membrane with a molecular weight cutoff that is significantly smaller than the antigen of interest.
  • Horizontal electrophoresis equipment
  • Additional reagents and equipment for spectrophotometric and colorimetric determination of protein concentration (unit 10.1; Simonian and Smith, )
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Figures

Videos

Literature Cited

Literature Cited
  Crawford, A.W. and Beckerle, M.C. 1991. Purification and characterization of zyxin, an 82,000‐dalton component of adherens junctions. J. Biol. Chem. 266:5847‐5853.
  Diehl, K.H., Hull, R., Morton, D., Pfister, R., Rabemampianina, Y., Smith, D., Vidal, J.M., and van de Vorstenbosch, C. 2001. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J. Appl. Toxicol. 21:15‐23. doi: 10.1002/jat.727.
  Fuller, S.A., Takahashi, M., and Hurrell, J.G. 1988. Fusion of myeloma cells with immune spleen cells. Curr. Protoc. Mol. Biol. 1:11.7.1‐11.7.4. doi: 10.1002/0471142727.mb1107s01.
  Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 97:10.2A.1‐10.2A.44. doi: 10.1002/0471142727.mb1002as97.
  Golde, W.T., Gollobin, P., and Rodriguez, L.L. 2005. A rapid, simple, and humane method for submandibular bleeding of mice using a lancet. Lab Anim. 34:39‐43. doi: 10.1038/laban1005‐39.
  Hornbeck, P., Winston, S.E., and Fuller, S.A. 1991. Enzyme‐linked immunosorbent assays (ELISA). Curr. Protoc. Mol. Biol. 15:11.2.1‐11.2.22. doi: 10.1002/0471142727.mb1102s15
  Simonian, M.H. and Smith, J.A. 2006. Spectrophotometric and colorimetric determination of protein concentration. Curr. Protoc. Mol. Biol. 76:10.1.1‐10.1A.9. doi: 10.1002/0471142727.mb1001as76
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