Determination of the Specific Antibody Titer

Helen M. Cooper1, Yvonne Paterson2

1 Walter and Eliza Hall Institute, Melbourne, Australia, 2 University of Pennsylvania, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 11.17
DOI:  10.1002/0471142727.mb1117s50
Online Posting Date:  May, 2001
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Abstract

The amount of specific antibody present in polyclonal antiserum, ascites fluid, or hybridoma supernatant can be quantitated by either solid‐phase radioimmunoassay (RIA) or by direct ELISA. In the solid‐phase assay described here, serially diluted antiserum is incubated in microtiter wells previously coated with the relevant antigen. Bound antibody is detected by employing 125I‐labeled anti‐immunoglobulin antibodies. The amount of specific antibody in the antiserum is then determined from a standard curve generated with a specific antibody of known concentration. The unknown antiserum and the standard antibody are assayed in parallel. The support protocols describe the chloramine T and IODO‐GEN procedures for radioiodination of the anti‐immunoglobulin reagent. The use of the solid‐phase RIA procedure to determine the light‐chain and heavy‐chain isotypes present in polyclonal antisera and fluids containing monoclonal antibodies is also described.

     
 
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Table of Contents

  • Basic Protocol 1: Solid‐Phase Radioimmunoassay (RIA) for Determination of Antibody Titer
  • Support Protocol 1: Iodination of Anti‐Immunoglobulin Reagent Using Chloramine T
  • Support Protocol 2: Iodination of Anti‐Immunoglobulin Reagent Using IODO‐GEN
  • Support Protocol 3: Determination of Antibody Isotypes
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Solid‐Phase Radioimmunoassay (RIA) for Determination of Antibody Titer

  Materials
  • Antigen
  • recipeCoating buffer (see recipe)
  • Control antigen (non‐cross‐reactive protein)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • recipe[125I]anti‐immunoglobulin reagent (see recipe; Ab must be specific for the species in which the test antibody was raised—e.g., [125I]goat anti‐mouse for mouse hybridoma or [125I]goat anti‐rabbit for rabbit sera)
  • Wash buffer (unit 11.2)
  • 1% bovine serum albumin in phosphate‐buffered saline (BSA/PBS)
  • Standardized antibody solution (5 mg/ml in recipediluting buffer)
  • recipeDiluting buffer (see recipe)
  • 96‐well microtiter plates compatible with γ counter such as Wallac ScintiStrips
  • Repeater pipet (e.g., Eppendorf with disposable Combi‐tips)
  • Multichannel pipet
  • Automated γ counter that counts 96‐well plates such as a Wallac MicroBeta TriLux

Support Protocol 1: Iodination of Anti‐Immunoglobulin Reagent Using Chloramine T

  Materials
  • Sephadex G‐25 (Amersham Pharmacia Biotech), washed and equilibrated in PBS (see appendix 22 for PBS)
  • DEAE‐Sephadex (Amersham Pharmacia Biotech), washed and equilibrated in PBS
  • Mixed bed resin (Bio‐Rad #AG 501‐X8), washed and equilibrated in PBS
  • 1% bovine serum albumin in phosphate‐buffered saline (BSA/PBS)
  • Protein to be iodinated (1 mg/ml, preferably in recipeiodinating buffer)
  • recipeIodinating buffer (see recipe)
  • recipeSaturated tyrosine solution (see recipe)
  • Na[125I] (1 mCi in 10 to 5 µl of 0.1 M NaOH, specific activity 17 Ci/mg; NEN Life Sciences)
  • recipeChloramine T solution (see recipe)
  • Plastic tubing (3 mm i.d., 10 cm long) with clamps
  • Silanized glass wool (unit 5.6)
  • Small column stand
  • 1‐ml syringes equipped with 22‐G needles
  • Plastic collection tubes with caps
  • Lead pig

Support Protocol 2: Iodination of Anti‐Immunoglobulin Reagent Using IODO‐GEN

  Materials
  • IODO‐GEN (Pierce #28600T)
  • Methylene chloride
  • recipeIODO‐GEN–coated glass test tubes (Pierce #28601T or see recipe)
  • Drierite
Additional reagents and equipment for iodination of anti‐immunoglobulin reagent using chloramine T (see protocol 2)

Support Protocol 3: Determination of Antibody Isotypes

  • Isotype standards (0.1 µg/ml)
  • recipeIsotyping reagents (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
   Goding, J.W. 1978. Use of staphylococcal protein A as an immunological reagent. J. Immunol. Meth. 20:241‐253.
   Hudson, L. and Hay, F.C. 1980. Practical Immunology, 2nd ed., Blackwell Scientific Publishers, Oxford.
Key Reference
   Mishell, B.B. and Shiigi, S.M. (eds.) 1980. Solid‐phase radioimmune assays. In Selected Methods in Cellular Immunology, pp. 373‐397. W.H. Freeman, N.Y.
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