Identification of Human Tumor Antigens by Serological Analysis of Recombinant cDNA Expression Libraries (SEREX)

Matthew J. Scanlan1

1 Memorial Sloan‐Kettering Cancer Center, New York, New York
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 20.7
DOI:  10.1002/0471142735.im2007s65
Online Posting Date:  March, 2005
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Abstract

The structural definition of human tumor antigens recognized by the autologous host has been a long‐standing challenge in tumor immunology. The growing list of human tumor antigens recognized by autologous antibodies or cytotoxic T lymphocytes provides convincing evidence for immune recognition of cancer by the host of origin, as well as attractive targets for vaccine‐based approaches to cancer therapy. In this regard, an approach called SEREX (serological analysis of recombinant cDNA expression libraries) has broad applicability to the analysis of the humoral immune response to cancer antigens. This method involves immunoscreening cDNA libraries prepared from tumor specimens with sera from cancer patients in order to identify gene products recognized by IgG antibody. Clones identified by SEREX can be directly sequenced, providing immediate structural definition of the antigenic target, and their expression profiles can be readily determined, providing information regarding their tissue distribution. Application of this technique has led to the discovery of a number of provocative tumor antigens.

Keywords: SEREX; Tumor Antigens; Serum Antibody Detection Array (SADA); Gene Discovery; Immunotherapy; Cancer Vaccines

     
 
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Table of Contents

  • Basic Protocol 1: Immunoscreening cDNA Expression Libraries with Human Sera
  • Support Protocol 1: Preabsorbing Human Sera to Remove Antibodies that React with E. coli and Phage Proteins
  • Alternate Protocol 1: Serum Antibody Detection Arrays (SADA)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunoscreening cDNA Expression Libraries with Human Sera

  Materials
  • E. coli XL‐1 Blue MRF′ (Stratagene) growing on agar plate
  • LB medium (unit 10.18) containing 12.5 µg/ml tetracycline (added from 12.5 mg/ml tetracycline stock in 95% ethanol)
  • LB medium (unit 10.18) containing 10 mM MgSO 4 (added from sterile 10 mM stock) and 0.2% (w/v) maltose (added from sterile 10% stock)
  • 10 mM MgSO 4 in H 2O
  • NZY (or NZCYM) agar plates, 150‐mm and 100‐mm (see recipe)
  • NZY (or NZCYM) top agarose (see recipe)
  • Lambda bacteriophage cDNA expression library derived from human tumor specimen or human tumor cell line (e.g., lambda ZAP family of vectors from Stratagene, custom prepared according to manufacturers instructions or purchased commercially; see )
  • 1 M isopropyl‐1‐thio‐β‐D‐galactopyranoside (IPTG)
  • Tris‐buffered saline/Tween 20 solution (TBST), pH 7.5 (see recipe)
  • Tris‐buffered saline (TBS), pH 7.5 (see recipe)
  • 5% (w/v) nonfat dry milk (NFDM) in TBS (see recipe for TBS)
  • Serum from cancer patient, preabsorbed and diluted 1:200 to 1:1000 (see protocol 2)
  • Alkaline phosphatase–conjugated Fc fragment–specific goat anti–human IgG secondary antibody (Jackson Immunoresearch, cat. no. 109‐055‐008), diluted 1:3000 in TBS (see recipe) containing 1% (w/v) BSA
  • NBT/BCIP substrate solution (see recipe)
  • Suspension medium (SM; see recipe)
  • Chloroform
  • Orbital shaker
  • 250‐ml Erlenmeyer flask
  • Spectrophotometer
  • Tabletop centrifuge
  • 50°C water bath
  • 15‐ml culture tubes
  • 137‐and 82‐mm pure nitrocellulose membranes (Schleicher & Schuell)
  • 21‐G needles, sterile
  • Flat‐ended forceps
  • Washing trays
  • 150‐mm and 100‐mm petri dishes precoated with BSA (see recipe)
  • Widened Pasteur pipets: cut tips off with glass cutter or diamond knife to leave a widened end of ∼2 mm
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Support Protocol 1: Preabsorbing Human Sera to Remove Antibodies that React with E. coli and Phage Proteins

  • Empty (nonrecombinant) bacteriophage expression vector (e.g., lambda ZAP, Stratagene)
  • Coupling buffer: 0.1 M NaHCO 3/0.5 M NaCl, pH 8.3
  • CNBr‐activated Sepharose 4B (Amersham Biosciences)
  • 1 mM HCl
  • 1 M ethanolamine, pH 8.0
  • Acetate buffer: 0.1 M sodium acetate/0.5 M NaCl, pH 4.0
  • Tris‐buffered saline (TBS), pH 7.5, filter‐sterilized
  • 2% (w/v) sodium azide stock
  • 50 mg/ml gentamicin stock
  • 0.2% (w/v) nonfat dry milk solution supplemented with 0.02% sodium azide and 50 µg/ml gentamicin
  • 50‐ml centrifuge tubes
  • Probe sonicator

Alternate Protocol 1: Serum Antibody Detection Arrays (SADA)

  • NZY (or NZCYM) agar plates, 150‐mm (see recipe; number needed depends on number of serum samples to be screened)
  • Purified bacteriophage clones encoding SEREX‐defined antigens (see )
  • Roll of pure nitrocellulose membrane (Schleicher & Schuell)
  • Sterile, round‐bottom 96‐ (or 384)–well plates
  • 96‐ (or 384)–pin replicator (Nunc)
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Figures

Videos

Literature Cited

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Internet Resources
   http://www2.licr.org/CancerImmunomeDB/
  The Cancer Immunome Database: An integrated database, search engine, and resource page focusing on SEREX‐defined antigens and T lymphocyte epitopes recognized by the immune system of cancer patients.
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