Animal Models for SLE

Philip L. Cohen1, Michael A. Maldonado1

1 University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 15.20
DOI:  10.1002/0471142735.im1520s52
Online Posting Date:  February, 2003
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Abstract

Systemic lupus erythematosus (SLE) in humans is characterized by inflammatory lesions in skin, joints, kidneys, the central nervous system (CNS), and elsewhere. The clinical manifestations are accompanied by autoantibodies to diverse self antigens, mainly but not exclusively derived from the cell nucleus. Autoantibodies are generally believed to cause most of the tissue damage, although direct injury via cell‐mediated immunity is also important. This unit describes protocols for the quantitation of SLE‐associated autoantibody levels in mouse serum: detection of anti‐chromatin antibodies, detection of anti‐single and anti‐double‐stranded DNA antibodies, and detection of rheumatoid factor. Support protocols for preparing chicken chromatin and dsDNA are included. Also included is an immunoglobulin allotype‐specific adaptation of the basic autoantibody ELISA which is useful for measuring antibody production in chimeric animals. The unit includes an ELISPOT protocol for quantitating cells producing anti‐chromatin, as well as a method for genotyping mice for the faslpr and fasLgld mutations.

     
 
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Table of Contents

  • Basic Protocol 1: ELISA of Mouse Sera for Detection of Anti‐Chromatin Antibodies
  • Basic Protocol 2: Detection of Antibodies to Single‐ and Double‐Stranded DNA
  • Basic Protocol 3: ELISA for Rheumatoid Factor
  • Basic Protocol 4: Preparation of Standard Curves for Detection of Allotype‐Specific Antibodies by ELISA
  • Alternate Protocol 1: ELISPOT Assay for Detection of Anti‐Chromatin Antibody Forming Cells
  • Support Protocol 1: Preparation of Chicken Chromatin by Hypotonic Lysis of Chicken Erythrocyte Nuclei
  • Support Protocol 2: Preparation of dsDNA
  • Support Protocol 3: PCR Based Genotyping of the faslpr and fasLgld Mutations
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: ELISA of Mouse Sera for Detection of Anti‐Chromatin Antibodies

  Materials
  • 3 µg/ml chicken chromatin stock solution (see protocol 6) in recipeBBS (see recipe)
  • recipeBorate‐buffered saline (BBS), pH 8.4 (see recipe)
  • recipeBorate‐buffered saline with BSA and Tween (BBT; see recipe)
  • recipeHigh‐titer anti‐chromatin control serum (see recipe)
  • Unknown serum sample(s) to be assayed for anti‐chromatin antibodies
  • recipeBorate‐buffered saline with 0.5% Tween (BBS‐T; see recipe)
  • Biotinylated goat anti‐mouse IgG, Fcγ‐fragment‐specific (IgG Fcγ‐BNHS), absorbed against bovine serum proteins (Jackson ImmunoResearch, cat. no. 115‐065‐071)
  • Avidin–alkaline phosphatase conjugate (Sigma, cat. no. A7294)
  • Alkaline phosphatase substrate (p‐nitrophenyl phosphate, disodium, hexahydrate; Sigma 104 brand)
  • recipe0.01 M diethanolamine (DEA), pH 9.8 (see recipe)
  • Flat‐bottom PVC microtiter plates (Dynex Technologies, no. 2801)
  • Microtiter plate reader (optional), preferably automated with computer interface and dual wavelength (e.g., Emax from Molecular Devices), or spectrophotometer
  • Additional reagents and equipment for ELISA (unit 2.1)

Basic Protocol 2: Detection of Antibodies to Single‐ and Double‐Stranded DNA

  Materials
  • 1 mg/ml stock solution in H 2O of calf thymus DNA, Type I (Sigma, cat. no. D1501), for detection of antibodies to ssDNA
  • 1 N NaOH
  • recipeBorate‐buffered saline (BBS), pH 8.4 (see recipe), cold (autoclaved BBS is required for detection of antibodies to dsDNA)
  • 0.1% (w/v) poly‐L‐lysine (Sigma)
  • dsDNA solution (see protocol 7; for detection of antibodies to dsDNA)
  • High‐titer anti‐ssDNA or anti‐dsDNA control serum (see recipe for recipeanti‐chromatin control sera)
  • Unknown serum sample(s) to be assayed for anti‐ssDNA or anti‐dsDNA antibodies
  • Additional reagents and equipment for ELISA detection of anti‐chromatin antibodies (see protocol 1)

Basic Protocol 3: ELISA for Rheumatoid Factor

  Materials
  • Target (coating antigen): clone MOPC‐31C, mouse IgG1a (Sigma, cat. no. M9035; other IgG1a isotype mAb or purified mouse IgG1a may be utilized)
  • recipeBorate‐buffered saline (BBS), pH 8.4 (see recipe)
  • recipeBorate‐buffered saline with BSA and Tween (BBT; see recipe)
  • High‐titer rheumatoid factor (RF)–containing control serum (see recipe for recipeanti‐chromatin control sera)
  • recipeBorate‐buffered saline with 0.5% Tween (BBS‐T; see recipe)
  • Biotinylated donkey anti‐mouse IgM F(ab′) 2fragment, µ‐chain specific (Jackson ImmunoResearch cat. no. 715‐066‐020)
  • Avidin–alkaline phosphatase conjugate (Sigma #A 7294)
  • Alkaline phosphatase substrate (p‐nitrophenyl phosphate, disodium, hexahydrate; Sigma 104 brand)
  • recipe0.01 M diethanolamine (DEA), pH 9.8 (see recipe)
  • Flat‐bottom PVC microtiter plates (Dynex Technologies, no. 2801)
  • Microtiter plate reader (optional), preferably automated with computer interface and dual wavelength (e.g., Emax, Molecular Devices), or spectrophotometer
  • Additional reagents and equipment for ELISA (unit 2.1)

Basic Protocol 4: Preparation of Standard Curves for Detection of Allotype‐Specific Antibodies by ELISA

  Materials
  • 1 µg/ml F(ab′) 2 fragment of donkey anti‐mouse IgG (Jackson ImmunoResearch, cat. no. 715‐006‐150)
  • recipeBorate‐buffered saline (BBS), pH 8.4 (see recipe)
  • recipeBorate‐buffered saline with BSA and Tween (BBT; see recipe)
  • Standard mAb: mouse mAb IgG2aa (clone G155‐178, PharMingen) or mouse mAb IgG2ab (clone C76‐47, PharMingen)
  • recipeBorate‐buffered saline with 0.5% Tween (BBS‐T; see recipe)
  • Rabbit anti‐mouse IgG2a, IgG2aa, and IgG2ab (Nordic Immunological Laboratories, available from Accurate Chemical and Scientific)
  • Alkaline phosphatase–conjugated donkey anti‐rabbit IgG F(ab′) 2 fragment (H+L; Jackson ImmunoResearch, cat no. 711‐056‐152)
  • Alkaline phosphatase substrate (p‐nitrophenyl phosphate, disodium, hexahydrate; Sigma 104 brand)
  • recipe0.01 M diethanolamine (DEA), pH 9.8 (see recipe)
  • Flat‐bottom PVC microtiter plates (Dynex Technologies, no. 2801)
  • Microtiter plate reader (optional), preferably automated with computer interface and dual wavelength (e.g., Emax from Molecular Devices), or spectrophotometer
  • Additional reagents and equipment for ELISA (unit 2.1)

Alternate Protocol 1: ELISPOT Assay for Detection of Anti‐Chromatin Antibody Forming Cells

  Materials
  • Chicken chromatin (see protocol 6)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Single‐cell suspension of mouse spleen cells (unit 3.1) or other cells to be tested for antibody production (e.g., anti‐chromatin hybridoma cells)
  • RPMI‐10: complete RPMI medium ( appendix 2A) containing 10% FBS
  • Blocking solution: combine 5 g Carnation nonfat dry milk with 100 ml PBS
  • Alkaline phosphatase–labeled anti‐mouse κ (Southern Biotech, cat. no. 1179‐04)
  • Substrate (BCIP/NBT): dissolve Sigma Fast tablet (Sigma cat. no. B5655) in 10 ml distilled H 2O by letting stand 1 hr; filter through 0.22‐µm syringe filter if necessary
  • MultiScreen‐IP plate (Millipore, cat. no. MAIPN4510)
  • Multichannel pipettor
  • Additional reagents and equipment for ELISPOT assays (unit 7.14)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All tissue culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Preparation of Chicken Chromatin by Hypotonic Lysis of Chicken Erythrocyte Nuclei

  Materials
  • Chicken blood in Alsever's solution (from any local supplier or Colorado Serum Company; http://www.colorado‐serum.com; cat no. C51153)
  • Buffer A: 0.08 M NaCl/0.02 M EDTA, pH 7.5
  • 1.5% (v/v) Triton X‐100 in buffer A
  • 1.7 M and 2.25 M sucrose (gradient quality) in buffer A
  • 0.1 M EDTA
  • 50 mM Tris⋅Cl, pH 7.9 ( appendix 2A)
  • 50‐ml centrifuge tubes
  • Sonicator (any general‐purpose instrument will be adequate)
  • 25 × 89–mm polyallomer ultracentrifuge tubes (Beckman)
  • Additional reagents and equipment for quantitation of DNA by spectrophotometry ( appendix 3L)

Support Protocol 2: Preparation of dsDNA

  Materials
  • Calf thymus DNA, Type I (Sigma, cat. no. D1501)
  • 20× SSC (unit 10.6), sterile
  • 24:1 chloroform:isoamyl alcohol
  • 95% ethanol, –20°C
  • recipe0.1 M acetate buffer, pH 5 (see recipe)
  • 0.1 M ZnCl 2
  • 15,000 U/ml S1 nuclease (Amersham Biosciences)
  • 250‐ml centrifuge tubes (Corning 25350‐250)
  • Tabletop centrifuge
  • Dialysis membrane (MWCO 3500)
  • Additional reagents and equipment for dialysis ( appendix 3H) and quantitation of DNA by spectrophotometry ( appendix 3L)
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Figures

Videos

Literature Cited

Literature Cited
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