Detection of Covalent Binding

Mary K. Bruno1, Steven D. Cohen1

1 University of Connecticut, Storrs, Connecticut
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.3
DOI:  10.1002/0471140856.tx0203s00
Online Posting Date:  May, 2001
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Abstract

Immunochemical detection of xenobiotics covalently bound to cellular proteins can provide information about toxic mechanism and is more specific than the alternative radiochemical studies. Both immunoblotting and immunohistochemical methods are used to pinpoint the target protein(s) and to identify the tissue targets. Also included in this unit are protocols for synthesizing artificial antigens, immunizing suitable host species, and using noncompetitive and competitive ELISA assays to characterize the antibodies produced.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of Covalent Binding by Immunoblotting
  • Basic Protocol 2: Detection of Covalent Binding by Immunohistochemistry
  • Support Protocol 1: Preparation of APAP Antigen
  • Support Protocol 2: Immunization and Serum Collection to Prepare Anti‐APAP Polyclonal Antibodies
  • Support Protocol 3: Preparation of NAPQI‐Protein Adducts as Coating Antigen for ELISA
  • Support Protocol 4: Detection of Anti‐APAP Antibody by Noncompetitive Enzyme‐linked Immunosorbent Assay (ELISA)
  • Support Protocol 5: Characterization of APAP Antibody by Competitive ELISA
  • Support Protocol 6: Construction of APAP Affinity Column
  • Support Protocol 7: Affinity Purification of APAP Antiserum
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Detection of Covalent Binding by Immunoblotting

  Materials
  • SDS‐PAGE gel with resolved proteins
  • Transfer buffer (see recipe)
  • Methanol
  • Coomassie brilliant blue solution (see recipe)
  • Destaining solution (see recipe)
  • Blocking buffer (see recipe)
  • Primary antibody against protein‐xenobiotic adduct ( protocol 4), affinity‐purified if necessary (see protocol 8)
  • Tris‐buffered saline (TBS; see recipe) containing 0.05% (w/v) Tween 20 (store up to 1 week at room temperature)
  • Secondary antibody: 125I‐, alkaline phosphatase–, or horseradish peroxidase–conjugated IgG raised against the host animal used for production of the primary antibody
  • 10 mg/ml nitroblue tetrazolium (prepare fresh)
  • 25 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl‐phosphate (BCIP) in N,N‐dimethylformamide (prepare fresh)
  • Developing buffer (see recipe)
  • 4‐chloro‐1‐naphthol solution (see recipe)
  • 30% hydrogen peroxide (store up to 1 month at 4°C)
  • Chemiluminescence‐based detection system: e.g., ECL Kit (Amersham) or SuperSignal Kit (Pierce Chemical)
  • Trans‐Blot apparatus (Bio‐Rad) including:
  •  Fiber pads
  •  Transfer tank
  •  Cassette gel holder
  • Transfer membranes: nitrocellulose (0.2 to 0.45 µm pore size) or polyvinylidene difluoride (PVDF; 0.45 µm pore size)
  • 23 × 33–cm and 21.5 × 21.5–cm glass baking dishes
  • Whatman no. 1 filter paper
  • Platform shaker or rocker
  • Kodak X‐Omat AR or equivalent 8 × 10–in. (20.32 × 25.4–cm) X‐ray film
  • Lead autoradiography cassette with intensifying screen
  • Additional reagents and equipment for SDS‐PAGE ( appendix 3A)

Basic Protocol 2: Detection of Covalent Binding by Immunohistochemistry

  Materials
  • Tissue sections (1 mm thickness) containing protein‐xenobiotic adducts
  • 0.9% NaCl ( appendix 2A)
  • 50%, 95%, and 100% ethanol
  • Xylene
  • Tris‐buffered saline (TBS; see recipe)
  • 0.5% pepsin in 0.1 N HCl (prepare fresh), prewarmed to 37°C
  • Nonimmune serum from species in which secondary antibody was raised
  • Primary antibody: anti‐APAP (see protocol 4), affinity purified (see protocol 8)
  • TBS (see recipe) containing 0.5% bovine serum albumin (BSA)
  • AS/AP Universal Rabbit Detection System (Bio‐Can Scientific; or equivalent detection system employing alkaline phosphatase–conjugated anti‐rabbit secondary antibody)
  • Double‐strength (2×) Gill's hematoxylin (see recipe or purchase from Polyscientific)
  • Ammonium hydroxide
  • Aqueous mounting medium (CrystalMount from BioMedia or equivalent)
  • Coverslips
  • Tissue‐processing cassettes
  • Microwave oven with temperature probe
  • Programmable automated tissue processor (e.g., Model LX‐120, Innovative Medical Systems)
  • Equipment for paraffin embedding and sectioning (e.g., dedicated histology laboratory)
  • Vacuum desiccator
  • Poly‐L‐lysine‐coated glass microscope slides (see recipe)
  • 60°C and 80°C drying ovens
  • Coplin jars
  • Glass staining pan with metal slide rack

Support Protocol 1: Preparation of APAP Antigen

  Materials
  • p‐aminobenzoic acid (PABA)
  • 0.1 N HCl, 4°C
  • 1% (v/v) sodium nitrite
  • Source of starch (e.g., 1% starch solution, starch paper, or slice of potato) saturated with freshly prepared 50 mM potassium iodide
  • Acetaminophen (APAP; Sigma; minimum purity, 99%)
  • 0.5 M carbonate/bicarbonate buffer, pH 9.0 (see recipe)
  • 3[H]acetaminophen ([3H]APAP; 15.5 Ci/mmol, uniformly labeled; must be custom‐synthesized)
  • 1.0 N and 0.1 N NaOH
  • N,N‐dimethylformamide
  • Keyhole limpet hemocyanin (KLH)
  • Isobutyl chloroformate (Aldrich)
  • Phosphate‐buffered saline (PBS), pH 7.0 to 7.2 (see recipe)
  • 6 N perchloric acid
  • 50 mg/ml bovine serum albumin (BSA; store up to 24 hr at 4°C)
  • 80% methanol containing 5 mM unlabeled APAP, ice‐cold
  • Nitrogen source
  • Tissue solubilizer (e.g., Solvable from NEN Life Science or Amplify from Amersham; optional)
  • Acetone/dry ice or ethanol/dry ice bath
  • Lyophilizer
  • 6000 to 8000 MWCO dialysis tubing (pretreated; see recipe) and dialysis clamps

Support Protocol 2: Immunization and Serum Collection to Prepare Anti‐APAP Polyclonal Antibodies

  Materials
  • Female New Zealand white rabbits (3.5 to 4.5 kg)
  • APAP‐PABA‐KLH conjugate (see protocol 3)
  • Adjuvant (Adjuvax from Alpha‐Beta Technology, Hunter's Titermax from CytRx, or RAS from RIBI Immunochemical Research)
  • 20‐ and 22‐G needles
  • 15‐ and 30‐ml Corex glass centrifuge tubes
  • Refrigerated centrifuge
  • 0.5‐ml glass syringes
  • Additional reagents and equipment for detection of anti‐APAP antibodies by ELISA (see protocol 6)

Support Protocol 3: Preparation of NAPQI‐Protein Adducts as Coating Antigen for ELISA

  Materials
  • Acetonitrile
  • Sodium sulfate, anhydrous
  • Acetaminophen (APAP; Sigma; minimum purity 99%)
  • [3H]acetaminophen ([3H]APAP; 15.5 Ci/mmol; must be custom‐synthesized)
  • Silver (I) oxide
  • Bovine serum albumin (BSA) or aldolase
  • Phosphate‐buffered saline (PBS), pH 7.4 (see recipe)
  • 10 mg/ml BSA in PBS (see recipe for PBS)
  • 1 N perchloric acid
  • 80% methanol, ice‐cold
  • 50 mM carbonate/bicarbonate buffer, pH 9.6 (see recipe)
  • 5‐ to 7‐ml glass screw‐cap vials
  • 6000 to 8000 MWCO dialysis tubing (pretreated; see recipe) and dialysis clamps

Support Protocol 4: Detection of Anti‐APAP Antibody by Noncompetitive Enzyme‐linked Immunosorbent Assay (ELISA)

  Materials
  • Coating antigen: 2 mg/ml NAPQI‐protein adduct solution (see protocol 5) and unmodified control protein solution (see protocol 5, step )
  • PBST (see recipe)
  • 1% (w/v) BSA or 0.2% to 0.5% (w/v) gelatin in 50 mM carbonate/bicarbonate buffer, pH 9.6 (see recipe)
  • Immune and preimmune rabbit serum to be assayed for anti‐APAP antibodies (see protocol 4)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Horseradish peroxidase (HRP)–conjugated anti‐rabbit IgG (or IgG made against other host animal used in primary antibody production)
  • 2,2′‐azino‐di‐(3‐ethylbenzthiazoline) sulfonic acid (ABTS)
  • 50 mM sodium citrate, pH 4.0 (see recipe)
  • 30% hydrogen peroxide (store up to 1 month at 4°C)
  • 37 mM sodium cyanide (optional)
  • 96‐well flat‐bottom polystyrene or polyvinyl chloride (PVC) microtiter plates
  • Multichannel pipettor
  • Microtiter plate reader with variable‐wavelength filters

Support Protocol 5: Characterization of APAP Antibody by Competitive ELISA

  • Competitors: e.g., p‐aminophenol, BSA‐NAPQI, APAP, 2,6‐dimethylacetaminophen, or 3,5‐dimethylacetaminophen

Support Protocol 6: Construction of APAP Affinity Column

  Materials
  • Epoxy‐activated Sepharose 6B (Amersham Pharmacia Biotech)
  • 0.1 M NaOH, pH ∼11.8
  • Acetaminophen (APAP; Sigma; minimum purity, 99%)
  • 0.1 M sodium borate, pH 8.0 (see recipe), containing 0.5 M NaCl
  • 0.1 M sodium acetate, pH 4.0 (see recipe)
  • 1 M ethanolamine, pH 9.0 (see recipe)
  • Phosphate‐buffered saline (PBS), pH 7.4 (see recipe)
  • 0.04% (w/v) sodium azide in PBS (see recipe for PBS)
  • Sintered‐glass funnels (coarse or medium) and filter flasks
  • 50‐ml conical plastic centrifuge tubes with caps
  • Platform shaker (e.g., Nutator from Becton Dickinson Primary Care Diagnostics)
  • 1.0 × 10.0–cm glass column (e.g., Econo‐Column from Bio‐Rad or Kontes‐Flex column from Kontes Glass) and 2‐way stopcock valve

Support Protocol 7: Affinity Purification of APAP Antiserum

  Materials
  • Antiserum to APAP (see protocol 4)
  • Saturated ammonium sulfate (SAS; see recipe)
  • 2.0 N NaOH
  • Phosphate‐buffered saline (PBS; see recipe)
  • 1% (w/v) barium chloride
  • APAP affinity column (see protocol 8)
  • Acetaminophen (APAP; Sigma; minimum purity, 99%)
  • 0.04% (w/v) sodium azide in PBS (see recipe for PBS)
  • 15‐ml Corex glass centrifuge tubes
  • Refrigerated centrifuge
  • 8,000 to 10,000 MWCO dialysis tubing (pretreated; see recipe) and clamps
  • Fraction collector
  • Spectrophotometer
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Figures

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Literature Cited

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