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Thiol-Reactive Compounds Prevent Nonspecific Antibody Binding in Immunohistochemistry

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Thiol-Reactive Compounds Prevent Nonspecific Antibody Binding in Immunohistochemistry Laboratory Investigation (2006) 86, 526–533 & 2006 USCAP, Inc All rights reserved 0023-6837/06 $30.00 www.laboratoryinvestigation.org Thiol-reactive compounds prevent nonspecific antibody binding in immunohistochemistry Arlin B Rogers, Kathleen S Cormier and James G Fox Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA, USA Nonspecific antibody binding is the primary source of confounding background in immunohistochemistry (IHC). Based on observed patterns of background staining, and the known spontaneous reduction of immunoglobulin disulfide bonds in vivo and in vitro, we tested the hypothesis that nonspecific antibody binding in IHC is mediated by sulfhydryl interactions. Coincubation of primary antibodies with reduced glutathione (GSH), L-cysteine, iodoacetic acid, Ellman’s reagent and other thiophilic reagents in pH 8 tris-EDTA (TE) buffer inhibited background staining. In contrast, oxidized glutathione (GSSG) exerted no effect. When empirically optimized, coincubation of GSH with primary antibodies significantly improved IHC signal:noise ratio. Tissue preincubation with mercaptans, soft and borderline metals, and other sulfhydryl-reactive reagents also inhibited background staining, but at the expense of target sensitivity. ELISA results confirmed direct binding between murine serum antibodies and GSH in a nonantigen-dependent manner. In summary, thiol- reactive compounds prevent nonspecific antibody binding in IHC. We propose a mechanism whereby nonspecific background resulting from formation of disulfide bridges and other sulfhydryl bonds between primary antibodies and tissue side groups is interrupted by prior exposure to thiol-reactive reagents such as GSH. These findings provide a molecular basis to improve the specificity of IHC and other immunoassays, and hold implications for antibody-based clinical diagnostics and therapeutics. Laboratory Investigation (2006) 86, 526–533. doi:10.1038/labinvest.3700407; published online 13 March 2006 Keywords: antibody specificity; immunohistochemistry; glutathione; sulfhydryl compounds Immunohistochemistry (IHC) is an important labo- as method of tissue fixation and antigen retrieval, ratory tool used to demonstrate specific molecules target quantity and availability, avidity of the in tissue sections. Unfortunately, nonspecific anti- primary antibody for its cognate antigen, assay body binding in IHC can produce high background kinetics like temperature and fluid dynamics, and staining, resulting in inconclusive target elucidation visualization strategy. Critically, IHC requires anti- that hinders interpretation.1 Implicated causes of body reactivity with antigenic motifs that may be nonantigenic antibody adherence include hydro- altered or rigidly conformed by fixation, all in a phobic protein interactions, ionic and electrostatic spatially and biochemically complex 3-dimensional forces, aldehydes in overfixed specimens, endo- environment. For these reasons, primary antibody genous Fc receptor binding and antigen diffusion.2 concentrations used in IHC are necessarily high. Endogenous biotins and insufficiently quenched Concentrated antibody solutions incubated under tissue enzymes are additional potential sources of static conditions may result in off-target labeling, inappropriate signaling in IHC.3–5 Strategies to producing the unwanted background staining reduce background include tissue pretreatment with familiar to all experienced immunohistochemists. serum from the secondary antibody or an unrelated Here, we demonstrate that thiol-reactive com- species, or application of protein-rich solutions pounds can prevent the nonspecific antibody binding containing bovine serum albumin, casein or that produces background, and describe a tech- skim milk.5 However, these approaches frequently nique that significantly improves IHC signal-to-noise fail to adequately suppress nonspecific staining. ratio. Success of IHC depends on many variables such Correspondence: Dr AB Rogers, DVM, PhD, Division of Compara- Materials and methods tive Medicine, Massachusetts Institute of Technology, 16-849, Cambridge, MA 02139, USA. Tissue Processing and Histologic Evaluation E-mail: [email protected] Received 14 October 2005 revised and accepted 1 February 2006; From our in-house laboratory rodent colony, tissues published online 13 March 2006 from healthy sentinel mice were collected at Thiols and nonspecific antibody binding AB Rogers et al 527 scheduled necropsy, fixed overnight in 10% intense background staining in the absence of neutral-buffered formalin or 100% ethanol, and blocking. routinely processed and embedded. Additional specimens were collected in OCT compound (Sakura Finetek USA, Torrance, CA, USA), flash Tissue and Antibody Blocking frozen in liquid nitrogen, and stored at À701C. For background blocking experiments, deparaffi- Tissues evaluated included pancreas, liver, kidney, nized and rehydrated tissues were incubated for thymus with surrounding mediastinal connective 1 h before IHC with a variety of test compounds (all tissues, skin and gastrointestinal tract. In addition Sigma or Pierce Biotechnology, Rockford, IL, USA) to routine hematoxylin and eosin (H&E), selected including methyl and ethyl mercaptan (15% v:v in tissues were stained with Sudan black B or periodic H O), iodomethane, formaldehyde (2% v:v) and acid-Schiff (PAS; with or without diastase digestion) 2 paraformaldehyde (0.2% v:v), iodoacetic acid and by routine histologic methods.6 iodoacetamide (0.5 M), N-ethyl maleimide (0.04 M), and the heterobifunctional crosslinking reagent 4-(N-maleimidomethyl) cyclohexanecarboxylic acid Immunohistochemistry N-hydroxysuccinimide ester (SMCC; 3 mM). In another series of experiments, tissues were incubated For all IHC experiments we followed a standard for 20 min with log dilutions (ranging from B8nM operating procedure, varying only the pretreatment to 40 mM) of metal chloride salts solubilized in of the primary antibody solution or tissue section ethanol. These included cobalt(II), copper(II), before the assay. Epitope recovery for formalin- iron(II) and (III), lead(II), and mercury(II) chloride. fixed tissues was accomplished by heating slides For antibody blocking studies, test reagents in pH 6 citrate-buffered Target Retrieval Solution (3–90 mM) were coincubated with primary antibodies (DAKOCytomation, Carpinteria, CA, USA) in a rice for 1 h on ice. Compounds evaluated (all Sigma) steamer for 20 min followed by benchtop cooling, or included reduced glutathione (GSH) and oxidized by incubating in a commercial trypsin protease glutathione (GSSG), L-cysteine, n-acetyl-L-cysteine, digestion solution (DAKO) at 371C for 20 min. iodoacetic acid, and 5,5-dithiobis(2-nitrobenzoic On an automated immunostainer (i6000, Biogenex; acid) (DTNB; Ellman’s reagent). Buffers used San Ramon, CA, USA), deparaffinized and rehy- for antibody dilution and blocking reagent drated tissue sections were circumscribed with a coincubation were phosphate-buffered saline PAP pen and sequentially overlaid with the follow- (PBS; pH 7.4) and 10 mM tris-HCl pH 8, 1 mM ing reagents: 3% H O in PBS  5 min (repeated), 2 2 ethylenediaminetetraacetic acid (1  tris-EDTA; TE egg white avidin (1 egg white in 100 ml PBS, buffer). Buffer pH was verified with a meter and coarsely filtered)7  5 min, 0.2% biotin (Sigma, St adjusted as needed before use. Louis, MO, USA)  5 min, primary antibody  1h, biotinylated species-appropriate secondary anti- body (H&L; Sigma)  30 min, 1:100 streptavidin- Glutathione ELISA peroxidase (DAKO)  10 min, 3,30-diaminobenzi- dine (DAB; DAKO)  5 min, and Gill’s hematoxylin In order to evaluate antibody behavior directly counterstain  1 min. Slides were washed 5  with in a sulfhydryl-rich compartment, we added to PBS/Tween-20 (Sigma) between steps. For fluoro- a standard in-house murine parvovirus (MPV) genic IHC, AlexaFluor 488-conjugated secondary enzyme-linked immunosorbent assay (ELISA) antibodies (Molecular Probes/Invitrogen, Carlsbad, microplate wells coated only with GSH (Sigma). CA, USA) were applied and slides mounted with Sera from mice in our surveillance colony, and antifade Vectashield plus DAPI (Vector Laboratories, from known positive and negative controls, Burlingame, CA, USA). When mouse primary were diluted 1:800 and applied to wells coated with antibodies were applied to mouse tissues, we MPV antigens or GSH only. Additional wells used the ARK kit (DAKO). Photomicrographs were treated with fetal calf serum (FCS) or PBS were taken with a Nikon DXM1200 camera in place of murine serum. After primary incubation (chromogenic) or Zeiss Axioskop system and washing, wells were incubated sequentially (fluorogenic). We tested a variety of primary with biotinylated monoclonal anti-mouse immuno- antibody sources including rabbit anti-human CD3 globulin (BD Pharmingen, San Diego, CA, USA), and pan-cytokeratin (DAKO), iNOS (NOS2, extravidin-peroxidase (Sigma), and 2,2-azino-bis Santa Cruz Biotechnology, Santa Cruz, CA, USA), (3-ethylbenzothiazoline-6-sulfunoic acid) diammo- mouse anti-human c-myc (Novocastra, New- nium salt (ABTS substrate, KPL, Gaithersburg, MD, castle-upon-Tyne, UK) and goat anti-Treponema USA) for color development. Optical density (OD) pallidum (Novocastra; used on archival rabbit development at 405/592l was recorded by an ELISA tissue with T. paraluis-cuniculi). During initial plate reader (Dynatech MR7000, Dynatech Labora- screening, primary antibody concentrations were tories Inc., Chantilly, VA, USA). Results were
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