Chun Xie1, Zhiyan Liang, Sooghee Chang, Chandra Mohan. 1. Simmons Arthritis Research Center, Department of Internal Medicine/Rheumatology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Abstract
OBJECTIVE: The autoantibody specificities that dominate the deposits in lupus kidneys remain unclear. Reasoning that previously utilized elution buffers such as acidic glycine and ammonium thiocyanate may not have been maximally effective in eluting all Ig deposits from the kidneys, this study was conducted to experiment with a stronger dissociating agent, urea-glycine. METHODS: Seven antinuclear antibody-positive, nephritic female (SWR x NZB)F(1) (SNF1) lupus mice were selected for the elution study. Deposited Ig was eluted from their kidneys using 3 different elution buffers: 0.15M glycine-HCl buffer, 1.3M ammonia thiocyanate/0.15M glycine-HCl buffer, and 5M urea/0.15M glycine-HCl buffer. All eluates were tested for specificity against a variety of nuclear and glomerular antigens. RESULTS: Compared with conventional elution buffers, the urea-based regimen eluted severalfold more IgG and IgM antinuclear antibodies from the kidneys of nephritic SNF1 lupus mice. IgG anti-double-stranded DNA (anti-dsDNA) antibodies were not only the most prevalent species in these renal deposits, they were also heavily enriched in the kidneys, relative to the corresponding serum levels. A substantial fraction of the anti-single-stranded DNA and antihistone/DNA (but not antihistone) reactivity in these eluates was due to cross-reactive anti-dsDNA antibodies. No reactivity with SSA, SSB, Sm, RNP, Jo-1, Scl-70, or ribosomal P antigens could be demonstrated in these eluates. Importantly, the urea-glycine eluates differed from the conventional eluates in having significantly greater reactivity to glomerular substrate and laminin. CONCLUSION: This novel urea-based elution provides further support for the dominance of antibodies in lupus kidneys, with strong polyreactivity to DNA and glomerular substrate.
OBJECTIVE: The autoantibody specificities that dominate the deposits in lupus kidneys remain unclear. Reasoning that previously utilized elution buffers such as acidic glycine and ammonium thiocyanate may not have been maximally effective in eluting all Ig deposits from the kidneys, this study was conducted to experiment with a stronger dissociating agent, urea-glycine. METHODS: Seven antinuclear antibody-positive, nephritic female (SWR x NZB)F(1) (SNF1) lupus mice were selected for the elution study. Deposited Ig was eluted from their kidneys using 3 different elution buffers: 0.15M glycine-HCl buffer, 1.3M ammonia thiocyanate/0.15M glycine-HCl buffer, and 5M urea/0.15M glycine-HCl buffer. All eluates were tested for specificity against a variety of nuclear and glomerular antigens. RESULTS: Compared with conventional elution buffers, the urea-based regimen eluted severalfold more IgG and IgM antinuclear antibodies from the kidneys of nephritic SNF1 lupusmice. IgG anti-double-stranded DNA (anti-dsDNA) antibodies were not only the most prevalent species in these renal deposits, they were also heavily enriched in the kidneys, relative to the corresponding serum levels. A substantial fraction of the anti-single-stranded DNA and antihistone/DNA (but not antihistone) reactivity in these eluates was due to cross-reactive anti-dsDNA antibodies. No reactivity with SSA, SSB, Sm, RNP, Jo-1, Scl-70, or ribosomal P antigens could be demonstrated in these eluates. Importantly, the urea-glycine eluates differed from the conventional eluates in having significantly greater reactivity to glomerular substrate and laminin. CONCLUSION: This novel urea-based elution provides further support for the dominance of antibodies in lupus kidneys, with strong polyreactivity to DNA and glomerular substrate.
Authors: Amjad Mustafa; Steven D Holladay; Sharon Witonsky; Kurt Zimmerman; Christopher M Reilly; D Phillip Sponenberg; Danielle A Weinstein; Ebru Karpuzoglu; Robert M Gogal Journal: Toxicol Sci Date: 2009-08-10 Impact factor: 4.849