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J Immunol Methods. 2005 Jan;296(1-2):83-93. Epub 2004 Dec 2.

Mimotopes for lupus-derived anti-DNA and nucleosome-specific autoantibodies selected from random peptide phage display libraries: facts and follies.

Author information

1
Nephrology Research Laboratory, Nijmegen Center for Molecular Life Sciences and Division of Nephrology (545), University Medical Center, 6500 HB Nijmegen, The Netherlands.

Abstract

Autoantibodies against chromatin are the most characteristic serological feature in SLE patients. Anti-dsDNA and nucleosome-specific antibodies are associated with glomerulonephritis, the most serious manifestation of SLE. Identification of peptides mimicking conformational epitopes (so-called mimotopes) on the nucleosome recognized by these antibodies is of considerable interest. Using an approach similar to that used previously to characterize mimotopes for anti-DNA autoantibodies, we have selected and identified a mimotope for a nucleosome-specific autoantibody (#32) by screening a random peptide phage display library. However, the reactivity of monoclonal antibody (mAb) #32 with the selected mimotope (MIMO#0) in ELISA was dependent on the blocking reagents used. Using nonfat dry milk (5%), mAb #32 clearly bound to MIMO#0, but using fetal bovine calf serum (FCS) (5%), there was no binding. Furthermore, again dependent on the blocking reagent used in ELISA, the selected mimotope MIMO#0 was not only recognized by the selecting antibody mAb #32, but also by a large number of other monoclonal anti-DNA, anti-histone and nucleosome-specific autoantibodies (NSA). We could demonstrate that the selected mimotope was able to bind directly to nucleosomal material (DNA/histone complexes) and labeled DNA. This finding was extended to other previously identified mimotopes for anti-DNA autoantibodies. We conclude that nucleosomal material (DNA/histone complexes), derived from reagents used during the mimotope selection procedure, resulted in the selection of DNA-binding peptides from the phage display library, rather than mimotopes. In addition, we could demonstrate that blocking reagents greatly influence the reactivity of anti-DNA, anti-histone and nucleosome-specific autoantibodies in ELISA. Development of blocking reagents devoid of nucleosomal material (DNA/histone complexes) is urgently needed for assay systems in which anti-nuclear autoantibodies are tested.

PMID:
15680153
DOI:
10.1016/j.jim.2004.10.010
[Indexed for MEDLINE]

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