General linear mixed models were used to examine the relationship between OD450 levels at varying dilutions for each patient group (normal controls, pSLE patients with nephritis, and pSLE patients without nephritis) taking into account the nature of repeated sampling for some pSLE patients. IgG3 levels could LRE1 distinguish with good sensitivity the 13 pSLE patients with a history of nephritis from the 8 non-renal pSLE patients. High-titer anti-matrigel IgG, IgA, IgM or IgG3 did not correlate with positive anti-double stranded DNA, but defined an overlapping subset of patients. Conclusion The addition of anti-basement membrane antibody testing to serologic testing in pSLE may help to monitor disease activity or to define important subsets of patients with risks for specific disease manifestations. Committee on Immunologic Testing Guidelines, assays measuring anti-dsDNA Abs predicted a diagnosis of SLE with a weighted mean sensitivity of 57%, specificity of 97% [10]. The presence of high-titer anti-dsDNA Abs predicted the presence of active renal disease in SLE patients with a weighted mean sensitivity of 86% and a specificity of 45%. Titers of anti-dsDNA Abs correlate with the degree of renal injury in SLE, but only to a limited extent[10]. Recently, there has been renewed interest in anti-basement membrane (BM) Abs, due to new findings reported in the NZB/W F1 mouse model of lupus[4]. This model displays loss of tolerance, auto-Ab generation, and inflammatory kidney injury comparable to that seen in patients with SLE. Genetic variation in the F1 mice leads to variable production of auto-Abs of varying specificities that correspond in differing degrees of nephritis[11]. Anti-dsDNA Ab titers are not predictive LRE1 of subsequent nephritis in the NZB/W F1. However, among 69 monoclonal Abs originating from the mouse strain, there was a perfect correlation between Abs that bound to BM antigens with high affinity and those that accumulated in glomeruli and caused significant proteinuria after injection into non-immune mice[4]. An ELISA was used with matrigel as a surrogate for detecting mouse Abs that bound to BM antigens. Although anti-matrigel Ab titers have not been rigorously tested as a diagnostic tool in human SLE, there is some promising data. Multiplex analysis of circulating auto-Abs in a single center cohort of 37 adults with SLE showed a correlation between the presence of high IgG titer anti-matrigel Abs, anti-DNA Abs (ssDNA, dsDNA, chromatin), and higher total and renal SLE disease activity scores (SLEDAI scores)[12]. In order to determine whether heightened reactivity to BM antigens occurs in pediatric SLE patients, reactivity to matrigel in human plasma and serum was developed. Children with and without lupus were tested to assess whether a correlation exists between anti-BM Ab titer and a clinical diagnosis of pSLE, a history of nephritis, or reactivity to anti-dsDNA. Titers of IgG, IgM, IgA and Ig3 anti-BM Abs were measured in all samples. Associations between ACR criteria and clinical findings were also explored. METHODS Matrigel (BD Biosciences) LRE1 was used as a surrogate BM preparation. Matrigel is derived from an EHS sarcoma cell line and is composed primarily of laminin (55%), collagen type IV (30%), entactin (8%), and heparin sulfate proteoglycans (5%). An anti-matrigel ELISA for detection of mouse Abs was performed as previously described 4. Briefly, high protein-binding 96-well microtiter plates (Nunc Apogent) were coated with Matrigel at 500 mcg /well in cold PBS (4C). After gelling at 4C overnight, serum or plasma samples were added at 2-fold serial dilutions. Bound immunoglobulin from samples was detected using a biotin-conjugated goat anti-mouse IgG polyclonal Ab (Invitrogen) diluted 1:20000, followed by streptavidinCHRP (R&D systems) diluted 1:500, and TMB substrate (Pierce). Optical density was measured at 450nm (OD450) using a SpectraMax i3 plate reader (Molecular Devices). The protocol was changed for detection Cops5 of anti-matrigel Abs in humans by substituting anti-human IgG polyclonal Ab (Invitrogen), also diluted at 1:20000. For anti-Matrigel IgA, IgM, and IgG3 ELISAs, the same protocol was followed, except for the use of a biotin-conjugated goat anti-human IgA diluted at 1:5000, anti-human IgM diluted at 1:2000, or anti-human Ig-G3 diluted at 1:1000 (Invitrogen). Testing of individual patient samples on individual occasions by two different experimenters (A.O. and A.S.) produced results that were highly reproducible with low inter-assay variation (CV 10C15%). End-point titers were decided as the dilution at which binding was no greater than the non-specific binding measured from normal human serum. For anti-dsDNA Ab ELISA, plates were coated in poly-L-lysine 5 mcg/well, and then.