Plates coated with 5.0 g/ml gave much better separation between positive and unfavorable sera. and proposed that they might contribute to injury because SGC-CBP30 the antigen is usually expressed in the plasma membrane of glomerular endothelial cells.13,14 Antibodies to hLAMP-2 were originally discovered in 16 of 17 patients with piFNGN by Western blotting in a systematic search for SGC-CBP30 autoantibodies to neutrophil or glomerular membrane proteins.13 We found a similarly high prevalence in a subsequent cohort of 84 patients with active piFNGN.14 Patients autoantibodies commonly bind two epitopes, one of which (P41-49) is shared with the bacterial adhesin FimH with which they cross-react. Injection of antibodies Rabbit Polyclonal to RCL1 to the LAMP-2 extracellular domain induced piFNGN in WKY rats as did immunization with FimH that acted as molecular mimic and provoked synthesis of antibodies to rat LAMP-2. Thus, antibodies to LAMP-2 cause piFNGN in rodents, which raises the issue whether they are similarly pathogenic in humans. Robust assays are required to investigate this further, and development of suitable assays for antibodies to hLAMP-2 has been challenging because of the difficulty in obtaining pure preparations of appropriately glycosylated native or recombinant antigen,15,16 a problem shared with other glycosylated membrane proteins such as the membranous nephropathy antigen, phospholipase A2 receptor.17 Recombinant membrane proteins often need modification to produce soluble substrates for ELISA, and inappropriate glycosylation can affect accessibility of epitopes recognized by patients autoantibodies. Only one other group has reported the development of assays for anti-hLAMP-2 antibodies and they have challenged our conclusions.18 In this study, we characterize three assays for antibodies to hLAMP-2 in human sera and show that they give highly concordant results. In applying them to new European cohorts from three different centers, we confirm that antibodies to hLAMP-2 are highly prevalent in patients with piFNGN both at presentation and during clinical relapse. Results of sequential measurements after the start of treatment provide a possible explanation for the disparity between our findings and those of Roth Expressed hLAMP-2 for Western Blotting and ELISA Most patients autoantibodies bind epitopes in the protein backbone of the extracellular domain not occluded by glycosylation in native neutrophil and glomerular hLAMP-2.13,14 Consequently, we induced recombinant unglycosylated hLAMP-2 truncated to 342 amino acids of the full extracellular domain as GST fusion protein in (Figure 1A). After purification on Glutathione-Sepharose, hLAMP-2/GST fusion protein runs as a single band of approximately 65 kD on SDS-PAGE SGC-CBP30 (Figure 1B), whose identity was confirmed by immunoblot with antibodies to hLAMP-2 and GST. It also binds IgG in sera from patients with antibodies to hLAMP-2 but not controls (Figure 1C). Patients sera were diluted 1:100 to give the best binding/background ratio (Figure 1D). Open in a separate window Figure 1. cDNA constructs, generation, and quality control of recombinant hLAMP-2. (A) Representation of cDNA encoding hLAMP-2A with the 28 amino acid leader peptide (LP), 347 amino acid extracellular domain, 24 amino acid transmembrane domain (TM), and 11 amino acid cytoplasmic domain (Cytopl). The two extracellular domain constructs were utilized to express soluble hLAMP-2 in (hLAMP-2/GST) and mammalian cells (hLAMP-2sol). Both contain the leader peptide but not the transmembrane domain or cytoplasmic tail. hLAMP-2sol expressed in mammalian cells results in an appropriately glycosylated soluble protein exported into the culture supernatant via the default secretory pathway in mammalian cells. The hLAMP-2 cytoplasmic tail contains the signal that directs its retrieval from the plasma membrane to lysosomes. The critical tyrosine was mutated to a histidine in hLAMP-2H (Y/H), which targets it to the plasma membrane when expressed in ldlD cells. (B) Purified hLAMP-2/GST SGC-CBP30 runs as a single 65-kD band on SDS-PAGE and silver stain. Fractions of high purity were pooled and identity of hLAMP-2 was confirmed with an antibody reactive with hLAMP-2 only. SGC-CBP30 (C) Purified hLAMP-2/GST (10 g/ml) was separated by SDS-PAGE and transferred onto PVDF before probing with specific antibodies to hLAMP-2 (932b), which bound exclusively to the fusion protein. Antibody to GST (anti-GST) recognized both fusion protein and free GST. A human serum (1:100 dilution) containing anti-hLAMP-2 antibodies (Pat. 6) also bound the fusion protein, whereas serum from a healthy control (Healthy Co 50) did not. Serum from a patient with renal disease (Dis. Co 8) contained antibodies to GST. Secondary antibodies alone were negative (anti-human, anti-rabbit). (D) Sensitivity of the Western blot was assessed from doubling dilutions (1:50 to 1 1:400) of the standard positive control (Stand pos Co) used.