Journal: PLoS ONE

Article Title: An Anti-Phospholipase A 2 Receptor Quantitative Immunoassay and Epitope Analysis in Membranous Nephropathy Reveals Different Antigenic Domains of the Receptor

doi: 10.1371/journal.pone.0061669

Figure Lengend Snippet: Amino Acid Sequences of Consensus PLA 2 R Epitopes Identified by SPOT.

Article Snippet: Western immunoblots were performed on transfected cell lysates (described below) with a commercial goat anti-PLA 2 R (Acris Antibodies, Herford, Germany), mouse anti-GFP (Santa Cruz) and patient sera as described above.

Techniques:

Journal: PLoS ONE

Article Title: An Anti-Phospholipase A 2 Receptor Quantitative Immunoassay and Epitope Analysis in Membranous Nephropathy Reveals Different Antigenic Domains of the Receptor

doi: 10.1371/journal.pone.0061669

Figure Lengend Snippet: Panel A: The median of ALBIA fluorescence units of anti-PLA 2 R positive IMN samples were compared to IMN samples negative for anti-PLA 2 R antibodies and control samples (normal healthy controls and unrelated inflammatory disease controls).Fluorescence values of anti-PLA 2 R positive samples were significantly higher than values of anti-PLA 2 R negative samples and of controls (p-values<0.0001). [Median with interquartile range]. Panel B: ALBIA readings were analyzed according to antibody titres determined on CB-IIF. Samples with a high titre on CB-IIF had often a high fluorescent value on the bead-based assay but ALBIA readings did not correlate with CB-IIF titres. [Whiskers: 2.5–97.5 percentile]. Panel C: A ROC curve is a graphical plot illustrating the performance of a binary classifier system and is used to evaluate diagnostic tests. Sensitivity (fraction of true positives out of positives) is plotted versus 1-specificity (the fraction of false positives out of negatives). Here, our established ALBIA using HEK cell lysates is compared to the EUROIMMUN IIF-CBA. The EUROIMMUNIIF-CBA is a commercially available immunoassay for anti-PLA 2 R and therefore defined the outcome (anti-PLA 2 R positive vs. anti-PLA 2 R negative).Thus, the IIF-CBA perfectly classifies patients with high sensitivity and specificity. With an area under the curve (AUC) of 0.978, the ALBIA is very close to the CB-IIF assay.

Article Snippet: Western immunoblots were performed on transfected cell lysates (described below) with a commercial goat anti-PLA 2 R (Acris Antibodies, Herford, Germany), mouse anti-GFP (Santa Cruz) and patient sera as described above.

Techniques: Fluorescence, Bead-based Assay, Diagnostic Assay

Journal: PLoS ONE

Article Title: An Anti-Phospholipase A 2 Receptor Quantitative Immunoassay and Epitope Analysis in Membranous Nephropathy Reveals Different Antigenic Domains of the Receptor

doi: 10.1371/journal.pone.0061669

Figure Lengend Snippet: Grey scale heat map representation of results from SPOT used to detect PLA 2 R epitopes. Peptide membranes were probed with 10 randomly selected IMN samples that had previously been tested by IIF-CBA for anti-PLA 2 R antibodies (7 positive (IMN+) and 3 negative (IMN-)), as well as 5 normal healthy controls (NHC). The positive control rabbit antibody to PLA 2 R reacted with the expected peptide used as the immunogen. Consensus epitopes and their respective PLA 2 R domains derived from this analysis are illustrated in and summarized in .

Article Snippet: Western immunoblots were performed on transfected cell lysates (described below) with a commercial goat anti-PLA 2 R (Acris Antibodies, Herford, Germany), mouse anti-GFP (Santa Cruz) and patient sera as described above.

Techniques: Positive Control, Derivative Assay

Journal: PLoS ONE

Article Title: An Anti-Phospholipase A 2 Receptor Quantitative Immunoassay and Epitope Analysis in Membranous Nephropathy Reveals Different Antigenic Domains of the Receptor

doi: 10.1371/journal.pone.0061669

Figure Lengend Snippet: All of the determinants identified by epitope mapping were located in the extracellular domain of PLA 2 R and are ∼10 to 25 aa long. Only one epitope is not in the C-type lectin like domains of the receptor. [C-R,cysteine-rich region; FNII, fibronectin type II domain; CTLDs, C-type lectin like domains; N, N-terminal end; C, C-terminal end].

Article Snippet: Western immunoblots were performed on transfected cell lysates (described below) with a commercial goat anti-PLA 2 R (Acris Antibodies, Herford, Germany), mouse anti-GFP (Santa Cruz) and patient sera as described above.

Techniques:

Journal: PLoS ONE

Article Title: An Anti-Phospholipase A 2 Receptor Quantitative Immunoassay and Epitope Analysis in Membranous Nephropathy Reveals Different Antigenic Domains of the Receptor

doi: 10.1371/journal.pone.0061669

Figure Lengend Snippet: Panel A: Different concentrations of a mixture of PLA 2 R derived peptides were used to inhibit the reactivity to the PLA 2 R whole molecule in an addressable laser bead assay. The reactivity showed a significant, dose dependent inhibition. The inhibition with a control peptide (GE-1) was significantly lower. All values are expressed as residual reactivity after inhibition (in %) compared to the sample without inhibitor or control. Panel B: The peptide mixture together with seven individual PLA 2 R peptides and a control peptide were used at a concentration of 126 µg/mL. Besides the peptide mixture, peptide 3 and peptide 4 showed inhibition of anti-PLA 2 R antibodies. All values are expressed as residual reactivity after inhibition (in %) compared to the sample without inhibitor or control. Panel C: The peptide mixture together with seven individual PLA 2 R peptides and a control peptide were used at a concentration of 126 µg/mL. Besides the peptide mixture, peptide 3 and peptide 4 showed inhibition of anti-PLA 2 R antibodies. All values are expressed as ALBIA median fluorescence intensities (MFI) or titer by indirect immunofluorescence on cell-based assay (IIF-CBA).

Article Snippet: Western immunoblots were performed on transfected cell lysates (described below) with a commercial goat anti-PLA 2 R (Acris Antibodies, Herford, Germany), mouse anti-GFP (Santa Cruz) and patient sera as described above.

Techniques: Derivative Assay, Inhibition, Concentration Assay, Fluorescence, Immunofluorescence, Cell Based Assay

Journal: PLoS ONE

Article Title: Autoantibodies against Phospholipase A 2 Receptor in Korean Patients with Membranous Nephropathy

doi: 10.1371/journal.pone.0062151

Figure Lengend Snippet: Clinical characteristics of patients with idiopathic MN at the time of kidney biopsy according to anti-PLA 2 R reactivity.

Article Snippet: A goat polyclonal Ab against PLA 2 R (Sigma-Aldrich, St. Louis, MO, USA) was used at a dilution of 1∶400 to confirm the location of the PLA 2 R band.

Techniques:

Journal: PLoS ONE

Article Title: Autoantibodies against Phospholipase A 2 Receptor in Korean Patients with Membranous Nephropathy

doi: 10.1371/journal.pone.0062151

Figure Lengend Snippet: Human glomerular extracts were electrophoresed, and serum samples of idiopathic membranous nephropathy patients were used as the primary antibody at a dilution of 1∶100. The band detected by immunoblotting with commercial anti-PLA 2 R antibody was used to indicate the position of PLA 2 R. Serum samples from a number of patients with idiopathic MN (except MN5, MN9, and MN10) and the commercial anti-PLA 2 R antibody demonstrated positive bands at approximately 185kD.

Article Snippet: A goat polyclonal Ab against PLA 2 R (Sigma-Aldrich, St. Louis, MO, USA) was used at a dilution of 1∶400 to confirm the location of the PLA 2 R band.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Autoantibodies against Phospholipase A 2 Receptor in Korean Patients with Membranous Nephropathy

doi: 10.1371/journal.pone.0062151

Figure Lengend Snippet: A: Proteinuria was more severe in patients with higher anti-PLA 2 R level. *Dunn’s multiple pairwise comparison test after Kruskal-Wallis test. B Severity of hypoalbuminemia was increased depending on the anti-PLA 2 R levels. **ANOVA test with polynomial contrast. C: The proportion of patients with nephrotic range proteinuria was increased according to the anti-PLA 2 R levels. *** Mantel-Haenszel χ 2 test.

Article Snippet: A goat polyclonal Ab against PLA 2 R (Sigma-Aldrich, St. Louis, MO, USA) was used at a dilution of 1∶400 to confirm the location of the PLA 2 R band.

Techniques:

Journal: PLoS ONE

Article Title: Autoantibodies against Phospholipase A 2 Receptor in Korean Patients with Membranous Nephropathy

doi: 10.1371/journal.pone.0062151

Figure Lengend Snippet: The proportion of the high-risk group was gradually increased as anti-PLA 2 R levels increased. * Mantel-Haenszel χ 2 test.

Article Snippet: A goat polyclonal Ab against PLA 2 R (Sigma-Aldrich, St. Louis, MO, USA) was used at a dilution of 1∶400 to confirm the location of the PLA 2 R band.

Techniques: