Journal: Journal of Innate Immunity

Article Title: Development and Characterization of Novel ELISAs for the Specific Quantification of the Factor H-Related Proteins 2, 3, 4, and 5

doi: 10.1159/000545139

Figure Lengend Snippet: Calibration and sigmoidal shape of the standard curve. a–d The immunoassays consist of a standard curve (calibration curve; depicted in purple) based on EDTA plasma. The standards were calibrated against the calibrator material that was either a recombinant human factor H-related (rhFHR) protein or a previously calibrated serum pool (depicted in black). The FHR-2 assay was calibrated using rhFHR-2. Assays for FHR-3, -4, and -5 were calibrated against a previously calibrated serum pool. e – h Standard curves for all immunoassays ranged from 0.156 to 10 ng/mL. For each assay, elongated curves are shown to visualize the sigmoidal dose-response curves. The coefficient of determination ( R 2 ) was obtained using a non-linear regression curve fit.

Article Snippet: This serum pool was used to calibrate a pool of healthy human EDTA plasma samples purchased from BioIVT (New York, NY, USA) for each assay to be used as a standard in the ELISAs.

Techniques: Clinical Proteomics, Recombinant

Journal: Journal of Innate Immunity

Article Title: Development and Characterization of Novel ELISAs for the Specific Quantification of the Factor H-Related Proteins 2, 3, 4, and 5

doi: 10.1159/000545139

Figure Lengend Snippet: Reproducibility of assay development and assay performance. a–d Batch-to-batch comparison. For each ELISA two independently produced batches were compared for their ability to quantify the FHR protein level in EDTA plasma samples ( n = 4). e–h Inter-laboratory variation (variation in multiple determinations of a sample in several assay runs performed at different laboratories) was determined by comparison of the obtained data from seven different laboratories. Pearsons R correlation was used to determine whether the results from different laboratories correlate to each other. ELTE, Eötvös Loránd University (Hungary); UCM, Complutense University of Madrid (Spain); UMR, Philipps University Marburg (Germany); MICRO, Microcoat Biotechnology GmbH (Germany); UMCG, University Medical Center Groningen (the Netherlands); SAN, Sanquin (the Netherlands); HBT, Hycult Biotech (the Netherlands).

Article Snippet: This serum pool was used to calibrate a pool of healthy human EDTA plasma samples purchased from BioIVT (New York, NY, USA) for each assay to be used as a standard in the ELISAs.

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Produced, Clinical Proteomics

Journal: Journal of Innate Immunity

Article Title: Development and Characterization of Novel ELISAs for the Specific Quantification of the Factor H-Related Proteins 2, 3, 4, and 5

doi: 10.1159/000545139

Figure Lengend Snippet: Summary of assay characteristics of the novel FHR assays

Article Snippet: This serum pool was used to calibrate a pool of healthy human EDTA plasma samples purchased from BioIVT (New York, NY, USA) for each assay to be used as a standard in the ELISAs.

Techniques: Intra Assay, Inter Assay, Clinical Proteomics

Journal: Journal of Innate Immunity

Article Title: Development and Characterization of Novel ELISAs for the Specific Quantification of the Factor H-Related Proteins 2, 3, 4, and 5

doi: 10.1159/000545139

Figure Lengend Snippet: a–d Matrix analysis A matrix analysis was performed by comparison of different sample types. The EDTA plasma sample (recommended sample type) was used as reference to determine the recovery in citrate plasma, heparin plasma and serum ( n = 2). A recovery between 80 and 120% (depicted in the gray area) is considered a good recovery, indicating minimal differences between sample types.

Article Snippet: This serum pool was used to calibrate a pool of healthy human EDTA plasma samples purchased from BioIVT (New York, NY, USA) for each assay to be used as a standard in the ELISAs.

Techniques: Comparison, Clinical Proteomics

Journal: Journal of Innate Immunity

Article Title: Development and Characterization of Novel ELISAs for the Specific Quantification of the Factor H-Related Proteins 2, 3, 4, and 5

doi: 10.1159/000545139

Figure Lengend Snippet: The effect of sample type and pre-analytical sample handling on observed FHR protein levels. The effect of sample handling on the assessment of factor H-related protein (FHR) levels is evaluated by testing the sample benchtop stability and the freeze-thaw stability. For benchtop stability testing, samples were stored at room temperature (RT) or on ice on indicated time intervals ranging from 10 min (min) to 16 h (h). The concentrations of each FHR was measured for each incubation time and sample concentrations were compared to the 10-min reference sample using a one-way ANOVA. A recovery between 80 and 120% is considered a good recovery (depicted in gray area). a–d The sample benchtop stability is performed on EDTA plasma samples ( n = 4). Comparison of the samples to the 10-min reference sample showed no significant differences. e–h A CHES panel ( n = 2) was used to determine the effect of sample type on benchtop stability. For each sample type the sample recovery was compared to the 10-min reference samples. i–l To determine the freeze-thaw stability, sample aliquots were exposed to 0–4 freeze-thaw cycles prior to assessing the FHR protein concentrations. A CHES panel ( n = 2) and several additional EDTA plasma samples ( n = 4) were evaluated. All samples were compared to a reference sample (no freeze-thaw cycles).

Article Snippet: This serum pool was used to calibrate a pool of healthy human EDTA plasma samples purchased from BioIVT (New York, NY, USA) for each assay to be used as a standard in the ELISAs.

Techniques: Analytical Sample Preparation, Incubation, Clinical Proteomics, Comparison