The C3NeF detection ELISA kit enables in vitro qualitative detection of C3NeF autoantibodies in heat-inactivated serum or plasma. C3NeF is an IgG autoantibody that binds to the Bb portion of the C3 convertase C3bBb(P), thereby stabilizing the complex and prolonging its half-life, resulting in sustained activation of the complement alternative pathway (AP). Sustained AP activation can contribute to disease and indeed the presence of C3NeF has been reported in several complement-mediated disorders, most prominently in C3 glomerulopathy, including dense deposit disease and C3 glomerulonephritis, but also in atypical hemolytic uremic syndrome and related conditions characterized by dysregulated alternative pathway activation.
The assay consists of a C3b coated plate to which functional Factor B (FB) and Factor D (FD) should be added for the formation of the C3bBb convertase to which the potentially present C3NeFs can bind. Bound C3NeFs are detected using a peroxidase-conjugated anti-human IgG antibody, which catalyzes conversion of the TMB substrate into a measurable color signal.
Application
Immuno assays
Use
The C3NeF detection ELISA kit is to be used for the in vitro qualitative determination of C3 nephritic factor (C3NeF) autoantibodies in heat-inactivated serum or plasma.
Principle
The C3NeF detection ELISA is a qualitative enzyme-linked immunosorbent assay with a working time of ~1½ hours.
The efficient format of a plate with twelve disposable 8-well strips allows free choice of batch size for the assay.
Samples are incubated in microtiter wells coated with C3b in the presence of Factor B and Factor D, allowing in situ formation of the C3bBb convertase and subsequent binding of C3 nephritic factors, if present. In parallel, samples are also incubated in C3b-coated wells in the absence of Factor B and Factor D, which serve as background controls for non-specific binding to C3b or the plate surface, ensuring that the measured signal specifically reflects antibody binding to the assembled C3bBb convertase.
Peroxidase conjugated detection antibody will bind to the captured C3NeFs.
Peroxidase conjugate will react with the substrate, tetramethylbenzidine (TMB).
The enzyme reaction is stopped by the addition of oxalic acid.
The absorbance at 450 nm is measured with a spectrophotometer.
OD values for each sample are calculated by subtracting the OD in the C3b wells from the OD in the C3bBb wells. Sample OD = ODC3bBb wells - ODC3b wells.
Samples are classified as C3NeF positive when the sample OD is above the ODcut-off and regarded negative when the sample OD is below the ODcut-off.
C3NeF positivity is determined using the predefined cut-off*.
*No positive control is provided with the assay. Therefore, laboratories should use internally collected C3NeF-positive and C3NeF-negative samples to validate the assay and monitor assay performance prior to routine sample testing. During assay validation, also an in-house cut-off value should be determined. The cut-off is established using at least 10–20 independent C3NeF-negative samples and is calculated as the mean optical density of the negative samples plus three times the standard deviation (ODcut-off = mean ODneg + 3 × SD ODneg). During routine testing, both in-house C3NeF-positive and C3NeF-negative samples should be included in each run as internal controls, alongside the provided negative (technical) control, to verify assay performance and cut-off robustness.
Comment regarding the evaluation of properdin-dependent C3NeFs:
Some C3 nephritic factors require properdin for stable binding to the C3 convertase (properdin-dependent C3NeFs), whereas others bind and stabilize C3bBb independently of properdin. Properdin dependence can be assessed by adding properdin (0.5 – 3.1 µg/mL; not provided) to the Factor B and Factor D mixture to allow formation of the C3bBbP complex and performing the assay according to the standard procedure. By incubating the same samples in parallel in C3bBb- and C3bBbP-coated wells, C3NeF binding can be directly compared under properdin-independent and properdin-dependent conditions. Please note that when measuring C3bBbP binding C3NeFs, also a cut-off OD should be set based on C3bBbP wells in C3NeF negative samples.
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