flowchart LR S[Serum dilution] --> W[Three technical replicate wells] STD[Standards + blanks + controls] --> OD[Plate optical density] W --> OD OD --> C[Calibration + dilution correction] C --> Q[Replicate and range QC] Q --> V[One participant sIgA estimate]
Protocol 8: serum sIgA by ELISA
protocol
sIgA
ELISA
serum
From triplicate wells and a calibration curve to one reviewed participant concentration
Assay role
ELISA converts antibody-dependent color intensity into an estimated serum sIgA concentration. It is a separate assay lane from sequencing.
| Raw data | Processed data | Final integration value |
|---|---|---|
| plate, well, replicate, OD, dilution | blank-corrected OD, back-calculated concentration, CV, flags | one reviewed concentration per participant |
The presentation’s seven-point 1.250-to-0 µg/ml curve and reported \(R^2=0.959\) describe assay development, not automatic plate acceptance. The laboratory must lock the kit, lot, sample dilution, incubation/read settings, calibration model, LLOQ/ULOQ, control limits, CV threshold, and repeat rules.
QC gate
Inspect standards and residuals, blank behavior, control recovery, saturation, dilutional parallelism, replicate precision, edge/plate effects, and censor flags. Do not silently turn values below LLOQ into zero or average a failed well into the result.
ImportantWhat the assay does not generate
It does not count mucosal plasma cells, sequence IgA, measure stool IgA coating, or directly report intestinal secretion. It produces an accessible serum concentration with assay uncertainty.