The secondary antibodies will bind to the captured human H-FABP.
After sample binding, unbound proteins and molecules were washed off with washing buffer, and a biotinylated detection antibody was added to the wells to bind to the captured albumin.
After each target analyte was bound to the capture probes and then to each corresponding biotinylated antibody, and finally to the versatile ECL signal probe, ECL signals were recorded using PMT or CCD as a detector.
Briefly, beads of defined spectral properties were conjugated to protein-specific capture antibodies and added along with samples (including standards of known protein concentration, control samples, and test samples) into the wells of a filter-bottom microplate, where proteins bound to the capture antibodies over the course of a 2-hour incubation.
A subsequent wash step removes all species not bound to the capture antibody (including analyte-label antibody complexes) and leads to a lower-than-expected signal during detection.
Note that in this case the micropatterns next to the cell give some contrast in the deflection image; we attribute the signal to shedded and crosslinked protein clusters, which are specifically bound to the capture antibody spots via GFP.
The purified EEHV-gB antigen was diluted in PBS containing 0.1 % Triton-X to a concentration of 750 ng/well and bound to the capture antibody for 1 h at 37 °C.
The sample is mixed with a detection cocktail consisting of a) capture beads coated with antibodies specific for the target(s) of interest and b) detection antibodies labeled with a fluorescent tag, which will be bound to the capture bead in the presence of the corresponding antigen.
Briefly, beads of defined spectral properties were conjugated to protein-specific capture antibodies, and then samples were added (including standards of known protein concentration, control samples, and test samples) into the wells of a filter-bottom microplate where proteins bound to the capture antibodies over the course of a 2 hour incubation.
Figitumumab bound to the capturing receptor was then detected using horseradish peroxidase-conjugated mouse anti-human IgG2.
