More available binding sites were found when Fab fragments were immobilized onto a microparticle than when intact mAbs were conjugated because of the smaller size and improved orientation (site-specific conjugation through cysteine residue) of the Fab fragment.
We obtained excellent linear response and a detection range covering more than three orders of magnitude in the miniature Fab fragment immunoassay of free and total PSA when the analyte was detected on a single microparticle.
The RecAll prostate-specific antigen (PSA)  assay, based entirely on site-specific recombinant Fab fragments for capturing and detecting the analyte, has been demonstrated previously (9).
The microparticles were coated with specific thiolated monoclonal antibodies (mAbs) or recombinant Fab fragments against PSA (H117-mAb-SH or H117-Fab-SH).
amount of Fab fragment, and kinetics) and initial serum sample tests were done with both tracer Fab fragments labeled with [Eu.
The stabilities of the biotinylated and lanthanide-labeled Fab fragments were studied by storing each Fab fragment (at concentrations between 126 and 243 mg/L) at 4 and 35 [degrees]C for 1 week, 3 weeks, and 3 months before they were used in the assay.
Because the Fab fragment accounts for only one-third of the IgG molecule, the coating of wells can be more efficient, and the site-specific biotinylation ensures that the fragments are in the correct orientation and able to bind antigen.
The small size of the Fab fragment may potentially give faster kinetics compared with the whole IgG molecule.
The dose of Fab fragments should be approximately equimolar to the total body digoxin load, which is determined according to the serum digoxin concentration and/or patient's medical history (6).
Because the unbound fraction of digoxin is the pharmacologically active form, its accurate and reliable measurement in serum collected at various times after administration of Fab fragments may be clinically important (18).
We believe that ultrafiltration of a portion of these samples followed by measurement of the unbound digoxin by an immunoassay (previously shown to be free of matrix-dependent error) might have been used to monitor the accuracy of these immunoassays in measuring unbound digoxin in the presence of Fab fragments.
Serum samples to which various concentrations of digoxin and Fab fragments have been added may not accurately represent samples collected from antidotetreated patients.