Fast dissociation kinetics may be, however, limiting for using Strep-tag®II for direct purification of target proteins from large volumes of diluted extracts like mammalian cell culture supernatants or in assay formats requiring extended washing like ELISA.
The optimal dilutions of the mycotoxin-RPE conjugates were determined in monoplex assay format and in all cases the highest dilution (approximately 1 ng reporter each, per well), while still retaining substantial absolute signal, gave the best dynamic range (data not shown).
Note that this SPR set-up is in reversed assay format compared to the applied immunoaffinity isolation method; however, in both methods identical immunochemistry is involved to dissociate the Mab-OTA complex.
In this assay format, mouse IgG was detected within a dynamic range of 100 ng/mL to 10 μg/mL.
However, neither non-opaque gonococci nor non-opaque meningococci were able to precipitate any of the CEACAM1 proteins in this assay format (Fig. 6E).
Also in this case, they could not detect any association of Opa-negative meningococci with CEACAM1 in this assay format, though the used construct encompassed the IgC2-like extracellular domains of CEACAM1 [46].
In this assay format, a divalent analysis model was used, and KD values of 37±9 nM and 44±7 nM were calculated for the chimeric and the murine antibodies, respectively (Table 2).
Having established that the MAbs were able to bind their target antigen in this assay format, we next tested the ability of the reference MAbs to precipitate intact virions under conditions similar to those used above without detergent solubilization.
Initially, calibration standard solutions containing individual mycotoxins were prepared and tested in monoplex assay format.
The results shown in Figure 5 clearly demonstrate that the clones expressed as bivalent scFv-AP exhibit an improved sensitivity in this assay format.
