2.2.1. Serum Titration

The polyclonal IgG fraction purified ZON-specific rabbit antisera obtained against ZON-CONA as an immunogen were titrated in the OWLS immunosensor setup using a protein-heterologous conjugate to bovine serum albumin (BSA) at a ZON-BSA concentration of 10 µg/mL as a sensor surface antigen. Purified antisera were injected onto this sensor surface at increasing concentrations (decreasing dilutions) to assess the binding affinity of the antibodies. Typical titration curves are shown in Figure 3, indicating the peak signals obtained in the flow-through system in 3.4 min upon injection, decreasing peak intensities with increasing serum dilution and optimal dilution (the highest dilution still allowing distinguishable signal) at a serum dilution of 1:2000. The use of more concentrated serum for further competitive measurements results in deteriorated method sensitivity, while lower antibody concentrations allow for less stable sensor performance.

**Figure 3.** Optimisation of serum dilution by recording sensor responses to polyclonal antiserum at various dilutions using a sensor surface modified with 10 µg/mL of zearalenone conjugate to bovine serum albumin. Serum dilutions at 1:500 (red line), 1:1000 (blue line), 1:2000 (purple line), 1:4000 (green line), 1:8000 (yellow line), and 1:16,000 (brown line).

The determination of the amount of polyclonal IgG applied is essential in both the direct and indirect (competitive) measurements, particularly in the latter as it is a rather sensitive equilibrium. As seen in Figure 3, when the IgG is applied at small concentrations (high dilutions, e.g., 1:8000 or 1:16,000), antibodies poorly saturate the sensor surface, and small, unstable sensor responses are obtained. On the contrary, in the case of high IgG concentrations (dilutions of 1:500 or 1:1000), although the signal obtained is well measurable (exceeding 100 or 50 arbitrary units, respectively), the surface becomes saturated, and it loses its sensitivity during the measurement of standards and samples. For the measurements, we chose an IgG concentration that is high enough to provide well-measurable signals (at least 20 arbitrary units). On the other hand, the IgG concentration should not be too high, so that the system remains sensitive enough to detect standards containing low amounts of the antigen. Taking the height and shape of the signals into consideration, in the case of competitive measurement of ZON, the dilution of the antibody solution was chosen to be 1:2000.
