*2.2. Enzyme-Linked Fluorescent Immunoassay (ELFIA) 2.2. Enzyme-Linked Fluorescent Immunoassay (ELFIA)*

### 2.2.1. Titration and Inhibition of the Antiserum 2.2.1. Titration and Inhibition of the Antiserum

Efficacy of the immunization was monitored by titration of the two rabbit antisera against the coating antigen, ZON conjugated to bovine serum albumin (BSA) (ZON–BSA) between 1:50 and 1:12,200 dilution factors. Microplates were coated with the BSA conjugate at concentrations of 1 to 5 µg/mL in coating buffer. Serum titers, defined as the serum dilution that binds 50% of the antigen under the given conditions, were determined for sera obtained from rabbits (rabbit 1 and rabbit 2). Only slight differences occurred between the efficacy of the two antisera: titer values were 1:828 and 1:448 for antisera from rabbit 1 and rabbit 2, respectively (Figure 2a). In the subsequent immunofluorescence assay experiments, antiserum from rabbit 1, showing somewhat higher affinity to the antigen, was applied. Accuracy and reproducibility of the measurements are better the nearer they are to the titer value, and thus antiserum was applied at a dilution factor of 1:1000 in Efficacy of the immunization was monitored by titration of the two rabbit antisera against the coating antigen, ZON conjugated to bovine serum albumin (BSA) (ZON–BSA) between 1:50 and 1:12,200 dilution factors. Microplates were coated with the BSA conjugate at concentrations of 1 to 5 µg/mL in coating buffer. Serum titers, defined as the serum dilution that binds 50% of the antigen under the given conditions, were determined for sera obtained from rabbits (rabbit 1 and rabbit 2). Only slight differences occurred between the efficacy of the two antisera: titer values were 1:828 and 1:448 for antisera from rabbit 1 and rabbit 2, respectively (Figure 2a). In the subsequent immunofluorescence assay experiments, antiserum from rabbit 1, showing somewhat higher affinity to the antigen, was applied. Accuracy and reproducibility of the measurements are better the nearer they are to the titer value, and thus antiserum was applied at a dilution factor of 1:1000 in the ELFIA tests.

the ELFIA tests. To avoid the risk of saturatization or weak signal detection in assays, optimizations of the coating antigen concentration and serum dilution factor were performed by checkboard titration. The coating antigen, ZON-60 -carboxymethyloxime–BSA conjugate, was applied at concentrations in the range of 0.3125–2.5 µg/mL against the antiserum from rabbit 1 at dilutions in the range of 1:3375–1:1000 dilution factor. All combinations were investigated uninhibited and under inhibition by 3.2 ng/mL of ZON, as well. The coating antigen concentration and the antiserum dilution factor consistently influenced the analytical parameters (Figure 2b). The analytical signal (relative fluorescence unit, RFU) increased with increasing concentrations of the coating antigen and decreased with increasing dilution of the serum. The addition of ZON at a concentration of 3.2 ng/mL resulted in an average 40.0% ± 0.1% inhibition of the assay signal.

*Toxins* **2021**, *13*, x FOR PEER REVIEW 5 of 18

**Figure 2.** Analytical characterization of antisera collected from two 3-month old female New-Zealand white rabbits: (**a**) titer curves of antisera from rabbit 1 () and rabbit 2 () (dilution factor range of 1:50–1:12,200) using a zearalenone-6′-carboxymethyloxime-bovine serum albumin conjugate as a coating antigen at 5 µg/mL; blocked with 1% gelatin in phosphate buffer saline; (**b**) checkboard titration of the antiserum from rabbit 1 using the coating antigen at concentrations in the range of 0.3125–2.5 µg/mL and the serum at various dilution factors (solid symbols, solid lines): 1:1000 (), 1:1500 (), 1:2250 (), 1:3375 (). Titration was also performed under the same conditions with the serum inhibited by 3.2 ng/mL of zearalenone at various dilution factors (hollow symbols, slashed lines): 1:1000 (), 1:1500 (), 1:2250 (), and 1:3375 (). **Figure 2.** Analytical characterization of antisera collected from two 3-month old female New-Zealand white rabbits: (**a**) titer curves of antisera from rabbit 1 () and rabbit 2 () (dilution factor range of 1:50–1:12,200) using a zearalenone-60 -carboxymethyloxime-bovine serum albumin conjugate as a coating antigen at 5 µg/mL; blocked with 1% gelatin in phosphate buffer saline; (**b**) checkboard titration of the antiserum from rabbit 1 using the coating antigen at concentrations in the range of 0.3125–2.5 µg/mL and the serum at various dilution factors (solid symbols, solid lines): 1:1000 (), 1:1500 (), 1:2250 (), 1:3375 (). Titration was also performed under the same conditions with the serum inhibited by 3.2 ng/mL of zearalenone at various dilution factors (hollow symbols, slashed lines): 1:1000 (2), 1:1500 (2), 1:2250 (2), and 1:3375 (2).
