Enrichment ELISA after four panning rounds

To obtain tau specific binding partners, 1x 10¹¹ phages from Ph.D.-12 Phage Display Peptide Library were added to a tau coated plate,the unbound phages were washed away and the bound phages were eluted by decreasing the pH. The eluted phages were amplified in E.coli ER2738 and taken for another panning round. Four panning rounds were performed, after each panning round the input titer and the output titer were calculated. The number of plaque forming units (pfu) was counted and phages were prepared to the next panning round by diluting to a concentration of 1x 10¹¹ phages.

4.2.1 Enrichment ELISA after four panning rounds

An enrichment ELISA was carried out to evaluate in which panning round the entire phage pool had the highest affinity to the tau. The wells were coated with tau protein and the amplified phage pools from all panning rounds were added to coated wells.

The bound phages were detected with anti-M13 HRP conjugated antibodies. The obtained signal intensity was proportional to the amount of bound HPR conjugated antibody and thus proportional to the amount of binding phages in the respective eluate. As negative controls, wells were filled with coating buffer and coating buffer containing 1% BSA.

The absorbance signal of wells containing tau, representing the binding, was significantly higher than that of negative control wells. This indicated the success of the selection (Figure 13).

As expected, the increase in the ELISA signal following the second round of biopanning is likely due to the presence of a greater abundance of target-binding phages in the third and fourth round pool. The enrichment ELISA can be estimated after rounds of selection by calculating the ratio of specific binding signal versus non-specific binding signal. Each ratio value is obtained using the absorbance of the target wells to the negative control wells. The binding ratio in this selection was more than 2 for all panning rounds (Table 8). A ratio threshold of 2 or greater indicates the likely presence of specific binding phages (Miersch et al., 2015).

To isolate single phage clones, the eluate of the later rounds of selection (i.e. rounds 3 and 4, which showed higher binding signal to the target), was infected into E.coli ER2738 and plated on IPTG/Xgal LB plates. The binding properties of the individual phage clones to the target were tested using single phage ELISA.

Figure 13: Enrichment ELISA showed the enrichment of tau specific phages during the affinity selection process. Wells were coated with 100 µg/ml tau protein, wells containing only buffer or buffer with 1% BSA were used as negative controls. After blocking, amplified phages from round one to four were added to the respective wells and incubated for 1h. The unbound phages were washed away and the bound phages were detected using anti-M13

HRP monoclonal antibody. After washing the unbound antibodies, the enzymatic transformation of the TMB substrate was measured at 450 nm.

Table 8: Binding ratio of tau monomer to the negative control (buffer) by enrichment ELISA.

Round of panning 1 2 3 4

Enrichment ELISA (A450 nm ratio) tau monomer: buffer

4.8 10 20.2 35.5

4.2.2 Single phage ELISA

Individual phage clones from the third and fourth panning rounds were picked from LB/IPTG/Xgal plates, amplified in E.coli ER2738 and, after several steps of precipitation and centrifugation, individual phage stocks were produced. The resulting phage stocks were added to tau coated wells to determine whether a selected phage clone binds to the target, without using a target-specific antibody. The detection was performed using anti-M13 HRP conjugated antibodies.

Single phage ELISA data was analyzed by comparing the absorbance of the wells coated with tau protein to the absorbance of the negative control wells. As shown in Figure 14, some phage clones showed a significantly higher absorption value in comparison to the negative controls, indicating binding to tau protein. These clones included clone numbers, e. g. 2, 3, 5 and 7. Other clones exhibited slightly stronger binding to their antigen compared to negative controls, e.g. clones 22, 26 and 27.

Phages such 9, 21 and 31 exhibited a relatively high binding to the negative control wells, potentially indicating a plastic binder phage.

The DNA of the positive clones, with the potentially highest binding to tau protein, was sequenced to determine the identity of the peptide expressed on the phage surface.

The binding properties of 96 clones from the third and fourth panning round were characterized. Figure 14 represents an example of the binding properties of 32 phage clones.

Figure 14: Identification of binding properties of individual randomly-selected phage clones to tau protein with single clone ELISA. Tau protein in concentration of 100 µg/ml was immobilized on the plate. As negative controls, wells were filled with the coating buffer.

After blocking, individual phage stocks were added to the respective wells and incubated for 1h. After 6 times of washing, phages were detected with anti-M13 HRP monoclonal antibody.

After washing the unbound antibodies, the enzymatic transformation of the TMB substrate was measured at 450 nm. The figure represents an example of 32 phage clones, approximately 96 phage clones were tested. Later, the synthesized peptides from the phage clones 4, 5, 17, 20, 23, 24, 25 and 29 were named as MM1, MM2, MM3, MM4, MM5, MM6, MM7 and MM8 respectively.