Quality control of phage display samples

3.6.1 Indirect ELISA of MPSIL0294 variants on neutravidin coated plates

In order to show that the various MPSIL0294 variants could bind a solid streptavidin coated surface no matter the method of immobilisation employed, the various forms of MPSIL0294 were subjected to ELISA probed with an anti-polyhistidine tag antibody as described in section

2.6.2. These ELISAs involved immobilising the various proteins onto the surface of manually

produced neutravidin coated plates thereby providing a useful analogue for the subsequent phage display procedure, except here we use neutravidin instead of streptavidin. Due to their structural similarities, neutravidin is often used interchangeably with streptavidin (the

difference between the two being the removal of streptavidin glycosylation sites). Especially as they both have very similar affinities for biotin and isoelectric points. While it was highly probable that MPSIL0294-avitag and MPSIL0294-V532C would successfully bind to the

neutravidin surface (as they are both biotinylated). It was unclear if MPSIL0294-SBP would also be able to do so, however due to neutravidin and streptavidin’s extreme functional similarity the SBP/neutravidin interaction was presumed.

The ELISA results for each of the three MPSIL0294 formats are represented graphically in

Figure 3.13 and show that while each of the protein formats follow a similar trend when

subjected to this test they have different magnitudes. MPSIL0294-V532C produces the highest A450nm trace and the highest reading in the presence of 50 µg/mL antigen. Intriguingly both the

chemically biotinylated MPSIL0294-V532C and the in vivo biotinylated MPSIL0294-avitag appear to suffer from a hook affect at the highest concentration (100 µg/mL) which would suggest a very high concentration of analyte on the surface. While this was deemed sufficient for this study due to time constraints, the success of these two proteins could be tested by repeating this ELISA with non-biotinylated samples. This effect doesn’t appear in the trace for MPSIL0294-SBP, possibly due to the fact that neutravidin was used as opposed to streptavidin. Nevertheless it appears as if each of the three detergent solubilised variants saturate the neutravidin coated plate at 50 µg/mL and is therefore suitable for phage display screening.

Figure 3.13 Quality control ELISA with detergent solubilised samples of the three MPSIL0294 variants: Each different MPSIL0294 variant was immobilised on a neutravidin coated plate in a serial dilution from 100 to 0.098

µg/mL and subjected to ELISA probed with an anti-polyhistidine tag antibody conjugated with HRP. Each concentration was performed in duplicate and the absorbance readings were measured spectrophotometrically after treatment with TMB stop solution followed by 0.5M H2SO4.

3.6.2 Analysis of detergent solubilised MPSIL0294 variants by Fluorescence- activated cell sorting

As a secondary method of assessing the three different MPSIL0294 variants ability to immobilize on a streptavidin coated surface they were each subjected to FACs analysis as described in section 2.6.3 using streptavidin coated magnetic Dynabeads® (Thermo scientific). A mouse anti-polyhistidine tag antibody was used as a primary while a goat anti-mouse antibody conjugated with DyLight 488 was used as a secondary for this analysis. The overlay histograms shown in Figure 3.14 show that there is distinctively more intense fluorescence on the Dynabeads® incubated with the MPSIL0294 variants compared with the negative control (which were blank beads). This increase in fluorescence represent an increase in secondary antibody and thus an increase in MPSIL0294 material immobilised on the Dynabeads®. It is therefore clear that all three of the detergent solubilised MPSIL0294 are successfully able to immobilise on the surface of the streptavidin coated beads, while maintaining the

accessibility of the polyhistidine tag to the antibody as they all show an increase in fluorescence when compared with the negative control (the blank beads). Two of the

detergent solubilised MPSIL0294 variants performed particularly well in this assay (in regard to their ability to immobilise on beads), namely MPSIL0294-SBP and MPSIL0294-avitag as they produce an identical level of fluorescence that almost matches the positive control (a

biotinylated, his-tagged protein extensively worked on by MedImmune which shall be referred to as ‘protein x’ from here on). ‘Protein x’ was bound to the beads at the same molar

concentration as the MPSIL0294 variants, however it is considerably smaller, therefore it is possible that the increase in fluorescence which ‘protein x’ exhibits in the FACs analysis is due to an increased amount of packing on the surface of the beads.

The overlay histogram in Figure 3.14 shows that the MPSIL0294-SBP peak is higher than the MPSIL0294-avitag, thus suggesting that it was better able to pack on the beads; although it appears that both are almost able to saturate the beads in a similar manner as the positive control. The Dynabeads modified with this variant exhibit a more homogeneous fluorescence profile with less of the Dynabeads exhibiting a lower fluorescence compared to MPSIL0294- avitag. MSPIL0294-V532C shows the least binding to streptavidin in this particular assay, but still shows that the protein is successfully able to immobilise onto the beads.

The three MPSIL0294 variants were immobilised onto the beads in the presence of 0.05% DDM as properly folded protein immobilised on the surface of the plate was required in order to isolate confirmation specific DARPins during phage display. However there is no evidence provided in this study that the protein, once immobilised, is in its native confirmation.

Figure 3.14: FACs analysis of detergent solubilised samples of the three MPSIL0294 variants with streptavidin coated beads: FACs analysis was performed on each of the detergent solubilised variants of MPSIL0294 mixed with magnetic streptavidin coated DynaBeads® (Thermo scientific). The primary antibody was against the polyhistidine tag while a goat anti mouse antibody conjugated with DyLight 488 was used as the secondary. A biotinylated, his-tagged ‘protein x’ was the positive control. Blank streptavidin coated magnetic beads were used as the negative control.

3.7 A Comparison of different methods of membrane protein