Native, cell-based antigen for antibody selection

At Abwiz Bio, we use a revolutionary approach that combines immunization with phage display. By harnessing the power of the rabbit immune response, we can obtain high affinity, high specificity antibodies that do not require affinity maturation. By using phage display, instead of hybridoma-based methods, we can select for the rare clones that possess desired functional properties. These clones would be missed by other systems that rely on screening alone. It is easy to obtain antibodies that work in ELISA, but it is much harder to obtain functional clones that work in a specific assay.

Phage display is compatible with peptide, recombinant protein, and cell-based, native antigen formats. Our platform can combine these antigens during phage panning in a unique way to obtain clones that recognize native antigen. We have successfully used this approach in our affinity maturation platform and to obtain antibodies that recognize cell-surface antigens. Below, we describe representative results for the development of a phospho-specific antibody for flow cytometry.

Case Study: Phospho-Jak2 (Y1007/1008)

In this study, we wanted to determine if we could elicit phospho-specific antibodies that recognize the native target protein and possess strong activity by flow cytometry. A phospho peptide was designed, synthesized, and used to immunize rabbits. The serum from the immunized rabbit was tested by flow cytometry for binding to cells treated with a cocktail that boosts dual phosphorylation of Jak2 at Tyr1007 and Tyr1008.

Flow cytometry analysis of rabbit serum
Immunized rabbit serum shows strong binding to treated cells that is blocked phospho-peptide only.

We observed strong binding to treated cells (with minimal binding to control cells) which was specifically blocked by phospho-peptide and not by an identical but non-phosphorylated control peptide. DNA libraries were constructed from the immunized rabbit.

Polyclonal phage screening

We observed strong binding to treated cells (with minimal binding to control cells) which was specifically blocked by phospho-peptide and not by an identical but non-phosphorylated control peptide. DNA libraries were constructed from the immunized rabbit.

Using our proprietary, optimized phage-Fab vector, phage panning was performed with three rounds of panning on phospho-peptide, followed by a fourth round of selection on either phospho-peptide or on treated cells. Phage populations were propagated before (Round 0, R0) or after (R1, R2, R3, R4-peptide, and R4-cells) each round of selection. To monitor the enrichment of phospho-specific phages, polyclonal phage ELISA was performed.

Phage ELISA showing enrichment of target-binding phages
Polyclonal phage ELISA shows phospho-specific clones emerging in round 2 and further enriched in rounds 3-4. All phage titers are normalized.

In our experience, enrichment of antigen-specific clones that possess activity by ELISA is relatively straightforward from immunized rabbit libraries and can be accomplished by a variety of methods including hybridoma-based systems. However, obtaining functional clones that possess desired activity, such as recognition of native antigen, is more difficult and where our RabWizTM platform, that pairs immunized libraries with the versatility of phage display-based selection, really shines.

In the above ELISA, it appears that four rounds of selection on phospho-peptide elicited the highest activity. However, we next tested for enrichment of clones recognizing the native antigen by using polyclonal phage in flow cytometry.

Phage flow showing enrichment of cell binding
Flow cytometry using polyclonal phage shows enrichment of cell binders only in the R4 phage population that was selected on three rounds of phospho-peptide antigen followed by a single round of cell-based antigen. All phage titers are normalized.

Only by using cell-based selection presenting the native antigen were we able to obtain functional antibodies that showed activity by flow cytometry. To further confirm this, individual clones were picked from the R4 populations and tested by ELISA and flow cytometry.

Monoclonal Fab screening

Peptide panning yielded Fabs with weak flow binding
Fabs from the R4-peptide population (4 rounds of panning on phospho-peptide only) were propagated in 96-well format and tested by ELISA on phospho-peptide or non-phospho peptide. Nearly all peptide-binding clones were phospho-specific. However, low activity was observed by flow cytometry.
Cell panning yielded Fabs with weak flow binding
Fabs from the R4-cells population (3 rounds of panning on phospho-peptide followed by a single round on cells) were propagated in 96-well format and tested by ELISA on phospho-peptide or non-phospho peptide. All peptide-binding clones were phospho-specific. Strikingly, strong activity was observed for nearly all by flow cytometry.

Monoclonal Fab screening clearly shows that (1) ELISA screening does not necessarily correlate with recognition of native antigen and (2) only by using native antigen for selection could these unique clones be identified. Further characterization was performed for the flow cytometry binders by DNA sequencing

CDR grouping of Fab clones
VL and VH sequences were obtained for the strongest binding flow-positive Fabs from both R4-peptide and R4-cells populations. ELISA values are reported, as are the median fluorescence intensity ratios (positive cells/control cells) for flow cytometry. Notably, the peptide-only selected clones possessed a major dominant cluster. The Fabs with the strongest flow activity possessed a unique CDR H3 and only emerged from the library after cell-based panning.

This data gives strong evidence for the robustness of our unique RabWizTM system. Traditional hybridoma-based systems that rely on screening directly from immunized rabbit libraries would have likely failed to elicit these rare clones. Only by using a platform that harnesses the power of both the rabbit immune system and phage-based selections could these functional clones be obtained. This product is currently available from our catalog.