Sensitive Protein Detection Using Site-Specifically Oligonucleotide-Conjugated Nanobodies

High-quality affinity probes are critical for sensitive and specific protein detection, in particular for detection of protein biomarkers in the early phases of disease development. Proximity extension assays (PEAs) have been used for high-throughput multiplexed protein detection of up to a few thousand different proteins in one or a few microliters of plasma. Clonal affinity reagents can offer advantages over the commonly used polyclonal antibodies (pAbs) in terms of reproducibility and standardization of such assays. Here, we explore nanobodies (Nbs) as an alternative to pAbs as affinity reagents for PEA. We describe an efficient site-specific approach for preparing high-quality oligo-conjugated Nb probes via enzyme coupling using Sortase A (SrtA). The procedure allows convenient removal of unconjugated affinity reagents after conjugation. The purified high-grade Nb probes were used in PEA, and the reactions provided an efficient means to select optimal pairs of binding reagents from a group of affinity reagents. We demonstrate that Nb-based PEA (nano-PEA) for interleukin-6 (IL6) detection can augment assay performance, compared to the use of pAb probes. We identify and validate Nb combinations capable of binding in pairs without competition for IL6 antigen detection by PEA.


Method S3.
Expression, isolation and affinity measurement of NbSORIL6 S4 Tables and Figures   Table S1.
Summary of Nbs and tag sequences S5 Table S2. Affinity comparison of Nbs with different tags S5 Table S3.
Summary of oligos sequences composition, length (nt) and modifications of oligos conjugated to the Ab and Nb used for PEA S6 Table S4. Equilibrium dissociation constants (KD) as determined by various models . SPR sensorgrams of single cycle kinetics measurenet for all four NbSORIL6 products interacting with the target IL6 antigen S11 Figure S6. Gel electrophoretic validation of unpurified products of sortasemediated oligo conjugation reactions of four different Nbs S12 Figure S7.
Comparison of the effect of dimerizing Nb probes. S13 Figure S8.
Gel validation of Ab-oligos conjugation by copper free click chemistry S14 References S15

Methods
Method S1. Expression, identification and selection of IL6-specific Nbs (NbSORIL6s . Four days after the last immunization, lymphocytes were extracted from the anticoagulated blood of the animal, and cDNA was prepared and used as a template in a two-step, nested PCR that amplified the gene fragments encoding the variable domain of the heavy-chainonly Abs. The amplified DNA fragments encoding Nbs were ligated into a pMECS phagemid vector and transformed into Escherichia coli TG1 cells (Lucigen), resulting into a cloned Nblibrary of 7.7x10 8 transformants of which 80% contained a phagemid with the expected insert size for a Nb. After three rounds of phage selection, 24 individual clones were cultured from each round of panning, and their periplasmic proteins were tested for specificity against recombinant human IL6 protein in an ELISA. 1 The ELISA-positive clones were selected for DNA sequencing (VIB Genetic Service Facility, Antwerp, Belgium) of their phagemid insert. The inserted DNA sequences were in silico translated into amino acid sequences, aligned according to the international ImMunoGeneTics database (http://imgt.cines.fr, accessed on 25 October 2019), and manually annotated. 2 Six different IL6-specific Nbs (NbIL6_5,15, 16, 21, 25 and 91) were selected and transformed in Escherichia coli WK6 cells for periplasmic expression. Following extraction from the periplasm by osmotic shock, these 6 Nbs (containing a HA and a His-tag at the C-terminus) were purified by IMAC and SEC. NbIL6_91 produced very poorly and could only be purified in small quantities. We further performed an enzymelinked immunosorbent assays (ELISA) to select the most suitable IL6 binders.

Method S2. NbIL6s clones' selection via enzyme-linked immunosorbent assays (ELISA).
A 96-well microplate (Thermo scientific Nunc Maxisorp™) was coated with 100 μl/well of IL6 antigen (at 2 μg/ml in ELISA coating buffer (0.1 M NaHCO3, pH 8.2)) and incubated overnight at 4°C. Next, the wells were rinsed 5 times using PBST20 buffer (PBS + 0.05% Tween-20), blocked for 120 min at room temperature (RT) with 200 µl skimmed milk powder in PBS (1% w/v) and again washed 5 times using PBST20 buffer. The NbIL6-HAHis6 were then added to the wells in a 4-fold serial dilution starting from a concentration of 8 µM to 30.5 pM in PBS. After 60 min incubation at RT, the plate was washed 10 times with PBST20 buffer. Detection of attached Nb was performed with a monoclonal mouse anti-HA Ab (Biolegend; 1 μg/ml) followed by a 2000-fold diluted goat anti-mouse IgG-alkaline phosphatase conjugated Ab, each for 60 min at RT. Finally, the plate was rinsed 5 times with PBST20 and the color was developed with 4-phenylphosphate disodium substrate (Sigma-Aldrich) (2 mg/ml) and read at 405 nm after 30 min of reaction.

Method S3. Expression, isolation and affinity measurement of NbSORIL6.
Recombinant protein binding reagents may be modified by site-directed conjugation, without risking inactivation of the affinity reagent. To allow coupling of the oligos required for PEA to the Nbs via a SrtA reaction, four IL6-specific Nbs (NbSORIL6s) were recloned to include a C-terminal sortase tag (NbSORIL6s). To this end, these Nbs were cloned into the pTEVSOR expression vector, containing a TEV-cleavage site (TENLYFQS), a sortase recognition site (LPETGG), a His6-tag and a C-tag (EPEA) downstream of the Nb (summary of Nbs and tag sequences in Supplementary Table S1). The Nb genes were amplified by PCR, and PstI and Eco91I (ThermoScientific) digestion fragments were ligated into the pTEVSOR vector cut with the same restriction enzymes. After expression and purification, the affinities for IL6 of these Nbs with or without sortase tags, were measured via SPR (Supplementary Table 2). The analyses revealed no major differences in binding kinetics when comparing the Nbs in their original form with C-terminal HA-His6-tag versus the sortase tag-modified variants (Supplementary Table 2 1.71 x 10 -9 NbSORIL6_5 2.03 x 10 -9 NbIL6_15 6.47 x 10 -9 NbSORIL6_15 8.14 x 10 -9 NbIL6_16 6.96 x 10 -9 NbSORIL6_16 7.41 x 10 -9 NbIL6_21 1.37 x 10 -8 NbSORIL6_21 1.0 x 10 -8 Supplementary Table S3. Summary of oligos sequences composition, length (nt) and modifications, designed for conjugation to the Ab and Nb used in PEA.  Table 1. S11 Figure S5.

Sequence (5´-> 3´) and modifications Suppliers
Supplementary Figure S5. Interactions between Nbs and IL6 captured on mAb that has been immobilized on the chip surface. Since the NbSORIL6 showed a slow dissociation from the target protein, a single-cycle kinetics experiment was performed. The sensorgram results, shown in red, were fitted to the different interaction models in black. The data were fitted to 1:1 and steady-state affinity models. The left column represents fitting to a 1:1 interaction mode and the right represents a steady state analysis. S14 Figure S8.
Supplementary Figure S8. Gel validation of Ab-oligos conjugation by copper free click chemistry. Ab-oligo conjugates were validated by agarose gel electrophoresis, bands in the red box in lanes 2, 3, 4, 5, 6 and 7 are free oligos while the Ab-ssDNA conjugates are in the blue box in lanes 4, 5, 6 and 7. In contrast to site-directed Nb-oligo conjugates, Ab-oligo conjugation produces multiple species with varying number of oligos conjugated to the individual Abs at different locations.