Single molecule studies of the native hair cell mechanosensory transduction complex

Hearing and balance rely on the conversion of a mechanical stimulus into an electrical signal, a process known as mechanosensory transduction (MT). In vertebrates, this process is accomplished by an MT complex that is located in hair cells of the inner ear. While the past three decades of research have identified many subunits that are important for MT and revealed interactions between these subunits, the composition and organization of a functional complex remains unknown. The major challenge associated with studying the MT complex is its extremely low abundance in hair cells; current estimates of MT complex quantity range from 3–60 attomoles per cochlea or utricle, well below the detection limit of most biochemical assays that are used to characterize macromolecular complexes. Here we describe the optimization of two single molecule assays, single molecule pull-down (SiMPull) and single molecule array (SiMoA), to study the composition and quantity of native mouse MT complexes. We demonstrate that these assays are capable of detecting and quantifying low attomoles of the native MT subunits protocadherin-15 (PCDH15) and lipoma HMGIC fusion partner-like protein 5 (LHFPL5). Our results illuminate the stoichiometry of PCDH15- and LHFPL5-containing complexes and establish SiMPull and SiMoA as productive methods for probing the abundance, composition, and arrangement of subunits in the native MT complex.


Introduction
The vertebrate sensations of hearing and balance are critical for everyday life, yet the molecular basis of these sensations remains largely unresolved.This is due to the challenges associated with studying the mechanosensory transduction (MT) complex, the protein machinery that is responsible for converting the mechanical stimulus associated with a sound or fluid movement into an electrical signal that is processed by the brain 1 .Numerous genetic and biochemical studies have identified MT subunits that are important for mechanosensory transduction, including the tip-link proteins, protocadherin-15 (PCDH15) and cadherin-23 (CDH23), which connect adjacent stereocilia and transduce force associated with stereocilia movement into ion channel opening 2,3 .Transmembrane channel-like proteins 1 and 2 (TMC1/2) are the likely pore-forming subunits of the ion channel and are located at the lower end of the tip-link [4][5][6] .Lipoma HMGIC fusion partner-like protein 5 (LHFPL5, also known as TMHS) [7][8][9] , transmembrane inner ear protein (TMIE) [10][11][12] , and calcium and integrin-binding proteins 2 and 3 (CIB2/3) 13,14 are predicted to assemble with TMC1/2 and PCDH15 to form a functional MT complex.Additional proteins, including transmembrane O-methyltransferase (TOMT) 15,16 , whirlin 17,18 , and myosin XVa 19,20 , also play an important role in hearing and possibly interact with MT subunits at different stages of complex assembly.
Structural and biochemical studies have shed light on interactions between some of these subunits and their probable organization in the vertebrate MT complex.Recombinantly-expressed PCDH15 binds to LHFPL5 and CDH23, and their respective protein complexes have been characterized by cryo-EM 9 and x-ray crystallography 21 .Peptides of TMC1 form a complex with CIB2 and CIB3 14,22 , and mutagenesis experiments in mouse hair cells suggest that TMIE directly interacts with TMC1 12 .In accordance with these studies, the recently elucidated structures of the native C. elegans TMC-1 and TMC-2 complexes revealed a dimeric complex that is composed of two copies of TMC-1/2, TMIE, and CIB2/3, the latter of which is known as CALM-1 in C. elegans 23 .Further, while numerous immunoprecipitation and yeast twohybrid experiments have suggested interactions between MT subunits 10,12,15,24,25 , these experiments have been hampered by the inability to express biochemically well-behaved TMC1 and TMC2 proteins.TMC1 and TMC2 do not migrate to the plasma membrane in heterologous cell lines 26 , preventing reconstitution of the vertebrate MT complex.To elucidate the composition and organization of the vertebrate MT complex, it is therefore necessary to use native tissue.
A well-known hurdle in studying the vertebrate MT complex is its extremely low abundance.On the one hand, electrophysiological experiments of mouse hair cells indicate that there are approximately two functional MT channels per stereocilium [27][28][29] , which amounts to about three attomoles of MT complex per mouse cochlea.On the other hand, photobleaching experiments report an average of 7.1 TMC1 molecules per stereocilia in the inner hair cells of P4 aged mice and the number of TMC1 molecules varies from ~8 to 20 depending on the location in the cochlea 30 .Electron tomography images of labeled PCDH15 on native stereocilia derived from P6-P9 aged mice indicate there are anywhere from 2, to more than 5, PCDH15 dimers per tip, plus many more PCDH15 molecules that are localized on the stereocilia shaft as lateral links 31 .Quantification and characterization of attomole quantities of protein has been hindered by the available techniques.Biochemical methods that are commonly used to analyze macromolecular complexes, such as western blots, mass spectrometry, or enzyme-linked immunosorbent assays (ELISAs) require femtomoles of material.Ultrasensitive ELISA 32 and immuno-PCR 33,34 techniques developed in recent years can detect low attomole quantities of protein, but these assays are limited in their ability to accurately measure protein quantity or assess interactions between proteins and their stoichiometry.
Single molecule studies offer many advantages in the study of low abundance macromolecular complexes 35 because they allow for the capture of small amounts of material directly from tissue extracts.
Here we describe the optimization of two single molecule assays, single molecule pull-down (SiMPull) 35 and single molecule array (SiMoA) 36 , to study the composition and quantity of native mouse MT complexes, demonstrating that we can detect single digit attomoles of PCDH15-and LHFPL5-containing complexes.Our results not only shed light on the composition of the native mouse MT complex, but also establish techniques to quantify low abundance complexes and to establish the stoichiometry of MT subunits.

Development of an ultrasensitive SiMPull assay using recombinant PCDH15/LHFPL5
Due to the extremely low abundance of the MT complex in mouse cochlea, we sought to develop an ultrasensitive assay that is capable of detecting attomoles of protein.We employed single molecule pulldown (SiMPull), a technique that combines a conventional pulldown assay with total internal reflection microscopy (TIRF) to examine individual complexes from cell or tissue extracts 35,37 .The first step of this assay is to capture MT complexes from a cell extract using a monoclonal antibody (mAb) immobilized on a coverslip.Next, a mAb or antibody fragment (Fab) that is linked to a fluorophore is used to detect the complex.Subunit interactions are assessed through colocalization experiments, wherein multiple mAbs/Fabs are labeled with different fluorophores and applied to the slide simultaneously.Subunit stoichiometry is measured by labeling a mAb/Fab with a single GFP or YFP molecule and photobleaching the imaging area.High affinity antibodies are therefore a key component of a successful SiMPull assay.
We generated antibodies directed against different regions of PCDH15 by immunizing mice and rabbits with constructs of recombinantly expressed PCDH15 and the PCDH15/LHFPL5 complex (Fig 1a).We obtained two antibodies with exceptional binding kinetics that recognize native PCDH15: 8D1, which binds to the EC11 domain of PCDH15, and 39G7, which recognizes the EC3 domain of PCDH15.8D1 and 39G7 both bind PCDH15 with a sub-nanomolar KD (Fig. 1b, Supp.Fig. 1) and 8D1 exhibits a remarkably fast on rate of 7 x 10 5 M -1 s -1 .Immunostaining of wild-type (WT) mouse cochlea with these antibodies produces robust fluorescence that is localized to stereocilia tips (Fig. 1c).The 39G7 mAb was recently employed in cryo-electron tomography experiments to elucidate the molecular structures and conformational states of PCDH15 on stereocilia 31 .Further, we characterized an anti-LHFPL5 antibody and found that it binds to LHFPL5 with ~1 nM affinity and similarly stains the tips of stereocilia in WT mice (Fig. 1b,c).
Determination of the amino acid sequence of the variable domains of these three antibodies allowed us to generate recombinantly expressed Fab constructs, protein constructs that facilitate single molecule photobleaching experiments.
To develop a SiMPull assay of sufficient sensitivity to detect attomole quantities of PCDH15 and LHFPL5, we used recombinantly expressed PCDH15/LHFPL5 complex as a control.Multiple antibody combinations were assessed for capture and detection, allowing us to determine that 8D1 is the best antibody for capturing protein from low concentration samples, likely due to its fast on rate and slow dissociation rate (Supp.Fig. 1a).Passivated slides were coated with biotinylated 8D1 to capture PCDH15/LHFPL5, which was then detected with an anti-PCDH15 39G7 Fab fused to GFP or with an anti-LHFPL5 mAb conjugated to Alexa647 (Fig. 2a).We routinely were able to detect 3.2 attomoles of PCDH15 and 0.8 attomoles of LHFPL5 with signal-to-noise about 5-fold above background (Fig. 2b), indicating that the assay is adequately sensitive to detect native PCDH15 and LHFPL5.The difference in sensitivity between PCDH15 and LHFPL5 is likely due to the increased brightness of the Alexa647 fluorophore relative to a GFP molecule.For comparison, we performed a western blot with the same quantity of protein and discovered that we can detect only 12 picomoles of LHFPL5 with the anti-LHFPL5 mAb (Supp.Fig 2).8D1 and 39G7 both recognize a folded, three-dimensional epitope and are unsuitable for western blot.
Next, we analyzed the composition and stoichiometry of the recombinantly expressed PCDH15/LHFPL5 complex to confirm that it forms a heteromeric complex, as expected from structural studies 9 .Application of 10 µL of 100 pM PCDH15/LHFPL5 complex to the sample chamber, followed by simultaneous application of the 39G7-GFP and anti-LHFPL5-Alexa647 antibodies, enabled assessment of the degree of colocalization between PCDH15 and LHFPL5.Recombinant LHFPL5 colocalizes with PCDH15 62% of the time (Fig. 2c), consistent with the expectation that the two proteins are found in the same complex.Although PCDH15 and LHFPL5 form a stable, homogenous complex, their lack of 100% colocalization can be attributed to a number of factors, including incomplete antibody binding, incomplete fluorophore labeling, and dissociation of the PCDH15/LHFPL5 complex.
To determine the stoichiometry of PCDH15 and LHFPL5, we captured the complex with 8D1 and, in separate experiments, labeled PCDH15 with 39G7 conjugated to GFP and labeled LHFPL5 with the anti-LHFPL5 Fab conjugated to GFP.The imaging area was photobleached for one minute and the resulting movies were analyzed to determine stoichiometry.Approximately 68% of 39G7 Fab-GFP molecules and 68% of anti-LHFPL5 Fab-GFP molecules bleached in two steps, demonstrating that PCDH15 and LHFPL5 are present in two copies each.These results demonstrate that we have developed a highly sensitive, robust assay that is capable of detecting and defining the stoichiometry of native PCDH15-and LHFPL5containing complexes.

Native PCDH15 and LHFPL5 form a heterotetrametric complex
While tremendous progress has been made in identifying the proteins that are necessary for MT in hair cells, the composition of the MT complex and organization of subunits within the complex is unknown.It is suspected that native PCDH15 forms a complex with LHFPL5 based on data from biochemical experiments and mutagenesis studies in mice 7,9 .However, the interaction between native PCDH15 and LHFPL5 has never been demonstrated and it is unclear what portion of the PCDH15 population associates with LHFPL5.
To address these questions and demonstrate the utility of the SiMPull assay in detecting native MT subunits, we pulled down native PCDH15-containing complexes from mouse cochlea.Cochlea were homogenized in a buffer supplemented with a non-ionic detergent and the supernatant was applied to slides coated in the 8D1 antibody.Simultaneous application of the 39G7 Fab-GFP and anti-LHFPL5-Alexa647 mAb allowed us to measure colocalization between PCDH15 and LHFPL5.Approximately 58% of LHFPL5 subunits colocalized with PCDH15, indicating that the majority of PCDH15 molecules are in complex with LHFPL5 (Fig. 3a).Importantly, we did not observe a statistically significant signal above background for either PCDH15 or LHFPL5 when supernatant derived from the cochlea of PCDH15 knockout mice were applied to the slide.We similarly performed photobleaching experiments to assess the stoichiometry of native PCDH15 and LHFPL5.Approximately 59% of 39G7 Fab-GFP molecules and 63% of LHFPL5 molecules bleached in two-steps, indicating that both proteins are present in two copies.Our results indicate that PCDH15 and LHFPL5 form a heterotetrametric complex in hair cells and demonstrate that the SiMPull assay can be used to probe the assembly and stoichiometry of native MT subunits from solubilized mouse cochlea.

Single molecule quantitation of native PCDH15 and LHFPL5
Many different approaches have been employed to estimate the number of MT subunits per stereocilia, all of which have relied on counting subunits in images of stereocilia.Freeze-etch electron microscopy images of stereocilia tips from bullfrogs and guinea pigs suggest that there is only one tip-link per stereocilia 38 .
Cryo-electron tomograms of stereocilia with gold nanoparticle-labeled PCDH15 indicate that there are 2, to more than 5, PCDH15 molecules per stereocilia tip and dozens more PCDH15 per shaft, amounting to greater than 15 attomoles per utricle.High resolution confocal microscopy images of fluorescently-labeled TMC1 similarly suggest that there are ~8-20 TMC1 per stereocilia, or ~24-60 attomoles of TMC1 per cochlea.While these methods have provided insight into the abundance of MT subunits, they are complicated by damage to the stereocilia during sample preparation and background staining of the labeled antibody.These methods are also labor intensive, requiring acquisition of dozens of high-resolution images.
While SiMPull can be used to estimate the number of MT subunits, its accuracy is limited, in part due to the sample application method.The small volume of the SiMPull sample chamber requires iterative applications of the homogenized cochlea sample, introducing room for error.Further, the data from dilution experiments of recombinant PCDH15/LHFPL5 were not ideally linear (Fig. 2b).We therefore developed an ultrasensitive assay to accurately estimate the abundance of individual MT subunits in a mouse cochlea or utricle using single molecule array (SiMoA) 36 .
In several respects, SiMoA is similar to SiMPull in that MT subunits in cell or tissue lysate are first captured on an antibody-coated surface, which for SiMoA is a paramagnetic bead, and then subsequently detected with a second antibody that is labeled with -galactosidase (Fig. 4a).Following assembly of immunocomplexes on beads, the beads are loaded into an array of femtoliter-sized reaction wells, each of which can accommodate a single bead, and the -galactosidase substrate is applied.Beads that possess an enzyme-labeled immunocomplex generate a relatively high local concentration of fluorescent product that is contained within the femtoliter reaction well, thus amplifying the signal and allowing for detection of single complexes.
To develop the SiMoA assay, we used recombinant PCDH15/LHFPL5 complex and the same antibodies that were used in SiMPull assays (Fig. 1b).Paramagnetic beads were functionalized with 8D1 and incubated either with recombinant protein sample or, following method optimization, cochlea supernatant that was prepared identically to SiMPull experiments.The beads were washed extensively and incubated with biotinylated 39G7 or anti-LHFPL5 mAbs, followed by a streptavidin--galactosidase conjugate (Fig. 4a).Using the SR-X Biomarker Detection System, the beads were isolated in arrays of 50 femtoliter reaction wells and the number of fluorescent wells were quantitated.We found that we were able to reliably detect 2 attomoles of PCDH15 and 4 attomoles of PCDH15/LHFPL5 using this assay, well within the range needed for detection of native MT subunits (Fig. 4b).
We applied the SiMoA assay to the quantification of native PCDH15 and LHFPL5 from cochlea and utricle supernatant (Fig. 4c).We found that there are an average of 55 attomoles of PCDH15 per cochlea and 25 attomoles per utricle.The individual measurements of PCDH15 abundance spanned a wide range, from 31-76 attomoles per cochlea and 9-55 attomoles per utricle, likely due to experimental variability, coupled with variations in mouse age.The number of PCDH15 molecules per stereocilia changes dramatically during hair bundle development up to post-natal day 9 (ref 39), so measurements of PCDH15 abundance will change due to small differences in mouse age.A signal above background was not detected for PCDH15 derived from cochlea of knockout animals.The number of PCDH15/LHFPL5 complexes, measured by detecting PCDH15-captured complexes with an anti-LHFPL5 antibody, fell within a similar range, 18 attomoles per cochlea and 21 attomoles per utricle.These data suggest that the majority of native PCDH15 is in complex with LHFPL5, in accordance with data obtained from the SiMPull experiments.

Discussion
We have developed two single molecule assays for the detection, quantitation, and characterization of native MT subunits.We applied these assays to the native PCDH15/LHFPL5 complex isolated from mouse cochlea and utricles, demonstrating the exceptional sensitivity and utility of these assays for studying extremely low abundance proteins.The results of our SiMPull assay indicate that the majority of native PCDH15 is bound to LHFPL5 and the two proteins form a stable heterotetrameric complex.SiMoA quantitation reveals that there are an average of 18 attomoles of PCDH15/LHFPL5 complex and 55 attomoles of PCDH15 per cochlea, the first quantitative measurement of MT complex subunits.
The SiMPull and SiMoA assays have substantial potential for uncovering the molecular composition and organization of the MT complex, which have eluded scientists for decades.
Immunoprecipitation and yeast two-hybrid experiments suggest multiple interactions between MT subunits, but these results are clouded by an inability to reconstitute the complex in heterologous cell lines.
The SiMPull assay overcomes this barrier by isolating individual MT complexes from native cochlea and enabling analysis of their stoichiometry through photobleaching experiments, as well as their interactions with other subunits through colocalization experiments.These tools have the potential to elucidate the organization of the MT complex, as well as to report on the distribution of MT complexes if the subunit composition varies.Indeed, a recently-developed bead-based SiMPull assay indicates complex formation between TMC1 and LHFPL5, although colocalization and stoichiometry measurements were not possible due to the assay format 40,41 .An additional application of SiMPull and SiMoA is the study of hair bundle assembly.Information regarding the developmental progression of protein expression, as well as data surrounding protein-protein interactions, are needed to understand how the tightly coordinated process of hair bundle assembly occurs 42 .
There are, however, several limitations to these methods, the most importance of which is their reliance on high affinity antibodies.The assays cannot be employed if antibodies with good binding kinetics and affinities do not exist and there is no alternate way to capture the target protein, such as with a genetically-engineered tag.Additionally, it is possible that isolation of the complex results in the loss of weakly or transiently bound partners.Although we use gentle solubilization conditions, there is always a possibility that the removal of a protein from its native environment disrupts transient interactions.Further, both assays probe the composition of complexes derived from the entire cochlea, not just what is present at the stereocilia membrane.It is possible that the functional, fully-assembled MT complexes on stereocilia are only a fraction of the total complexes and that 'differently assembled variants' are present as intermediates or alternative species.

Construct design
The PCDH15 and LHFPL5 constructs correspond to the canonical Mus musculus sequences as recorded in the UniProt database.The PCDH15 construct was synthesized by Genscript and the LHFPL5 construct is the same construct used in previous studies 9 .All constructs were cloned into the pEG BacMam vector under control of the CMV promoter, allowing expression by infection using Baculovirus produced in Sf9 cells.Details regarding the sequences, fluorophores, and affinity tags are described in Figure 1.

Expression and purification of PCDH15 in complex with LHFPL5
The recombinant PCDH15/LHFPL5 complex was expressed and purified as described in Ge et al 9 .Briefly, HEK293 tsa201 cells were co-infected with PCDH15 and LHFPL5 BacMam viruses at a MOI of 1:1.
Cultures were supplemented with 10 mM sodium butyrate 12 hr post-infection and transferred to 30°C.
Cells were harvested 48 hr post-infection and lysed in buffer containing 100 mM Tris pH 8.0, 150 mM NaCl, 1% (w/v) digitonin and protease inhibitors for 2 hr at 4°C.The solubilized material was incubated with Strep-Tactin resin, washed with buffer A containing 20 mM Tris pH 8.0, 150 mM NaCl, 0.07% (w/v) digitonin and eluted with buffer A plus 5 mM desthiobiotin to remove free His-tagged PCDH15.The elution was then incubated with TALON resin, washed with buffer A plus 10 mM imidazole to remove free streptagged LHFPL5, and further eluted with buffer A plus 200 mM imidazole.The complex was then further purified by size exclusion chromatography in buffer A. Peak fractions were collected and analyzed by SDS-PAGE.

Expression and purification of antibody fragments
The anti-PCDH15 39G7 monoclonal antibody was generated as described in Elferich et al. 31 .The anti-LHFPL5 monoclonal antibody was obtained from Abcam and the amino acid sequence of the variable domain was determined by mass spectrometry.The anti-LHFPL5 construct was synthesized by Genscript.
The DNA sequences encoding the heavy and light chains of the variable domains of the 39G7 and anti-LHFPL5 antibodies were cloned into the pEG BacMam vector for baculovirus expression in HEK293 tsa201 cells.An mVenus tag and 8xHis tag were added to the C-terminus of the heavy chain in both constructs.Cells were infected with 39G7 or anti-LHFPL5 BacMam viruses at a MOI of 1:1.Cultures were supplemented with 10 mM sodium butyrate 12 hr post-infection and transferred to 30°C.Cells were harvested 96 hr post-infection and the cell media was filtered and concentrated to 200 mL.Concentrated media was incubated with TALON resin, washed with TBS plus 10 mM imidazole, and eluted with TBS plus 200 mM imidazole.The antibody fragments were further purified using size exclusion chromatography in PBS.The affinities of the 8D1, 39G7 and anti-LHFPL5 monoclonal antibodies for their antigen were determined with biolayer interferometry using an OctetRED384 instrument.

Cochlea and utricle solubilization
Cochlea and utricle samples were prepared by homogenizing cochlea or utricles from post-natal day 6 mice on ice with a pestle homogenizer in lysis buffer consisting of 1% C12M, 0.2% CHS, 50 mM Tris, 40 mM NaCl, 10 mM KCl, 1 mM EDTA, 1 mM protease inhibitors (0.8 µM aprotinin, 2 µg/ml leupeptin and 2 µM pepstatin), 0.2 mg/mL BSA, pH 8.0.20 L of lysis buffer was added per cochlea.Homogenized cochlea were incubated for 1 hr at 4 °C and insoluble material was pelleted by centrifuging for 10 minutes at 14,000 rpm.The supernatant was collected and used immediately for SiMPull or SiMoA experiments.

Single molecule pulldown
Coverslips and glass slides were prepared as described in Jain et al 37 .In brief, the coverslips and glass slides were extensively cleaned, passivated, and coated with methoxy polyethylene glycol (mPEG) and 2% biotinylated PEG.A flow chamber was created by drilling 0.75 mm holes in the quartz slide and placing double-sided tape between the holes.A coverslip was placed on top of the slide and the edges were sealed with epoxy, creating tiny flow chambers.0.25 mg/mL streptavidin was then applied to the slide, allowed to incubate for 5 minutes, and washed off with T50 BSA buffer consisting of 50 mM Tris pH 8.0, 50 mM NaCl, 0.25 mg/mL BSA.Anti-PCDH15 8D1 capture antibody was biotinylated using NHS-PEG4-biotin (Thermo Fisher CAS# 21330) and anti-LHFPL5 antibody was labeled with Alexa647 using a Zip Alexa Fluor Rapid Antibody Labeling Kit (Thermo Fisher CAS#Z11235).Excess dye and biotin were removed using two Zeba spin desalting columns per labeling reaction and labeling was confirmed using fluorescence-detection size exclusion chromatography (FSEC) 43 .Biotinylated 8D1 at approximately 7 g/mL was applied to the slide, allowed to incubate for 10 minutes, and washed off with buffer B containing 0.005% C12M, 0.001% CHS, 50 mM Tris, 40 mM NaCl, 10 mM KCl, 0.1 mM EDTA, 0.2 mg/mL BSA, pH 8.0.For recombinant PCDH15/LHFPL5 complex, the sample was diluted 50 pM in buffer B and incubated in the chamber for 5 minutes.For cochlea supernatant, the sample was applied to the chamber in 10 L increments, with a 5 minute incubation per application.Following sample application, the chamber was washed and 3 g/mL fluorophore-labeled detection antibody was applied to the chamber for 5 minutes.
The chamber was imaged immediately after the final wash step using total internal reflection (TIRF) microscopy and 15 images at different locations were collected per chamber.Photobleaching movies were collected by exposing the imaging area for 2 minutes.

Single molecule array
Recombinant PCDH15/LHFPL5 complex and cochlea samples were quantitated using the Quanterix homebrew assay kit and a Quanterix SR-X Biomarker Detection System.Anti-PCDH15 8D1 was conjugated to paramagnetic beads for capturing analyte and anti-PCDH15 39G7 mAb or anti-LHFPL5 mAb was biotinylated for analyte detection according to instructions in the homebrew assay kit.The 2-step method was employed wherein beads, sample, and detection antibody were incubated simultaneously for 30 minutes with continuous shaking at room temperature.Sample binding buffer consisted of 1% C12M, 0.2% CHS, 1% casein in PBS, 1 mM EDTA, protease inhibitors, and 10 g/mL mouse IgG.After incubation, the beads were washed with buffer C containing 0.005% C12M in PBS.120 pM streptavidin beta-galactosidase (SBG) was applied for 7 minutes and beads were washed again with buffer C, followed by a short wash with SiMoA wash buffer B (buffer composition is proprietary), and allowed to dry for 10 minutes.Dried plates were then placed in the SR-X for substrate application and analysis.All samples were run in duplicate.

Figure 1 :
Figure 1: Characterization of reagents used for SiMPull and SiMoA assay development.

Figure 4 :
Figure 4: SiMoA assay development and quantification of native PCDH15 and the PCDH15/LHFPL5