Journal: Analytical Chemistry

Article Title: Overcoming Lot-to-Lot Variability in Protein Activity Using Epitope-Specific Calibration-Free Concentration Analysis

doi: 10.1021/acs.analchem.3c05607

Figure Lengend Snippet: Variability in the percent activity of distinct sLAG3 reagent lots may account for perceived discrepancies in kinetic fits of the respective binding parameters. (A) A pie chart depicting the active and inactive species that may proportionally change between reagent lots, producing lot-to-lot discrepancies in assay responses, using domains of PDB: 7TZG . (B) The molar active concentrations reported for different epitopes from Lot1 of sLAG3 calibrator (Bradford: n = 2, Capture-ProteinG: n = 7, Capture-ProteinA: n = 2, Capture-CM5: n = 2, Detection(u)-CM5: n = 2, Detection(s)-CM5: n = 2). The Capture-CM5 and Detection(s)-CM5 chips were calibrated with a ProteinG-CM5 chip from the same chip lot, while the Detection(u)-CM5 chip was calibrated with a ProteinA-CM5 chip. Capture mAb was biotinylated, Detection(u) mAb was unconjugated, and Detection(s) mAb was sulfo-tagged. (C) An expansion of the data shown in panel (B) including all three sLAG3 lots, comparing each active concentration value to the respective lot’s Bradford total protein concentration to measure the percent activity of each epitope in each lot. (D) Biolayer interferometry was performed to measure the binding kinetic on-rates of the sLAG3 reagent lots to the capture or detection mAbs. Apparent lot-to-lot discrepancies measured using Bradford-defined concentrations were much less prominent when sLAG3 lots were instead defined by mAb-specific active concentrations ( n = 2). (E) The respective dissociation constants from the fits in panel (D) similarly demonstrate that using the active concentrations of each sLAG3 reagent leads to increased lot-to-lot agreement compared with using the total concentration.

Article Snippet: Additionally, the active concentrations for the capture mAb epitope measured using a CM5, ProteinA, or ProteinG chip were similar for each lot of sLAG3, emphasizing the utility of independently calibrating each flow cell/sensor ( Figure B,C).

Techniques: Activity Assay, Binding Assay, Concentration Assay, Protein Concentration

Journal: Analytical Chemistry

Article Title: Overcoming Lot-to-Lot Variability in Protein Activity Using Epitope-Specific Calibration-Free Concentration Analysis

doi: 10.1021/acs.analchem.3c05607

Figure Lengend Snippet: Defining sLAG3 calibrator lots by the capture or detection epitope active concentration moderately unifies immunoassay responses over total protein concentration. (A) Venn diagram representing the overlap of protein species with active capture mAb and/or detection mAb epitopes. (B) MSD titration of three sLAG3 lots, defining each lot by their respective Bradford concentration, capture mAb concentration (Capture-ProteinG), or detection mAb concentration (Detection(s)-CM5). Pairwise %CV comparisons were conducted using data from a prior sLAG3 bridging study, containing responses from 50 patient serum samples. These signals were reinterpolated using the standard curves shown above to have %CVs spanning the clinically relevant range of MSD signals. The overall %CVs for the three lots (100* stdev of all three/mean of all three) were 42.4% for Bradford, 19.0% for capture mAb, and 18.5% for detection mAb.

Article Snippet: Additionally, the active concentrations for the capture mAb epitope measured using a CM5, ProteinA, or ProteinG chip were similar for each lot of sLAG3, emphasizing the utility of independently calibrating each flow cell/sensor ( Figure B,C).

Techniques: Concentration Assay, Protein Concentration, Titration

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) The 25 a.a. peptide used to immunize the mice and location in the full-length protein. B) Western blot showing the specificity of each hybridoma clone selected on HEK cells lysate. Cells were transfected with either an aSyn encoding plasmid alone or in combination with a PLK2 kinase encoding plasmid. A plasmid encoding the mutated aSyn-S129G that cannot be phosphorylated at the serine 129 was also used. A condition with no transfection was used as a negative control. Commercial antibodies against total and pS129-aSyn were used as comparison (ab51253 against pS129-aSyn, and CST2628 against total aSyn). C) Representative images of immunofluorescence on HEK cells that were transfected with the plasmids described above using each hybridoma clone media as a primary antibody solution. Antibody-specific signal is shown in white, DAPI is shown in blue. Scale bar = 50μm.

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Western Blot, Transfection, Plasmid Preparation, Negative Control, Comparison, Immunofluorescence

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) Representative images of immunofluorescence using our purified monoclonal antibodies on human Parkinson’s disease (n=3), and control (n=2) substantia nigra sections. Antibody-specific signal is shown in white while TH is shown in red. Commercial antibodies were used as a comparison (CST2628 against total aSyn, and BioLegend 825701 against pS129-aSyn). B) Representative images of immunofluorescence using our purified monoclonal antibodies on human MSA (n=3), DLB (n=3) and control (n=2) cortex sections. SN = Substantia nigra; MSA = multiple system atrophy; DLB = dementia with Lewy bodies. Scale bar = 100μm.

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Immunofluorescence, Purification, Control, Comparison

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A-D) Real-time binding curves of 833D4 and 73B3 IgGs to aSyn and pS129-aSyn measured using surface plasmon resonance (SPR). aSyn monomers were coated on the sensor chip, and the antibodies were flown over at different concentrations. The response is shown in resonance units, which is proportional to the number of bound antibodies to the chip. E) Association (Ka), dissociation (Kd) and equilibrium (KD) constants of both antibodies towards aSyn and pS129-aSyn. F) and G) A Thioflavin T (ThT) aggregation assay was performed by incubating aSyn monomers in the presence of ThT with agitation for seven days at 37°C (n=3). F) Relative fluorescence units (RFU) of ThT correlating to the level of β-sheets structures of aSyn monomers in the presence of 833D4 or an unspecific IgG. G) RFU of ThT with pS129-aSyn monomers in the presence of 73B3 or an unspecific IgG.

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Binding Assay, SPR Assay, Fluorescence

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) Illustration of the structure of a scFv generated from a monoclonal IgG. B) Illustration of the scFv construct cloned inside a scAAV expression plasmid. C) Dot blot performed by blotting aSyn or pS129 aSyn monomers (and BSA as a negative control) on a nitrocellulose membrane. Membranes were either incubated with cell media from HEK cells previously transfected with each pscFvs, or with the purified monoclonal IgGs. Results show that both scFvs maintained the same specificity as their parental IgG. D) ELISA performed by coating the plate with purified monomers of aSyn or pS129-aSyn (or BSA as a negative control) showing the specificity of scFv-833D4 and scFv-73B3. Again, the cell media from HEK cells previously transfected with each pscFvs was used, and commercial antibodies to total (CST2628) and pS129-aSyn (Wako 015-25191) were used as positive controls (n=2).

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Generated, Construct, Clone Assay, Expressing, Plasmid Preparation, Dot Blot, Negative Control, Membrane, Incubation, Transfection, Purification, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) Representative images of pS129-aSyn immunostaining in the midbrain, PAG/pons and medulla of M83 mice injected with PFFs and with scAAVs encoding scFv-833D4, scFv-73B3, and scFv-control. B), C), D) and E) Quantification of the pS129-aSyn staining as a percentage of the area measured (n=6/group). One-way ANOVA with Dunnett’s multiple comparison test *p<0.05. **p<0.01. F) Representative images of pS129-aSyn and total aSyn in western blot on medulla tissue lysate. G) Quantification of pS129-aSyn normalized on total aSyn. One-way ANOVA with Dunnett’s multiple comparison test *p<0.05. H) Quantification of total aSyn normalized on actin (n=3-4/group). One-way ANOVA with Dunnett’s multiple comparison test. (n=3-4/group).

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Immunostaining, Injection, Control, Staining, Comparison, Western Blot

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A-C) Aged M83 homozygous mice were injected intravenously with an AAV-CapB10-scFv-IRES-GFP. This construct allows the expression of a secreted HA-tagged anti-aSyn scFv (833D4) and a non-secreted GFP protein. Mice were sacrificed 3 weeks post-injection. B) Representative confocal image taken in the cerebellum showing HA (scFv) in white and GFP in green. Red arrows point to neurons that are HA + /GFP - and thus have internalized the scFv. C) Confocal image showing the colocalization of the scFv (HA) and pS129-aSyn in a GFP-negative neuron from the medulla. D) Confocal images showing the colocalization of the scFv (HA, white) and Iba1 (red), indicating the internalization of scFvs by microglia. E) Representative images of human iPSCs-derived dopaminergic neurons (identified with a TH staining) infected with a scAAV2/retro encoding either scFv-833D4, scFv-73B3 or control scFv (visualized with Myc tag staining) and exposed for 2 hours to human aSyn PFFs tagged with Alexa Fluor 488. Scale bar = 10μm. F) Quantification with corrected total cell fluorescence (CTCF) of Alexa Fluor 488 intensity inside TH+ neurons (n=6 coverslips/condition, from 3 independent differentiations). One-way ANOVA with Dunnett’s multiple comparison test, *p<0.05, **p<0.01.

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Injection, Construct, Expressing, Derivative Assay, Staining, Infection, Control, Fluorescence, Comparison

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) The 25 a.a. peptide used to immunize the mice and location in the full-length protein. B) Western blot showing the specificity of each hybridoma clone selected on HEK cells lysate. Cells were transfected with either an aSyn encoding plasmid alone or in combination with a PLK2 kinase encoding plasmid. A plasmid encoding the mutated aSyn-S129G that cannot be phosphorylated at the serine 129 was also used. A condition with no transfection was used as a negative control. Commercial antibodies against total and pS129-aSyn were used as comparison (ab51253 against pS129-aSyn, and CST2628 against total aSyn). C) Representative images of immunofluorescence on HEK cells that were transfected with the plasmids described above using each hybridoma clone media as a primary antibody solution. Antibody-specific signal is shown in white, DAPI is shown in blue. Scale bar = 50μm.

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Western Blot, Transfection, Plasmid Preparation, Negative Control, Comparison, Immunofluorescence

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) Representative images of immunofluorescence using our purified monoclonal antibodies on human Parkinson’s disease (n=3), and control (n=2) substantia nigra sections. Antibody-specific signal is shown in white while TH is shown in red. Commercial antibodies were used as a comparison (CST2628 against total aSyn, and BioLegend 825701 against pS129-aSyn). B) Representative images of immunofluorescence using our purified monoclonal antibodies on human MSA (n=3), DLB (n=3) and control (n=2) cortex sections. SN = Substantia nigra; MSA = multiple system atrophy; DLB = dementia with Lewy bodies. Scale bar = 100μm.

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Immunofluorescence, Purification, Control, Comparison

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A-D) Real-time binding curves of 833D4 and 73B3 IgGs to aSyn and pS129-aSyn measured using surface plasmon resonance (SPR). aSyn monomers were coated on the sensor chip, and the antibodies were flown over at different concentrations. The response is shown in resonance units, which is proportional to the number of bound antibodies to the chip. E) Association (Ka), dissociation (Kd) and equilibrium (KD) constants of both antibodies towards aSyn and pS129-aSyn. F) and G) A Thioflavin T (ThT) aggregation assay was performed by incubating aSyn monomers in the presence of ThT with agitation for seven days at 37°C (n=3). F) Relative fluorescence units (RFU) of ThT correlating to the level of β-sheets structures of aSyn monomers in the presence of 833D4 or an unspecific IgG. G) RFU of ThT with pS129-aSyn monomers in the presence of 73B3 or an unspecific IgG.

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Binding Assay, SPR Assay, Fluorescence

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) Illustration of the structure of a scFv generated from a monoclonal IgG. B) Illustration of the scFv construct cloned inside a scAAV expression plasmid. C) Dot blot performed by blotting aSyn or pS129 aSyn monomers (and BSA as a negative control) on a nitrocellulose membrane. Membranes were either incubated with cell media from HEK cells previously transfected with each pscFvs, or with the purified monoclonal IgGs. Results show that both scFvs maintained the same specificity as their parental IgG. D) ELISA performed by coating the plate with purified monomers of aSyn or pS129-aSyn (or BSA as a negative control) showing the specificity of scFv-833D4 and scFv-73B3. Again, the cell media from HEK cells previously transfected with each pscFvs was used, and commercial antibodies to total (CST2628) and pS129-aSyn (Wako 015-25191) were used as positive controls (n=2).

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Generated, Construct, Clone Assay, Expressing, Plasmid Preparation, Dot Blot, Negative Control, Membrane, Incubation, Transfection, Purification, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A) Representative images of pS129-aSyn immunostaining in the midbrain, PAG/pons and medulla of M83 mice injected with PFFs and with scAAVs encoding scFv-833D4, scFv-73B3, and scFv-control. B), C), D) and E) Quantification of the pS129-aSyn staining as a percentage of the area measured (n=6/group). One-way ANOVA with Dunnett’s multiple comparison test *p<0.05. **p<0.01. F) Representative images of pS129-aSyn and total aSyn in western blot on medulla tissue lysate. G) Quantification of pS129-aSyn normalized on total aSyn. One-way ANOVA with Dunnett’s multiple comparison test *p<0.05. H) Quantification of total aSyn normalized on actin (n=3-4/group). One-way ANOVA with Dunnett’s multiple comparison test. (n=3-4/group).

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Immunostaining, Injection, Control, Staining, Comparison, Western Blot

Journal: bioRxiv

Article Title: Virally encoded single-chain antibody fragments targeting alpha-synuclein protect against motor impairments and neuropathology in a mouse model of synucleinopathy

doi: 10.1101/2024.09.09.612044

Figure Lengend Snippet: A-C) Aged M83 homozygous mice were injected intravenously with an AAV-CapB10-scFv-IRES-GFP. This construct allows the expression of a secreted HA-tagged anti-aSyn scFv (833D4) and a non-secreted GFP protein. Mice were sacrificed 3 weeks post-injection. B) Representative confocal image taken in the cerebellum showing HA (scFv) in white and GFP in green. Red arrows point to neurons that are HA + /GFP - and thus have internalized the scFv. C) Confocal image showing the colocalization of the scFv (HA) and pS129-aSyn in a GFP-negative neuron from the medulla. D) Confocal images showing the colocalization of the scFv (HA, white) and Iba1 (red), indicating the internalization of scFvs by microglia. E) Representative images of human iPSCs-derived dopaminergic neurons (identified with a TH staining) infected with a scAAV2/retro encoding either scFv-833D4, scFv-73B3 or control scFv (visualized with Myc tag staining) and exposed for 2 hours to human aSyn PFFs tagged with Alexa Fluor 488. Scale bar = 10μm. F) Quantification with corrected total cell fluorescence (CTCF) of Alexa Fluor 488 intensity inside TH+ neurons (n=6 coverslips/condition, from 3 independent differentiations). One-way ANOVA with Dunnett’s multiple comparison test, *p<0.05, **p<0.01.

Article Snippet: Briefly, aSyn or pS129-aSyn monomers (Proteos #RP-003 or #RP-004) diluted in 10 mM NaAc (pH 5.5) were immobilized on a CM5 sensor chip using maleimide EDC/NHS coupling.

Techniques: Injection, Construct, Expressing, Derivative Assay, Staining, Infection, Control, Fluorescence, Comparison