phosphorylation site-specific antibodies  (ABclonal Biotechnology)

Journal: medRxiv

Article Title: Pittsburgh plasma p-tau217: classification accuracies for autosomal dominant and sporadic Alzheimer’s disease in the community

doi: 10.1101/2025.05.03.25326526

Figure Lengend Snippet: Each panel displays the results of sandwich ELISAs, utilizing either the p-tau217-specific rabbit monoclonal antibody or the anti-tau mouse monoclonal antibody, clone Tau5, for capture. Detection was performed using a biotinylated anti-tau mouse monoclonal antibody, clone Tau12 – the same antibody used for detection in the eventual Pittsburgh p-tau217 assay. The assays tested varying concentrations of recombinant tau441 (non-phosphorylated tau441 (2N4R) isoform) or its GSK3beta-phosphorylated variant (p-tau441) either alone (A and B) or against a set concentration (0.1 μg/ml) of a synthetic peptide (C-E). These peptides, corresponding to tau441 amino acids 210 to 224 (SRTPSLPTPPTREPK), were linked to an N-terminal cysteine via a peptide bond and phosphorylated at the residues specified in the illustrations in the figure. Panels (A-B) depict the binding profiles of the p-tau217 and Tau5 antibodies to recombinant non-phosphorylated tau441 (A) and phosphorylated tau441 (B). Panels (C-E) show the binding profiles of the p-tau217 and Tau5 antibodies to phosphorylated tau441 in the presence of synthetic peptides phosphorylated exclusively at threonine-217 (C), peptides phosphorylated jointly at serine/threonine 210, 212, 214, and 217 (D), and non-phosphorylated peptides (E).

Article Snippet: We chose to compare the Pittsburgh and ALZpath p-tau217 assays as like-for-like methods in the sense that: (1) they both use Simoa technology; (2) both assays use monoclonal antibodies that bind p-tau217 specifically without cross-reactivity to neighboring phosphorylation sites, as seen with the Janssen p-tau217+ assay for example; and (3) they both partner their p-tau217 antibody with an N-terminal phosphorylation-independent detection antibody.

Techniques: Recombinant, Variant Assay, Concentration Assay, Binding Assay

Journal: medRxiv

Article Title: Pittsburgh plasma p-tau217: classification accuracies for autosomal dominant and sporadic Alzheimer’s disease in the community

doi: 10.1101/2025.05.03.25326526

Figure Lengend Snippet: Section of middle temporal gyrus from an AD case neuropathologically diagnosed as Braak stage VI, immunohistochemically stained with p-tau217 antibody. A dense network of immunoreactive structures is present throughout the cortical laminae illustrated at low magnification in (A). The dashed red box in (A) delineates the area shown at higher magnification in panel (B). The dashed red boxes in (B) delineate areas illustrated at higher magnifications in panels (C, superficial lamina) and (D, deep lamina). The dashed red boxes in (C) and (D) delineate areas illustrated at higher magnifications in panels (E) and (F), respectively. Immunoreactive clusters of dystrophic neurites in neuritic plaques are most numerous in superficial lamina, while immunoreactive neurofibrillary tangles and neuropil threads are numerous throughout the cortical laminae. Scale bars = 500 µm (A), 200 µm (B), 100 µm (C, D), and 50 µm (E, F).

Article Snippet: We chose to compare the Pittsburgh and ALZpath p-tau217 assays as like-for-like methods in the sense that: (1) they both use Simoa technology; (2) both assays use monoclonal antibodies that bind p-tau217 specifically without cross-reactivity to neighboring phosphorylation sites, as seen with the Janssen p-tau217+ assay for example; and (3) they both partner their p-tau217 antibody with an N-terminal phosphorylation-independent detection antibody.

Techniques: Staining

Journal: medRxiv

Article Title: Pittsburgh plasma p-tau217: classification accuracies for autosomal dominant and sporadic Alzheimer’s disease in the community

doi: 10.1101/2025.05.03.25326526

Figure Lengend Snippet: Section of hippocampus from an AD case neuropathologically diagnosed as Braak stage VI, immunohistochemically stained with p-tau217 antibody. Numerous immunoreactive neurofibrillary tangles are present in the CA fields, immunoreactive dystrophic neurites in neuritic plaques are abundant in the dentate gyrus stratum moleculare, and immunoreactive neuropil threads are distributed throughout the CA fields and dentate gyrus illustrated in a low magnification composite in panel (A). The dashed red box in (A) delineates the area (field CA1 and dentate stratum moleculare) shown at higher magnification in panel (B). The dashed red boxes in (B) delineate areas illustrated at higher magnifications in panels (C) and (D). The dashed red boxes in (C) and (D) delineate areas illustrated at higher magnifications in panels (E) and (F), respectively. Immunoreactive neurofibrillary tangles embedded in a mesh of neuropil threads are the most prominent features of the CA1 area. Many immunoreactive neurons had a classic flame-shaped tangle morphology, while others appeared to contain more homogeneous immunoreaction and morphology of intracellular tangles.

Article Snippet: We chose to compare the Pittsburgh and ALZpath p-tau217 assays as like-for-like methods in the sense that: (1) they both use Simoa technology; (2) both assays use monoclonal antibodies that bind p-tau217 specifically without cross-reactivity to neighboring phosphorylation sites, as seen with the Janssen p-tau217+ assay for example; and (3) they both partner their p-tau217 antibody with an N-terminal phosphorylation-independent detection antibody.

Techniques: Staining

Journal: medRxiv

Article Title: Pittsburgh plasma p-tau217: classification accuracies for autosomal dominant and sporadic Alzheimer’s disease in the community

doi: 10.1101/2025.05.03.25326526

Figure Lengend Snippet: Dilution linearity of the p-tau217 assay in pooled plasma samples (A). For each matrix, the plots show the measured AEB signal in three unique samples with variable levels of the biomarker. Three equal-volume aliquots of each sample were prepared and measured diluted 2-, 4-, or 8-fold with the assay diluent. Samples were run in duplicates. (B) Percentage recovery of the p-tau217 in pooled plasma sample spiked with assay calibrator of concentrations 5,10 and 20 pg/ml. Samples at each concentration were assayed in duplicates. The dashed line represents the acceptable recovery range between 80-120%. (C) Assay specificity of the newly developed plasma p-tau217 assay by competitive inhibition. The plot shows the results of competitive inhibition experiments of p-tau217 concentrations in a pooled plasma sample that was either untreated or inhibited with the addition of different amounts (range: 0.1-200 ug/ml) of a synthetic peptide bearing phosphorylation at the threonine-217 site. Data points represent mean concentration of plasma p-tau217, measured in duplicates in two independent experiment runs and error bars represent ± SD. AEB, average enzyme per bead.

Article Snippet: We chose to compare the Pittsburgh and ALZpath p-tau217 assays as like-for-like methods in the sense that: (1) they both use Simoa technology; (2) both assays use monoclonal antibodies that bind p-tau217 specifically without cross-reactivity to neighboring phosphorylation sites, as seen with the Janssen p-tau217+ assay for example; and (3) they both partner their p-tau217 antibody with an N-terminal phosphorylation-independent detection antibody.

Techniques: Clinical Proteomics, Biomarker Discovery, Concentration Assay, Inhibition, Phospho-proteomics

Journal: medRxiv

Article Title: Pittsburgh plasma p-tau217: classification accuracies for autosomal dominant and sporadic Alzheimer’s disease in the community

doi: 10.1101/2025.05.03.25326526

Figure Lengend Snippet: (A) Correlations between Pitt and ALZpath p-tau217 concentration. The correlation coefficient was calculated using Spearman rank-based correlation. The red line indicates the least square regression line. (B) Boxplot distributions of the p-tau217 levels in ADAD participants with pathogenic PSEN1 mutations, sporadic AD participants, and control. P-values were derived from post-hoc tests following Kruskal-Wallis, with Bonferroni corrections for multiple comparisons.

Article Snippet: We chose to compare the Pittsburgh and ALZpath p-tau217 assays as like-for-like methods in the sense that: (1) they both use Simoa technology; (2) both assays use monoclonal antibodies that bind p-tau217 specifically without cross-reactivity to neighboring phosphorylation sites, as seen with the Janssen p-tau217+ assay for example; and (3) they both partner their p-tau217 antibody with an N-terminal phosphorylation-independent detection antibody.

Techniques: Concentration Assay, Control, Derivative Assay

Journal: medRxiv

Article Title: Pittsburgh plasma p-tau217: classification accuracies for autosomal dominant and sporadic Alzheimer’s disease in the community

doi: 10.1101/2025.05.03.25326526

Figure Lengend Snippet: A β -PET positivity. (A) Distribution of plasma p-tau217 concentrations according to Aβ-PET status in the MYHAT-NI cohort at baseline. P-values were determined using the Wilcoxon Rank-Sum test. (B) Plasma p-tau217 levels in Aβ-negative (CL<15) to low-burden Aβ (CL15-25) and Aβ-positive (CL>25) in the MYHAT-NI cohort at baseline. P-values were determined from post-hoc tests following Kruskal-Wallis, with Bonferroni corrections to account for multiple comparisons. (C-D) AUC of Pittsburgh p-tau217 assay measurements for distinguishing Aβ-from Aβ+ participants across the full cohort (C), as well as in cognitively normal participants (D) in the MYHAT-NI and HCP cohorts.

Article Snippet: We chose to compare the Pittsburgh and ALZpath p-tau217 assays as like-for-like methods in the sense that: (1) they both use Simoa technology; (2) both assays use monoclonal antibodies that bind p-tau217 specifically without cross-reactivity to neighboring phosphorylation sites, as seen with the Janssen p-tau217+ assay for example; and (3) they both partner their p-tau217 antibody with an N-terminal phosphorylation-independent detection antibody.

Techniques: Clinical Proteomics

Journal: medRxiv

Article Title: Pittsburgh plasma p-tau217: classification accuracies for autosomal dominant and sporadic Alzheimer’s disease in the community

doi: 10.1101/2025.05.03.25326526

Figure Lengend Snippet: (A) Distribution of plasma p-tau217 concentrations according to tau-PET status in the MYHAT-NI cohort at baseline. P value was determined using the Wilcoxon Rank-Sum test. (B) Plasma p-tau217 levels across AT groups at baseline in the MYHAT-NI cohort. P values were based on post-hoc tests following Kruskal-Wallis, with Bonferroni corrections. (C-D) AUC of Pittsburgh p-tau217 assay measurements for distinguishing tau PET-from tau PET+ participants across the full cohort (C), as well as in cognitively normal participants (D) in the MYHAT-NI cohort.

Article Snippet: We chose to compare the Pittsburgh and ALZpath p-tau217 assays as like-for-like methods in the sense that: (1) they both use Simoa technology; (2) both assays use monoclonal antibodies that bind p-tau217 specifically without cross-reactivity to neighboring phosphorylation sites, as seen with the Janssen p-tau217+ assay for example; and (3) they both partner their p-tau217 antibody with an N-terminal phosphorylation-independent detection antibody.

Techniques: Clinical Proteomics