![Specificity of 1G2 antibodies. a) Direct ELISA on Tau2N4R or Tau peptides coated plates for analysis of 1G2 binding to antigens. Peptides comprised amino acid sequence 172–184 of Tau 2N4R containing T175 and T181, respectively and amino acid sequence 228–240 of Tau 2N4R containing T231. Additional peptide sequences 172–184 phosphorylated at position T175 [T175Pi] or phosphorylated at position T181 [T181Pi] and peptide 228–240 phosphorylated at position T231 [T231Pi] were tested, respectively. Bound 1G2 was detected using anti-mouse-IgG antibody HRP conjugted followed by TMB staining. b) Sandwich ELISA was used for inhibition of Tau2N4R capturing by 1G2 antibody by competition with peptides of amino acid sequence 172–184 containing T175 and T181 and amino acid sequence 228–240 containing T231 compared with inhibition effects of these peptides with different phosphorylations at position T175 [T175Pi], T181 [T181Pi], or T231 [T231Pi], respectively. Captured antigen by 1G2 was detected using 7E5 antibody HRP conjugated followed by TMB staining.](https://www.researchgate.net/publication/308390555/figure/fig2/AS:11431281313627831@1741125478483/Specificity-of-1G2-antibodies-a-Direct-ELISA-on-Tau2N4R-or-Tau-peptides-coated-plates_Q320.jpg)
October 2024
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34 Citations
Background: Virtually nothing is known about a potential diagnostic role of non-phospho-epitopes of Tau (Non-P-Tau) in cerebrospinal fluid (CSF). Objective: To establish and analytically and clinically characterize the first assay capable to measure concentrations of Non-P-Tau in human CSF. Methods: An antibody (1G2) was developed that selectively binds to the Tau molecule non-phosphorylated at the positions T175 and T181, and was used in establishing a sandwich ELISA capable to measure Non-P-Tau in human CSF, following analytical and clinical validation of the method. Results: The 1G2 antibody shows decreasing reactivity to tau peptides containing phosphorylation mainly at positions T175 and T181. Detection limit of the assay is 25 pg/ml; the coefficients of variation (CVs) of the optical densities of the repeated standard curves were between 3.6–15.9%. Median intra-assay imprecision of double measurements was 4.8%; inter-assay imprecision was in the range of 11.2% – 15.3%. Non-P-Tau concentrations are stable in the CSF samples sent to distinct laboratories under ambient temperature; inter-laboratory variation was approximately 30%. The Non-P-Tau CSF concentrations were highly significantly increased in patients with Alzheimer’s disease in stage of mild cognitive impairment or dementia (AD/MCI, n = 58, 109.2±32.0 pg/mL) compared to the non-demented Controls (n = 42, 62.1±9.3 pg/mL, p < 0.001). At the cut-off of 78.3 pg/mL, the sensitivity and the specificity were 94.8% and 97.6%, respectively. Conclusion: For the first time, an assay is reported to reliably measure concentrations of non-phosphorylated Tau in human CSF.
![Figure 1: HAPP and pHBACE are processed, transported and localized similarly to their endogenous counterparts APP and BACE. A) Western blots of the lysates and media of cells cultured for 48 h and transfected with pHAPP or pHBACE or untransfected (wt). An immunoblot of a pHAPP-transfected cell lysate displayed an ∼170 kDa band, corresponding to mature pHAPP, and an ∼140 kDa band, corresponding to immature pHAPP. For the pHBACE-transfected cell lysates, an ∼80 kDa band corresponding to pHBACE was detected. The loading control (GAPDH) was detected in all cells. An ∼155 kDa band, corresponding to pHsAPP, was detected in the pHAPP cell media. The images of the blots of pHAPP in the lysate (green) and the media (red) are merged. B) Western blot intensity profiles of the bands and molecular weight markers in the pHAPP-transfected cell lysate (green) and media (red) according to the distance traveled. The bottom graph shows the plotted and interpolated molecular weight marker bands according to their associated molecular weights. Based on this interpolated curve, the exact molecular weights of mature pHAPP (168.6 kDa), immature pHAPP (144.1 kDa) and pHsAPP (158.9 kDa) were determined. C) The normalized (1 = 10.81 pg/mL) concentration of Aβx–40 in the cultured HeLa cell media at 48 h. The Aβx–40 concentrations in untransfected controls and pHAPP- or pHBACE-transfected cells, respectively, are depicted. These measurements were performed via particle sorting-based multiplexing. Overexpression of either protein caused a significant increase in the Aβx–40 concentration compared to non-transfection [Aβx–40untransfected = 10.81 pg/mL (±0.50 pg/mL), Aβx–40pHAPP = 134.73 pg/mL (±2.52 pg/mL), Aβx–40pHBACE = 17.62 pg/mL (±0.84 pg/mL); pTTEST(pHAPP) = 9.0 × 10−11, pTTEST(pHBACE) = 1.9 × 10−5, N = 8]. D) Representative immunocytochemistry images of untransfected and pHAPP- or pHBACE-transfected HeLa cells labeled using an anti-BACE or anti-APP antibody, respectively. The cells were imaged via TIRF microscopy. APP and BACE were predominantly detected as clusters in both the untransfected and transfected cells, as shown in greater detail in the insets. The number of clusters per 3 µm-sided square was not significantly different between the transfected and untransfected cells [numberwtAPP = 5.85 (±0.45), numberpHAPP = 5.25 (±0.22), pTTEST(APP) = 0.19; numberwtBACE = 5.96 (±0.68), numberpHBACE = 6.24 (±0.65), pTTEST(BACE) = 0.40; N = 5]. Examples of the colocalization of immunolabeling and transfected expression protein fluorescence are shown in the insets [Mander's overlappHBACE = 0.97 (±0.01), Mander's overlappHAPP = 0.97 (±0.01); N > 3]. The arrows indicate the annular accumulation of APP.](https://www.researchgate.net/publication/272840988/figure/fig3/AS:272608566444041@1442006355074/HAPP-and-pHBACE-are-processed-transported-and-localized-similarly-to-their-endogenous_Q320.jpg)
![Figure 3: The APP and BACE exocytosis events display distinct kinetics. A) Normalized average fluorescence profiles of pHAPP exocytosis based on TIRF microscopy [τpHAPP = 125.3 seconds (±23.8 seconds), N > 5]. The curve is corrected for bleaching. B) Influence of various inhibitors on the fluorescence decay after pHAPP-containing vesicle fusion events. The normalized average fluorescence profiles of cells treated with DMSO (blue), Galardin (green), Dyngo 4A (red) and the BACE inhibitor (gray) are shown after correction for bleaching. The bar graph displays the relative reductions in the decay rate based on the linear fit to the fluorescence profile after the fusion events for each inhibitor compared to the DMSO control. The BACE inhibitor reduced the linear decay rate by ∼3.48% (±0.15%); Galardin reduced the decay rate by ∼26.38% (±0.04%); and Dyngo 4A reduced the decay rate by ∼1.75% (±0.11%) (N > 3). Only the inhibitory effect of Galardin was significant (pBACE-Inh. = 0.38, pGalardin = 7.05 × 10−5, pDyngo 4A = 0.56). C) Normalized average fluorescence profiles of pHBACE exocytosis based on TIRF microscopy (N > 5). The fluorescence profile displayed different kinetics from pHAPP [τpHBACE = 2.28 seconds (±0.48 seconds); pTTEST = 1.10 × 10−8; n > 8]. D) Normalized average fluorescence profiles of the ROIs centered at the fusion site and their corresponding neighboring regions in the pHAPP- and pHBACE-transfected cells (n = 20). The fluorescence profiles were normalized to evaluate the delay in the increases in fluorescence. Relative to the amplitude of the fluorescence increase in the central ROI for pHAPP or pHBACE, those of the first neighboring ROIs (red plot) were ∼10% for pHAPP and 25% for pHBACE, and those of the second neighboring ROIs (purple plot) were ∼2.5% for pHAPP and 12.5% for pHBACE. E) The MSD for pHAPP (green dots) and pHBACE (red dots). The slope of the linear fit (gray line) corresponds to the diffusion coefficient (DBACE = 0.47 µm2/second, n > 20). F) HeLa cells transfected with pHAPP were perfused with a pH 7.5 extracellular solution for 30 seconds, followed by perfusion with a pH 5.5 solution for 20 seconds and a final perfusion with a pH 7.5 solution for 10 seconds. The circles indicate fusion events that withstood the acidic pulse, whose appearances in the different phases of the experiment are depicted below. The arrows indicate fusion events during the acidic pulse. Bottom: the mean fluorescence profile of the fusion events, as indicated by the green circle, is shown.](https://www.researchgate.net/publication/272840988/figure/fig1/AS:272558943633434@1441994524584/The-APP-and-BACE-exocytosis-events-display-distinct-kinetics-A-Normalized-average_Q320.jpg)
