Peptides including Tau (1–45), Tau (1–22), Tau (12–34), Tau (23–44), p-Tau231-KLH, p-Tau231-BSA, p-Tau217-KLH, p-Tau217-BSA, p-Tau181-KLH, and p-Tau181-BSA were synthesized by TGpeptide. Novel Peptides Tau (1–22)-Tau (224-pT231-240), Tau (1–22)-Tau (210-pT217-227), and Tau (1–22)-Tau (174-pT181-191) were synthesized as calibrators by Sangon. They are abbreviated as Tau (1–22)-pT231, Tau (1–22)-pT217, and Tau (1–22)-pT181. The sequences of these peptides are shown in Table 1. Tau-441 was procured from Sigma and GSK3β-phosphorylated Tau-441 was provided by SignalChem. Moreover, Streptavidin, Horseradish Peroxidase Conjugated from Thermo Scientific and Goat F(ab’)2 Anti-Mouse IgG (Fab’)2 (HRP) from Abcam were employed in pivotal experimental procedures. Mice strains including BALB/C and C57BL/6 were procured from Zhuhai BesTest Bio-Tech Co., Ltd., while SP2/0 cells were sourced from Shenzhen TOP Biotechnology Co., Ltd. Additionally, the Easy-Sep Mouse CD138 Pos Selection Kit and Big EasySep Magnet were purchased from STEMCELL. Reagents such as the ELISA coating buffer, ELISA stop solution, single-component TMB chromogenic solution, 20 × PBS buffer, and DMSO were obtained from Solarbio.

Following immunizations with various forms of Tau protein in BALB/C and C57BL/6 mice, we evaluated the immune response of the mice using indirect ELISA and selected those with the highest titers. Utilizing a standard protocol, we fused splenocytes with SP2/0 myeloma cells at a ratio of 3:1 using polyethylene glycol (PEG) 4,000, resulting in the successful generation of hybridomas (Groth and Scheidegger, 1980). Under incubation conditions of 37°C with 5% CO₂, cells were cultured in HAT medium (containing hypoxanthine, aminopterin, and thymidine) for 10 days. We subsequently employed indirect ELISA to screen for hybridomas producing mAbs against various forms of Tau. Positive hybridoma cells were then sub-cloned thrice and cultured in HT medium (containing hypoxanthine and thymidine). All animal experiments were approved by the Ethics Committee of Shenzhen Bay Laboratory.

Using an indirect ELISA, we assessed serum titers, epitopes, and the EC50 affinity. Initially, we diluted various Tau proteins in coating buffer, then coated them onto a 96-well microplate (100 μL/well) and incubated overnight at 4°C. After incubation, the wells were washed three times with PBST, followed by a blocking step using 2% BSA buffer (200 μL/well) at 37°C for 1.5 h. After washing, diluted mouse serum or mAb was added to each well (100 μL/well) and incubated at 37°C for 1 h. Subsequent washing was followed by the addition of diluted Goat F(ab’)2 Anti-Mouse IgG (Fab’)2 (HRP) as the secondary antibody (100 μL/well), with an incubation at 37°C for 30 min. After a further washing step, 100 μL of freshly prepared TMB substrate solution was added to each well, and the ELISA plate was allowed to react at room temperature for 8 min. The reaction was then quenched using an ELISA stop solution (100 μL/well). Finally, absorbance was measured at a wavelength of 450 nm using a multifunctional microplate reader.

The HPLC analysis of the peptide segments was performed on a Shimadzu LC-20A system using a NanoChrom Chromcore TM120 C18 and a Shim-pack GIST (4.6 × 250 mm, 5 μm) column. The mobile phase is comprised of A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The gradient elution conditions for peptide segments were sequentially set at 0.01–20 min (21–41% B), 0.01–20 min (23–43% B), and 0.01–20 min (23–43% B), respectively. For this analysis, a 0.1 mg sample was dissolved in 0.5 mL of 10% or 20% ACN and 90% or 80% H₂O. The injection volume for the samples was 30 μL, with a flow rate set at 1.0 mL/min. The absorbance of peptides was detected at 214 nm.

Peptide analysis using LC–MS was performed on a Shimadzu LCMS-2020. The mobile phase for the LC–MS consisted of 50% methanol and 50% water solution, with a flow rate of 0.2 mL/min. Each analysis had an injection volume of 1 μL. The data was processed using the LabSolutions software. The following mass spectrometry parameters were utilized during the analysis: Nebulizing Gas flow rate: 1.50 L/min; Drying Gas flow rate: 5.0 L/min; Interface temperature: 350°C; Desolvation line temperature: 250°C; and Heating block temperature: 300°C.

In the DAS-ELISA assay, capture antibodies were first diluted to 1 μg/mL with the ELISA coating buffer and coated onto a 96-well plate (100 μL/well), followed by an overnight incubation at 4°C. After the incubation, the wells were washed three times with PBST. A blocking step was then performed using 2% BSA buffer (200 μL/well) at 37°C for 1.5 h. Novel calibrators Tau (1–22)-pT231, Tau (1–22)-pT217, and Tau (1–22)-pT181 were initiated at concentrations of either 400 ng/mL or 4,000 ng/mL, followed by 2-fold serial dilutions. These diluted solutions (100 μL/well) were added to each respective well and incubated at 37°C for 2 h. After washing, biotin-labeled detection antibodies, diluted to 1 μg/mL (100 μL/well), were added and incubated at 37°C for 1 h. Following three washes, diluted Streptavidin and horseradish peroxidase conjugated secondary antibody (100 μL/well) was added and incubated at 37°C for 30 min. After the final washing step, 100 μL of the 3,3′,5,5’-Tetramethylbenzidine (TMB) substrate solution was added to each well for color development, and the ELISA plate was left to react at room temperature for 8 min. The reaction was terminated using the ELISA stop solution (100 μL/well), and absorbance was measured at 450 nm using a multifunctional microplate reader. A satisfactory 4PL curve fitting within the range was observed in all 60 assays. The standard curve represents three replicated independent experiments, and the LOD is determined by calculating the average of 10 blank values plus 2.5 times the standard deviation (SD).

SPR experiments were conducted on the Biacore 8 K+ instrument from Cytiva. Antigens were chosen as the capture ligands and antibodies were used as the analytes. Using the amine coupling method, antigens Tau-441, p-Tau231-BSA, p-Tau217-BSA, and p-Tau181-BSA were immobilized onto CM5 sensor chips with carboxymethyl dextran matrix, facilitating the analysis of binding kinetics between Tau mAbs and their respective antigens. Before the assay, 1× HBS-EP buffer was freshly prepared through a tenfold dilution of 10× HBS-EP+ buffer with ultrapure water, followed by filtering and degassing. The mAb solutions were then serially diluted in 1× HBS-EP buffer, yielding concentrations of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.125 nM. The association phase was set to 120 s, followed by a 120 s or 300 s dissociation phase. At a constant flow rate of 30 μL/min, samples were introduced to the chip surface using an automated sample handler. The surfaces were regenerated by a 60 s injection of 10 mM pH 2.0 glycine, and with the aid of BIA 2.0.1 (Cytiva) software, the dissociation constant (KD) and other kinetic parameters were estimated from the sensorgrams. After the experiments, chips were stored in 1 × HBS-EP buffer at 4°C.

All assays based on Simoa technology were conducted using the automated Simoa HD-X analyzer from Quanterix. All custom assay components for Simoa, including wash buffer 1, wash buffer 2, sample diluent, bead diluent, SBG diluent, discs, 96-well plates, sealing oil, cuvettes and other consumables were sourced from Quanterix Corp.

Initially, capture antibodies were conjugated to homebrew carboxylated paramagnetic beads. The buffer of the capture antibody was exchanged for the bead conjugation buffer using ultrafiltration tubes, followed by a 30-min activation of the beads with freshly prepared EDC. Subsequently, these activated beads were conjugated with the capture antibody for 2 h, followed by blocking and washing steps.

For the biotinylation of the detection antibody, its buffer was swapped for the biotinylation reaction buffer using ultrafiltration tubes. The mAb was then mixed with NHS-PEG4-Biotin from Thermo Fisher at a molar ratio of 40:1 and incubated for 30 min. Excess biotin was subsequently removed by using ultrafiltration tubes. The conjugated beads with the capture antibody and the biotinylated detection antibody were stored at 4°C until further use, with all experimental steps carried out at room temperature.

During the Simoa analysis, the capture antibody, detection antibody, and SBG were diluted to specified concentrations and transferred into plastic bottles. Concurrently, recombinant calibrators were serially diluted to the desired concentrations in the diluent and pipetted into a 96-well plate. All necessary reagents were then loaded onto the Simoa HD-X analyzer, and the analysis was executed using a two-step procedure. Ultimately, signals obtained from the beads were quantified in terms of the average enzyme per bead (AEB). For data analysis, we employed GraphPad Prism 9.5.1 software, utilizing a four-parameter logistic (4PL) curve with a weighting factor of 1/y² to plot the standard curves.

We used indirect ELISA to measure non-phosphorylated Tau (np-Tau) and p-Tau antibody titers after immunizing mice. Seven mice with high titers (1:32000) were chosen for fusion (Figure 1). High antibody titers appear to be characteristic of a potent immunological response in mice.

The formation of hybridomas in the cell culture wells was observed after fusing B lymphocytes with SP2/0 myeloma cells. The positive hybridoma clones were then identified using an indirect ELISA. After three subcloning procedures, all derived cell lines stably secreted mAbs. Following mAb production and purification, the epitopes of mAbs were investigated. Interestingly, there were 49 mAbs, mAb-1 ~ 49, that bound to np-Tau and 12 mAbs, mAb-50 ~ 61, that bound to p-Tau. As shown in Figures 2A,B, all 49 np-Tau mAbs are bound to Tau-441 and Tau (1–45). Further epitope refinement of three peptide segments: Tau (1–22), Tau (12–34), and Tau (23–44) revealed that all 49 np-Tau mAbs specifically targeted the N-terminal epitope 1–22 on human Tau protein, with amino acid numbering consistent with that full-length Tau 1–441 (Uniprot ID P10636-8) (Figure 2C, Supplementary Figure S1). The sequences of peptides are depicted in Table 1. Furthermore, epitope analysis of the 12 p-Tau mAbs revealed that nine of them (mAb-50 ~ 58) bound to p-Tau231, two of them (mAb-60 ~ 61) bound to p-Tau181, and mAb-59 bound to p-Tau217 (Figure 2D). Supplementary Table S1 shows the binding ability of the 12 p-Tau mAbs with diverse epitopes, and Figure 2E summarizes the binding epitopes of all mAbs. These mAbs with different epitopes exhibited the potential for Tau immunoassay development.

The amino acid sequence of the Tau-441 protein and synthesized Tau (1–22)-pT231, Tau (1–22)-pT217, and Tau (1–22)-pT181 peptides are depicted in Figure 3A. We used chemical synthesis to generate peptides with capture and detection antibody epitopes. The purity and homogeneity of the peptides were evaluated by HPLC. The chromatogram data showed that distinct, symmetrical, and distinguishable peaks were observed. The retention times of the major peaks representing the targeted peptides were 10.0, 9.6, and 5.3 min, respectively. Based on peak area analysis, the estimated purity for these three peptides was 95.8, 91.9, and 96.1%, respectively. The absence of substantial secondary peaks in the chromatogram highlighted the high degree of purity of the peptides (Figures 3B,D,F). Further analysis using LC–MS confirmed the molecular properties of peptides that are closely aligned with the predicted amino acid sequences (Figures 3C,E,G). The combined analysis using HPLC and LC–MS confirmed the high purity and integral molecular structure of the synthesized peptides. Based on two validation methods, we subsequently applied these three synthesized peptides to develop p-Tau231, p-Tau217, and p-Tau181 immunoassays.

To evaluate the synthetic Tau peptide, the binding affinities of 12 p-Tau mAbs to GSK3β-phosphorylated Tau-441 and the novel peptides Tau (1–22)-pT231, Tau (1–22)-pT217, and Tau (1–22)-pT181 were determined. Our findings showed that when the mAbs concentration is 100 ng/mL, it has a strong binding with our synthetic peptides, but GSK3β-phosphorylated Tau-441 has almost no binding (Figures 4A–L). The EC50 of mAbs binding to different p-Tau proteins is shown in Supplementary Table S2. These findings demonstrate the fact that the novel p-Tau peptides interact with the mAbs with a higher affinity and stability than the GSK3β-phosphorylated Tau-441. This increased affinity is essential for the development of ultra-sensitive DAS-ELISA.

To investigate the applicability of the Tau peptides as calibrators, we established immunoassays for p-Tau231, p-Tau217, and p-Tau181 using synthesized calibrators Tau (1–22)-pT231, Tau (1–22)-pT217, and Tau (1–22)-pT181. All possible combinations (a total of 588 pairs) by employing 49 np-Tau mAbs specific to Tau (1–22) as capture antibodies and 12 p-Tau mAbs as detection antibodies were investigated. The OD450 values of the 588 assays at a single concentration point are shown in Figure 5 and Supplementary Table S3. 60 assays with OD450 values greater than 2.0 from 588 assays were chosen for further concentration gradient pairing analysis. Especially in the detection antibodies of these 60 assays, all are mAb-53#, mAb-59#, and mAb-60#.

To further examine immunoassay sensitivity and linearity, we prepared a series of concentration gradients of the novel Tau (1–22)-pT231, Tau (1–22)-pT217, and Tau (1–22)-pT181 calibrators to establish 60 corresponding standard curves (Figure 6). For each phosphorylation site, two immunoassays were selected after evaluating the crucial selection criteria (Tables 2–4). The LODs of the six immunoassays were 0.47, 0.40, 2.94, 1.64, 0.41, and 0.37 ng/mL. These combinations are selected to be the most appropriate candidates for developing ultra-sensitivity Simoa immunoassays.

To verify whether our calibrators can be applied to ultra-sensitive platforms, we developed six immunoassays on the Simoa platform, which are Simoa-Assay 1–6: capture antibody mAb-53# and detection antibody mAb-32#; capture antibody mAb-53# and detection antibody mAb-36#; capture antibody mAb-59# and detection antibody mAb-36#; capture antibody mAb-59# and detection antibody mAb-45#; capture antibody mAb-26# and detection antibody mAb-60#; capture antibody mAb-28# and detection antibody mAb-60#, respectively (Figure 7). Exceptionally, the assays achieved LLOQs of 2.5, 2.4, 31.1, 32.9, 46.9, and 52.1 pg/ml, respectively. Our findings demonstrate that the Tau (1–22)-pT231, Tau (1–22)-pT217, and Tau (1–22)-pT181 calibrators can be used to achieve sensitive quantification of p-Tau levels.