METHODS

MSD’s electrochemiluminescence (ECL) detection technology uses SULFO-TAG labels that emit light upon electrochemical stimulation initiated at the electrode surfaces of MULTI-ARRAY and MULTI-SPOT microplates.

Electrochemiluminescence Technology

• Minimal non-specific background and strong responses to analyte yield high signal-to-background ratios.
• The stimulation mechanism (electricity) is decoupled from the response (light signal), minimizing matrix interference.
• Only labels bound near the electrode surface are excited, enabling non-washed assays.
• Labels are stable, non-radioactive, and directly conjugated to biological molecules.
• Emission at ~620 nm eliminates problems with color quenching.
• Multiple rounds of label excitation and emission enhance light levels and improve sensitivity.
• Surface coatings can be customized.

Immunization and antibody engineering activities were conducted to produce many new clones against Tau (pT217). Selected antibodies were labeled individually with biotin (to serve as a capture antibody) and TURBO-BOOST (to serve as a detection antibody for the ultrasensitive S-PLEX assay format), and they were tested in various combinations and orientations for their ability to detect pT217. The S-PLEX platform uses an enhanced, electrochemiluminescence reporter technology for detection. Antibody pairs were chosen that included both a pan-specific total Tau antibody and an antibody specific for the phosphorylated T217 site. Furthermore, antibodies were tested for specificity against non-phosphorylated Tau protein and the phosphorylated T217 site using a mutated pTau protein, in which the threonine was replaced with an alanine at position 217. After a final antibody pair was chosen, the assay was optimized and used to measure pT217 in serum and plasma from healthy and diseased samples.

RESULTS

Clone Selection for pT217

Table 1: Mice were immunized with KLH-conjugated peptide and resulting antibody clones were screened for reactivity against pT217 using 3 different Tau (total) capture antibodies. The 30 top-performing pT217 antibody clones are shown. Three antibody pairs were chosen for further screening (boxed) that had high ECL signal generation against phosphorylated Tau (orange) and a cell model that has natural expression of phosphorylated Tau (dark green), while having low reactivity against non-phosphorylated Tau (blue) and blanks (gray). ECL signal scale: blue>white>red.

Effect of Dephosphorylation of pT217

Table 2: The 3 top-performing antibody pairs for pT217 from Table 1 were selected for further characterization and compared with a Tau (total) pair in the U-PLEX format for screening using a calibrator concentration of 20,000 pg/mL. Phosphatase was used to remove phosphate groups from the pT217 calibrator and compared with one given “mock” treaent and the untreated calibrator. The total Tau assay did not show a reduction in signal, whereas all three pT217 antibody pairs showed a marked decrease in signal compared with the untreated or “mock” treated conditions (denoted in green text).

Antibody Orientation Comparison

Table 3: The 3 top-performing antibody pairs for pT217 were tested in the ultrasensitive S-PLEX format for screening as capture or detect using a calibrator concentration of 2,110 pg/mL. Using pT217-specific capture antibodies provides the best signal/background ratios and sensitivity overall. Both pT217 clones #1 and #2 maintained low background signals in the assay (denoted in green text). Several pairs show elevated background signals and reduced sensitivity (denoted in red text). eLLOD = estimated lower limit of detection.

Specificity Testing

Table 4: The 3 top-performing antibody pairs were tested in the S-PLEX format against non-phosphorylated Tau and phosphorylated Tau with threonine at the 217 position mutated to alanine (A217), using a calibrator concentration of 2,110 pg/mL. Both pT217 clones #2 and #3 have reduced specificity (denoted in red text). pT217 #1 was chosen as the final capture antibody and paired with the Tau (total) #1 antibody as the detect antibody (denoted in green text).

Concentration of pT217 in Plasma and Serum from Healthy and Diseased Samples

Figure 1: Both healthy and diseased (A) plasma or (B) serum samples were collected from two independent cohorts. pT217 was quantified using the optimal antibody pair in the S-PLEX format. Individual data points are shown. Dotted line indicates estimated lower limit of detection. *** indicates p < 0.001.

Correlation of pT217 Concentration with Alzheimer’s Severity

Figure 2: Serum was collected from a well-characterized cohort of diseased samples, and pT217 was quantified using the optimal antibody pair in the S-PLEX format. The concentrations of serum pT217 and the mini-mental state examination (MMSE) score were compared to determine the correlation of serum pT217 levels with disease severity using a linear regression. p < 0.001.

CONCLUSIONS

• An ultrasensitive assay for Tau (pT217) was developed.
• The assay is highly specific to Tau that is phosphorylated at the T217 site, while not recognizing total Tau.
• The assay is able to measure elevated levels of pT217 in diseased plasma and serum samples.