Antibodies opposing an epitope conserved among species can be utilized to produce a single assay for various species. The Human Neurology 4-Plex “A” (N4PA) and Human Neurology 3-Plex “A” (N3PA) Simoa® assay kits were analyzed for species cross-reactivity with non-human primate samples.
Materials and Methods
Cynomolgus (macaca fascicularis) samples supplied by BioIVT were utilized to assess non-human primate reactivity. 10 unique lots of plasma, 10 unique lots of serum, and one pooled CSF were analyzed.
Two multiplex assays were employed:
- Human Neurology 4-Plex “A” (N4PA) – Neurofilament-light (NF-Light), GFAP, UCH-L1, Tau
- Human Neurology 3-Plex “A” (N3PA) – Abeta40, Abeta42, Tau
The samples were analyzed and processed using the Simoa HD-1 analyzer. The samples were assessed for specificity, dilution linearity, spike and recovery, and normal concentration.
Normal Sample Testing
Ten plasma and 10 serum were diluted 4x in the kit sample diluent. A single pooled CSF was diluted 20x in the kit sample diluent.
The CSF sample was diluted from 20x to 2560x in the kit sample diluent to evaluate dilutional linearity.
Spike and Recovery
Ten serum lots were pooled in the same proportions and were then spiked with a pooled CSF. Ten plasma lots were similarly pooled and then spiked with a pooled CSF. The spiked plasma and serum were utilized to evaluate recovery.
Serum and Plasma Dilution Linearity
The CSF-spiked pooled plasma and serum samples were diluted from 2x to 256x in assay sample diluent to evaluate dilutional linearity.
Specificity was calculated by depleting the sample of analyte through the captured antibody. Depletion was carried out on 4x diluted pooled plasma, 4x diluted pooled serum, and a 20x diluted CSF. The samples were then incubated while rotating at room temperature with a 10x number of assay kit capture beads for one hour.
Specificity was determined utilizing kit capture beads at a 10x concentration. The change in signal was utilized to discover whether the sample reading is because of a false positive signal.
Further characterization can be carried out to elucidate the kit specificity with cynomolgus more. For example, the unconjugated detection and capture antibodies can be added to outcompete the biotinylated detector and kit capture beads.
To strip the sample of the protein, a third antibody can be utilized. Additionally, to discover if all analyte has fully been removed, an increased concentration of capture beads along with an increased incubation time can be used. Certain tested samples demonstrate poor or limited dilution linearity.
In some cases, this is because the dilution has approached the limit of quantification. The limit of background experiments in the sample type should be performed with samples removed of the analyte to distinguish nonspecific background signal. There may be matrix-specific cases that demonstrate a signal above the calibrator LOD that are caused by background signals that are matrix related.
Both N3PA and N4PA kits are reactive with plasma in cynomolgus (non-human primate) and Tau protein in CSF samples. Plasma and CSF give positive signals that are able to be depleted. CSF displays poor dilutional linearity.
As the plasma linearity is spiked with CSF, linearity may be expected to adhere to the same pattern, because the CSF in diluent serum samples read at significantly lower concentrations. CSF spiked into serum demonstrates extremely poor recovery. Its matrix-related effects are obvious with serum samples that affect the measurement of Tau.
In all three matrices, the N4PA kit is reactive with NF-L protein. Standard levels of CSF, plasma, and serum are all measurable above LOQ. The signal from each diminishes to background levels.
Diluted from 2x to 16x, plasma samples demonstrate similar linearity and then quickly drop off at 32x and higher. Serum samples diluted from 2x to 32x display similar linearity before suddenly dropping off at 64x and higher.
CSF linearly dilutes from 20x to 320x. Linearity cannot be assessed accurately, and greater dilutions are equal to or below the LOQ. NF-L plex is distinctly reactive to cynomolgus NF-L however the dilution linearity is poor.
In all three matrices, the N4PA kit is reactive with GFAP protein. Standard levels of CSF, plasma, and serum are all measurable above LOQ. The serum and plasma signal depletes similar to the calibrator blank values.
Demonstrating a high level of specificity, the CSF signal is depleted by >95%. CSF dilutes linearly across the whole range tested of 20x to 2560x. The plasma displays dilution linearity across the whole range tested 2x to 256x.
Serum shows linearity upon the initial dilution in the range of 4x to 256x. This shows that a 2x dilution has some interference that is matrix-related. The parameters tested display a great level of confidence in the reactivity of GFAP in cynomolgus samples.
The N4PA is not likely to be reactive with UCH-L1 protein. The testing displays signals above the kit LOQ, however, the signals demonstrate no level of depletion. The positive signal is likely to be caused by a non-specific interaction such as Heterophilic interference.
The positive signal from serum, CSF, and plasma does demonstrate partial dilutional linearity but as the signal does not deplete, the linearity is likely from the interferent.
In all three matrices, the N3PA kit is reactive with AB42 protein. Standard levels of CSF, serum, and plasma can all be measured above LOQ. 30% of the tested serum samples are above the kit LOQ. The signal from serum and plasma samples diminishes to background levels. CSF is decreased by >94% and demonstrates positive specificity.
CSF linearly dilutes from 20x to 640x. Greater dilutions are lower than the LOQ and cannot be evaluated for linearity. Plasma samples diluted from 4x to 16x displays linearity. Greater dilutions are under the LOQ and cannot be evaluated for linearity. The serum samples diluted from 4x to 32x support linearity.
The higher dilutions are under the LOQ and cannot be assessed for linearity. Both serum and plasma show poor recovery and spike when CSF is spiked into the sample. This is possibly related to the nature of Abeta proteins in CSF versus blood matrices.
In all three matrices, the N3PA kit is reactive with AB40 protein. Normal levels of CSF, plasma, and serum can all be measured above LOQ. The signal from serum samples diminishes to background levels.
While the values do not reduce to the value of the blank calibrator, plasma and CSF is reduced by >9.0% and displays specificity. Further depletion or alternative methods may provide a greater reduction in signal. CSF does not display linearity over the total range tested. The plasma samples, diluted from 2x to 64x, demonstrate linearity.
The greater dilutions are less than the LOQ and cannot be assessed for linearity. The serum samples diluted from 2x to 32x demonstrate linearity. The higher dilutions are under the LOQ and cannot be measured for linearity.
Possibly related to the nature of Abeta proteins in CSF versus blood matrices, both serum and plasma display poor recovery and spike when CSF is spiked into the sample.
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