Examining Species Cross-Reactivity in Mice and Rats

Antibodies in opposition to an epitope conserved among species can be utilized to develop 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 rat and mouse samples.

Materials and Methods


Mouse (CD-1) and rat (Sprague-Dawley) samples from a commercial supplier were employed to distinguish cross-reactivity. For each species, 10 unique lots of plasma, 10 unique lots of serum, and one pooled CSF were analyzed.

Two multiplex assays were utilized:

  1. Human Neurology 4-Plex “A” (N4PA) - Neurofilament-light (NF-Light), GFAP, UCH-L1, Tau
  2. Human Neurology 3-Plex “A” (N3PA) – Abeta40, Abeta42, Tau

The samples were evaluated and processed employing the Simoa HD-1 analyzer. The samples were analyzed for specificity, dilution linearity, spike and recovery, and normal concentration.

Normal Sample Testing

The 10 plasma and 10 serum were diluted 4x in the assay sample diluent. One pooled CSF was diluted 20x in the assay sample diluent.

CSF Linearity

The CSF sample was diluted from 20 to 2560x in the assay sample diluent to evaluate dilutional linearity.

Spike and Recovery

The spiked plasma and serum were used to evaluate recovery.

  1. The 10 serum lots were pooled in identical proportions and spiked with a pooled CSF.
  2. The 10 plasma lots were pooled in identical proportions and spiked with a pooled CSF.

Serum and Plasma Dilution Linearity

To evaluate dilutional linearity, the CSF-spiked pooled plasma and serum samples were diluted from 2x to 256x in the assay sample diluent.

Analyte specificity

Specificity was calculated by the immunodepletion of the analyte through the captured antibody. Depletion was carried out on 4x diluted pooled serum, 4x diluted pooled plasma, and 20x diluted CSF. The samples were then incubated in a rotational manner at room temperature, with a 10x number of assay capture beads for an hour.



The N4PA kit is reactive with NF-L protein in all three matrices in both mouse and rat, however, the serum exhibits issues that are matrix related. Average levels of CSF, plasma, and serum are all measurable above LOQ. The signal from each falls to below LOQ. The assay demonstrates restricted dilution linearity in CSF spiked plasma, where 4x to 32x are within 20% of each other, however, larger dilutions fall outside of range. The CSF spiked serum exhibits weak linearity across all dilutions.


The human N4PA kit shows reactivity with GFAP in rat CSF. Standard levels of rat CSF can be measured above LOQ and demonstrate average dilutional linearity. The signal from rat CSF falls to below LOQ. The N4PA kit is not adequately reactive with mouse CSF or with rat and mouse plasma and serum. Standard samples display signals under LOQ, and mouse CSF is only slightly above LOQ.

UCH-L1, Tau

The human N4PA kit is not adequately reactive with UCH-L1 and Tau in rat and mouse samples. Assays give signals that are undetectable in rat and mouse plasma and serum. CSF is not measurable for Tau. UCHL1 displays weak depletion of CSF signal with poor linearity, suggesting that the positive signal is unlikely to be specific.

ABeta42, ABeta40

There is a lack of measurable signal in CSF, serum, or plasma for both Abeta proteins in mouse or rat samples. The human N3PA kit is insufficiently reactive with Abeta42, Tau, or Abeta40 for quantitative measurement.

Assay Likely reactive with Mouse Unlikely reactive with Mouse Likely reactive with Rat Unlikely reactive with Rat
NF-L x   x  
GFAP   x x  
UCH-L1   x   x
Tau   x   x
ABeta42   x   x
ABeta40   x   x


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Last updated: Feb 1, 2020 at 8:26 PM


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