Novel methods of immunogenic antigen selection for serological diagnosis of Parelaphostrongylus tenuis infection

Abstract

This paper outlines methods used to identify novel antigens for use in the development of serological assays. Specifically, we applied these methods to a neurogenic parasitic nematode of cervids called Parelaphostrongylus tenuis. This parasite is of particular concern in both wild and domestic ungulates as it causes significant neurological signs and definitive diagnosis is only possible post-mortem, necessitating the development of serologic assays for antemortem diagnosis. Proteins extracted from P. tenuis organisms were affinity isolated using antibodies enriched from seropositive moose (Alces alces). The proteins were analyzed using mass spectrometry and liquid chromatography to obtain amino acid sequences that were then cross-referenced to open reading frames predicted from an assembled transcriptome. An antigen of interest was assessed for immunogenic epitopes and subsequently synthesized into 10-mer synthetic overlapping peptides representing these regions. These synthetic peptides were then assessed for reactivity against positive and negative moose sera and demonstrated potential use as a serological assay in diagnostic laboratories. Known negative moose sera revealed significantly lower optical density when compared to the positive samples (p < 0.05). This method serves as a pipeline for the construction of diagnostic assays of pathogens in both human and veterinary medicine.

Figure 1

Illustration of protocol for the purification of polyclonal antibodies from positive animals, Parelaphostrongylus tenuis protein extraction, and affinity purification of antigen. Created with BioRender.com 2022.

Figure 2

Immunogenic proteins were identified using liquid chromatography and mass-spectrometry and then cross-referenced to an assembled transcriptome. Identified proteins were subjected to epitope prediction using Bepipred Linear Epitope Prediction 2.0 and then 10-mer synthetic overlapping peptides were synthesized accordingly. These peptides were probed with positive sera to detect areas of high reactivity which would indicate immunogenic epitopes. Created with BioRender.com 2022.

Figure 3

Plate layout for 10-mer synthetic overlapping peptides 1–40. Peptides 1–20 were coated on Plate (A) with the exception of 5 which was omitted due to predicted low reactivity and replaced with a 1:5 dilution of peptide 15 in PBS to assess if concentration could be reduced further. Peptides 21–40 were coated onto Plate (B). Columns 11 and 12 on each plate were left to be PBS blank wells to control for background noise. Created with BioRender.com 2022.

Figure 4

Organization of samples on plates to assess difference in positive and negative control reactivity. Across both plates a total of 30 positive controls and a total of 32 negative controls were utilized for comparison of means analysis. Created with BioRender.com 2022.

Figure 5

Organization of samples on plates to assess difference in positive and negative control reactivity across several peptide combinations. Each plate contained Cocktail A which comprised of all four peptides so that subjective comparisons could be made across both plates. Created with BioRender.com 2022.

Figure 6

The 10-mer Synethetic ovelapping peptides (1–40) that represented predicted epitopes were probed with pooled P. tenuis positive moose sera from Minnesota. It should be noted that peptide 5 was omitted due to predicted low reactivity and replaced with a 1:5 dilution of peptide 15 in PBS to assess if concentration could be reduced further.