Optimization of Peptide-Based ELISA for Serological Diagnostics: A Retrospective Study of Human Monkeypox Infection

Abstract

Although smallpox has been eradicated, other diseases caused by virulent orthopoxviruses such as monkeypox virus (MPV) remain endemic in remote areas of western and central sub-Saharan Africa, and represent a potential biothreat due to international travel and/or inadvertent exposure. Unfortunately, extensive antigenic cross-reactivity among orthopoxviruses presents a challenge to serological diagnosis. We previously reported a 20mer peptide-based ELISA that identified recent MPV infection with > 90% sensitivity and > 90% specificity. However, the sensitivity of this approach was not determined with samples obtained at later time points after antibody titers had declined from their peak levels. To improve assay sensitivity for detecting MPV-specific antibodies at later time points, we compared diagnostic 20mer peptides to 30mer peptides. In addition, optimal 30mer peptides were tested in combination or after conjugating selected peptides to a carrier protein (bovine serum albumin) to further improve assay performance. An optimized combination of four unconjugated 30mer peptides provided 100% sensitivity for detecting MPV infection at 2-6 months post-infection, 45% sensitivity for detecting MPV infection at > 2 years post-infection, and 99% specificity. However, an optimized combination of two peptide conjugates provided 100% sensitivity for detecting MPV infection at 2-6 months post-infection, 90% sensitivity for detecting MPV infection at > 2 years post-infection, and 97% specificity. Peptide-based ELISA tests provide a relatively simple approach for serological detection of MPV infection. Moreover, the systematic approach used here to optimize diagnostic peptide reagents is applicable to developing improved diagnostics to a broad range of other viruses, and may be particularly useful for distinguishing between closely-related viruses within the same genus or family.

Identification of novel B-cell epitope in gp85 of subgroup J avian leukosis virus and its application in diagnosis of disease

“…1 , when we truncated the 3p-2 peptide to 83 WDPQELDILGSQ 94 and 75 QALNTTLPWDPQEL 88 which contain the minimal conserved epitope, the reactivity of the polypeptide decreased significantly although the OD 450 value was still positive. The similar results have been reported in previous study, which described that peptide length alters diagnostic sensitivity and specificity [ 15 ]. In the same report, the authors suggested that conjugation of peptides to BSA could improve assay sensitivity.…”

Identification of three immunodominant motifs with atypical isotype profile scattered over the Onchocerca volvulus proteome

“…Immunodiagnostic tests based on peptide serology offer several advantages over others that rely on more complex biological materials. The fact that synthetic peptides are not derived from biological material but are in fact chemically defined antigens greatly simplifies assay standardization and validation [ 52 ].…”

A Cross-Sectional Serosurvey of Anti-Orthopoxvirus Antibodies in Central and Western Africa

“…However, MPXV may also cause subclinical infections which might account for seroconversion in some people [ 42 ]. Promising MPXV-specific peptide-based ELISAs might be useful in differentiating between MPXV-specific antibodies and anti-OPV antibodies in the future [ 43 ].…”