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Problems with biotin-labelled virions as probes in poliovirus-specific mu-capture-IgM assays
S Valtanen1, M Roivainen, T Hovi
1Enterovirus Laboratory, National Public Health Institute, Helsinki, Finland.
Background:
We have previously developed a mu-capture-based radioimmunoassay (RIA) for detecting virus-specific IgM for the diagnosis of poliomyelitis. To probe captured IgM we used radiolabelled, purified preparations of representatives of each poliovirus serotype (Roivainen M, Agboatwalla M, Stenvik M, Rysa T, Akram DS, Hovi T. J Clin Microbiol 1993;31:2427-32). However, this assay is not directly applicable for wider use because preparation and handling of radioactive reagents is cumbersome and potentially hazardous.
Objectives:
To develop a non-radioactive modification of the assay retaining the number of steps and reagents to a minimum.
Study Design:
Replacement of radioactive labelling by in vitro biotinylation of purified virions, and detection of bound virions with horseradish peroxidase-conjugated streptavidin. To study sensitivity and poliovirus serotype-specificity, 129 sera and 115 CSF specimens from children with acute poliomyelitis were used in comparative tests with the in-house RIA. In addition, sera from 40 healthy adults and 11 paired sera from patients with non-polio enterovirus infection were used to assess specificity.
Results:
While results with the new test on specimens from clinically confirmed polio patients revealed some correlation with those obtained in the in-house RIA, studies on sera from healthy adults indicated, that non-specific binding of biotinylated virions is difficult to control. Moreover, examination of sera from patients with non-polio enterovirus infection suggested frequently occurring cross-reactivity between immune responses induced by polio- and other enterovirus infections. The latter were also seen in the RIA.
Conclusion:
Cross-reactive epitopes between poliovirus serotypes and between polioviruses and other enteroviruses may compromise the use of an assay for virus-specific IgM for poliovirus diagnosis. Biotinylation of the virions seemed to aggravate these problems.
Insights
Developing a non-radioactive assay for poliovirus IgM diagnosis proved challenging. Biotinylation of virions increased cross-reactivity issues, potentially compromising diagnostic accuracy for poliovirus and other enteroviruses.
Area of Science:
- Virology
- Immunology
- Diagnostic Assay Development
Background:
- A radioimmunoassay (RIA) was previously developed for detecting virus-specific IgM in poliomyelitis diagnosis.
- The original RIA utilized radiolabeled reagents, posing handling and safety challenges for wider application.
Purpose of the Study:
- To develop a non-radioactive version of the IgM capture assay.
- Minimize the number of steps and reagents in the modified assay.
Main Methods:
- Replaced radioactive labeling with in vitro biotinylation of purified poliovirions.
- Detected bound virions using horseradish peroxidase-conjugated streptavidin.
- Evaluated sensitivity and serotype specificity using patient samples (sera and CSF) and controls.
Main Results:
- The non-radioactive assay showed some correlation with the in-house RIA for polio patients.
- Non-specific binding of biotinylated virions was difficult to control in healthy adult sera.
- Frequent cross-reactivity was observed between poliovirus and other enteroviruses, also noted in the RIA.
Conclusions:
- Cross-reactive epitopes between poliovirus serotypes and other enteroviruses can hinder specific IgM-based poliovirus diagnosis.
- Biotinylation of virions appeared to exacerbate these cross-reactivity issues.