Multiplexed High-Throughput Serological Assay for Human Enteroviruses

Abstract

Immunological assays detecting antibodies against enteroviruses typically use a single enterovirus serotype as antigen. This limits the ability of such assays to detect antibodies against different enterovirus types and to detect possible type-specific variation in antibody responses. We set out to develop a multiplexed assay for simultaneous detection of antibodies against multiple enterovirus and rhinovirus types encompassing all human infecting species. Seven recombinant VP1 proteins from enteroviruses EV-A to EV-D and rhinoviruses RV-A to RV-C species were produced. Using Meso Scale Diagnostics U-PLEX platform we were able to study antibody reactions against these proteins as well as non-structural enterovirus proteins in a single well with 140 human serum samples. Adults had on average 33-fold stronger antibody responses to these antigens (p < 10−11) compared to children, but children had less cross-reactivity between different enterovirus types. The results suggest that this new high-throughput assay offers clear benefits in the evaluation of humoral enterovirus immunity in children, giving more exact information than assays that are based on a single enterovirus type as antigen.

Cross-Reactive Antibody Responses against Nonpoliovirus Enteroviruses

“…ELISA binding assays have previously been to test for the presence of cross-reactive anti-enterovirus antibodies in human polyclonal sera ( 16 – 19 , 26 , 40 – 42 ). Only EV-D68 was used in antibody neutralization assays in some of these studies.…”

“…Results of studies examining both polyclonal human sera and a recombinant monoclonal antibody against the VP-4 capsid protein of EV-A71 suggest that antibodies against one genotype of EV-A71 can block infection of cells in culture by other genotypes and serotypes of the virus, as well as Coxsackievirus A16 and A6 ( 38 , 39 ). Antibody cross-reactivity among enteroviruses has also been observed using peptide arrays derived from the capsid proteins of multiple enteroviruses, including EV-D68, poliovirus type 1/Mahoney, Coxsackievirus A4 and B1, and human rhinoviruses ( 26 , 40 , 41 ). Whether or not cross-reactive antibodies can neutralize viruses of different enterovirus species (e.g., EV-C and EV-D) has not been demonstrated.…”

IgA-Type Enterovirus Antibodies Are Increased among Adults and Children with Recently Diagnosed Type 1 Diabetes

“…Another limitation is the use of a synthetic common EV peptide instead of different EV antigens in individual antibody assays. However, because of the great number of different EVs, which have so far been demonstrated to be associated with T1D [ 40 ], our approach might even be more justified. This is also a strength of our study, as it allows comparing the summation of exposures to different EVs for controls and for participants with T1D.…”

Assessment of Enterovirus Antibodies during Early Childhood Using a Multiplex Immunoassay

“…Enzyme-linked immunoassays are faster, but there is considerable cross-reactivity between non-neutralizing antibodies that target different enteroviruses ( 15 – 17 ) and the general population, which has high levels of antibodies ( 18 ) due to the high prevalence of different kinds of enteroviruses. However, in our previous studies, we have observed that the antibody responses toward viral envelope protein 1 (VP1) proteins are more cross-reactive in adults than in children ( 19 ). We have also shown that the antibody responses toward enterovirus nonstructural protein proteases 2A and 3C are potential markers of acute infection ( 20 ).…”