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. 2019 Apr 24;4(2):e00236-19.
doi: 10.1128/mSphere.00236-19.

Development and Standardization of a High-Throughput Multiplex Immunoassay for the Simultaneous Quantification of Specific Antibodies to Five Respiratory Syncytial Virus Proteins

Rutger M Schepp  1 , Cornelis A M de Haan  2 , Deidre Wilkins  3 , Hans Layman  3 , Barney S Graham  4 , Mark T Esser  3 , Guy A M Berbers  5
Affiliations

Development and Standardization of a High-Throughput Multiplex Immunoassay for the Simultaneous Quantification of Specific Antibodies to Five Respiratory Syncytial Virus Proteins

Rutger M Schepp et al. mSphere. .

Abstract

Human respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in (premature) newborns and causes respiratory illness in the elderly. Different monoclonal antibody (MAb) and vaccine candidates are in development worldwide and will hopefully become available within the near future. To implement such RSV vaccines, adequate decisions about immunization schedules and the different target group(s) need to be made, for which the assessment of antibody levels against RSV is essential. To survey RSV antigen-specific antibody levels, we developed a serological multiplex immunoassay (MIA) that determines and distinguishes antibodies against the five RSV glycoproteins postfusion F, prefusion F, Ga, Gb, and N simultaneously. The standardized RSV pentaplex MIA is sensitive, highly reproducible, and specific for the five RSV proteins. The preservation of the conformational structure of the immunodominant site Ø of prefusion F after conjugation to the beads has been confirmed. Importantly, good correlation is obtained between the microneutralization test and the MIA for all five proteins, resulting in an arbitrarily chosen cutoff value of prefusion F antibody levels for seropositivity in the microneutralization assay. The wide dynamic range requiring only two serum sample dilutions makes the RSV-MIA a high-throughput assay very suitable for (large-scale) serosurveillance and vaccine clinical studies.IMPORTANCE In view of vaccine and monoclonal development to reduce hospitalization and death due to lower respiratory tract infection caused by RSV, assessment of antibody levels against RSV is essential. This newly developed multiplex immunoassay is able to measure antibody levels against five RSV proteins simultaneously. This can provide valuable insight into the dynamics of (maternal) antibody levels and RSV infection in infants and toddlers during the first few years of life, when primary RSV infection occurs.

Keywords: assay development; immunoassays; multiplex; respiratory syncytial virus.

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Figures

FIG 1
FIG 1
Linearity and range of standard sera. Seven sera were serially diluted to assess parallelism (dilutional linearity) and the dynamic range of the RSV-MIA for postfusion F, prefusion F, Ga, Gb, and N proteins.
FIG 2
FIG 2
Potency of reference and control sera. The potency of different standard and control sera was established using intraveneous immunoglobulin (IVIG) as a reference with assigned potencies of 400, 80, 12, 12, and 400 AU/ml for the postfusion F, prefusion F, Ga, Gb, and N proteins, respectively.
FIG 3
FIG 3
Specificity illustrated as inhibition with homologous and heterologous RSV proteins. Specificity of the five RSV proteins was assessed by depleting sera with an excess of homologous or heterologous proteins of the pentaplex MIA and compared to an uninhibited benchmark (100% MFI).
FIG 4
FIG 4
Specificity of MAbs. Specific MAbs were used to assess the conformation and cross-reactivity of five different RSV-MIA proteins after conjugation to beads.
FIG 5
FIG 5
Interassay variation illustrated in scatterplots, including the batch-to-batch comparison for prefusion F. Samples were tested independendly five times over 2 months using two different bead batches. The scatterplots (log-log) depict the variation (mean + range) of the assay for all RSV-MIA proteins.
FIG 6
FIG 6
Correlation between MIA and microneutralization (MN). (A) Scatterplots of the comparison between the MN assay (titers) and the RSV-MIA (AU/ml) for five RSV proteins (depicted separately). The vertical dotted line indicates the LLOQ of 1:10 in the MN. Samples below the LLOQ were assigned a titer of 1:5. The horizontal lines represent the estimated optimal cutoffs as a surrogate of positivity in the MN for the five RSV proteins evaluated in panel C. (B) ROC curves for the five RSV proteins plotting the sensitivity against the false-positive rate (1 – specificity) at various cutoff settings indicating the AUC. (C) ROC curves plotting the false-positive rate (FPR or 1 – specificity) and true-positive rate (TPR or sensitivity) as a function of threshold values. The dotted horizontal lines represent 95% sensitivity, and the vertical lines indicate the chosen cutoff considered an optimal trade-off between the true and false positivity rates.
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