Highly sensitive detection of the group A Rotavirus using Apolipoprotein H-coated ELISA plates compared to quantitative real-time PCR

Abstract

BackgroundThe principle of a capture ELISA is binding of specific capture antibodies (polyclonal or monoclonal) to the surface of a suitable 96 well plate. These immobilized antibodies are capable of specifically binding a virus present in a clinical sample. Subsequently, the captured virus is detected using a specific detection antibody. The drawback of this method is that a capture ELISA can only function for a single virus captured by the primary antibody. Human Apolipoprotein H (ApoH) or β2-glycoprotein 1 is able to poly-specifically bind viral pathogens. Replacing specific capture antibodies by ApoH should allow poly-specific capture of different viruses that subsequently could be revealed using specific detection antibodies. Thus, using a single capture ELISA format different viruses could be analysed depending on the detection antibody that is applied. In order to demonstrate that this is a valid approach we show detection of group A rotaviruses from stool samples as a proof of principle for a new method of capture ELISA that should also be applicable to other viruses.ResultsStool samples of different circulating common human and potentially zoonotic group A rotavirus strains, which were pretested in commercial EIAs and genotyped by PCR, were tested in parallel in an ApoH-ELISA set-up and by quantitative real-time PCR (qPCR). Several control samples were included in the analysis. The ApoH-ELISA was suitable for the capture of rotavirus-particles and the detection down to 1,000 infectious units (TCID50/ml). Subsets of diagnostic samples of different G- and P-types were tested positive in the ApoH-ELISA in different dilutions. Compared to the qPCR results, the analysis showed high sensitivity, specificity and low cross-reactivity for the ApoH-ELISA, which was confirmed in receiver operating characteristics (ROC) analysis.ConclusionsIn this study the development of a highly sensitive and specific capture ELISA was demonstrated by combining a poly-specific ApoH capture step with specific detection antibodies using group A rotaviruses as an example.

Prevalence and Molecular Genotyping of Group A Rotaviruses in Iranian Children

“…The majority of human infections are caused by viruses in group A, which can further be differentiated into 23G-and 32P-type, respectively, by VP7 and VP4 antigens [4,5]. Knowledge of the distribution of G and P genotypes, including detection of emerging genotypes, is crucial to rotavirus vaccination programmes [6].…”

Sensitive and Specific Quantitative Detection of Rotavirus A by One-Step Real-Time Reverse Transcription-PCR Assay without Antecedent Double-Stranded-RNA Denaturation

“…Most previously published RNA detection assays for RVA required a denaturation step prior to RNA addition to the RT or RT-PCR mixture (13,14,17,28,29,(31)(32)(33)35). Typically, RVA dsRNA and oligonucleotides are mixed in a reaction vessel and heated for 5 min at 95 to 97°C followed by 1 min incubation on ice.…”

“…qRT-PCR assays offer several advantages over traditional RT-PCR, including increased sensitivity, higher throughput, and faster turnaround time as well as quantification of viral loads. Several qRT-PCR assays have been developed for detection of RVA targeting VP2 (28),VP4 (29,30), VP6 (17,19,(31)(32)(33), VP7 (18,30), NSP3 (13)(14)(15)(16)34), and NSP4 (35) genes. Due to the genetic diversity of VP4 and VP6 gene segments, the NSP3 gene, encoded by genomic segment 7, has been shown to be the best target for detection of a wide variety of RVA genotypes (13-16, 34, 36).…”

Apolipoprotein H, an Acute Phase Protein, a Performing Tool for Ultra-Sensitive Detection and Isolation of Microorganisms from Different Origins

“…More recently, within the European USDEP project (www.usdep.eu), it has been shown that ApoH capture viruses including Hantavirus in both serum and urine from infected patients (Godoy et al, 2009), and very interestingly avoiding false negative generated by conventional and standard PCR methods (Fig. 10) and from stools rotavirus-infected patients permitting a very highly immune detection as sensitive as quantitative PCR (Adlhoch et al, 2011). Within the same project, many other viruses have been shown strongly bind to ApoH, including poxvirus virus, hepatitis C virus, pseudorabies virus, vesicular stomatitis virus, and Altogether these data clearly demonstrate that ApoH binds enveloped or non-enveloped DNA or RNA virus with high affinity permitting their ultrasensitive detection.…”