Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells

Abstract

Background Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. Objectives This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected human A549 cells in vitro. Study design A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10−1 to 10−8. Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. Results RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10−8). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10−7. The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10−6, while ISH detected the virus at dilutions of 10−4. Conclusions All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies.

Pathogenicity of streptococcus agalactiae in oreochromis niloticus

“…Among these, only ISH, IHC, and TEM can localize GBS in individual cells. The sensitivity of IHC varies with the type of antibody used [26]. ISH-PCR allows the amplification of target sequences within intact cells and combines high sensitivity with the ability to localize specific DNA in tissues.…”

Enteroviral Infections as a Trigger for Type 1 Diabetes

“…Proteomics was the next most sensitive technique, reaching the same dilution, followed by immunohistochemistry, which was able to detect viral protein up to the 10 −6 dilution, with variability between different antibodies. Lastly, ISH detected virus dilutions of 10 −4[24].Despite the good sensitivity of most of these techniques, one thing to keep in mind is that EVs are able to establish persistent infections, which are characterized (in vitro) by no evident cytopathic effect, expression of viral antigens in a low number of cells, production of viral particles at low titers, and secretion of cytokines and chemokines [25••]. An important consideration is that infected cell lines constitute an ideal testing sample and the sensitivity of these techniques on tissue samples, and especially pancreas, is expected to be lower.…”

“…of different techniques for CVB1 detection. Human A549 cells were infected (multiplicity of infection: 10-15) and harvested at 1, 2, 4, and 5 h post-infection[24]. Then, dilution series that ranged from 10 −1 to 10 −8 were prepared.…”

Large enteroviral vaccination studies to prevent type 1 diabetes should be well founded and rely on scientific evidence. Reply to Skog O, Klingel K, Roivainen M et al [letter]

“…This is a singular claim given that the fidelity of this antibody has been tested rigorously under the stringent conditions we use [15]. In addition, and as they suggested, we have also tested additional antibodies and find that the majority are much less sensitive than the Dako anti-VP1 reagent [16]. However, as noted in one earlier study [17], some additional antisera do label equivalent islet cells to those stained by the Dako clone in pancreas sections from people with type 1 diabetes.…”