Adaptations of a Competitive Enzyme-Linked Immunosorbent Assays for the Detection of Antibodies to Influenza A Virus in Horse Sera for Use in Wild Aquatic Birds

Abstract

We applied a competitive enzyme-linked immunosorbent assay for the detection of antibodies for influenza A in equine sera to their detection in sera from wild aquatic birds. Suboptimal results were obtained for the optical density (OD) of the monoclonal antibody (MAb) control and reproducibility between duplicate analyses in the initial assessment. It was therefore necessary to modify the assay to deliver increased reliability and reproducibility while maintaining adequate sensitivity. We optimized reagent concentrations to obtain optimal OD values (close to 2) for the monoclonal antibody control and used 2, 2'-Azino-bis: 3-Benzthiazoline-6-Sulphonic Acid as an alternative chromogen to potentially reduce variability in duplicate analyses. The original assay was compared with the optimized versions, with and without post coating, for the detection of avian influenza viral antibodies in 240 sera obtained from wild plumed whistling ducks. A separate analytical sensitivity study on diluted positive field sera of plumed whistling ducks and a test of antigen stability after post coating were also performed. Some quantitative differences were detected between the original and modified assays. The original assay recorded higher percentage inhibition results which were potentially indicative of increased sensitivity. However, when reagent concentrations were increased in the original assay to the same levels as used in the modified versions, there were no quantitative differences for practical purposes. The original assay produced a median (OD) value of 0.81 for the (MAb) controls that is at the limit of acceptability. By contrast, the modified assays always produced acceptable optical density values for MAb controls. Our overall results indicated the modified assays were potentially more reliable (OD values close to 2), and of adequate sensitivity compared to the original assay in the detection of avian influenza viral antibodies in wild bird sera. Although further optimization of antigen and MAb concentrations should also be considered to increase the sensitivity of a modified assay, while maintaining acceptable optical density values for the MAb control. Post coating had a minimal quantitative effect on the results and stabilized the plates for 214 days. We therefore recommend the incorporation of post coating.

Avian influenza in Australia: a summary of 5 years of wild bird surveillance

“…subtyping PCR and DNA sequencing, and/or microarray subtyping), with all H5‐ and H7‐positive samples characterised and confirmed at the CSIRO Australian Animal Health Laboratory . Serum samples were collected from wild birds and tested for antibodies to the influenza A virus nucleoprotein using a blocking ELISA (b‐ELISA) . All sampling of wild birds was approved by the relevant institutional animal ethics committees in each state/territory (details available on request).…”

Novel biosensing methodologies for ultrasensitive detection of viruses

“…Conventional virus diagnostic methods include virus isolation, immunouorescence microscopy, enzyme immunoassays and conventional qualitative polymerase chain reaction (PCR) techniques are becoming obsolete for routine clinical practices. Enzyme immunoassays such as enzyme-linked immunosorbent assay (ELISA), 4 enzyme-linked immunospotting (ELISPOT) 5 and others have been widely employed for high throughput analysis of multiple samples. However, the laboratory usage of these methods are limited by time-consuming analysis (3-5 h), false negative results attributed to low virus titre during the early stages of infection and the need to obtain multiple serum samples from patients for serological diagnosis during different phases of viral infection.…”

Electrochemical immunosensor for detection of antibodies against influenza A virus H5N1 in hen serum

“…The conventional methods most frequently used for detection of antibodies against influenza A virus are enzyme-linked immunosorbent assays (ELISA) (Chen et al, 2011;Ciacci-Zanella et al, 2010;Hoque et al, 2012;Lebarbenchon et al, 2012;Moreno et al, 2013), hemagglutination inhibition (HI) (Allwinn et al, 2010;Schultsz et al, 2009;Peng et al, 2007) and Western blot assay (WB) (Uyeki et al, 2012). Nevertheless, they are often laborious and time-consuming or need expensive instruments.…”