Development and Evaluation of a Nanoparticle-Based Immunoassay for Rotavirus Detection: A Suitable Alternative to
Margaret Oluwatoyin Japhet1,2, Adeogo Timilehin Bankole1, Temiloluwa Ifeoluwa Omotade1
1Department of Microbiology, Faculty of Science, Obafemi Awolowo University, Ile-Ife 220103, Osun State, Nigeria.
Insights
A new, low-cost nanoparticle immunoassay offers a machine-free method for diagnosing rotavirus, a leading cause of childhood diarrhea deaths. This diagnostic tool shows high sensitivity and specificity, improving public health interventions in resource-limited regions.
Area of Science:
- Nanotechnology
- Immunodiagnostics
- Public Health
Background:
- Diarrhea causes over 1 million deaths annually in children under five, with rotavirus being the primary culprit.
- Low- and middle-income countries face challenges in routine rotavirus diagnosis due to high costs and limited access to specialized equipment and personnel for methods like ELISA.
- Existing diagnostic methods such as Enzyme-Linked Immunosorbent Assay (ELISA) and molecular techniques are often inaccessible in resource-limited settings.
Purpose of the Study:
- To develop and evaluate a cost-effective, nanoparticle-based immunoassay for machine-free, routine rotavirus diagnosis.
- To optimize the conditions for cotton swab oxidation and aldehyde production for immunoassay kit development.
- To compare the diagnostic performance of the developed nanoassay against commercial ELISA and quantitative reverse transcription PCR.
Main Methods:
- Fourier-Transform Infrared Spectroscopy (FTIR) was used to confirm optimal oxidation conditions for cotton swabs.
- Lactoferrin (LF) was immobilized on activated cotton swabs to capture rotavirus antigen.
- The captured antigen was detected using colored nanobeads conjugated with rotavirus-specific monoclonal antibodies for visual identification.
Main Results:
- Optimal oxidation conditions were determined using 48 mg/mL NaIO4 in 0.1 M sodium acetate buffer at 35 °C for 9 hours.
- The nanoassay demonstrated superior performance compared to ELISA, with a sensitivity of 88% and specificity of 94% versus ELISA's 60% and 84%, respectively.
- The developed immunoassay achieved a positive-predictive value of 82% and a negative-predictive value of 96%.
Conclusions:
- The developed nanoparticle-based immunoassay provides a viable, low-cost, and machine-free solution for rotavirus detection.
- This novel diagnostic tool has the potential to significantly improve rotavirus surveillance and public health interventions in resource-limited settings.
- The high sensitivity and specificity of the nanoassay offer a reliable alternative for diagnosing rotavirus in areas with limited access to advanced diagnostic technologies.
Abstract:
Every year, diarrhoea is responsible for >1 million deaths in children with ages from 0 to 5 years, with rotavirus as the leading cause. The regions most affected lack routine rotavirus diagnosis due to high cost, lack of necessary equipment and shortage of trained-personnel for Enzyme-Link-Immunosorbent-Assay (ELISA) and molecular methods. We report the development and evaluation of a cheap, nanoparticle-based immunoassay for routine machine-free rotavirus diagnosis. In this work, optimal conditions for oxidation of cotton swabs and aldehyde production for kit development was confirmed by Fourier-Transform Infrared Spectroscopy (FTIR). Lactoferrin (LF) needed to bind the virus to the cotton swab was immobilised on activated cotton swabs, followed by the capture of commercial rotavirus antigen on LF-immobilised swabs. This was dipped in coloured nanobeads covalently coupled to rotavirus-group-specific monoclonal antibody for visual rotavirus detection. Subsequently, rotavirus detection by nanoassay, commercial ELISA and quantitative reverse transcription PCR were compared using same set of 186 stool samples and subjected to statistical analyses. Optimal oxidisation condition was observed using 48 mg/mL NaIO4 in 0.1 M sodium acetate buffer at 35 °C for 9 h. Rotavirus detection was confirmed visually by blue colour retention on swabs after several washings. Sensitivity, specificity, positive-predictive-value and negative-predictive-value of ELISA in rotavirus detection were 60%, 84%, 53% and 88%, respectively, while our immunoassay showed performance at 88%, 94%, 82% and 96%. This immunoassay will provide effective rotavirus public health interventions in low-and-middle-income countries with high morbidity/mortality.
