An in-house lateral flow immunoassay: Point of care test for covid-19 IGG/IGM antibodies detection

ABSTRACT

Aims & Objectives: The ''gold-standard'' test for the diagnosis of COVID-19 is real time PCR, not all cases with mild symptoms or asymptomatic cases are subjected to RT-PCR analysis. It is also not practically feasible in a country like India, considering the cost, number of subjects to be screened and the required technical expertise. The sero-conversion in case of COVID-19 generally occurs from 4th or 5th day after the onset of symptoms.To develop a cost-effective, rapid kit for detection of IgG and IgM antibodies to COVID-19 using gold nanoparticle labeled antigen/antibody. Patients/Materials & Methods: Standardization of the LFIA antibody test involved many different experiments. Different size of gold nanoparticles were prepared and used. These GNPs were then adjusted to different pH for optimal binding pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 using different buffers(PBS, Borate, carbonate, etc.). The pore size of membranes play an important role and thus different pore size membranes were use (5,8,10,12,15 lm). Reaction temperature of 37C and RT were checked for maximum sensitivity. Different concentrations of the proteins ranging from 0.4 mg/ml to 100 mg/ml were tried. For the same, serum samples of 15 patients who were COVID-19 RT PCR positive having symptoms of fever, sore throat, etc. were collected between 7 to 14 days post symptoms and of 40 patients at least 28 days post symptoms. All the results were cross checked and compared with ICMR validated Rapid tests and ELISAs for IgG and IgM. Results: Internal Validation The results are in good accordance to ICMR validated Rapid tests and ELISAs where in ssensitivity of 89.6% and 91% were obtained for IgG and IgM respectively. External Validation. 1 ICMR-National Institute of Virology 200 kits were sent for validation, Sensitivity, Specificity and cross reactivity with real-time RT-PCR positive for influenza A (H1N1) pdm09, influenza A (H3N2), human coronavirus OC43, rhinovirus, respiratory syncytial virus, influenza B, parainfluenza type 4, hepatitis B virus, hepatitis C virus, etc. was done by ICMR -NIV. Sensitivity was 87.5% and 90% for IgG and IgM, respectively and specificity was 97.12 for both. 2 Rajiv Gandhi Centre for Biotechnology (RGCB)-DBT 300 kits were sent for validation. Sensitivity was 96% and 90.67% and specificity was 97.12 for IgG and IgM, respectively. The working cost is around 120Rs. The kit has now gone to the Technology transfer and commercialization department at ICMR, New Delhi. Discussion & Conclusion: The antibody diagnosis approach is important in COVID-19 epidemic to diagnose the asymptomatic cases, to assess the magnitude of infection as well as to assess the number of people who are protected from infection, though it is still not clear about the protective effect of these antibodies for repeated infection. The in-house kit was found to be highly sensitive and specific with the working cost being 100-120 Rs.