Implementation of a rapid and accurate SARS-CoV-2 antigen detection kit capable of identifying Omicron mutants

In a recent study published in Vaccinesresearchers have developed an antigen detection kit to identify the concerning Omicron variant of severe acute respiratory syndrome (VOC) coronavirus 2 (SARS-CoV-2).

Background

The continued emergence of new coronavirus disease 2019 (COVID-19) vaccines threatens the effectiveness of vaccines and therapeutic agents aimed at mitigating COVID-19. Early diagnosis of COVID-19 and prompt treatment of those affected could help reduce the global burden of COVID-19. According to the WHO (World Health Organization), rapid antigen tests are complementary to the diagnosis of COVID-19. Immune colloidal gold chromatography would allow the detection of Omicron subvariants in a simple, rapid and cost-effective manner.

About the study

In the present study, researchers developed an antigen recognition kit based on colloidal gold immunochromatography (CGIA) assays to detect Omicron subvariants.

BALB/c murine animals treated with beta-propiolactone were vaccinated with Omicron’s core protein. After the fusion of immunological cells and myeloma cells, the team performed an ELISA (enzyme immunoassay) test to screen for cells capable of producing monoclonal antibodies. The antigen detection kit was prepared based on CGIA assays, and the anti-interference property, specificity and sensitivity were evaluated by stimulation of positive samples.

The nucleocapsid protein was considered the target antigenic molecule to vaccinate murine animals, and the team prepared highly stable and sensitive monoclonal antibodies using the hybridoma cell screening method. They prepared the immunochromatographic analysis test strips and the monoclonal antibodies were successfully matched to assemble the test strips.

The lowest concentration required to obtain positive reports was considered the lowest detection limit for the sensitivity of the test kit. Concentrations of inactivated SARS-CoV-2 cultures were identified using several nucleic acid identification reagents. The clinical scenario of COVID-19 patients was simulated by obtaining nasal swabs from healthy individuals and incorporating a dilute solution of inactivated SARS-CoV-2.

High-sensitivity antibody pairs were chosen, including antibodies capable of identifying the ancestral strain, Beta VOC, Delts VOC, and Omicron VOC strains, and antibodies without Omicron subvariant detection capability. Antibodies were incorporated onto a test strip to produce a card having two lines for detection.

The antibody failing to identify the Omicron subvariant was designated as T1, and that possessing the ability to detect the Omicron subvariant was designated as T2. Based on the results of the detection lines, subvariants of Omicron have been identified. In addition, 25.0 interfering substances, such as interferon-alpha, pure mucin, peramivir, oseltamivir, fluticasone and lopinavir, were used to perform the identification of the anti colloidal gold band. -interference.

Regarding the minimum detection limit allowed, the team selected three cultures of SARS-CoV-2, with titers of SARS-CoV-2 diluted to concentrations of 500.0 TCID₅₀ (50.0% tissue culture infective dose)/mL, 31 TCID₅₀/mL, 63 TCID₅₀/mL, 125.0 TCID₅₀/mL, 250.0 TCID₅₀/mL and 500.0 TCID₅₀/mL for initial identification. All dilutions were repeated over 20.0 times. The stability of the colloidal gold test strip was evaluated.

Oral cavity specimens, throat swab specimens, and nasal swab specimens were obtained from ≥ 90 individuals infected with novel coronavirus CoV (COVID-19 cases within one week of testing positive for novel coronavirus CoV) in Chengdu. Nasal swab specimens were tested with the strip, and oral and throat swab specimens were tested with the new CoV Nucleic Acid Identification Kit (2019-nCoV) based on fluorescent reading of the polymerase chain reaction (PCR) analysis].

Results

The sensitivity of antigen detection kit could reach 63.0 TCID₅₀/mL, with the good incorporation of several SARS-CoV-2 variants. No cross-reactions with commonly observed pulmonary pathogens have been observed. The sensitivity of the test remained unchanged under the various concentrations and interferences, indicating good stability and specificity of the antigen detection kit.

Antibody pair matching led to the detection of Omicron subvariants by antibodies, of which the sixth antibody pair showed the highest sensitivity, with a detection limit of 1.0:2 .0 × 10⁷. The culture detection limit of Omicron subvariants was 1.0:2.0×10⁵. It should be noted that the sensitivity of the colloidal gold identification card has reached levels similar to those of nucleic acid detection reagent tests. The results indicated that the antigen recognition kit met the requirements for clinical use.

Regarding the mixed subvariants of Omicron, the two detection lines did not give false positives. The results showed that 48 viral organisms and other pathogenic organisms did not interfere with the detection capabilities of the kit, with no false negative reports.

The minimum detection limit value of the colloidal gold kit for various virus strains was 63.0 TCID₅₀/mL, revealing the good incorporation of samples from different strains of SARS-CoV-2. Additionally, detection of dilute cultures of SARS-CoV-2 showed that by extending storage to >1.0 years, the test line color and quality control line of all test strips were unchanged, indicating real-time stability.

Conclusion

Overall, the study results highlighted the design details of a simple, highly sensitive, highly specific, rapid and accurate antigen detection kit based on CGIA technology that could make the difference between novel coronavirus infection and Omicron subvariants.