mAb-based bELISA developed to detect SARS-CoV-2 antibodies in a wide range of animal species

In a recent study published in msphereresearchers have developed a blocking monoclonal antibody (mAb)-based enzyme immunoassay (bELISA) to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in animals.

Background

Current diagnostic tests for SARS-CoV-2 are based on host antibodies or viral nucleic acids. SARS-CoV-2 shows a wide host range; Besides humans, the virus can infect various animals, such as cats, lions, tigers, deer, mink, dogs, and snow leopards. This is concerning, given the potential for transmission from humans to animals and vice versa.

It also presents challenges for the development of antibody tests because the commercial availability of host-specific secondary antibodies reagents is limited. Unlike conventional ELISA, bELISA can detect antibodies independent of host-specific secondary antibodies. Reports suggest that bELISA achieves similar levels of sensitivity to conventional ELISA and has increased specificity.

The study and the conclusions

In the present study, researchers generated a panel of anti-SARS-CoV-2 mAbs and developed a mAb-based bELISA to detect seroconversion in animals. The nucleocapsid (N) gene of the SARS-CoV-2 Wuhan strain has been cloned and recombinantly expressed. Mice were immunized with the recombinant protein to generate specific mAbs. Four mAbs (41-10, 86-12, 127-3 and 109-33) were obtained.

The researchers further included a previously described mAb (B61G11). Immunofluorescent assay (IFA) revealed the ability of the five mAbs to recognize protein N. In addition, the mAbs displayed variable reactivities to Vero cells infected with SARS-CoV-2 WA.1, B.1, P. 1, B.167.2 or B.1.1.7. mAbs, B61G11 and 127-3 had high reactivity, while 109-33 showed low reactivity; other mAbs had moderate reactivity.

Next, the team assessed the cross-reactivity of the mAbs with common CoVs from species susceptible to SARS-CoV-2. N proteins were expressed from two canine CoVs (CCoVs), four human CoVs (HCoVs), and two feline CoVs. IFA revealed the cross-reactivity of 86-12 with the N proteins of SARS-CoV-1, HCoV-NL63 and CCoV type 1, and of B61G11 with SARS-CoV-1 N. The remaining mAbs n did not show cross-reactivity with N proteins of these CoVs.

Additionally, the authors selected the mAb, 127-3, to develop bELISA, given its strong reactivity to SARS-CoV-2 variants and its inability to cross-react with common CoVs or SARS-CoV-1. . They established serum standards using samples from 24 cats 14 days after SARS-CoV-2 infection; this served as a positive control. Similarly, sera from SARS-CoV-2-negative cats served as a negative control.

Positive controls were stratified into high, medium, and low positive standards based on optical density in indirect ELISA. The team optimized the bELISA conditions using control sera. After optimization, the percent inhibition (PI) of bELISA ranged from 75% to 85%, 55 to 65%, and 35% to 45% for high, medium, and low positive controls, respectively. bELISA PI was approximately 0% for the negative control.

Then, negative and highly positive control standards were used to estimate the analytical sensitivity. A statistical difference between the positive and negative controls was evident at the highest dilution, 1:128. However, the 1:4 dilution maximized the distinction between negative and positive results while minimizing background interference and was therefore selected.

The sensitivity and specificity of bELISA were assessed using samples with known antibody status. A bELISA PI threshold of 17.6% yielded maximum sensitivity and specificity of 97.8% and 98.9%, respectively. The area under the receiver operating characteristic curve was 0.998, implying high bELISA accuracy. In addition, the authors confirmed the reproducibility of the test by using positive control sera in the medium.

The team explored the dynamics of anti-N responses in cats infected with SARS-CoV-2 B.1, Delta or Omicron. Sera were obtained at several time points. The test detected an anti-N response seven days post infection (dpi) for Delta or B.1 infection and 14 dpi for Omicron infection. The highest antibody response was observed for Delta infection. Additionally, three dogs from a pet clinic that exhibited respiratory symptoms were tested using bELISA. Two dogs were positive for antibodies against SARS-CoV-2.

conclusion

In summary, the researchers developed a panel of anti-N protein mAbs and selected an appropriate mAb (127-3) to develop bELISA. This mAb demonstrated strong reactivity to several SARS-CoV-2 variants without cross-reacting with N proteins of common CoVs or SARS-CoV-1, leading to a high specificity of the assay (98.9%). The test was also highly sensitive (97.8%), similar to commercial serology tests, and may be useful in monitoring COVID-19 in animal populations and identifying potential reservoirs.