A rapid point of care immunoswab assay for SARS-CoV detection

Abstract

The emergence of severe acute respiratory syndrome (SARS) resulted in several outbreaks worldwide. Early tests for diagnosis were not always conclusive in identifying a SARS suspected patient. Nucleocapsid protein (NP) is the most predominant virus derived structural protein which is shed in high amounts in serum and nasopharyngeal aspirate during the first week of infection. As part of such efforts, a simple, easy to use immunoswab method was developed by generating a panel of monoclonal antibodies (MAbs), Bispecific MAbs and chicken polyclonal IgY antibody against the SARS-CoV nucleocapsid protein (NP). Employing the MAb-based immunoswab, an NP concentration of 200 pg/mL in saline and pig nasopharyngeal aspirate, and 500 pg/mL in rabbit serum were detected. BsMAb-based immunoswabs detected an NP concentration of 20 pg/mL in saline, 500 pg/mL in rabbit serum and 20-200 pg/mL in pig nasopharyngeal aspirate. Polyclonal IgY-based immunoswabs detected an NP concentration of 10 pg/mL in pig nasopharyngeal aspirate providing the most sensitive SARS point of care assay. Results show that the robust immunoswab method of detecting SARS-CoV NP antigen can be developed into an easy and effective way of identifying SARS suspected individuals during a future SARS epidemic, thereby reducing and containing the transmission. The key feature of this simple immunoswab diagnostic assay is its ability to detect the presence of the SARS-CoV antigen within 45-60 min with the availability of the body fluid samples.

Bispecific Antibodies for Diagnostic Applications

“…Our laboratory has developed a highly sensitive, rapid, and simple BsMAbbased immunoswab assay for early detection of SARS-CoV (Kammila et al 2008). The assay is designed to detect the most abundant and conserved viral antigen nucleocapsid protein NP (Di et al 2005;Hiscox et al 1995;Lau et al 2004;Rota et al 2003;Suresh et al 2008) because its presence in different body fluids (serum, urine, nasopharyngeal aspirate, throat wash samples, and saliva) is suggestive of current infection.…”

“…The obtained anti-SARS-CoV Â anti-HRPO BsMAb was then purified by a novel, dual sequential affinity chromatography method (Protein-G column followed by m-aminophenyleboronic acid agarose column), which was developed by our laboratory ). In the immunoswab assay, easy-to-use swabs were first coated with anti-SARS-CoV monoclonal antibody to capture NP in the test sample followed by detection with BsMAb (Kammila et al 2008). Refer to Fig.…”

“…Further, the sensitivity of NP detection was high in BsMAb-based assay compared to a parallel MAb-based assay (20 pg/mL vs. 200 pg/mL in saline; 20-200 pg/mL vs. 200 pg/mL in pig nasopharyngeal aspirate) indicating that the BsMAb-based immunoassay works better than its MAb-based counterpart. Furthermore to determine the robustness of the assay, swabs pre-coated with capture antibody were tested after storage for 2-, 6-, 10-, and 14-week time periods under different conditions (Kammila et al 2008). The BsMAb-based immunoswabs showed good sensitivity for NP detection even on week 14 at 200 pg/mL (4 C storage) and 500 pg/mL (RT and À20 C storage) indicating a good shelf-life for the immunoswabs.…”

Detection of Methicillin-Resistant Staphylococcus aureus Using a Specific Anti-PBP2a Chicken IgY Antibody

“…A large amount of IgY (100-200 mg/egg) can be extracted at a low cost from the egg yolks of hens immunized via a simple procedure (11). Many IgY-based applications are currently under development (12)(13)(14)(15). This study focused on its lower reactivity with protein A since IgY lacks a structurally different Fc region (16).…”

Development, Characterization, and Application of Monoclonal Antibodies against Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

“…Using different combinations of MAb, bispecific MAb, and IgY, we have developed a pointof-care immunoswab assay with high sensitivity (9). Thus, our new MAbs could be useful reagents for diagnostics, as well as functional analysis of NP during infection.…”