A Rapid Colloidal Gold Immunochromatographic Assay for the Diagnosis of Coronavirus Disease 2019
The global outbreak of Coronavirus Disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has led to widespread pneumonia and severe acute respiratory syndrome. While China has managed to control the epidemic through stringent measures, the number of confirmed cases and deaths continues to rise in other countries. SARS-CoV-2 is notably more contagious than its predecessors, the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV). Rapid and accurate identification of the virus is crucial for controlling the spread of the disease. Currently, computed tomography imaging and hematologic parameters serve as initial markers for COVID-19 infection, while reverse transcription-polymerase chain reaction (RT-PCR) for viral nucleic acid detection is the gold standard for clinical diagnosis. However, RT-PCR is time-consuming, requires specialized laboratories and trained technicians, and is susceptible to variations in specimen quality. In contrast, the detection of serum-specific antibodies using colloidal gold immunochromatography assay (GICA) offers a rapid, highly sensitive, and equipment-free alternative, making it ideal for point-of-care testing.
This study aimed to develop efficient GICA kits for the detection of SARS-CoV-2-specific immunoglobulin (Ig) M and IgG antibodies in the blood of COVID-19 patients. Two types of detection kits were designed: Kit A uses a single test line to capture specific IgM and IgG antibodies targeting the N protein of SARS-CoV-2, while Kit B, an advanced version of Kit A, features separate test lines for IgG and IgM antibodies to distinguish between them. Both kits utilize colloidal gold-labeled N protein of SARS-CoV-2 as the antigen. For the control line, rabbit IgG and goat anti-rabbit IgG antibodies were selected as the antigen and antibody, respectively.
The development of the GICA detection kits involved the preparation of colloidal gold-labeled N protein and rabbit IgG. Briefly, 10 mL of colloidal gold solution (40 nm) was mixed with 65 mL of potassium carbonate (0.1 mol/L). Recombinant SARS-CoV-2 N protein antigen was quickly added to the mixture, which was then incubated for 30 minutes at room temperature. Subsequently, 100 mL of 20% bovine serum albumin solution was added, and the mixture was incubated for an additional 30 minutes. The mixture was centrifuged at 12,000 r/min for 20 minutes and washed with 5 mL of phosphate-buffered saline (PBS; 50 mmol/L, pH 7.4). The precipitate was dissolved in 1.0 mL of colloidal gold buffer. The antigen was loaded and printed on a conjugation pad using specific printing conditions. Similarly, the mouse anti-human IgM/IgG antibody and goat anti-rabbit IgG antibody were diluted with PBS and printed on a polyvinyl chloride sheet with nitrocellulose. The printed film was dried in a 37°C drying box for 6 hours. All prepared materials were assembled according to the design scheme.
To evaluate the sensitivity and specificity of the developed kits, a clinical study was conducted using serum samples from 43 confirmed COVID-19 patients and 45 patients with unrelated diseases. All blood samples were collected from the First Affiliated Hospital and Pulmonary Hospital of Shanxi Medical University, Taiyuan, China, between February 7 and 20, 2020. The study adhered to the Declaration of Helsinki and was approved by the Human Ethics Committee of Shanxi Medical University. For the detection of SARS-CoV-2-specific antibodies, 10 mL of the specimen was loaded into the sample pad, followed by the addition of 80 mL of dilution solution. Test results were determined qualitatively after 15 minutes based on the coloration of the IgM and/or IgG line and the control line. All procedures were performed in a biosafety level 3 laboratory. The interpretation of test results was as follows: the presence of visual color in the control line and IgM and/or IgG test line indicates the presence of SARS-CoV-2-specific IgM and/or IgG antibodies in the specimen. If only the control line is chromogenic, it indicates the absence of IgM and IgG antibodies. No chromogenic reaction in the control line signifies an invalid test, possibly due to poor kit quality or other unknown reasons.
Representative test results of Kit B demonstrated that visual bands at both the detection line and control line were observed in COVID-19 patient samples but not in control samples. Tests were also performed using Human IgG Monoclonal Antibody to Nucleocapsid Protein Solution Reference Material of SARS-CoV-2, and the results were confirmed by western blot. The findings showed that the sensitivities of Kit A and Kit B were 88.3% and 93.0%, respectively, with a specificity of 100% for both kits. These results indicate that rapid and accurate detection of SARS-CoV-2 IgG and IgM antibodies can be achieved using the GICA kits. The higher sensitivity of Kit B is attributed to the separately coated detection lines, which effectively capture IgM or IgG antibodies against SARS-CoV-2. To minimize operator bias, a blind method was employed during the testing process. All blood specimens were numbered and handled by a dedicated manager before testing, and sample information was kept confidential from the operators until the test was completed. It is important to note that the sensitivity and specificity of the pilot study may be overestimated due to the small sample size.
In summary, this study successfully developed two rapid, sensitive detection kits for SARS-CoV-2-specific IgG and IgM antibodies using colloidal gold immunochromatographic technology. These kits hold significant potential for the clinical diagnosis and epidemiologic surveillance of COVID-19. The rapid detection assay offers a practical solution for timely identification of the virus, aiding in the control and management of the ongoing pandemic.
doi.org/10.1097/CM9.0000000000000922
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