A colorimetric lateral flow immunoassay based on oriented antibody immobilization for sensitive detection of SARS-CoV-2

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The high human-to-human transmission and rapid evolution of SARS-CoV-2 have resulted in a worldwide pandemic. To contain SARS-CoV-2, it is essential to efficiently control the transmission of the virus through the early diagnosis of infected individuals, including asymptomatic people. Therefore, a rapid and accurate assay is vital for the early diagnosis of SARS-CoV-2 in suspected individuals. In this study, we developed a colorimetric lateral flow immunoassay (LFIA) in which a CBP31-BC linker was used to immobilize antibodies on a cellulose membrane in an oriented manner. The developed LFIA enabled sensitive detection of cultured SARS-CoV-2 in 15 min with a detection limit of 5 × 10⁴ copies/mL. The clinical performance of the LFIA for detecting SARS-CoV-2 was evaluated using 19 clinical samples validated by reverse transcription-polymerase chain reaction (RT-PCR). The LFIA detected all the positive and negative samples accurately, corresponding to 100% accuracy. Importantly, patient samples with low viral loads were accurately identified. Thus, the proposed method can provide a useful platform for rapid and accurate point-of-care testing of SARS-CoV-2 in infected individuals to efficiently control the COVID-19 pandemic.

Keywords: Antibody; COVID-19; Lateral flow immunoassay; Orientation; SARS-CoV-2; Sensitive.

Fig. 1

Schematic of the CBP31-BC-based LFIA for the detection of SARS-CoV-2. The LFIA strips consist of a sample pad, conjugate pad, cellulose membrane, and absorbent pad. The test line placed on the cellulose membrane contains a capture antibody with immobilized CBP31-BC to detect the SARS-CoV-2 spike antigen. CBP31-BC alone is used for the control line. The developed LFIA can sensitively detect the SARS-CoV-2 spike antigen within 15 min.

Fig. 2

Sensitivity analysis of the CBP31-BC-based LFIA. a) Photographic images showing the results of the LFIA strip for different SARS-CoV-2 RBD concentrations. The cellulose membrane was photographed using a smartphone at 15 min after sample loading, and the test line intensity was analyzed using ImageJ. b) The normalized test line intensity of the LFIA for different SARS-CoV-2 RBD concentrations. The data were fitted to a logistic four-parameter curve with a high correlation coefficient of 0.9914. All the data are presented as the mean of at least three independent measurements, and the error bars represent the standard deviation from the mean. The LOD was calculated as previously described by Holstein et al .

Fig. 3

Specificity analysis of the CBP31-BC-based LFIA. a) Photographic images for the cross-reactivity of the LFIA against SARS-CoV-2 RBD, SARS-CoV S1, MERS-CoV S1, and CoV-H229E S1 antigens. The cellulose membrane was photographed using a smartphone at 15 min after sample loading, and the test line intensity was analyzed using ImageJ. b) The normalized test line intensity of the LFIA for SARS-CoV-2 RBD, SARS-CoV S1, MERS-CoV S1, and CoV-H229E S1 antigens. The dotted line indicates no sample. All the data are represented as the mean of at least three independent measurements, and the error bars represent the standard deviation from the mean. P-values: *P ≤ 0.05, * *P ≤ 0.01, * **P ≤ 0.001.

Fig. 4

Laboratory confirmation of the CBP31-BC-based LFIA using clinical samples. a) Photographic images showing the results of the LFIA strip for different cultured SARS-CoV-2 concentrations. b) Photographic images showing the results of the LFIA strips for samples from clinical COVID-19 patients. The cellulose membrane was photographed using a smartphone at 15 min after sample loading, and the test line intensity was analyzed using ImageJ. c) The normalized test line intensity of the LFIA for different cultured SARS-CoV-2 concentrations. The black dotted line indicates the cutoff value (the mean of 3 blank samples + 3 standard deviations). All the data are represented as the mean of at least three independent measurements, and the error bars represent the standard deviation from the mean. d) Confusion matrices for evaluating the performance of the LFIA using nasophyngeal swab samples from COVID-19 patients. P-values: ns > 0.05, *P ≤ 0.05, * *P ≤ 0.01, * **P ≤ 0.001.