| dc.description.abstract | Background: Lateral flow assay (LFA) is a prevalent methodology in diagnosis due to its rapid,
convenient, and affordable characteristics. However, the comparatively low sensitivity of the assay
is a considerable disadvantage. A wide variety of optimization and modification in the LFA
platform has been investigated, including secondary antibodies, biotin-streptavidin binding, silver
nanoparticles, the substitution of electrochemical luminescence or modification of the flow, etc.
This study aims to improve the colorimetric SARS-CoV-2 LFA's sensitivity by the capture
antibodies orientation protein A due to its high affinity to the Fc region of antibodies.
Method: The gold nanoparticles (AuNPs) based colorimetric LFA test for SARS-CoV-2 was
designed based on the sandwich ELISA principle. Monoclonal antibodies (Anti-SARS-CoV-2 N
mouse antibodies 7B3) and detection antibodies (Anti-SARS-CoV-2 Nucleocapsid Recombinant
Rabbit Monoclonal Antibody 3E8A5) are used to capture the SARS-CoV-2 protein N. For capture
antibody orientation, protein A is added to the nitrocellulose membrane before dotting the capture
antibodies. Different capture antibodies and protein A ratios ranging from 1:3 to 1:0.5 (Molar:
Molar ratio) were investigated. To test the effectiveness and limit of detection (LoD) of the
designed system, half-strip models, including nitrocellulose membrane with pre-immobilized
protein A/capture antibody and wicking pad, were assembled. Samples containing protein N at
different concentrations (1 µg/mL, 100 ng/mL, 10 ng/mL, 1 ng/mL to 100 pg/mL) were mixed
with AuNPs-detection antibody conjugates at 1:5 (v/v ratio) then loaded to the half-strips. Finally,
the full-strip fabrication procedure is processed with the combination of the modified half-strip
fabrication procedure (without sample loading step), conjugate pad, and sample pad pretreatment.
Band signals were analyzed by both naked eyes and ImageJ mean Grayscale Value (GSV)
measurement. All experiments, except for the conjugation characterization and the full-strip
effectiveness evaluation, were triplicated. For statistical analysis, the Student's t-test and One-way
ANOVA with the Tukey HSD post-hoc multiple comparison test were used for the variance
comparisons across the means of data groups. The results were considered statistically significant
when p-value < 0.05. Result: A molar ratio 1:1 was shown to be the optimal capture antibody: protein A ratio as there
was no excess protein A. In the initial half-strip fabrication, the strips faced the problem of slow
intensity signal revealing as positive signal reached GSV = 221.320 ± 0.645, which is extremely
close to the GSV = 231.997 ± 1.885 of the negative control and is difficult to distinguish from the
negative sample by the naked eye. With the modification in non-specific blocking methodology
by directly mixing BSA 2.5% (w/v) to the sample mixture instead of pretreating on the membrane,
the signal band of the half-strip reveals a denser pink color with a clear signal at GSV = 209.506
± 1.041 compared to the negative threshold and the signal of the strip before being optimized (p
value < 0.05). In the limit of detection experiments, LFA strips without protein A and
commercialized kit (eDiagnosis Covid-19 antigen Rapid Test Kit) reach a similar antigen
concentration of 1 ng/mL as the limit of detection. In the presence of protein A, the limit of
detection of the LFA reached the antigen concentration of 100 pg/mL, with a stable GSV of
216.296 ± 1.071, being significantly different from and much lower than the intensities of negative
results threshold, without protein A half-strip, and commercialized test kit (p-value < 0.05). The
results indicate protein A has a significant impact on enhancing the sensitivity of the SARS-CoV-
2 half-strip. The functional full-strip is also fabricated subsequently and can distinguish positive
and negative samples with clear signal bands.
Conclusion: Protein A is the key factor for the 10-times enhancement of the Protein N SARS
CoV-2 detecting Lateral Flow Test strip's sensitivity as the Limit of Detection decreases from 1
ng/mL to 100 pg/mL antigen concentration. | en_US |
