The principles of LFA differ depending on the reagents used. We assembled the LFA with reference to CONAN developed by Yoshimi et al. 1.
The strip used was pre-installed with AuNPs with rb anti FITC antibody on the conjugate pad, streptavidin on the control line (C-line), and anti rb antibody on the test line (T-line). FITC-ssDNA-biotin was used as a reporter 2. When CRISPR-Cas3 is activated, the ssDNA within the reporter molecule is cleaved, producing FITC-labeled reporter fragments and biotin-labeled reporter fragments. Using this, both the T-line and C-line turn red if the test is positive, and only the C-line turns red if the test is negative.
For more information about the principle of LFA, see Proposed Implementation_Selection of Amplification and Quantitative Detection Module.
For more information about the actual experimental procedure of LFA, see Experiments.
We conducted an experiment to confirm the performance of the strip used. The purpose of this experiment was to confirm that only the C-line turns red when a reaction solution containing only FITC-ssDNA-biotin is used.
According to the product guide 2, if the concentration of FITC-ssDNA-biotin is not appropriate or the viscosity of the buffer is low, the T-line may turn red even if ssDNA is not cleaved. Therefore, we referred to the product guide and adjusted the concentration of FITC-ssDNA-biotin to 1.00 pM/LFA. A comparison was made between using the assay buffer that comes with the product as is and adding 5% polyethylenglycol (PEG) to the assay buffer.
The product used this time is designed to be used by standing the strip vertically, immersing the bottom part in the developing solution, and using the developing solution to flow the reaction solution. However, we are convinced that POIROT would be easier to use by placing the strip horizontally so that the user did not have to hold the strip vertically during the reaction. So we conducted an experiment with the strip horizontal. We kept the strip horizontal without touching it by folding a piece of paper to create a stand like the one pictured below.
After 5 min of development, the following bands were obtained. The upper strip had no PEG added to the buffer, and the lower strip had PEG added to the buffer.
When PEG was not added to the assay buffer, not only the C-line but also the T-line became colored. On the other hand, when 5% PEG 6000 was added to the buffer, the T-line did not turn red and only the C-line turned red. When performing quantitative determination using this product, it was shown that it is necessary to increase the viscosity of the assay buffer by adding PEG, etc.
We conducted an experiment to determine whether LFA functions when connected from the amplification system. First, we thought about connecting from a simple 1step-SDA.
A case where primer2 of multistep-SDA was added to the reaction solution at a final concentration of 5 nM was compared with a case where primer2 was not added (NC).
After 10 min of development, the following bands were obtained. The upper strip is NC and the lower strip is when primer2 was added.
Even when primer2 was added, no T-line coloration was observed.
The graph below shows the fluorescence change measured using an FQ probe up to 20 minutes later when 1step-SDA was connected to Cas3. Regardless of whether the final concentration of ds-template is 8 nM or 16 nM, an increase in fluorescence intensity can be confirmed when the final concentration of primer2 is 4 nM.
The conditions for this experiment were an incubation time of 20 min, ds-template (12 nM), and primer2 (5 nM) or NC (primer2: 0 M). From the above graphs, it is expected that a sufficient amount of ssDNA would be cleaved when the primer is added, and no cleavage would occur when the primer2 is not added. That is, if primer2 was present, both the C-line and T-line would be colored, and if primer2 was not present, only the C-line would be colored. However, in reality, even when primer2 was added, the T-line did not turn red.
Furthermore, the color of the C-lines were extremely pale compared to when PEG 6000 was not added to the assay buffer in experiment 1. AuNPs with rb anti FITC antibody flowing on the conjugate pad are captured by either C-line or T-line. Therefore, if the T-line is not colored, it is expected that the C-line will be strongly colored. However, in this experiment, the coloring of the C-line was weak even though the T-line was not colored. From this, it is thought that the binding between FITC-ssDNA-biotin or FITC-labeled reporter fragment and AuNPs with rb anti FITC antibody did not occur sufficiently on the conjugate pad, and the AuNPs with rb anti FITC antibody remained on the conjugate pad.
In the product used this time, it was suggested that the negative control only works by increasing the viscosity of the assay buffer. However, if the viscosity of the assay buffer is made too high, the reporter and AuNPs with rb anti-FITC antibody will not be able to bind in sufficient quantities on the conjugate pad, and the T-line may not be colored even under conditions that should be positive. When incorporating this product into POIROT, it is necessary to optimize the viscosity of the buffer.
Yoshimi, K., Takeshita, K., Yamayoshi, S., Shibumura, S., Yamauchi, Y., Yamamoto, M., Yotsuyanagi, H., Kawaoka, Y., & Mashimo, T. (2022). CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus.iScience 25, 103830, 1-13. https://doi.org/10.1016/j.isci.2022.103830
Milenia Biotec GmbH. (n.d.). CRISPR/Cas-based Detection Methods and HybriDetect: Universal Test Strips - Individual Readout. https://www.milenia-biotec.com/uploads/2019/07/improved-CRISPR-readout_final-1.pdf