The strategy for our Cerebrospinal Fluid (CSF) Rhinorrheas detection consists of two essential parts. First, to avoid the false positive result from the sample, the sialo Transferrin (sTF) in the sample needs to be removed by Siglec-1 lectin. Second, for detecting the presence of beta-2-Transferrin (bTF), the conjugated colloidal gold nanoparticles (AuNP)-bTF complex need to be captured by the anti-TF antibodies immobilized on nitrocellulose (NC) membrane. To verify the capability of CSF detection, the following experiment is taken place.
For the first part of the experiment, we used BL21 E. coli to synthesize the Bioactive Siglec-1 lectin-His tag. The His tag connected with Siglec-1 lectin ensures that the lectin can bind to Nickel beads in the Deletion Chamber. We then pass the bTF samples, and sTF samples with different concentrations through the Deletion Chamber, and the concentration of each sample before and after the Deletion Chamber is verified by Enzyme-Linked Immunosorbent Assay (ELISA).
For the second part of the experiment, we also used the BL21 E.coil to synthesize bTF-mCherry complex (bTF-mC), which is used in the following NC membrane test. First, Colloidal gold-antibody (AuNP-antibody) is mixed with bTF-spiked solutions and sTF-spiked solutions, and the mixed solutions are added to the test strip. The presence of the red line indicates that the anti-TF antibody is not able to distinguish between bTF and sTF, which substantiates the necessity of a Deletion Chamber. Next, the bTF-mC complex synthesized from the bacteria is spiked into to solution and mixed with the sTF respectively. After conjugated with AuNP-antibody solution, the samples are added to the NC membrane. By using the spectrometer, the trace of bTF on the NC membrane can be visualized due to the connected mCherry, and we therefore prove that the presence of the red line after the Deletion Chamber is induced by bTF, not sTF.
The result of the two parts all shows that our Point of Care (POC) strategy for CSF Rhinorrheas test kit is essential, and capable.
Figure 1. Function of the AuNP and the Test Strip
Figure 2. Construction of four plasmids
We designed 4 recombinant plasmids: pET28a-bTF, pET28a-mCherry, pET28a-bTF-mCherry and pET28a-Siglec-1. In order to construct four recombinant plasmids, we first amplified bTF (2094bp), mCherry (711bp), and Siglec-1 (5130bp) fragments using PCR. As shown in Figure 2A-B, compared to the 15K maker, these three genes were successfully amplified. Through a double enzyme digestion, we successfully obtained a linearized plasmid pET28a (Figure 2C), which was then linked to bTF, mCherry, and Siglec-1 fragments, respectively. The map of the recombinant plasmid after ligation is shown in Figure 2D-G.
Figure 3. Results of plasmid transformation, identification, and sequencing
Next, the four plasmids were transformed into Escherichia coli BL21 (DE3), respectively. After overnight cultivation, the transformants successfully grew on LB plates (Figure 3B). We conducted colony PCR identification on the transformants, and the results are shown in the Figure 3A and 3C. From Figure 3A, it can be seen that the pET28a-beta-2-Transferrin and pET28a-mCherry plasmid transformants were positive. Then we found that 9 out of 16 samples were found to be positive in pET28a-beta-2-Transferrin-mCherry E.coli (DH5α) and only 1 positive sample in pET28a-beta-2-Transferrin-mCherry E.coli (BL21) by PCR identification (Figure 3C). To further validate the construction of the plasmid, we sequenced the positive transformants, and the sequencing results showed that the gene sequences were all correct, indicating the successful construction of the plasmid (Figure 3D).
After centrifugation, the bacteria samples are hypersonic-lysed for breaking the cell structures. The samples then go through filter tubes containing nickel columns. Due to the double 6 His tag on the proteins, they bind to the nickel web on the column. Next, the proteins are washed by His A solution, His B solution respectively. The His B solution that washed the protein are then collected.
We purified the target protein using His tags and validated the protein expression and purification results through SDS-PAGE. As shown in Figure 4, we successfully expressed the beta-2-Transferrin (79 kD), mCherry(30 kD), beta-2-Transferrin-mCherry(107 kD) and Siglec-1 protein (183 kD).
Figure 4. SDS-PAGE results of four protein expression and purification
(P represents “Precipitation”, S represents “Supernatant”, T represents “Flow through”, and E represents “Eluent”)
In order to deplete sTF which is the protein that would affect the testing of the target protein bTF, a deletion chamber is constructed. Siglec-1 is immobilized with Nickel beads and its function is tested with ELISA and test strips.
The ELISA assay allows for the detection of a specific antigen, thereby indicating the presence of a particular protein. However, it is unfortunate that the presence of antigens in sTF and bTF cannot be distinguished by ELISA testing as shown in figure 5. Thus, this underscores the critical importance of utilizing the deletion chamber, as the presence of sTF would likely lead to false-positive results and compromise the accuracy of diagnosing CSF rhinorrhea.
Figure 5. ELISA could use same antibody to detect sTF and bTF
In order to evaluate the depletion capability of the deletion chamber on sTF concentration, solutions with varying concentrations of sTF are utilized to assess the performance of the chamber. A concentration gradient is established using this approach, and the result is presented in figure 6. The concentration of sTF exhibits a substantial reduction and approached near-zero levels after undergoing reaction within the deletion chamber. This outcome serves as compelling evidence supporting the effectiveness of the deletion chamber in depleting sTF concentration.
Figure 6. sTF concentration before (x-axis) and after (y-axis) deletion chamber
To assess the impact of the deletion chamber on both bTF and sTF, bTF and sTF solutions are passed through the deletion chamber separately. The concentration of these solutions before the deletion and after the deletion are measured via ELISA, as shown in figure 7.
From the result in figure 7, we can see that there is no significant change on the concentration of bTF. On the contrary, the concentration of sTF has a substantial reduction. This confirms that bTF is barely affected by the deletion chamber.
Figure 7. concentration of sTF and bTF before and after (sTF+DC, bTF+DC), respectively
In order to assess the practical efficacy of the deletion chamber, a simulated test was conducted using six nitrocellulose membranes in equal size. Anti-TF antibodies are applied to the test membranes. Solutions containing bTF-mC, sTF, and a combination of bTF-mC and sTF are prepared, with half of each solution being processed using the Deletion Chamber. Equal volumes of each solution are then mixed with an AuNP solution and added to the six separate membranes prepared for testing.
According to our hypothesis, the unprocessed solutions are expected to react with the antibodies, yielding a red band on the test strip, whereas no reaction is anticipated for the sTF solution that has been processed. Approximately 10 minutes after the time of solution added to the membrane, the appearance of red bands confirms our hypothesis, as depicted in the accompanying image. This experiment will provide strong evidence on the efficiency and availability of our product, while also further verifying the effectiveness of the deletion chamber.
When the antibody on the NC membrane captures collodial gold-TF complex, the complex of colloidal gold moves slower/doesn’t move as antibodies applied on NC membrane are immobilized. On the contrary, those colloidal gold that does not bind with TF goes continuously. Thus, when there is a successful capture of TF, there will be two bands visible, the solvent front band, and the antigen band. The antigen band, i.e., the band after the band on the solvent front, proves a successful capture of sTF by the antibody on NC membrane and on the colloidal gold. By measuring the color intensity of the mCherry of the secondary band, we can therefore determined the cause of the band.
Figure 8: bTF-mC sample, bTF-mC sample with deletion chamber processed (bTF-mC+DC)
As we can see in figure 8, there are antigen bands on the test strip, which proves that antibodies and colloidal gold can be used for visualization of the detection of bTF. Furthermore, the spectrometer experiment also proves that the Deletion Chamber barely affects the result for bTF detection. Under the spectrometer, the color intensity of mCherry presented in both of the bands are visibly similar, which proves that the appearing red line is caused by bTF, and the Deletion Chamber has a minor effect on the depletion of bTF.
Figure 9: sTF sample, sTF sample with deletion chamber processed
In figure 9, the presence of a antigen band in Strip sTF shows that sTF can factually affect the test to cause a false positive result.
Figure 10: Result of the test on the bTF-mC solution mixed with sTF after going through the deletion chamber
Comparing to figure 8 and 9, in figure 10 we can see that the sample bTF with sTF will result in the same antigen band which echoes the concern of false positive results caused by sTF. After DC, sTF shall be removed per the figure 9 result while the bTF can be still detected by presenting a positive antigen band, therefore, we can conclude that the deletion chamber can successfully eliminate the effect from sTF in the real context. With this conclusion, we believe our upgraded CSF Rhinorrheas detection kit has a very promising future.
As we have proved that the strategy works for the CSF rhinorrhoea test kit, improvement and industrialization of making the test kit is possible.
Firstly, as we must ensure that all reactants react (e.g. colloidal gold-antibody binds with bTf; sTf is immobilized by nickel beads), we might be assigned longer times for reactants to react than what is really required. Regarding reducing manual inaccuracies, the common to immobilize antibodies on an NC membrane is by use of a dispenser machine, which in our experiment, we find not as necessary as using a pipettor directly applying on the NC membrane to demonstrate the success of the experiment is enough. Regarding the system, every system listed, e.g. the volumetric ratio between colloidal gold solution, PBS, and sample, can be readjusted to lower the cost for a test and to maintain, even improve the accuracy of the test.
Regarding making it into a product, we can consider assembling the test kit into hardware, which can be achieved by simply adapting our NC membrane on absorption pads, etc., just like a common COVID-19 Rapid Antigen Test’s non-biological components. Then, we may think about the test in the market, which must pass through testing real clinical samples to apply a Receiver Operating Characteristic (ROC) analysis, and a calculation of Youden's index may be required. Stringent clinical examinations and assessments then will be carried out.
Then, as the product is proven to be acceptable to the public, industrial large-scale production can then be considered.