Cerebrospinal fluid (CSF) rhinorrheas occurs when there is a fistula between the dura and the skull base and discharge of CSF from the nose^[1]. CSF leak most commonly occurs following trauma (80-90 % of cases) and the majority of cases presenting within the first three months. Other etiologies include postoperative defect (10 %), spontaneous leak (3-4 %), tumor, and inflammation^[2]. This can cause low-pressure headaches, neck pain, ringing in the ear, and occasionally, loss of smell or taste. A leak can be detrimental to brain blood supply and function and can increase the risk of direct trauma to brain parenchyma due to loss of fluid cushion. Open communication of the subarachnoid space with CSF leak also presents a pathway for life-threatening CNS infection, including meningitis. Therefore, the presence of signs and symptoms of a CSF leak indicates the need for further evaluation and management ^[4]. 

The current diagnostic methods of CSF rhinorrhea including CT, MRI, beta-2-Transferrin (bTF) assay and Glucostix test strips ^[1], are relatively time-consuming, expensive, complex. The current cost of bTFtest is around $37.90 ^[5] which is relatively expensive and there is no method at all to provide a Point-of-Care (POC) diagnosis, which means all current existing tests requires laboratory equipment. This causes delayed diagnosis and may lead to serious consequences because of the risk of an ascending infection which could produce fulminant meningitis. Making the thing worse, the public still lacks of awareness to the CSF rhinorrhea. According to the data we collected via a questionnaire, 74 % of 325 people are uninformed about CSF and only 7 % of them understand what CSF rhinorrhea is. Consequently, raising the awareness of CSF rhinorrhea, as well as a convenient, accurate and cheap test kit that can test for CSF rhinorrhea is urgently needed.

To give a faster and more accurate method to detect CSF rhinorrhea, we have come up with an original idea based on an immunochromatographic assay (ICA) test design. Our product is a breakthrough, a change from zero to one. It is the first product that ordinary people can use to diagnose CSF rhinorrheas without aid of laboratory apparatuses and staff. Additionally, we have first created a deletion chamber that can remove the greatest factor that would cause misdiagnosis which is sialo transferrin (sTF), a protein that has a similar structure to bTF. According to our experiment, the concentration of sTF reaches nearly 0 after being processed by the deletion chamber. This would largely increase the accuracy of the diagnosis of CSF rhinorrhea.

 

Beta-2-Transferrin (bTF) has the potential to be the specific biomarker of CSF rhinorrheas only when it is distinguished with Sialo Transferrin (sTF). sTF is a major component that present in both CSF and sialo. The minor differences in the Transferrin (TF) based glycan chains make it difficult to distinguish from sTF, which may lead to sTF binding with anti-TF antibody to create a false positive result ^[3].

In this study, we introduce an immunochromatographic assay (ICA) method to a detect the presence of bTF. The test kit is composed with 3 parts, the deletion chamber, the AuNP chamber, and the nitrocellulose test strip. The simulated sample of CSF is sent to the deletion chamber with immobilized Siglec-1 synthesized by bacteria to remove sTF. After that, the treated sample is sent to the AuNP chamber and conjugated with the AuNP. Finally, the conjugated sample is added to the nitrocellulose membrane immobilized with anti-TF antibodies. The red line on the test line indicated the positive result.

 

 

Figure 1. Product design of our CSF rhinorrheas ICA detection assay with special deletion chamber

 

Firstly, we produced the siglec-1 lectin using BL21 E.coli. We constructed of plasmid pET28a-siglec-1 through restriction enzyme and ligation,and following transferred into E. coli. After culturing, we chose positive monoclonal and extracted the plasmids to sequence it. Upon protein IPTG inducing expression through E. coli BL21, we conducted ultrasonication, centrifugation and a Ni-NTA Resin kit for protein purification. The purified Siglec-1 is then immobilized on the Nickel beads for the use of sTF deletion.

Figure 2. Experiment flowchart

 

We then established the Deletion Chamber and verified the effectiveness of Deletion Chamber (DC) for removing sTF. The deletion chamber was built by the nickel beads immobilized with Siglec-1. Next, the spiked sTF sample, spiked bTF sample, and spiked sTF-bTF mixture with different concentrations are “processed” by the deletion chamber respectively. The concentration of each sample before the DC and after DC is measured by ELISA.

Finally, we constructed the test strip in semi-practical situation. We constructed bTF-mCherry plasmids and transformed into BL21 E. coli for bTF-mcherry protein expression. Using the anti-TF antibody and nitrogen cellulose membrane, we then built up simple test strip. By conjugating the anti-TF antibody with AuNP-streptavidin can we obtain AuNP-anti-TF antibody, and we conjugated the bTF-mcherry samples with AuNP-anti-TF antibody before and after DC, respectively. The conjugated samples are then dripped on the test strip and the color change is observed, and the spectrometer is used on the bTF-mCherry to trace the result.

Figure 3. Workflow of the deletion chamber on the test strip

 

 

By utilizing synthetic biology technology, the vital part of the Deletion Chamber, Siglec-1, a protein that traps sTF, can be effectively synthesized through E. coli and allows trance of the antigen on the NC membranes. Meantime, bTF-mcherry complex was produced for observing the path of the AuNP-bTF through a spectrometer, which enableed us to examine the effect of the test strip.

The ultimate impact of these proteins is firmly demonstrated by the data of ELISA and the AuNP assay. This innovation creates an unprecedented accuracy of the CSF rhinorrhea test by eliminating the most confusion caused by sTF and the possibility of misdiagnosis is diminished to a large extent. It would largely help those patients and high-risk groups to prevent delay and inaccurate diagnosis of CSF rhinorrheas and further decrease the risk of further infectious and more severe diseases. 

Through this project, we hope to raise more attention from the public to CSF rhinorrhea as well as other brain injuries and diseases that were easily ignored, and contribute with a great example of a convenient, cheap, and accurate diagnostic method that would benefit the society and improve public health.

 

References:

[1] Abuabara A. Cerebrospinal fluid rhinorrhoea: diagnosis and management. Med Oral Patol Oral Cir Bucal. 2007 Sep 1;12(5):E397-400. PMID: 17767107.

[2] Iffenecker C, Benoudiba F, Parker F, Fuerxer F, David P, Tadié M, Bobin S, Doyon D. Place de l'IRM dans l'exploration des fistules de liquide céphalo-rachidien [The place of MRI in the study of cerebrospinal fluid fistulas]. J Radiol. 1999 Jan;80(1):37-43. French. PMID: 10052036.

[3] Kwon SJ, Zhang F, Dordick JS, Sonstein WJ, Linhardt RJ. Detection of cerebrospinal fluid leakage by specific measurement of transferrin glycoforms. Electrophoresis. 2015;36:2425-2432

[4] Severson M, Schaurich CG, Strecker-McGraw MK. Cerebrospinal Fluid Leak. [Updated 2023 Mar 6]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK538157/

[5] Oakley GM, Alt JA, Schlosser RJ, Harvey RJ, Orlandi RR. Diagnosis of cerebrospinal fluid rhinorrhea: an evidence-based review with recommendations. Int Forum Allergy Rhinol. 2016 Jan;6(1):8-16. doi: 10.1002/alr.21637. Epub 2015 Sep 15. PMID: 26370330.