Awarded Amount$1,000,000DiseaseTuberculosisInterventionDiagnosticDevelopment StageConcept DevelopmentCollaboration PartnersAsahi Kasei Corporation , Biopromic AB
Introduction and Background of the Project
Despite intense efforts to eliminate malaria, tuberculosis and a number of other infectious diseases only moderate to low success has been achieved. Thus, highlighting the necessity to expand and refine current eradication strategies, in particular those targeting the early detection in endemic areas.
Point-of-care (POC) tests dramatically enhanced physician's ability to diagnose patients’ diseases rapidly and accurately at a bed side or even by the patient himself. Nevertheless, currently there is only a small number of infections POC tests available and they represent a fraction of the actual demand for this kind of diagnostics (WHO TPP for infectious disease diagnostics).
The greatest challenge in POC diagnostics is the very low concentration of antigens in the sample which are easily accessible.
Current LFA devices offer analytical sensitivity above 1-5ng/ml. Taking into account that in case of tuberculosis and malaria patients’ concentration of antigens in the samples is usually below 100pg/ml there is a clear need for more sensitive LFA systems to be developed.
In this project, we plan to develop a new Lateral Flow Assay (LFA) platform aiming at up to 50x improvement of antigen detection from current industry standard.
Achieving such a high analytical sensitivity of the LFA tests requires significant improvement and integration of multiple components of the LFA system. .
To achieve this goal we will combine, integrate and optimize over 5 technologies which were developed by the project partners, in 3 phases of the project.
- Cross-partner technology testing and detailed TPP development where we develop a detailed TPP for the platform. While TPP for each of the technologies exist (or the products are already implemented on the market) a product combining multiple of them would require a separate, balanced approach.
- Technical feasibility testing of combined technologies and their modifications to work within the platform where at the end of this phase we expect all the potential modifications to be complete and a platform to be ready for the prototype phase.
- Development of a LFA prototype test and its initial testing
In this final stage of the project we aim to build two prototypes of POC-LFA tests which utilize the solutions from phase 2 and validate the designs on laboratory and patient
How can your partnership (project) address global health challenges?
Aside from effective vaccines a major global health need is a fast, cheap and sensitive DIY-type diagnostics for TB and malaria.
The inability to diagnose or monitor numerous diseases rapidly is a major cause of prolonged sickness or death regardless of the economic status of a country or the patient. Ability of testing for TB and Malaria outside of the centralized laboratory system without the need for high-end equipment is the ultimate goal in the development of infectious disease diagnostics.
To our knowledge our platform would be uniquely able to improve the sensitivity of the tests without the need for read-out equipment, allowing for true bed-side diagnostics.
Early detection of active cases in case of TB and asymptomatic malaria patients is the key to disease eradication. Currently TB patients are infectious months before the diagnosis with sputum sample can be achieved. In case of malaria it has been described that during periods of limited to no transmission, e.g. the dry season, P. falciparum commonly resides inside its human host in very low numbers and this small minority of malaria infected individuals is sufficient to maintain or re-initiate malaria transmission. Therefore sensitive low cos PoC is a global health must.
As our long-term goal is to establish a platform allowing for other diseases to be diagnosed in the same manner as the global health impact of this project, in our opinion, will be significant. We aim to move the infectious disease diagnostics from the centralized lab setting closer to the patient. Ideally, allowing first contact medical staff to perform the tests immediately on site.
What sort of innovation are you bringing in your project?
There are multipole novel technologies which both partners will utilize in this project
- NanoAct Cellulose beads - Highly colored cellulose nano beads for LFIA labels enable to obtain improved sensitivity, stability and reproducibility over existing colored labels such as colloidal gold and colored latex.
- Nonwoven Pads – new nonwoven pads made of continuous nonwoven and significantly reduce lint comparing to many short fiber materials and increase the flow rate or the amount of liquid adsorption.
- New proprietary conjugation technology able to specifically immobilize mAbs to uniquely coated nanoparticles possessing different surface chemistries. Resulting mAb-particle conjugates are stable and superior in terms of antigen binding efficiency.
- Signal intensification– a technology allowing for significant visual readout increase of the signaling particles on the Lateral Flow membrane
Role and Responsibility of Each Partner
Due to the fact that both Asahi-Kasei and Biopromic bring in sets of their respective technologies into the platform, both parties will be responsible equally for the technical development with each taking a lead in the modification of its respective technologies.
Asahi Kasei will be primarily responsible for the membrane and pad materials as well as for the NanoAct particles and Biopromic is taking charge of conjugation and signal enhancement. Additionally, Asahi-Kasei will be responsible for LFA builds and optimization while Biopromic will develop and provide necessary antibodies.
Project management will be handled by a steering committee consisting of two representatives from each party.
1. Project objective
Current tests available in the market for diagnosis of tuberculosis (TB) are time consuming (1-5 weeks), expensive and not sensitive to detect all cases and because of this many TB infected individual goes undiagnosed, which in turn infect more and more people. Malaria is a life-threatening disease caused by parasites. Several rapid diagnostics tests (RDT) in the market, are not enough sensitive to detect all infected people, especially people with low parasitemia level. The main objective of this project was to develop a rapid, sensitive, accurate and affordable Lateral-Flow Assay (LFA) tests for the diagnosis of tuberculosis and malaria.
2. Project design
【TB】Lipoarabinomannan (LAM) is a TB biomarker excreted into the urine. However, low abundance of LAM in the urine as well as the presence of naturally occurring inhibitory/ interfering components affects the accuracy of the RDT. Biopromic proprietary technologies for the removal of the inhibitors, LAM enrichment and highly sensitive monoclonal antibodies were adapted together with the Asahikasei Corporation unique detector beads (NanoAct™) to develop an ultrasensitive LFA test for TB. 【Malaria】For malaria LFA test, new sample preparation method developed to detect HRP2 and LDH biomarker both in spiked urine sample and Blood standard.
3. Results, lessons learned
【TB】During this project, more than 50 different LFA prototypes were developed for LAM TB using more than 12 different antibodies (10 monoclonal + 2 polyclonal) having different specificity covering various epitopes within the LAM molecules. Although all the prototypes manifest high sensitivity with native LAM-spiked urine, only few of them detecting LAM in the clinical samples. This clearly indicates that LAM wasn’t excreted into the urine as an intact molecule rather than as fragments. One of the prototypes (sensitivity 50 pg/ml) was used for testing a small number of well-characterized clinical samples (tuberculosis patients without HIV infection), and this resulted in 72% sensitivity and 92% specificity. In this study, Alere TB determine (Abbott) was used as a benchmark, the sensitivity of this test was 2% and specificity 100%.
The Biopromic- Asahikasei LFA test sensitivity in TB+/HIV- is superior to Alere TB test. Our recently developed prototype has 5 times higher sensitivity (10 pg/ml) than the previous prototype, and this will be used for testing about 1000 TB patient clinical samples during the new project. From this project, we have learned that antibody selection for the generation of RDT test should be based on testing clinical samples rather than native LAM spiked samples, and antibody qualities and sensitivities are crucial for maximizing accuracy and hence increasing case finding.
【Malaria】Six HRP2 based prototypes out of 81 and 5 LDH based prototypes out of 25 were selected for further evaluation. Two of each prototype showed promising results with parasite infected blood samples and they were further investigated.
In this project, SD-Bioline (Abbott), which is classed as best was employed as a benchmark.
Our prototype detected 20 parasites/µl compared to 50 parasites/ µl for SD-Bioline. After employing the proprietary sample preparation technology, we were able to detect 2 parasites/ µl based on HRP2 biomarker, which means 25 times higher sensitivity than SD-Bioline. Unfortunately, we were not able to acquire the new Abbott uRDT test to be used as a benchmark (the uRDT is available only in the African countries). In addition, when LDH is used as a malaria biomarker, our newly developed RDT showed 16 times higher sensitivity (100 parasites/µl) than the best test in the market (1600 parasites/µl).
Development of LFA platform for improving sensitivity of Point-of-Care assays for infectious disease with main focus on Tuberculosis and Malaria.