Introduction:

The basic idea of our project was to detect trace amounts of a specific antigen, for example, tumor markers. In order to be able to detect these antigens, we have incorporated an intermediate step into the basic principle of an immunoassay: the amplification part. This method should enable the visualization of binding events for smallest concentrations of the antigen. The first area of application for our project idea is at the general practitioner offices. With our detection system they are able to use it as a reliable way to confidentially screen for trace amounts of antigens, making it ideal for a diagnosis in early stages of esophageal cancer. "It is important to note that the antigens to be detected are approved tumor markers" - Jörg Meckoni, general practitioner. For the implementation of our project idea, we had to think about how to combine all our components in a suitable system to ensure the simplest application possible. Our self-amplifying detection system consists of three individual components. The detection component consists of scFv-fragments that bind our target antigen. Thereby an active protease is produced that can activate the other two subsystems of our detection system. The amplification component amplifies the signal by producing more active protease through Inteins, which also activates the reporter component. Causing a positive feedback loop finally, protease activity releases fluorescence, which we want to measure. This design of our assay also eliminates the need for secondary antibodies, which makes our detection system easier and faster compared to ELISA. [1].
All these components should function in a cell-free environment and must be made usable in a manageable form. For this purpose, we have developed a box in which all three components are combined. This is done via built-in pumps, which bring the sample together with our three components for detection. (More details can be found on the hardware page.) This saves us the time-consuming pipetting steps that would have to be carried out by an extra person. In addition, we can reduce application time and eliminate human error when combining the components.

Application areas:

One advantage of our system is that the scFv-fragments are interchangeable, which enables the system to be applicable for other antigens such as proteins and even entire pathogens as well [1]. This significantly expands our field of application. The first application would be as already written, by general practitioners. They could use our detection kit for a better assessment of diagnoses. An example would be the detection of potential biomarkers for the early detection of esophageal cancer. A major problem in the diagnosis of esophageal cancer is the occurrence of mostly nonspecific symptoms at an already advanced stage of the disease [2]. IPO5 (Importin 5) is a nuclear transport protein, which as an individual protein would be a good potential biomarker [3]. It is significantly elevated in the blood serum of esophageal cancer patients and are therefore the perfect target [3]. Here it should be noted, however, that this protein is not yet an approved tumor marker for esophageal cancer. Thus, if a primary care physician suspects esophageal cancer, he or she could take a blood sample and test it by using our detection kit to further isolate possible causes of such symptoms. If the suspicion is confirmed, the patient can be quickly referred to a specialist, which would save valuable time in cancer treatment. In addition to biomarkers for tumor diseases, our system can also be applied in other areas. As soon as a suitable scFv fragment are available, our system can detect a wide variety of antigens, since only the detection component has to be adapted. For example, viruses or allergens can also be detected.

Costs and marketing:

To compete in the assay market, our detection kit must be more affordable than conventional diagnostic products. Currently, ELISA (enzyme-linked immunosorbent assay) and FIA (fluorescent immunoassay) are some detection technologies for Tumor markers e.g. [4].

Below is a possible price calculation of our detection kit:

In order to establish a basic calculation, we spoke to an expert. She was able to give us some helpful tips, but also many aspects that we would have to consider if we wanted to bring our product to market.

Material costs

Direct material costs:
Filter: 17.90€ --> own experience
Pumps: 26.51€, Status 09/25/22 -->
Costs in print: data from Martin Johann
Fixation tray: 0.20€
hose adapter: 0.22€
cell phone holder: 1.22€
drawer: 1.31€
box housing: 5.46€
Material overheads:
20.5% from direct material costs = 10.83€
Total costs: 63.65€

Cost absorption by the statutory health insurance funds:

Before our test can be used in GP practices, it must be clarified whether it is covered by statutory health insurance in Germany [5]. Whether our test would be covered as a service is decided by the G-BA (Gemeinsame Bundesausschuss) [5]. Physicians, hospitals and therapists are members of the G-BA, which decides on the coverage of the service. As soon as it is decided that our test will be provided as a contractual service, it will be covered by the insurance companies [5]. If the G-BA rejects our test, it would be offered as an individual health service [5]. Individual health services (IGeL) are services that are not included in the catalog of services provided by the statutory health insurance funds but are part of contractual medical care [6]. These services are usually still being evaluated by the G-BA or there is not yet sufficient evidence of benefit [6]. IGeL are often offered by physicians as additional preventive examinations [6]. The costs of services that exceed the scope of the health care mandate must be paid privately by the patient [6]. Based on a discussion with a general practitioner, we need to provide an economical, accurate and easy-to-use test that will be covered by statutory health insurance in Germany. However, our test would probably be offered as an IGeL until further data is available.

References

1. Ahmad, Z. A., Yeap, S. K., Ali, A. M., Ho, W. Y., Alitheen, N. B. M., & Hamid, M. (2012). scFv Antibody: Principles and Clinical Application. In Clinical and Developmental Immunology (Vol. 2012, pp. 1–15). Hindawi Limited. https://doi.org/10.1155/2012/980250
2. Short MW, Burgers KG, Fry VT. Esophageal Cancer. Am Fam Physician. 2017 Jan 1;95(1):22-28. PMID: 28075104
3. Watt, P. J., Okpara, M. O., Wishart, A., Parker, M. I., Soares, N. C., Blackburn, J. M., & Leaner, V. D. (2021). Nuclear transport proteins are secreted by cancer cells and identified as potential novel cancer biomarkers. In International Journal of Cancer (Vol. 150, Issue 2, pp. 347–361). Wiley. https://doi.org/10.1002/ijc.33832
4. Immunoassays. (n.d.). Berthold Technologies GmbH & Co.KG. Retrieved October 9, 2022, from https://www.berthold.com/de/bioanalytik/wissen/glossar/immunoassays/
5. Wer entscheidet über die Leistungen der gesetzlichen Krankenkassen? (2022, May 31). Die Techniker. Retrieved October 12, 2022, from https://www.tk.de/techniker/leistungen-und-mitgliedschaft/informationen-versicherte/leistungen/weitere-leistungen/praevention/igel/entscheidung-aufnahme-leistungen-in-katalog-der-gkv-2008880?tkcm=ab
6. IGeL - Bundesgesundheitsministerium. (n.d.). App Title. Retrieved October 12, 2022, from https://www.bundesgesundheitsministerium.de/service/begriffe-von-a-z/i/igel.html