SECTION I: Testing
|1||Has the R & D result been tested?|
The following question is replied according to the reply in question 1
|1a||In what mode has the result been tested?
• Pilot Application
• Alpha/BETA testing
|1b.||Please describe and discuss the testing results|
|1c||Describe what type of testing does the R&D result need?|
|We participated as Partner in the ZF-TOOLS Consortium “High-throughput Tools for Biomedical Screens in Zebrafish”, funded by the European Commission as part of its Sixth Framework Programme (EC Contract LSHG-CT-2006-037220). We determined the transcriptome of Mycobacterium marinum-infected zebrafish by high-throughput sequencing. Within the set of fully representative transcripts we identified in databases 815 EST-mRNAs with statistically significant expressional alteration. Of these genes homology search identified 397 functionally known human orthologues. Among these human orthologues140 genes were associated with known diseases of Mendelian inheritance of which 72 were over- and 68 under-expressed. We analyzed the clinical presentations to be associated with each of these genes and evaluated the degree by which these cases recapitulated elements of TB pathomechanism.
These R&D results need testing gene expressional alterations by discovery of novel biomarkers associating with human tuberculosis. These TB-associated consistently and differentially expressed protein markers in human blood serum are suitable for diagnostic purposes, as the selected markers provide close link to TB, course of treatment of the disease and predict the clinical outcome.
Western analysis and ELISA testing will be used during determination of concentrations of protein markers in a diagnostic clinical trial, involving TB patients and matching healthy control persons. Western and ELISA testing belong to conventional laboratory measurements.
|1d.||What is the time needed for testing?|
In a clinical diagnostic study, we will first quantify alterations of selected proteins expressed in human blood serum from twenty TB patients and twenty sex and age matched controls. System validations will include determination of linear dynamic ranges of protein markers as recombinant proteins using Western and ELISA analyses. Concentrations of marker proteins in TB and control subjects will then be determined. Digitized Western and ELISA signal intensities will be analyzed by the Student’s t-test to determine if values in TB and control samples are significantly different. For these experiments we will develop targeted custom ELISA kits. In order to refine statistical analysis corrected for sex and age of the cohorts, double-sided t-test, variance-analysis and khi-square probe will be used. Results collectively will establish the elements of a TB-signature expression pattern. Once the signature pattern/fingerprint has been established, we continue to similarly tests other selected candidate proteins and validate data in large Eastern European TB and control cohorts.
We will attempt to address ethnicity-based variations in expression values in large cohorts enrolled through our established international network of collaborators, involving populations from Africa, Asia and Europe as well as samples of the WHO TB specimen bank. We anticipate that these TB signature proteins normalized to ethnic variations are suitable for developing the prototype of a diagnostic platform. We expect to generate new knowledge and establish the diagnostic value for a set of novel biomarkers for the detection of TB infection.
Development and large-scale production of ELISA-pairs of antibodies for the established prototype of the diagnostic tool. Aggressive marketing on the potential market. Introducing the product to the market.
Production of the diagnostic tool in medium scale, 10 000 platforms a year in the own laboratory of the participants. Initial steps toward establishment of own plant depending on the demand of the market.
|1e.||What is the cost needed for testing?|
|Year 1: 130 000 Euro
Year 2: 130 000 Euro
Year 3: 100 000 Euro
Year 4: 150 000 Euro
Year 5: 300 000 Euro
SECTION 2: Current Stage of Development
|2a||To what extent does the development team have technical resources for supporting the production of a new product? (Researchers, human resources, hardware, etc. )|
|The development team consists of three university professors, two postdocs, two predoctoral fellows and two PhD students. Two laboratory technicians will be hired.
All three professors have their own well-equipped laboratories where the project can be started immediately.
The development phase is based technically on Western- and ELISA methods that are parts of the technical repertoire of the participating laboratories.
|2b||What are the technical issues that need to be tackled for full deployment, if needed?|
|There are no technical issues to be tackled for full deployment during biomarker selection and prototype development.
|2c||What additional technical resources are needed for the production of this new product?|
|This step follows the 2+1 years of development phase.
Basically, a production line for ELISA plate construction is needed, based on
Initial production of the diagnostic tool in own laboratories to supply marketing demands and pilot testing by the potential partners, free of charge.
Production in the diagnostic tool en gross in own/hired laboratory with a capacity of 10000 platforms/year; establishment of own plant will be decided, depending on the demand of the market for the product.
|2d||Overall assessment of the current stage of technical development.|
|Ongoing activities in the field aiming identification of biomarkers associated with TB utilize elements host-pathogen interaction. PCR-based methods detect the presence of the pathogen in body fluids; operating the system, however, needs skilled personnel and positive diagnosis often refers to latent infection. S. Spivack, V. Gupta, and M. Schnitzer are developing diagnostic tools based on the presence of the pathogen. K. Dimitrov is working on a nanotechnology approach, whereas J. Fisk developes a phage-based system. C. Holm-Hansen is searching a saliva-based assay test. T.H.M. Ottenhof, M.L. Gennaro utilize host response to pathogen antigens in holistic approaches by antibody target recognition. S. Krishna developed a SELDI-TOF-based identification of diagnostic markers for tuberculosis by serum profiling.
The current standard diagnostic methods used in the developing world fail to detect more than half of the active TB cases. There is, therefore, a pressing need for a simple, rapid, inexpensive, yet highly accurate TB diagnostic tests that could save hundreds of thousands of human lives world wide.
There is no reliable TB diagnostic tool available on the market. Technical development in industrialized countries concentrates on producing TB diagnostic tools. However, estimates of the WHO expect these tools not earlier than 2015.
Our proposal is based on biomarkers with low diagnostic value for each serum protein, but in combination of poorer markers a high specificity and selectivity of TB diagnosis will be achieved by multiple marker screening. Our appearance on the market is expected by 2015-2016.
SECTION 3: Deployment
|3a||Define the demands for large scale production in terms of|
|Antibody pairs for quantitative ELISA detection
Printer, placing primary antibodies on the surface of wells in ELISA-plates
Reagents for conjugated, secondary antibodies
Sterile tools for blood cupping
ELISA reader with evaluation software
Normal laboratory equipped with ELISA plate producing machines for low-medium level of production
Pharmaceutical-level product plant, depending on market demands
|Own staff for low-medium level of production
Normal industrial organization at the level of mass production:
SECTION 4: Overall Assessment
|1||What is you overall assessment of the technical feasibility of the research result?|
|The easily accessible blood serum is the most complex human proteome present in any other sample. The dynamic range covers 10 orders of magnitude in concentration separate albumin and rarest proteins measured clinically. The serum proteome has the promise in revolutionizing disease diagnostics and in monitoring therapy. Scientific evidences suggest that serum protein abundance, alterations in concentrations is indicative of many, if not most, human diseases. The current clinical diagnosis, however, uses a few dozens of proteins routinely, and introduction of new protein markers for clinical diagnosis has declined to less than one per year. Infections and diseases induce immediate immune- and cellular responses that include persistent modulation of gene expression both at transcriptional and translational levels.
Our project proposal is based on finished and published high throughput transcriptome analysis of a natural tuberculosis model that provided a large set of biomarkers with altered expression. We selected dozens of candidate biomarkers by their abundance in blood serum, literature search and possible association with the pathomechanism of TB.
A series of Western and ELISA experiments will include the determination of abundance of markers in 20-30 serum in TB patients in comparison with healthy controls. A multivariate protein panel will be established by these results and utilized for prototype development of the TB diagnostic tool. Reference intervals will be adjusted by fluctuations of values in the study population and by ethnic variations.
The proposal has the advantage by utilizing conventional laboratory methods established and used routinely in labs of the participants that can easily be implemented in resource-poor areas as well.
All of these considerations above argue for a feasible research result that establishes the development of the TB diagnostic tool in form of a multivariate protein panel.
KEYWORDS QUANTITATIVE ASSESSMENT (0-5).
|Please put X as appropriate.||1||2||3||4||5|
|Adequacy of testing activity undertaken so far||X|
|Adequacy and availability of technical resources of the development team||X|
|Current development stage||X|
|Overall technical feasibility||X|