A new study conducted by the DDS Diagnostic research team in partnership with Horia Hulubei National Institute for R&D in Physics and Nuclear Engineering (IFIN-HH) was published on Monday, 21st of June 2021 in Nature – Scientific Reports, a renowned scientific journal, where the most relevant research projects are published. The results of the study called “The influence of sugar–protein complexes on the thermostability of C-reactive protein (CRP)” generate a major contribution to the development of optimal positive controls for in vitro diagnostic rapid tests produced by DDS Diagnostic, and they bring along a great impact on the accuracy of these tests.
The purpose of the study was to evaluate the influence of sugar-protein complexes on the stability and functionality of C-reactive protein (CRP), after constant exposure to high temperatures. CRP is a plasma protein. Because it has an essential role in the evolution of chronic inflammation, it is a biomarker used for the diagnosis of several important medical conditions, such as ulcerative colitis, cardiovascular diseases, metabolic syndrome.
C-reactive protein can be determined with in vitro diagnostic rapid tests, which are often based on an antigen-antibody reaction for protein detection. The bonds that connect the antigen and the antibody can be influenced by various factors, which highlights the importance of a positive control to avoid erroneous results. In vitro diagnostic rapid tests are usually stored at room temperature and they do not need special storage conditions, but numerous factors might interfere with their stability, for example the high temperatures measured during summer. Therefore, the positive control should resist in various conditions, including higher temperatures.
The development of the positive control for the in vitro diagnostic rapid test for CRP started with a determination of the optimal concentration of C-reactive protein which should be used. Sucrose was used as a factor which increases the stability of the protein.
The results of the study confirmed that sucrose increases protein stability. After an exposure to 40°C, the protein was not structurally affected, but a loss of immunogenicity was observed, probably caused by conformational modifications. The samples produced using the optimal protein-sucrose ratio remained active even after 22 days of exposure to 40°C. The study also demonstrated that the exposure of CRP-sucrose to Gamma radiations can improve their stability at high temperature, especially 2kG doses, an effect that was not previously described in scientific literature.
The interaction of CRP with sucrose was evidentiated using steady time and time resolved fluorescence. CRP can generate endogenous fluorescence because it contains tryptophan, phenylalanine and tyrosine residues. The main amino acid responsible for CRP fluorescence is tryptophan. The results of the study demonstrated that there are more than two classes of tryptophan, which have different degrees of accessibility for quencher molecule (which decreases the intensity of the fluorescence of a particular substance). Adding higher and higher concentrations of sucrose to the solution determines a gradual decrease of the fluorescence, until a plateau phase is reached. Molecular docking was used to highlight the bonding site of CRP and sucrose which is located near a tryptophan residue, which could explain the phenomenon of fluorescence extinction.
The results of the DDS Diagnostic research team can be used to develop positive controls with high stability for qualitative and quantitative detection of C-reactive protein, with high efficacy and low costs. Using a positive control not only helps determine the test performance, but also significantly cuts off the erroneous results. The importance of the study and the relevance of its results is reflected by the publishing of the scientific work in a worldwide high impact scientific journal. The full article is available here.