SARS CoV-2 nucleocapsid-based diagnostic tests and serological response in allergic children

RT-PCR remains the primary method of diagnosing SARS-CoV-2. Serological diagnosis of COVID-19 is simple and does not require complex techniques and equipment, rendering it suitable for rapid detection and massive screening. However, serological tests cannot confirm SARS-CoV-2, and results will be false negative when antibody concentrations fall below detection limit. Serological testing is important method for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) infection. Antigen-based tests show high specificity, but sensitivity is relatively low. For development and further improvement of both serological and antigen-based tests, availability of recombinantly producted SARS CoV-2 antigens is needed and during the periods of high demend in the pandemic, it was a limited factor both for research and diagnostic tests production by national producers of tests. Nucleocapsid (N) protein is the most abundant virus derived protein and strong immunogen. We aimed to find its efficient, low-cost production. SARS-CoV-2 recombinant fragment of nucleocapsid protein (rfNP; 58-419 aa) was expressed in E. coli in soluble form, purified and characterized biochemically and immunologically. Purified rfNP has secondary structure of full-length recombinant N protein, with high percentage of disordered structure (34.2%) and of beta-sheet (40.7%). rfNP was tested in immunoblot using sera of COVID-19 convalescent patients. Cost-effective approach for soluble recombinant N protein fragment production was developed, with reliable IgG and IgM antibodies detection of SARS-CoV-2 infection. ELISA was optimized with sera of RT-PCR confirmed positive symptomatic patients and healthy individuals. IgG detection sensitivity was 96% (47/50) and specificity 97% (67/68), while IgM detection was slightly lower (94% and 96.5%, respectively). Application of serological ELISA for seropositivity detection in 200 allergic children in comparison to non-allergic did not show a bias towards any of the tested groups. Furthermore, we have optimized production of rfNP at a large scale to raise specific antibodies to N-protein in rabbit and mice and develop an in house ELISA for quantification of N protein in biological fluids. Test was clinically validated in 200 PCR positive patients and showed a sensitivity of around 50%

Serological Elisa test development at the INEP Institute

The COVID-19 diagnostic tools are categorized into two main groups of Nucleic Acid (NA)- based and protein-based tests. To date, nucleic acid-based detection has been announced as the gold-standard strategy for coronavirus detection; however, protein-based tests are promising alternatives for rapid and large-scale screening of susceptible groups. During the first months, no rapid and reliable detecting tool was readily available to adequatly respond to the requirement of massive testing. The aim was to develope cost-effective, sensitive and rapid screening mechanisms for the detection of immune response to SARS-CoV2 (which causes COVID-19), based on the principle of ELISA. The institute INEP has developed tests for detection of IgM and IgG SARS-CoV-2 specific antibodies based on S and N proteins of virus intended to monitor different phases of natural infection, ELISA test for IgG detection in naturale infection based on the use of exclusively domestic components of the ELISA kit (including proteins produced in Serbia, Faculty of Chemistry) and a test specifically designed to monitor the effects of immunization (determination of IgG antibodies specific for the RBD domain of S protein). The tests were independently validated at the relevant laboratories in the country and abroad, and compared to an FDA/WHO approved tests of a major test producers. All testing for validation was carried out on samples collected before COVID19 (negative controls), PCR confirmed COVID19 samples (positive controls) and potencialy cross-reactive samples (other pathogens and autoimmune diseases). The antibody tests showed high levels of sensitivity and specificity and extremely low background noises. The kits are extremely stable, have a shelf life of 1 year and opened kits are usable up to 3 months at storing temperatures of 5°

Comparative analysis of functionality of spray dried whey protein hydrolysates obtained by enzymatic and microbial hydrolysis

The aim of this study was to examine the bioactive potential of hydrolysate powders produced by enzymatic and microbial hydrolysis of whey proteins followed by spray drying, in order to reveal which one of these processes result in a product with significantly improved functional properties. Hydrolysate powders produced by the two different biotechnological processes were compared based on their antioxidant (DPPH and FTC), antibacterial as well as erythrocyte membrane stabilizing activities. The performed tests revealed that the concentration of at least 178.4 mg mL(-1) of the whey protein hydrolysate powder, produced by tryptic digestion, could inhibit the process of lipid peroxidation by 50 %, suppress the microbial contamination caused by S. aureus ATCC25923, B. cereus ATCC 11778 and L. monocytogenes, and provide the antioxidant and membrane stabilizing activities greater than 50 %. On the other hand, the hydrolysate powder obtained by whey fermentation at the concentration of at least 811.5 mg mL(-1) achieved 50 % of all tested bioactivities, with the emphasis on the significantly more pronounced antibacterial activity against all tested strains. In that sense, tryptic hydrolysis could be highlighted as an optimal process that provides production of the whey hydrolysate with pronounced bioactive properties that could be considered as a very promising natural food supplement