ABSTRACT
The SARS-CoV-2 coronavirus, which causes respiratory syndrome COVID-19, has a protein nucleocapsid that envelops the viral ssRNA. The main protein of the nucleocapsid is the Np protein, which presents limited homology with nucleoproteins of other coronaviruses and therefore turns out to be an attractive antigen for the development of specific anti-Np antibodies. These antibodies can be used for the development of diagnostic systems that allow the detection of the viral antigen in infected individuals from saliva samples. In this context, our group has developed a labelling system called FasTAG®, which allows the immobilization of recombinant proteins on the surface of Gram+ formaldehyde inactivated bacteria. In this system, the recombinant proteins expressed in heterologous systems are fused to the C-terminal domain of S-Layer proteins of Lactobacillus sp. Then, the intrinsic affinity this domain possesses for the membranes of Gram+ bacteria is used for the immobilization of the recombinant proteins of interest. In this way, it is possible to purify specific antibodies against an antigen of interest. Based on the above, the objective of this work was to evaluate the functionality of the FasTAG® system to purify specific anti-Np antibodies. For this, the recombinant protein Np-FasTAG® was incubated for 12 hours at 4 °C with a matrix made up of B. subtilis inactivated with 3% formaldehyde. Next, for the optimization of the protein fixation process to the matrix, a compound factorial design was carried out, the variables of which were: formaldehyde concentration (0.5-1.5-2.5% v/v) and time of incubation (15-30-45 minutes). The optimal condition was determined as the one that minimizes the detachment of the Np protein and maximizes the detachment of the specific antibodies. Turning out to be the optimal condition for the elaboration of the affinity matrix 2.5% v/v of formaldehyde and 15 minutes. Then, in order to evaluate the application of the affinity matrix in the purification of specific antibodies, it was incubated for 1 hour with polyclonal antibodies obtained from chicken egg yolks and the serum of goats immunized with the Np antigen. Next, to study the elution conditions of the antibodies, a compound factorial design was performed using variables: pH, time, and SDS concentration. The best elution condition was obtained for pH 10.5 and 15 minutes. Subsequently, the purified antibodies were evaluated by SDS-PAGE and ELISA. As a result, it was possible to purify 3.5 μg of anti-Np IgG and 3.1 μg of anti-Np IgY per mg of resin. Finally, the set of experiments carried out here demonstrate the potential and functionality of this system for the purification of specific anti-Np antibodies and their use for diagnostic purposes.
ABSTRACT
This research claims that the four countries that make up the Pacific Alliance (PA) (Chile, Colombia, Mexico, and Peru) will have an economic recovery in 2021, as evidenced by increases in their respective gross domestic products (GDP). Furthermore, it argues that it is possible to calculate both production decline for those countries during 2020 and recovery reported for the following year. The corresponding calculations were based on economic and socio-political contexts that influenced the measures adopted in the four PA countries in face of the COVID-19 pandemic;in particular, measures that began in early March 2020. The greatest economic reactivation would happen in Mexico, although it is noteworthy that this particular result is closely related to the performance of the US economy, which acts as a natural market for the aforementioned Latin American country. © 2021 Universidad Católica de Colombia.