PURIFICATION OF SPECIFIC ANTIBODIES AGAINST SARS-COV-2 PROTEIN NP USING THE FasTAG® SYSTEM

HARGUINDEGUY INÉS; HIRIART Y; CAVALITTO SF; CAVELLO I; ORTIZ GE

Caba

Congreso; SAIB-SAIMGE 2021; 2021

SAIB

The SARS-CoV-2 coronavirus, which causes respiratory syndrome COVID-19, has a protein nucleocapsid that envelops theviral ssRNA. The main protein of the nucleocapsid is the Np protein, which presents limited homology with nucleoproteins ofother 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 infectedindividuals from saliva samples. In this context, our group has developed a labelling system called FasTAG®, which allowsthe immobilization of recombinant proteins on the surface of Gram+ formaldehyde inactivated bacteria. In this system, therecombinant proteins expressed in heterologous systems are fused to the C-terminal domain of S-Layer proteins ofLactobacillus sp. Then, the intrinsic affinity this domain possesses for the membranes of Gram+ bacteria is used for theimmobilization of the recombinant proteins of interest. In this way, it is possible to purify specific antibodies against an antigenof interest. Based on the above, the objective of this work was to evaluate the functionality of the FasTAG® system to purifyspecific anti-Np antibodies. For this, the recombinant protein Np-FasTAG® was incubated for 12 hours at 4 ºC with a matrixmade up of B. subtilis inactivated with 3% formaldehyde. Next, for the optimization of the protein fixation process to thematrix, 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 thedetachment of the Np protein and maximizes the detachment of the specific antibodies. Turning out to be the optimal conditionfor the elaboration of the affinity matrix 2.5% v/v of formaldehyde and 15 minutes. Then, in order to evaluate the applicationof the affinity matrix in the purification of specific antibodies, it was incubated for 1 hour with polyclonal antibodies obtainedfrom chicken egg yolks and the serum of goats immunized with the Np antigen. Next, to study the elution conditions of theantibodies, a compound factorial design was performed using variables: pH, time, and SDS concentration. The best elutioncondition was obtained for pH 10.5 and 15 minutes. Subsequently, the purified antibodies were evaluated by SDS-PAGE andELISA. 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 setof experiments carried out here demonstrate the potential and functionality of this system for the purification of specific antiNp antibodies and their use for diagnostic purposes.