Full factorial designs of experiments to optimize an ELISA that detects SARS-CoV-2 anti-RBD antibodies in breast milk

In this work, an ELISA was optimized through the application of full factorial designs of experiments, to detect IgA antiRBD antibodies to SARS-CoV-2 in breast milk of puerperal women who received, during pregnancy, three doses of the Abdala anti-COVID-19 vaccine, created on the protein subunit platform. Based on a preliminary non- optimized protocol, the interactions between the factors and levels, at the step of RBD antigen coating of the plate and in the reaction of the antigen with the breast milk antibodies, were evaluated; the dilution factor to apply the enzyme conjugate in the assay was also determined. The optimized ELISA protocol was established with the best combination of factors and levels, with which it was possible to increase the signal to noise ratio of the assay by 4.67 times compared to the non- optimized one. The new protocol will guarantee the detection of SARSCoV-2 IgA anti-RBD antibodies in breast milk samples with a low content of these antibodies and that would probably have an absorbance signal close to the assay blank if the non-optimized ELISA were applied.