Desarrollo de un ELISA basado en la proteína de nucleocápside recombinante para la detección de anticuerpos contra SARS-CoV-2
Fecha
2023-06-14
Autores
Rodríguez Araba, Evelyn María
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Editor
Universidad Nacional, Costa Rica
Resumen
El síndrome respiratorio agudo severo coronavirus 2 (SARS-CoV-2) es la
causa de la enfermedad por coronavirus 2019 (COVID-19), una enfermedad
altamente contagiosa que afecta principalmente al sistema respiratorio y se divide
en categorías, según la gravedad, a saber, en asintomático, leve, moderado y
grave.
El primer objetivo del presente estudio fue producir la proteína de la
nucleocápside (proteína N) recombinante de SARS-CoV-2 para ser utilizada en un
inmunoensayo (ELISA) para evaluar la respuesta de anticuerpos de tipo IgG
(H+L), IgM, IgA, IgG1, IgG2, IgG3 e IgG4 en tres grupos de pacientes con
diferentes días de evolución de la enfermedad (Grupo 1: <15 días, grupo 2: 16-25
días y grupo 3: >25 días). La secuencia de codificación de esta proteína se
optimizó para la expresión en E. coli y se subclonó en el plásmido pET24a(+), lo
que dio como resultado una proteína etiquetada con 6xHis de 46 kDa. La
purificación de la proteína recombinante se realizó por cromatografía de afinidad
con metales inmovilizados y se evaluó en inmunoblot y ELISA. Un total de 200
sueros prepandémicos y 204 sueros de pacientes con SARS-CoV-2 (confirmados
por técnica molecular), se utilizaron para establecer el punto de corte, la
sensibilidad y la especificidad del ensayo. Las sensibilidades de los ELISA para
detectar los niveles séricos de anticuerpos de las clases IgG (H+L), IgM, IgA y la
subclase IgG1 contra la proteína N fueron 81,4%, 83,3%, 81,9% y 84,8%, y las
especificidades 90,1%, 77,6%, 88,1% y 85,6%, respectivamente. Se determinó
mayor sensibilidad de los inmunoensayos en el Grupo 1, excepto para IgM. Se
determinó un aumento significativo de la media de las unidades ELISA (UE) para
IgG (H+L), IgA e IgG1 en el Grupo 1, mientras que para el IgM se determinó un
aumento de estos valores en el Grupo 2. Anticuerpos de tipo IgG4 se detectaron
solamente en 12/204 muestras. No se logró estandarizar los ELISA para IgG2 e
IgG3.
El segundo objetivo del trabajo fue investigar títulos de anticuerpos de las
clases IgG (H+L), IgA e IgM y subclases IgG1 e IgG4 dirigidos contra la proteína N de SARS-CoV-2 en pacientes según la gravedad de la enfermedad, sexo y edad.
Se analizaron 204 sueros de pacientes con COVID19, 92 hombres y 112 mujeres,
los cuales se agruparon en tres grupos etarios: 18-35 años (n = 69), 36-60 años (n
= 70), >61 años (n = 65), y en cuatro grupos de pacientes: pacientes con
enfermedad grave (n = 57), pacientes con enfermedad moderada (n = 49) y
pacientes con enfermedad leve (n = 50), además, un grupo de pacientes que
resultaron positivos durante el periodo de dominancia de la variante ómicron (n =4
8) y que se encontraban hospitalizados con enfermedad grave. Los anticuerpos
IgG (H+L) e IgA mostraron las medias de UE más altos. Los pacientes graves
mostraron niveles de anticuerpos significativamente más altos (p<0.05) en
comparación a los pacientes moderados, leves y pacientes positivos durante el
periodo de dominancia de la variante ómicron. No se determinó correlación entre
el sexo y los niveles de anticuerpos. En cuanto a las edades solamente se
determinó diferencia significativa (p <0,05) para el anticuerpo IgM. Se pudo
demostrar una correlación moderada para IgM e IgG(H+L) (r= 0,6672, p<0,0001),
IgM e IgA (r= 0,5487, p<0,0001), e IgG(H+L) e IgA (r= 0,6620, p<0,0001) en los
pacientes graves. Se logró demostrar que niveles séricos de anticuerpos contra la
proteína N se correlacionan significativamente con la gravedad de la infección por
COVID-19 y que en la enfermedad grave los altos títulos de diversos anticuerpos
están correlacionados. Los inmunoensayos desarrollados en el presente estudio
son adecuados para la detección de anticuerpos contra la proteína N del SARS-
CoV-2 y podrían usarse para monitorear la respuesta inmune y determinar las
infecciones naturales.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), a highly contagious disease that mainly affects the respiratory system and is divided into categories according to severity, namely asymptomatic, mild, moderate, and severe. The first objective of the present study was to produce the recombinant nucleocapsid protein (N protein) of SARS-CoV-2 to be used in an immunoassay (ELISA) to evaluate the IgG (H+L), IgM, IgA, IgG1, IgG2, IgG3 and IgG4 antibody response in three groups of patients with different days of disease evolution (Group 1: <15 days, group 2: 16-25 days and group 3: >25 days). The coding sequence of this protein was optimized for expression in E. coli and subcloned into the pET24a(+) plasmid, resulting in a 46 kDa 6xHis-tagged protein. Purification of the recombinant protein was performed by immobilized metal affinity chromatography and evaluated in immunoblot and ELISA. A total of 200 prepandemic sera and 204 sera from SARS-CoV-2 patients (confirmed by molecular technique), were used to establish the cut-off point, sensitivity and specificity of the assay. The sensitivities of the ELISAs for detecting serum levels of IgG (H+L), IgM, IgM, IgA and IgG1 subclass antibodies against the N protein were 81.4%, 83.3%, 81.9% and 84.8%, and the specificities were 90.1%, 77.6%, 88.1% and 85.6%, respectively. Higher sensitivity of immunoassays was determined in Group 1, except for IgM. A significant increase in mean ELISA units (EU) for IgG (H+L), IgA and IgG1 was found in Group 1, while for IgM an increase in these values was found in Group 2. IgG4 antibodies were detected in only 12/204 samples. ELISAs for IgG2 and IgG3 could not be standardized. The second objective of the work was to investigate antibody titers of IgG (H+L), IgA and IgM classes and IgG1 and IgG4 subclasses directed against the N protein of SARS-CoV-2 in patients according to disease severity, sex and age. We analyzed 204 sera from patients with COVID19, 92 males and 112 females, which were grouped into three age groups: 18-35 years (n = 69), 36-60 years (n = 70), >61 years (n = 65), and into four groups of patients: patients with severe disease (n = 57), patients with moderate disease (n = 49) and patients with mild disease (n = 50), plus a group of patients who tested positive during the period of omicron variant dominance (n =4 8) and who were hospitalized with severe disease. IgG (H+L) and IgA antibodies showed the highest mean EUs. Severe patients showed significantly higher antibody levels (p<0.05) compared to moderate, mild and positive patients during the period of omicron variant dominance. No correlation between sex and antibody levels was determined. In terms of age, only significant differences were found. (p<0.05) for IgM antibody. A moderate correlation could be demonstrated for IgM and IgG(H+L) (r= 0.6672, p<0.0001), IgM and IgA (r= 0.5487, p<0.0001), and IgG(H+L) and IgA (r= 0.6620, p<0.0001) in severe patients. We were able to demonstrate that serum levels of antibodies to protein N correlate significantly with the severity of COVID-19 infection and that in severe disease high titers of various antibodies are correlated. The immunoassays developed in the present study are suitable for the detection of antibodies against the N protein of SARS-CoV-2 and could be used to monitor the immune response and determine natural infections.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), a highly contagious disease that mainly affects the respiratory system and is divided into categories according to severity, namely asymptomatic, mild, moderate, and severe. The first objective of the present study was to produce the recombinant nucleocapsid protein (N protein) of SARS-CoV-2 to be used in an immunoassay (ELISA) to evaluate the IgG (H+L), IgM, IgA, IgG1, IgG2, IgG3 and IgG4 antibody response in three groups of patients with different days of disease evolution (Group 1: <15 days, group 2: 16-25 days and group 3: >25 days). The coding sequence of this protein was optimized for expression in E. coli and subcloned into the pET24a(+) plasmid, resulting in a 46 kDa 6xHis-tagged protein. Purification of the recombinant protein was performed by immobilized metal affinity chromatography and evaluated in immunoblot and ELISA. A total of 200 prepandemic sera and 204 sera from SARS-CoV-2 patients (confirmed by molecular technique), were used to establish the cut-off point, sensitivity and specificity of the assay. The sensitivities of the ELISAs for detecting serum levels of IgG (H+L), IgM, IgM, IgA and IgG1 subclass antibodies against the N protein were 81.4%, 83.3%, 81.9% and 84.8%, and the specificities were 90.1%, 77.6%, 88.1% and 85.6%, respectively. Higher sensitivity of immunoassays was determined in Group 1, except for IgM. A significant increase in mean ELISA units (EU) for IgG (H+L), IgA and IgG1 was found in Group 1, while for IgM an increase in these values was found in Group 2. IgG4 antibodies were detected in only 12/204 samples. ELISAs for IgG2 and IgG3 could not be standardized. The second objective of the work was to investigate antibody titers of IgG (H+L), IgA and IgM classes and IgG1 and IgG4 subclasses directed against the N protein of SARS-CoV-2 in patients according to disease severity, sex and age. We analyzed 204 sera from patients with COVID19, 92 males and 112 females, which were grouped into three age groups: 18-35 years (n = 69), 36-60 years (n = 70), >61 years (n = 65), and into four groups of patients: patients with severe disease (n = 57), patients with moderate disease (n = 49) and patients with mild disease (n = 50), plus a group of patients who tested positive during the period of omicron variant dominance (n =4 8) and who were hospitalized with severe disease. IgG (H+L) and IgA antibodies showed the highest mean EUs. Severe patients showed significantly higher antibody levels (p<0.05) compared to moderate, mild and positive patients during the period of omicron variant dominance. No correlation between sex and antibody levels was determined. In terms of age, only significant differences were found. (p<0.05) for IgM antibody. A moderate correlation could be demonstrated for IgM and IgG(H+L) (r= 0.6672, p<0.0001), IgM and IgA (r= 0.5487, p<0.0001), and IgG(H+L) and IgA (r= 0.6620, p<0.0001) in severe patients. We were able to demonstrate that serum levels of antibodies to protein N correlate significantly with the severity of COVID-19 infection and that in severe disease high titers of various antibodies are correlated. The immunoassays developed in the present study are suitable for the detection of antibodies against the N protein of SARS-CoV-2 and could be used to monitor the immune response and determine natural infections.
Descripción
Maestría en Enfermedades Tropicales para optar al grado Magister Scientae
Palabras clave
ELISA, COVID-19 (ENFERMEDAD), EPIDEMIOLOGIA, EPIDEMIOLOGY, ENFERMEDADES BRONQUIALES, BRONCHIAL DISEASES