RESUMO
RESUMEN Se evaluó el uso de partículas magnéticas acopladas a proteína L para la concentración y purificación de anticuerpos monoclonales inmunoglobulina M (mIgM) contra Taenia solium. Se evaluaron tres métodos de concentración y diferentes tiempos de elución y se optimizó la proporción de partículas a la proporción de mIgM. Demostramos que: 1) con el uso partículas magnéticas no se requiere de una concentración previa de mIgM, lo que disminuye la manipulación de los anticuerpos y mejora la recuperación, 2) se puede omitir el uso de un tampón de unión, ya que el pH de la mayoría de los sobrenadantes de cultivo celular son neutros, y 3) se necesitan tiempos de elución más largos (~45 minutos) para aumentar la recuperación a un nivel mayor a 80%. El estudio demuestra que el uso de partículas magnéticas acopladas a proteína L es una herramienta simple y eficiente para la concentración y purificación de mIgM.
ABSTRACT The use of L protein coupled magnetic particles for the concentration and purification of immunoglobulin M (mIgM) monoclonal antibodies against Taenia solium was evaluated. Three concentration methods and different elution times were evaluated and the ratio of particles to the ratio of mIgM was optimized. It is demonstrated that: 1) with the use of magnetic particles, a previous concentration of mIgM is not required, which reduces the manipulation of the antibodies and improves the recovery, 2) the use of a binding buffer can be omitted, since the pH of most cell culture supernatants are neutral, and 3) longer elution times (~ 45 minutes) are needed to increase recovery to a level greater than 80%. The study demonstrates that the use of L protein-coupled magnetic particles is a simple and efficient tool for mIgM concentration and purification.
Assuntos
Animais , Imunoglobulina M , Taenia solium , Fenômenos Magnéticos , Anticorpos Monoclonais , Imunoglobulina M/imunologia , Taenia solium/imunologia , Anticorpos Monoclonais/isolamento & purificaçãoRESUMO
Objectives To express and characterize NS1 of Indonesian-specific DENV2 virus in Pichia pastoris (P. pastoris). Methods A codon optimized synthetic gene derived from the DENV-2 NS1 amino acid sequences was synthesized commercially and inserted into the P. pastoris pPICZαA expression vector. The recombinant DENV-2 NS1 protein was purified by Ni-NTA affinity chromatography, and its antigenicity was tested. Results The recombinant DENV-2 NS1 protein was secreted as a protein with a molecular weight of ∼45 kDa, and the optimal expression condition was achieved by induction with 2% (v/v) methanol for 72 h. The purified recombinant DENV-2 NS1 protein was able to interact with a monoclonal antibody of NS1 in a commercial rapid test. Conclusions The resulting recombinant DENV-2 NS1 protein produced in P. pastoris KM71 is a potential candidate for use in the development of a dengue diagnostic kit and vaccine.