ABSTRACT
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.
Subject(s)
Animals , Immunoglobulin M , Taenia solium , Magnetic Phenomena , Antibodies, Monoclonal , Immunoglobulin M/immunology , Taenia solium/immunology , Antibodies, Monoclonal/isolation & purificationABSTRACT
The target of this study is to assess and evaluate the use of S. mansoni and S. haematobium microsomal antigens [MAMA and HAMA] in FAST-ELISA and EITB for immunodiagnosis of schistosomiasis and to compare their findings with those of the dipstick [DS] assay as a practical screening test for field sites. Screening of the S.mansoni-infected sera [Kafr El-Sheikh site] with FAST-MAMA and EITB-MAMA, showed sensitivity levels of 98.3% and 100%, respectively. Meanwhile, screening of these sera with DS assay, resulted in the same sensitivity as the EITB assay. Screening of S. haematobium-infected sera [Giza site] with FAS-TMAMA showed a low sensitivity [80.5%] as compared to the FAST-HAMA where the sensitivity was much better, reaching 95%. About 97% of sera from parasitologically S. haematobium positive subjects sera, which were FAST-HAMA positive, recognized Gp23 in both EITB and DS assays. All participants from Giza site were parasitologically negative for S. mansoni. Sixty one percent of them were positive in FAST-MAMA and 45%-23% of them recognized Gp30 in EITB and DS assays, respectively. This lower percentage in DS assay could be a much more accurate estimation of S.mansoni antibodies in such S. haematobium endemic site. On comparing the EITB findings with that of the DS assay, the results showed that all negative sera in MAMA and HAMA blots were also negative in the DS assay. Ninety six percent of the positive HAMA blots were also positive in DS assay indicating nearly the same sensitivities of the two assays for Gp23 reactivity. About fifty two percent of the mixed infection sera, which weakly recognized Gp30 in MAMA blots, were totally negative in DS assay thus indicating the purity and specificity of the DS-assay antigen is much higher than that used in EITB. DS assay is sensitive, specific, reproducible and highly effective in screening patients with either schistosome infections. Also, DS assay is economic, rapid and lends itself for use under field conditions