ABSTRACT
Con base en estudios realizados previamente en los cuales se identificaron los residuos críticos para la unión de la secuencia 21KNESKYSNTFINNAYNMSIR40 del antígeno MSP-2 del Plasmodium falciparum, se diseñaron y sintetizaron dos secuencias de pseudopéptidos amida reducida en las cuales se sustituyó un enlace peptídico normal por su isóstero ψ[CH2-NH] entre los residuos fenilalanina-isoleucina y entre los residuos isoleucina-asparagina, para dar lugar a los análogos codificados ψ-128 (forma monomérica), ψ-129 (forma polimérica), ψ-130 (forma monomérica) y ψ-131 (forma polimérica). Con los péptido-miméticos obtenidos en forma de polímero se inmunizaron ratones BALB/c para generar anticuerpos monoclonales que presentaron isotipo IgM. Mediante ensayos controlados de inmunización in vitro se indujo el cambio isotipo de los clones reactivos aislados de los hibridomas obtenidos de manera reproducible. Las inmunoglobulinas aisladas se ensayaron por su capacidad funcional neutralizadora de la infección controlada in vitro de cepas de Plasmodium de roedores a glóbulos rojos. Los resultados obtenidos evidencian el papel que pueden tener los anticuerpos inducidos por péptido-miméticos Plasmodium en ensayos de infección realizados en modelos animales de experimentación.
Based on previous studies in which those residues being critical for Plasmodium falciparum binding to red blood cells (RBCs) through the antigenic sequence (21KNESKYSNTFINNAYNMSIR40) from the merozoite surface antigen-2 (MSP-2) were identified, we have designed and synthesized two reduced amide pseudopeptide sequences based on the 31IN32 binding motif. Synthesized peptidomimetics, possess each one a modified peptide bond that presented as a ψ[CH2-NH] reduced amide isoster bond, to allowing the Phe-Ile modified aminoacid pair allowing pseudopeptides coded ψ-128 (monomer form) and ψ-129 (polymer form) and the Ile-Asn modified aminoacid pair for pseudopeptides coded ψ-130 (monomer form) and ψ-131 (polymer form). By using the polymer forms of both peptido-mimetics as immunogens, monoclonal antibodies were produced in BALB/c mice. These Ig showed an IgM isotype. The isotype antibody switching was lead by in vitro immunization of the original hybridomas. Isolated immunoglobulins were tested for their functional in vitro activity, on a infection controlled experiment of rodent Plasmodium strains infecting red blood cells. Obtained results reveal the role played by antibodies to peptido-mimetic in infection assays performed further on animal experimental models.
Subject(s)
Antigen-Antibody Complex , Immunization , PlasmodiumABSTRACT
It is difficult to immunize human lymphocytes in vitro by conventional cell culture methods. Activation of lymphocytes requires not only specific antigen stimulation but also delicate cell to cell interaction. If the cellular organization could be maintained in culture system, lymphocytes could be immunized in vitro with higher frequency. For the purpose of in vitro immunization of human lymphocytes, we used slice culture system which could maintain morphological and functional organization. Human tonsils resected from eleven -year old boy were evenly divided into two pieces, and one was cultivated in conventional cell culture and the other in slice culture system. In the former system, tonsilar mononuclear cells, separated by Ficoll -Hypaque density gradient centrifugation, were cultivated in RPMI 1640 supplemented with 10% human type AB serum in the cell density of 5 x10 6 /ml. In the latter, tonsillar tissues were sliced into small pieces of 8 mm 3 , and were cultivated in Waymouth MB 752/1 medium supplemented with 10% Human type AB serum, gassed under 5% CO2 and 95% O2 at 37 C. After stabilized for one hour, each system wasw challenged with 50 microgram/ml of KLH or 100 microgram/ml of LPS. At 3, 6, 12 and 24 hours after antigen challenge, culture supernatants were assayed for the specific antibody by ELISA, and cells or tissues were analyzed for the expression of CD23 by flow cytometry. The result showed that tonsilar B lymphocytes in slice culture system expressed CD23 as early as 3 hours after antigen challenge, while those in cell culture system expressed CD23 from 6 hours after challenge. Specific antibodies were detected only in supernatants of slice culture system from 6 hours after challenge. These results suggested thathuman lymphocytes could be immunized in vitro if the cellular organization was maintained.
Subject(s)
Humans , Male , Antibodies , B-Lymphocytes , Cell Communication , Cell Count , Cell Culture Techniques , Centrifugation, Density Gradient , Enzyme-Linked Immunosorbent Assay , Ficoll , Flow Cytometry , Immunization , Lymphocytes , Palatine TonsilABSTRACT
No abstract available.
Subject(s)
Animals , Mice , Antibody Formation , Bone Marrow Cells , Bone Marrow , Culture Media, Conditioned , ThymocytesABSTRACT
We adapted one of the in-vitro immunization methods to induce antibody responses and confirmed the success of the immunization by enzyme-linked immunosorbent assay (ELISA) without hybridization. We have previously reported several methods of in-vitro immunization using different conditioned media. Here we introduce another method of in-vitro immunization using a mixture of three types of supernatant (thymocytes, and adherent and non-adherent splenocytes of mouse). Splenocytes were immunized in-vitro by a recombinant human growth hormone (rhGH) with the above conditioned media, and the results by ELISA showed a much higher optical density than the other in-vitro immunization methods that we had previously reported. Humoral responses as a result of in-vitro immunization to soluble antigens were easily confirmed by ELISA using the above-conditioned media. This finding indicates that any other conditions thought to be critical by other researches may not be essential for in-vitro immunization.