ABSTRACT
1. INTRODUCCIÓNLas inmunodeficiencias primarias son un grupo de más de 400 enfermedades, en las cuales el sistema inmune pierde sus funciones de reconocimiento de patógenos o funciona de forma inapropiada. Algunas de ellas son relativamente comunes; mientras otras son raras. Estas enfermedades son en ocasiones de por vida, debilitantes y costosas1,2.Sin embargo, muchos progresos se han hecho desde su des-cripción original en el año de 1952. Se han dado grandes pasos en cuanto a su entendimiento de las Inmunodeficiencias Pri-marias a nivel genético, de sus características, y tratamiento. Algunos tipos afectan un único tipo de célula; otros afectan más de un componente del sistema inmune2,3.Tomando en cuenta que la aproximación es entre 1-2% de la población, a nivel país se puede decir que un aproximado entre 170 000 a 340 000 pacientes en el país no cuentan con un diagnóstico y muchos mueren por falta de este. El número de afiliados al Instituto Ecuatoriano de Seguridad Social hasta julio de 2021 es de 3 672,611 por lo que se considera que un estimado de 36 726 a 73 452 pacientes podrían presentar este tipo de enfermedades y requerir de atención por infecciones a repetición, enfermedad autoinmune y enfermedades linfopro-liferativas, además de que sin un tratamiento específico po-drían fallecer debido a infecciones graves o tener discapacidad permanente, lo que implica mayor carga para el sistema de Seguridad Social en subsidios y menores ingresos. Ecuador, cuenta con 86 pacientes diagnosticados, según la base de datos de la Sociedad Latino-Americana de Inmunodeficiencias4.Algunas terapias, como la de reemplazo para inmunoglobu-linas, a la que es tributaria más del 60% de estas patologías permite que la esperanza de vida y la morbilidad casi alcancen a aquellos que no presentan la enfermedad57.
1. INTRODUCTIONPrimary immunodeficiencies are a group of more than 400 diseases, in which the immune system loses its pathogen recog-nition functions or functions inappropriately. Some of them are relatively common, while others are rare. These diseases are sometimes lifelong, debilitating, and costly1,2. However, much progress has been made since its original description in 1952. Great strides have been made in understanding Primary Immunodeficiencies at the genetic level, their characteristics, and treatment. Some types affect only one type of cell; others affect more than one component of the immune system2,3. Considering that the approximation is between 1 to 2% of the population, at the country level we could say that approximately between 170 000 to 340 000 patients in the country do not have a diagnosis and many die due to lack of it. The number of social security affiliates until July 2021 is 3 672,611, so we could consider that approximately 36 726 to 73 452 patients could present this type of disease and require care for recurrent infections, autoimmune disease and lymphoproliferative diseases, in addition to the fact that without specific treatment they could die due to serious infections or have permanent disability, which implies a greater burden for the social security system in subsidies and lower income. Currently the country has 86 diagnosed patients, according to the database of the Latin American Society of Immunodeficiencies4. Many of the therapies, such as immunoglobulin replacement therapy, to which more than 60% of these pathologies are de-pendent, allow life expectancy and morbidity to almost reach those who do not have the disease 57.
Subject(s)
Humans , Male , Female , Immunization, Passive , Primary Immunodeficiency Diseases , Immunologic Deficiency Syndromes , Antibodies , Antibodies/immunology , Antibody-Producing Cells , Therapeutics , IgA Deficiency , Common Variable Immunodeficiency , Diagnostic Techniques and Procedures , Hormone Replacement Therapy , Agammaglobulinemia , Diagnosis , Ecuador , Allergy and Immunology , Hyper-IgM Immunodeficiency Syndrome , Antibody FormationABSTRACT
ObjectiveTo establish the system for the isolation of peripheral single antibody-secreting cells (ASCs) and single cell RT-PCR system, and to clone highly specific human hepatitis B monoclonal antibody efficiently and rapidly. MethodsThree patients with HBV infection in the recovery stage were enrolled. Peripheral B cells were collected and activated by HBcAg Peptide pool. Flow sorting was performed to obtain memory ASCs (CD19+CD10-IgG+CD27+), the limiting dilution method was used to obtain single cells, single cell RT-PCR was used to obtain cDNA, and nested PCR was used for the amplification of the fragment in the constant region of the heavy chain of antibody. PCR product was purified and cloned to pEASY-T1 simple cloning vector for sequencing and identification. ResultsThe results of ELISA showed that a high level of IgG was observed in all three patients with HBV infection in the recovery stage. There were significant differences in peripheral IgG level between patients and healthy volunteers (both P＜0.05). The results of flow sorting showed that the proportion of B cells was higher than 94% and memory ASCs were isolated. A total of 24 ASCs containing at least one cell with normal morphology were selected, and after identification, 14 were successfully amplified, with a size of about 200 bp, which was consistent with expected results. The analysis of the sequence of the heavy chain of antibody showed successful amplification of the fragment in the constant region of the antibody. ConclusionThe system for the isolation of single ASCs and single cell RT-PCR system are successfully established and the sequence of the heavy chain of antibody is obtained, which lays a solid foundation for the high-throughput production of human hepatitis B monoclonal antibodies in future.