RESUMO
Culture filtrate and cell extracts from Mycobacterium bovis cultures contain molecules which could promote protective immunity to tuberculosis in animals. Different protein fractions of M. bovis cultures were obtained by elution electrophoresis and were tested in experimentally infected cattle. The fractions that elicited gamma interferon (IFN-gamma) responses were resolved by two-dimensional gel electrophoresis, and individual proteins were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The open reading frames were cloned, expressed as their recombinant forms, and retested with naturally and experimentally infected animals. Eleven protein fractions were highly reactive, from which the Rv1636, HspX, Rv0138, Rv2524, EsxI, and Rv3740 recombinant proteins were obtained. EsxI and HspX were the antigens most recognized by the IFN-gamma release assay. In summary, a proteomic approach allowed the identification of novel antigens useful for the diagnosis of bovine tuberculosis.
Assuntos
Antígenos de Bactérias/análise , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/imunologia , Mycobacterium bovis/química , Mycobacterium bovis/imunologia , Animais , Bovinos , Eletroforese em Gel Bidimensional , Imunoensaio , Interferon gama/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tuberculose Bovina/diagnósticoRESUMO
The affinities (Ka) and association rate constants (kon) of 23 mouse (BALB/c) anti-lysozyme mAbs obtained after short and prolonged immunizations have been measured by plasmon resonance techniques. The affinities for the 23 Abs, measured using their Fab, range from Ka = 1.1 x 10(7) to 1.4 x 10(10) M-1. There is no significant correlation between time or dose of immunization and affinity or association rates, indicating no time- or dose-dependent maturation of the response within the doses and times that were explored. IgMs are produced early and late in the response, with intrinsic affinities <10(5) M-1. Two independently derived mAbs, D44.1 (short term) and F10.6.6 (from a longer term response), result from identical or nearly identical somatic recombination events of germline gene segments. F10.6.6 has more mutations and a higher affinity constant (Ka = 1.4 x 10(10) M-1) than D44.1 (Ka = 1.1 x 10(7) M-1). Although higher affinities may result from an accumulation of mutations, they do not correlate with the length and dose of immunogenic challenge.
Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Muramidase/imunologia , Animais , Galinhas , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
A 37-year-old woman with a previous history of allergic rhinitis and bronchial asthma presented local and generalized allergic reactions to bovine, porcine and human insulins. She developed ketoacidosis, high insulin requirements and transient hypoglycemic episodes. Several desensitizing schedules were applied in order to induce tolerance to human insulin therapy. In addition, the following parameters of the humoral immune response were measured in different serum samples taken within 13 days after one of the anaphylactic episodes: insulin antibodies (binding range = 29.4-47.8%; cutoff = 2.6%), total IgE (range = 500-850 IU/ml; normal values = 3.7-269 IU/ml), specific IgE (range = 0.42-0.83 PRU/ml; class 2) and subclasses of specific IgG (IgG1 = 97.2 SDS; IgG2 = 41.1 SDS; IgG3 = 9.9 SDS; IgG4 = 0.3 SDS, on day 1). A binding capacity of 31.8 IU insulin/l obtained by Scatchard analysis was in agreement with episodes of elevated insulin requirements and hypoglycemia. A high anti-insulin IgG/IgE ratio, along with high levels of specific IgG1 antibodies, suggested that the latter antibodies could be involved in the development of anaphylactic episodes.
Assuntos
Hipersensibilidade a Drogas/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Insulina/imunologia , Adulto , Feminino , Humanos , Imunoglobulina G/classificaçãoRESUMO
On type 1 newly diagnosed and on insulin treated diabetic patients, anti-insulin autoantibodies (IAA) and antibodies (IA) having the same specificity are respectively induced. Such immune response may be evaluated either by radiobinding assay (RBA) or enzyme-linked immunosorbent assay (ELISA). Both methodologies have been compared at previous International Workshops, which pointed out discrepancies in results. In this work, IAA/IA prevalence was assessed by displacement RBA and ELISA, in normal subjects, type 2 (treated with hypoglycaemic agents), insulin treated and newly diagnosed type 1 diabetic patients. Results showed a lack of RBA-ELISA agreement. An attempt was then made to determine whether such results were, at least in part, attributable to iodination site in Tyr-A14. For this purpose parallel RBA assays were carried out by using radiolabelled insulin at A14 and A19 Tyr residues. Control sera and samples from insulin treated and type 1 newly diagnosed diabetic patients were tested. Our results suggest that labelling position is not involved in artifactual binding of tracers, at least as a systematic phenomenon. In the majority of cases the variability in RBA-ELISA signal ratios are best explained in terms of differences in the basic principles operating in both methods instead of artifacts due to tracer preparation.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Anti-Insulina/imunologia , Insulina/imunologia , Radioisótopos do Iodo , Ensaio Radioligante/métodos , Tirosina , Adolescente , Adulto , Idoso , Autoanticorpos/análise , Autoanticorpos/imunologia , Sítios de Ligação de Anticorpos/imunologia , Criança , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 2/imunologia , Feminino , Humanos , Anticorpos Anti-Insulina/análise , Masculino , Pessoa de Meia-IdadeRESUMO
The existence of homologous anti-human growth hormone (anti-hGH) and heterologous anti-bovine growth hormone (anti-bGH) humoral immune responses in hypopituitary patients under hGH therapy has been reported previously. In order to study the influence of the hormone source, both responses were compared by radiobinding assays performed with [125I]hGH or [125I]bGH as tracers. Fifty-seven hypopituitary patients treated with extractive hGH, recombinant methionyl hGH or authentic recombinant hGH were studied. A very low incidence of heterologous antibodies was found in patients under recombinant hGH therapy, contrary to the high incidence observed in patients treated with extractive hGH preparations. In addition, immunochemical studies performed with a synthetic peptide (hGH 44-128) indicated that this peptide exhibited, in the anti-bGH/[125]bGH radioimmunoassay system, higher reactivity than the native hGH, suggesting that such a fragment resembled an altered conformation of the hormone. The high heterologous response elicited only by the extractive hGH along with the behaviour of the hGH 44-128 fragment supports the fact that the extraction and purification procedures in extractive preparations may alter slightly the structure of the hGH molecule and trigger a heterologous immune response.
Assuntos
Hormônio do Crescimento/imunologia , Adolescente , Animais , Anticorpos/análise , Formação de Anticorpos , Especificidade de Anticorpos , Bovinos , Criança , Pré-Escolar , Feminino , Hormônio do Crescimento/análogos & derivados , Hormônio do Crescimento/uso terapêutico , Hormônios/uso terapêutico , Hormônio do Crescimento Humano , Humanos , Hipopituitarismo/tratamento farmacológico , Imunoquímica/métodos , Lactente , Pessoa de Meia-Idade , Proteínas Recombinantes/uso terapêutico , Especificidade da EspécieRESUMO
Using reliable displacement radiobinding assay (RBA) and ELISA, the existence of anti-human growth hormone autoantibodies (hGHAA) was confirmed in idiopathic hypopituitary patients with growth impairment. Six of 35 hypopituitary patients (17.1%) and 1/85 (1.2%) control children proved positive for hGHAA by RBA (>control mean + 3 SD). IgG isotype-hGHAA by ELISAIgG (> control mean + 3 SD) were positive for 6/34 (17.7%) and 3/85 (3.5% hypopituitary and control children, respectively. Due to an asymmetry to the right of the ELISAIgG distribution, an alternative cutoff based on a nonparametric method was obtained, and positive results for hypopituitary children increased to 10/34 (29.4%). Three of 34 hypopituitary patients but no control children were positive for hGHAA of IgM isotype. The hGHAA were detected in children with or without perinatal problems. These autoantibodies may represent markers of a major autoimmune process involving a portion of the anterior pituitary and may contribute to the development of hypopituitarism in over 15% of hypopituitary children.