RESUMEN
The staphylococcal superantigen toxic shock syndrome toxin 1 (TSST-1) induces massive cytokine production, which is believed to be the key factor in the pathogenesis of TSS. The temporal sequence and kinetics of both proinflammatory and anti-inflammatory cytokines induced by TSST-1 in human peripheral blood mononuclear cells were investigated. A panel of loss-of-function single-amino-acid-substitution mutants of TSST-1, previously demonstrated to be defective in either major histocompatibility complex (MHC) class II binding (G31R) or T-cell receptor (TCR) interaction (H135A, S14N), was studied in parallel to further elucidate the mechanisms of cytokine secretion. Wild-type recombinant (WT r) TSST-1 induced a biphasic pattern of cytokine secretion: an early phase with rapid release of proinflammatory cytokines (especially gamma interferon, interleukin-2 [IL-2], and tumor necrosis factor alpha [TNF-alpha]) within 3 to 4 h poststimulation, and a later phase with more gradual production of both proinflammatory (IL-1beta, IL-12, and TNF-beta) and anti-inflammatory (IL-6, IL-10) cytokines within 16 to 72 h poststimulation. G31R, which is defective in MHC class II binding, induced a cytokine profile similar to that of WT rTSST-1, except that secretion of the early-phase proinflammatory cytokines was delayed and production of IL-1beta and IL-12 was markedly reduced. In contrast, mutant toxins defective in TCR interaction either demonstrated complete absence of any cytokine secretion during the entire observation period (H135A) or resulted in complete abolishment of IL-2 and other early-phase proinflammatory cytokines, while secretion of IL-10 appeared unaffected (S14N). Neither WT rTSST-1 nor the mutant toxins induced IL-4 or transforming growth factor beta. Our data indicate that effective TCR interaction is critical for the induction of the early-phase proinflammatory cytokine response, thus underscoring the importance of T-cell signaling in TSS.
Asunto(s)
Toxinas Bacterianas , Citocinas/metabolismo , Enterotoxinas/inmunología , Leucocitos Mononucleares/inmunología , Superantígenos , Adulto , Enterotoxinas/genética , Enterotoxinas/farmacología , Humanos , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Interleucinas/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Linfotoxina-alfa/metabolismo , Mutación , Células TH1/inmunología , Células Th2/inmunología , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Staphylococcal superantigens (sAgs) including toxic shock syndrome toxin-1 (TSST-1) and related enterotoxins are exoproteins with unique immunobiological properties. They bind to major histocompatibility complex (MHC) class II molecules of antigen-presenting cells outside the peptide groove, and induce massive proliferation of T cells bearing specific V beta determinants. This tri-molecular interaction leads to uncontrolled release of various proinflammatory cytokines especially interferon-gamma (IFN-gamma) and tumor necrosis factor-a (TNF-alpha), the key cytokines causing sAg-mediated shock. The effector T cells involved in this hyper-immune response are predominantly of the T helper-1 (Th1) phenotype. There is also some evidence that polarization to a Th2 response with the production of classical anti-inflammatory cytokines (such as interleukins IL-4 and IL-6) also occurs. Moreover, the emergence of a novel regulatory T cell (Tr1) subset, producing mainly IL-10 but little or no IL-2 and IL-4, has recently been described following repeated sAg stimulation. In this review, the current knowledge regarding the regulation of T helper cell subsets in response to staphylococcal sAgs is critically evaluated, and the role of various cytokines which directly influence T cell differentiation and polarization is summarized. Particular emphasis is directed towards pro-inflammatory as well as anti-inflammatory and regulatory effector functions during toxic shock. Based on this review, we propose that a delayed production of IL-10 by Tr1 cells may be the most prominent driving force in the down-regulation of the Th1 hyper-immune response, and the critical determinant for the eventual recovery of the host.
Asunto(s)
Staphylococcus/inmunología , Superantígenos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Citocinas/inmunología , Humanos , Activación de Linfocitos , Choque Séptico/epidemiología , Choque Séptico/inmunología , Choque Séptico/patologíaRESUMEN
Staphylococcal toxic shock syndrome is caused by a family of related superantigens that includes toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxins (SEs) A, B, and C. The cross-inhibitory activity against SEA by a novel anti-TSST-1 monoclonal antibody (MAb), MAb5, which also cross-inhibits SEB-induced superantigenic activities, was investigated. MAb5 blocked SEA binding to human monocytes, cross-neutralized SEA-induced T cell mitogenesis and TNF-alpha secretion in human peripheral blood mononuclear cells, and prevented lethality in mice. Epitope mapping revealed that MAb5 binds to residues SEA(154-161) within the central alpha helix that is structurally highly conserved among TSST-1, SEA, and SEB. The cross-inhibitory activity of MAb5 is likely due to steric hindrance of this conserved motif, although the precise function of this motif shared among related staphylococcal superantigens remains to be further elucidated.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Toxinas Bacterianas , Enterotoxinas/inmunología , Staphylococcus aureus/inmunología , Superantígenos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Enterotoxinas/antagonistas & inhibidores , Enterotoxinas/química , Enterotoxinas/farmacología , Mapeo Epitopo , Femenino , Humanos , Inductores de Interferón/antagonistas & inhibidores , Inductores de Interferón/inmunología , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Superantígenos/química , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Toxic shock syndrome (TSS) is primarily caused by toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin B (SEB). These toxins belong to a family of pyrogenic toxin superantigens (PTSAgs) produced by Staphylococcus aureus and exhibit several shared biological properties, including the induction of massive cytokine release and Vbeta-specific T-cell proliferation. The crystal structures of most PTSAgs are now published, and they demonstrate a striking similarity in conformational architecture even though their primary protein sequences are different. Despite these structural and immunobiological similarities, no cross-reactivity between TSST-1 and other PTSAgs has been demonstrated in serological or neutralization assays. Our laboratory has developed a neutralizing murine anti-TSST-1 monoclonal antibody (MAb5) which displayed cross-reactivity with SEB by enzyme-linked immunosorbent assay. The aim of the present study was to evaluate whether MAb5 can also cross-neutralize SEB-induced superantigenic activities in vitro. MAb5 was found to partially inhibit SEB-induced T-cell mitogenesis (63%) and tumor necrosis factor alpha (TNF-alpha) secretion (70%) in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner, while an isotypic anti-TSST-1 monoclonal antibody showed no effect. Epitope mapping revealed that MAb5 bound to TSST-1 residues 47 to 56 ((47)FPSPYYSPAF(56)) and to SEB residues 83 to 92 ((83)DVFGANYYYQ(92)), sequences that located in different regions of these toxins and are structurally dissimilar. SEB peptide (83)DVFGANYYYQ(92) was synthesized and found to also inhibit SEB-induced mitogenesis and TNF-alpha secretion in human PBMC. Our results demonstrate for the first time that MAb5 binds to different epitopes on TSST-1 and SEB that appear functionally important in inducing T-cell mitogenesis and TNF-alpha secretion in vitro.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Toxinas Bacterianas , Enterotoxinas/inmunología , Activación de Linfocitos/efectos de los fármacos , Superantígenos/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo/efectos de los fármacos , Sitios de Unión , Reacciones Cruzadas , Mapeo Epitopo , Humanos , Modelos Moleculares , Choque SépticoRESUMEN
Staphylococcal toxic shock syndrome toxin-1 (TSST-1) is implicated in the pathogenesis of superantigen-mediated shock. We previously identified TSST-1 residues G31/S32 to be important for major histocompatibility complex (MHC) class II binding, as well as superantigenic and lethal activities. However, the site-directed TSST-1 mutant toxin, G31R, could still induce mitogenesis and low-level TNF alpha secretion, suggesting that additional MHC class II binding sites other than G31/S32 may exist. In the current study, a TSST-1-neutralizing monoclonal antibody, MAb5, was found to inhibit TSST-1 binding to human peripheral blood mononuclear cells, neutralize TSST-1-induced mitogenesis and cytokine secretion, and protect against TSST-1-induced lethality in vivo. Epitope mapping revealed that MAb5 bound to TSST-1 residues 51-56 (T(51-56); 51YYSPAF56). Peptide T(51-56) was synthesized and found to also inhibit TSST-1 binding to human monocytes as well as TSST-1-induced mitogenesis, cytokine secretion, and lethality in vivo. This T(51-56) epitope, located within the beta 3/beta 4 loop, and the previously identified G31/S32 epitope, within the beta 1/beta 2 loop of TSST-1, are separated within the primary sequence, but spatially juxtaposed to each other. Collectively, these findings suggest that a discontinuous epitope comprising of regions within both the beta 1/beta 2 and beta 3/beta 4 loops, are critical for MHC class II binding, and the consequent superantigenic and lethal activities of TSST-1.
Asunto(s)
Toxinas Bacterianas , Enterotoxinas/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Staphylococcus aureus/metabolismo , Superantígenos/metabolismo , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/metabolismo , Células Cultivadas , Citocinas/metabolismo , Enterotoxinas/química , Mapeo Epitopo , Femenino , Antígenos de Histocompatibilidad Clase II/química , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Mitosis , Modelos Moleculares , Monocitos/citología , Monocitos/metabolismo , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Conejos , Choque Séptico/inducido químicamente , Choque Séptico/mortalidad , Superantígenos/químicaRESUMEN
It has previously been reported that staphylococcal enterotoxin A (SEA) is frequently co-expressed with toxic shock syndrome toxin-1 (TSST-1) in menstrual Toxic Shock Syndrome (MTSS)-associated Staphylococcus aureus. It was hypothesized that co-production of SEA and TSST-1 might yield a more virulent strain than one that produced TSST-1 but not SEA. To test this hypothesis, a TSST-1+/SEA- derivative of S. aureus RN3984 (TSST-1+/SEA+) was constructed by plasmid integration, and the isogenic pair were introduced into a D-galactosamine sensitized mouse model of lethal shock. At 72 h, 27 out of 30 (90%) mice inoculated with the parental strain died, as compared to 21 out of 30 (70%) mice inoculated with the isogenic derivative (P=0.05, Fisher's exact test; 1-tailed; 95% confidence limits, 0.80-20.80). Our results suggest that co-production of SEA with TSST-1 does enhance the ability of this strain of S. aureus to induce lethal shock in vivo. This enhanced virulence could be due to an additive or synergistic activity of the toxin combination on T cell proliferation and cytokine production in the animal model.
Asunto(s)
Toxinas Bacterianas , Enterotoxinas/biosíntesis , Choque Séptico/metabolismo , Choque Séptico/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/patogenicidad , Superantígenos , Animales , Southern Blotting , Modelos Animales de Enfermedad , Enterotoxinas/genética , Femenino , Galactosamina/metabolismo , Galactosamina/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , Plásmidos , Recombinación Genética , Choque Séptico/mortalidad , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/mortalidad , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , VirulenciaRESUMEN
Since menstrual toxic shock syndrome (MTSS) is associated with a predominant clone of Staphylococcus aureus which produces both toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A (SEA), we sought to clarify the role of TSST-1 in a tampon-associated vaginal infection model in New Zealand White (NZW) rabbits, using isogenic tst+/sea+ S. aureus mutants in which tst was inactivated by allelic replacement. Rabbits infected with the tst-/sea+ strain became ill within 3 days, with fever, weight loss, conjunctival hyperemia, and lethargy. Mortality was significantly higher with the tst+/sea+ strain compared to its tst-/sea+ isogenic derivative (4/13 vs. 0/14; p < 0.05, Fisher's exact test, 2-tailed). Mean fever index was higher (p < 0.005; t test, 2-tailed) and weight loss more sustained among survivors in the tst+/sea+ group. Furthermore, culture filtrates from the tst+/sea+ strain induced a significantly greater response in mitogenesis and TNF alpha secretion from rabbit splenocytes in vitro compared to the tst-/sea+ isogenic derivative. Thus, regardless of the role of SEA, TSST-1 significantly contributed to both morbidity and mortality in this tampon-associated vaginal infection model in NZW rabbits. This is the first demonstration of the potential role of TSST-1 and SEA in the pathogenesis of MTSS with a MTSS-associated clinical S. aureus strain in a relevant animal model.
Asunto(s)
Toxinas Bacterianas , Modelos Animales de Enfermedad , Enterotoxinas/genética , Choque Séptico/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus , Superantígenos , Animales , Enterotoxinas/análisis , Enterotoxinas/metabolismo , Enterotoxinas/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Immunoblotting , Mutación , Conejos , Choque Séptico/inmunología , Choque Séptico/mortalidad , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/mortalidad , Staphylococcus aureus/química , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Factor de Necrosis Tumoral alfa/análisis , VirulenciaRESUMEN
BACKGROUND: The majority of menstrual toxic shock syndrome (MTSS) cases are caused by a single clone of Staphylococcus aureus that produces both toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin A (SEA). OBJECTIVE: To determine whether the two superantigens interact to cause an enhancement of biological activity in human peripheral blood mononuclear cells (PBMCs). DESIGN: PBMCs from nine healthy donors were stimulated with TSST-1 or SEA, either alone or in combination at their minimum effective concentrations. SETTING: In vitro study. INTERVENTIONS: Human PBMCs were stimulated in vitro with TSST-1 (1 pg/mL), SEA (0.1 pg/mL) or combination for 20 to 72 h. Mitogenic response was determined by [(3)H]-thymidine incorporation. PBMC culture supernatants were assayed for the presence of tumour necrosis factor-alpha (TNFα), interleukin (IL)-1ß and IL-6 by ELISA. MAIN RESULTS: The combination of TSST-1 and SEA induced significantly greater mitogenesis in human PBMCs compared with either toxin alone (P<0.05, paired Student's t test, two-tailed). Similarly, the production of TNFα in culture supernatants was significantly greater in the combination of TSST-1 and SEA compared with either TSST-1 or SEA alone (P<0.05). In contrast, no enhancement in the levels IL-1 or IL-6 was observed. CONCLUSIONS: These data suggest that the co-production of TSST-1 and SEA by S aureus may provide some biological advantage to the organism throughs an enhanced effect of these superantigens on T cell activation and TNF secretion.
RESUMEN
Random mutagenesis with hydroxylamine was done on toxic shock syndrome toxin-1 (TSST-1) to identify the amino acid residues critical for binding to major histocompatibility complex (MHC) class II molecules of human monocytes. A double mutant with amino acid substitutions of glycine 31-->arginine and aspartic acid 184-->asparagine (G31R.D184N) demonstrated markedly reduced binding to human monocytes and induction of mitogenesis or cytokine secretion. Site-directed mutagenesis revealed that G31R, but not D184N, was at least 4 orders of magnitude less active than wild type recombinant (r) TSST-1 in these biologic activities and did not induce lethal shock in mice. The global structure of G31R remained highly similar to wild type rTSST-1 as evidenced by circular dichroism spectroscopy and binding to anti-TSST-1 polyclonal and monoclonal antibodies. These studies identified TSST-1 residue 31 as critical for binding to MHC class II molecules and for the consequent superantigenic and lethal properties of TSST-1.
Asunto(s)
Toxinas Bacterianas , Enterotoxinas/genética , Enterotoxinas/fisiología , Antígenos de Histocompatibilidad Clase II/genética , Superantígenos/genética , Animales , Anticuerpos Antibacterianos/inmunología , Ácido Aspártico/genética , Dicroismo Circular , Clonación Molecular , Citocinas/metabolismo , Enterotoxinas/inmunología , Femenino , Glicina/genética , Antígenos de Histocompatibilidad Clase II/fisiología , Humanos , Hidroxilamina , Hidroxilaminas/farmacología , Immunoblotting , Leucocitos Mononucleares , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Mutagénesis , Mutagénesis Sitio-Dirigida , Plásmidos , Mutación Puntual , Análisis de Secuencia de ADN , Superantígenos/fisiologíaRESUMEN
An improved method for producing highly purified toxic shock syndrome toxin 1 (TSST-1) by preparative isoelectric focusing in a Bio-Rad Rotofor cell and then chromatofocusing is described. Purification to homogeneity was confirmed by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 50 micrograms of protein was loaded), by immunoblotting with polyclonal rabbit antiserum raised against the crude culture supernatant used for purification, and by autoradiography after iodination and SDS-PAGE. Biologic activity was demonstrated by mitogenicity and cytokine induction (tumor necrosis factor alpha [TNF-alpha], interleukin 1-beta [IL-1 beta], and IL-6) of human peripheral blood mononuclear cells (PBMCs) and by lethality in New Zealand White rabbits following subcutaneous infusion. In contrast to commercial TSST-1 preparations, our TSST-1 preparation required the presence of both monocytes and T cells for the induction of TNF-alpha and IL-1 beta from human PBMCs. A 46-kDa contaminating protein in the commercial TSST-1 preparation, identified as staphylococcal lipase, was likely responsible for the induction of TNF-alpha and IL-1 beta from human monocytes in the absence of T cells, a biologic activity falsely attributed to purified TSST-1. Our improved purification procedure for TSST-1 provides a high yield and is both more rapid and less labor intensive than previously reported methods. Furthermore, our studies clearly demonstrate the need for stringent methods of purity assessment of TSST-1 preparations before ascribing to them their potent biologic activities.
Asunto(s)
Toxinas Bacterianas , Enterotoxinas/aislamiento & purificación , Staphylococcus aureus/patogenicidad , Superantígenos , Adulto , Animales , Línea Celular , Enterotoxinas/toxicidad , Femenino , Humanos , Interleucina-1/biosíntesis , Interleucina-6/biosíntesis , Conejos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Highly purified staphylococcal toxic shock syndrome toxin 1 (TSST-1) was tested for its ability to induce the cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) from fractionated human peripheral blood mononuclear cells prepared from seven healthy donors. Highly purified monocytes alone or T lymphocytes alone did not produce TNF or IL-1 when incubated with TSST-1 at 37 degrees C for up to 72 h. However, the addition of 10 micrograms of TSST-1 per ml to a 1:1 mixture of monocytes and T cells resulted in significant TNF (predominantly TNF-alpha) and IL-1 beta production after 24 h at 37 degrees C. The nature of the monocyte/T-cell interaction did not appear to involve gamma interferon (IFN-gamma), since 10 micrograms of rabbit anti-IFN-gamma per ml did not neutralize TNF-alpha production after TSST-1 induction. Similarly, L243, a monoclonal antibody to HLA-DR which blocks TSST-1 binding to monocytes, did not inhibit TNF-alpha production following TSST-1 induction. However, direct contact between monocytes and T cells was required, since physical separation of cells in double-chamber culture wells abolished TNF-alpha secretion after TSST-1 stimulation. Furthermore, paraformaldehyde fixation of either monocytes or T cells prior to addition to viable T cells or monocytes, respectively, also abolished TNF-alpha secretion, suggesting that aside from cell contact, soluble factors were also involved. Our results suggest that cytokine production involves more than binding of TSST-1 to its receptor on monocytes alone and that cell contact with T cells and the release of a soluble factor(s) other than IFN-gamma may be essential for the induction of cytokines by this toxin.
Asunto(s)
Toxinas Bacterianas , Enterotoxinas/farmacología , Interleucina-1/biosíntesis , Monocitos/inmunología , Superantígenos , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Antiinfecciosos Locales/farmacología , Comunicación Celular/fisiología , Pruebas Inmunológicas de Citotoxicidad , Ensayo de Inmunoadsorción Enzimática , Formaldehído/farmacología , Humanos , Técnicas In Vitro , Interferón gamma/fisiología , Polímeros/farmacologíaRESUMEN
Rotavirus was identified in 256 (28.5%) of 899 hospitalized children with diarrhea during a 12-month period in Hong Kong. Fourteen electropherotypes were identified, and the appearance of each occurred in a sequential manner during the study period. One patient was shown to be mixedly infected with two prevalent electropherotypes. Sequential stool specimens from another case showed the appearance of an extra RNA band during the course of the diarrheal episode.
Asunto(s)
ARN Viral/análisis , Infecciones por Rotavirus/microbiología , Rotavirus/clasificación , Preescolar , Diarrea/microbiología , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Hong Kong , Humanos , Lactante , Recién Nacido , Rotavirus/genética , Rotavirus/aislamiento & purificación , Infecciones por Rotavirus/epidemiologíaRESUMEN
The immunofluorescent antibody test and immunocytochemical method were employed to study the surface antigens of Angiostrongylus cantonensis obtained from infected rats, mice and guinea pigs. Positive results with intense fluorescence and brownish peroxidase staining were observed on the cuticular surface of A. cantonensis recovered from rats 22 days (late cerebral phase) and 34 days (lung phase) post-infection when tested with antisera against host (normal rat serum) antigens as well as crude extracts of A. cantonensis. However, host antigens were not observed on the surface of the nematode recovered from the brain of mice and guinea pigs 15 days post-infection.
Asunto(s)
Angiostrongylus/inmunología , Antígenos Helmínticos/análisis , Antígenos de Superficie/análisis , Metastrongyloidea/inmunología , Infecciones por Nematodos/inmunología , Angiostrongylus/crecimiento & desarrollo , Animales , Antígenos Helmínticos/inmunología , Antígenos de Superficie/inmunología , Encéfalo/inmunología , Encéfalo/parasitología , Técnica del Anticuerpo Fluorescente , Cobayas , Técnicas para Inmunoenzimas , Masculino , Ratones , Infecciones por Nematodos/parasitología , Ratas , Ratas Endogámicas , CaracolesRESUMEN
The crude adult worm extract of Angiostrongylus cantonensis was subjected to a series of affinity chromatography for selective removal of host antigens as well as cross-reactive components with other helminths. The purified fraction designated as AC(p) with molecular weights ranging from 10,000-42,000 was found to contain antigenic components specific to A. cantonensis as determined by immunoelectrophoresis and enzyme-linked immunosorbent assay.
Asunto(s)
Angiostrongylus/inmunología , Antígenos Helmínticos/aislamiento & purificación , Metastrongyloidea/inmunología , Animales , Antígenos Helmínticos/análisis , Antígenos Helmínticos/inmunología , Cromatografía de Afinidad , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/análisis , Inmunoelectroforesis , Peso MolecularRESUMEN
Electrophoresis on SDS gel and analytical isoelectric focusing showed that a crude extract of Angiostrongylus cantonensis consisted of at least 40 protein components with molecular weights ranging from 13 000-70 000 and isoelectric points of pI values ranging from 3.7-10.0. Crossed-immunoelectrophoresis with a hyperimmune antiserum to A. cantonensis showed at least 40 different antigenic components in the crude worm extract which were cross-reactive with those of Ascaris suum, Metastrongylus apri and Toxocara canis. Using preparative isoelectric focusing, the somatic worm preparation was divided into 13 equal fractions, of which 3, 4 and 5, with pI values of 3.7, 4.0 and 4.45 respectively, were later shown by immunoelectrophoretic techniques and enzyme-linked immunosorbent assay to contain antigens specific to A. cantonensis.