RESUMO
BACKGROUND: Decontaminating and reusing filtering facepiece respirators (FFRs) for healthcare workers is a potential solution to address inadequate FFR supply during a global pandemic. AIM: The objective of this review was to synthesize existing data on the effectiveness and safety of using chemical disinfectants to decontaminate N95 FFRs. METHODS: A systematic review was conducted on disinfectants to decontaminate N95 FFRs using Embase, Medline, Global Health, Google Scholar, WHO feed, and MedRxiv. Two reviewers independently determined study eligibility and extracted predefined data fields. Original research reporting on N95 FFR function, decontamination, safety, or FFR fit following decontamination with a disinfectant was included. FINDINGS AND CONCLUSION: A single cycle of vaporized hydrogen peroxide (H2O2) successfully removes viral pathogens without affecting airflow resistance or fit, and maintains an initial filter penetration of <5%, with little change in FFR appearance. Residual hydrogen peroxide levels following decontamination were within safe limits. More than one decontamination cycle of vaporized H2O2 may be possible but further information is required on how multiple cycles would affect FFR fit in a real-world setting before the upper limit can be established. Although immersion in liquid H2O2 does not appear to adversely affect FFR function, there is no available data on its ability to remove infectious pathogens from FFRs or its impact on FFR fit. Sodium hypochlorite, ethanol, isopropyl alcohol, and ethylene oxide are not recommended due to safety concerns or negative effects on FFR function.
Assuntos
Infecções por Coronavirus/prevenção & controle , Descontaminação/normas , Desinfetantes/administração & dosagem , Reutilização de Equipamento/normas , Peróxido de Hidrogênio/administração & dosagem , Dispositivos de Proteção Respiratória/virologia , Hipoclorito de Sódio/administração & dosagem , Guias como Assunto , Humanos , Raios UltravioletaRESUMO
BACKGROUND: In pandemics such as COVID-19, shortages of personal protective equipment are common. One solution may be to decontaminate equipment such as facemasks for reuse. AIM: To collect and synthesize existing information on decontamination of N95 filtering facepiece respirators (FFRs) using microwave and heat-based treatments, with special attention to impacts on mask function (aerosol penetration, airflow resistance), fit, and physical traits. METHODS: A systematic review (PROSPERO CRD42020177036) of literature available from Medline, Embase, Global Health, and other sources was conducted. Records were screened independently by two reviewers, and data was extracted from studies that reported on effects of microwave- or heat-based decontamination on N95 FFR performance, fit, physical traits, and/or reductions in microbial load. FINDINGS: Thirteen studies were included that used dry/moist microwave irradiation, heat, or autoclaving. All treatment types reduced pathogen load by a log10 reduction factor of at least three when applied for sufficient duration (>30 s microwave, >60 min dry heat), with most studies assessing viral pathogens. Mask function (aerosol penetration <5% and airflow resistance <25 mmH2O) was preserved after all treatments except autoclaving. Fit was maintained for most N95 models, though all treatment types caused observable physical damage to at least one model. CONCLUSIONS: Microwave irradiation and heat may be safe and effective viral decontamination options for N95 FFR reuse during critical shortages. The evidence does not support autoclaving or high-heat (>90°C) approaches. Physical degradation may be an issue for certain mask models, and more real-world evidence on fit is needed.
Assuntos
Infecções por Coronavirus/prevenção & controle , Descontaminação/normas , Reutilização de Equipamento/normas , Guias como Assunto , Temperatura Alta , Dispositivos de Proteção Respiratória/virologia , Raios Ultravioleta , HumanosRESUMO
Inadequate supply of filtering facepiece respirators (FFRs) for healthcare workers during a pandemic such as the novel coronavirus outbreak (SARS-CoV-2) is a serious public health issue. The aim of this study was to synthesize existing data on the effectiveness of ultraviolet germicidal irradiation (UVGI) for N95 FFR decontamination. A systematic review (PROSPERO CRD42020176156) was conducted on UVGI in N95 FFRs using Embase, Medline, Global Health, Google Scholar, WHO feed, and MedRxiv. Two reviewers independently determined eligibility and extracted predefined variables. Original research reporting on function, decontamination, or mask fit following UVGI were included. Thirteen studies were identified, comprising 54 UVGI intervention arms and 58 N95 models. FFRs consistently maintained certification standards following UVGI. Aerosol penetration averaged 1.19% (0.70-2.48%) and 1.14% (0.57-2.63%) for control and UVGI arms, respectively. Airflow resistance for the control arms averaged 9.79 mm H2O (7.97-11.70 mm H2O) vs 9.85 mm H2O (8.33-11.44 mm H2O) for UVGI arms. UVGI protocols employing a cumulative dose >20,000 J/m2 resulted in a 2-log reduction in viral load. A >3-log reduction was observed in seven UVGI arms using >40,000 J/m2. Impact of UVGI on fit was evaluated in two studies (16,200; 32,400 J/m2) and no evidence of compromise was found. Our findings suggest that further work in this area (or translation to a clinical setting) should use a cumulative UV-C dose of 40,000 J/m2 or greater, and confirm appropriate mask fit following decontamination.
Assuntos
Infecções por Coronavirus/prevenção & controle , Desinfecção/normas , Reutilização de Equipamento/normas , Guias como Assunto , Máscaras/normas , Exposição Ocupacional/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Raios Ultravioleta , Betacoronavirus , COVID-19 , Eficiência , Humanos , SARS-CoV-2 , Segurança/normasRESUMO
Shifts in manure phosphorus (P) chemical forms and pool sizes induced by water treatment residuals and industrial mineral by-products are largely undefined. We conducted a manure P fractionation study to determine mechanisms of reduction of dissolved reactive phosphorus (DRP) in poultry manure upon mineral by-product additions. The effects of composting on the P immobilization efficacy of the by-products were determined using laboratory self-heating composting simulators. The mineral by-products included an aluminum-water treatment residual (Al-WTR) and an iron-rich titanium-processing by-product. The noncomposted manure averaged 0.11 g g(-1) of total P as DRP forms. The by-products significantly reduced manure DRP, by an average of 39 and 48% in the Al- and the Fe-treated manure, respectively. The by-products also reduced the 0.5 M NH4F-extractable phosphorus (FEP) fraction. Shifts in P forms between FEP and 0.1 M NaOH-extractable phosphorus (SHEP) depended upon the Al and Fe contents of the by-products while the combined FEP + SHEP pool remained constant. Phosphate sorption measurements supported the observations that the Fe-rich by-product was more effective at reducing manure DRP and enhancing the formation of SHEP forms at the expense of FEP than the Al-WTR. Composting had no effect on the efficacy of either by-product to reduce DRP. Potential mechanisms of enhanced P stabilization in treated manure upon composting included chemical shifts from the DRP and FEP fractions to the citrate-bicarbonate-dithionite extractable P fraction. Thus, the choice of P immobilization agents affected the stability of immobilized P forms and should be taken into consideration in developing manure processing and nutrient stabilization methods.
Assuntos
Bactérias Aeróbias/fisiologia , Esterco , Fósforo/química , Fósforo/metabolismo , Poluentes do Solo/análise , Animais , Disponibilidade Biológica , Conservação dos Recursos Naturais , Poluição Ambiental/prevenção & controle , Aves Domésticas , Eliminação de Resíduos , SolubilidadeRESUMO
This study demonstrates that in vivo exposure to cigarette smoke (CS) and in vitro treatment of long-term bone marrow cultures (LTBMCs) with nicotine, a major constituent of CS, result in inhibition of hematopoiesis. Nicotine treatment significantly delayed the onset of hematopoietic foci and reduced their size. Furthermore, the number of long-term culture-initiating cells (LTC-ICs) within an adherent layer of LTBMCs was significantly reduced in cultures treated with nicotine. Although the production of nonadherent mature cells and their progenitors in nicotine-treated LTBMCs was inhibited, this treatment failed to influence the proliferation of committed hematopoietic progenitors when added into methylcellulose cultures. Bone marrow stromal cells are an integral component of the hematopoietic microenvironment and play a critical role in the regulation of hematopoietic stem cell proliferation and self-renewal. Exposure to nicotine decreased CD44 surface expression on primary bone marrow-derived fibroblastlike stromal cells and MS-5 stromal cell line, but not on hematopoietic cells. In addition, mainstream CS altered the trafficking of hematopoietic stem/progenitor cells (HSPC) in vivo. Exposure of mice to CS resulted in the inhibition of HSPC homing into bone marrow. Nicotine and cotinine treatment resulted in reduction of CD44 surface expression on lung microvascular endothelial cell line (LEISVO) and bone marrow-derived (STR-12) endothelial cell line. Nicotine treatment increased E-selectin expression on LEISVO cells, but not on STR-12 cells. These findings demonstrate that nicotine can modulate hematopoiesis by affecting the functions of the hematopoiesis-supportive stromal microenvironment, resulting in the inhibition of bone marrow seeding by LTC-ICs and interfering with stem cell homing by targeting microvascular endothelial cells.
Assuntos
Células da Medula Óssea/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Receptores de Hialuronatos/análise , Nicotina/farmacologia , Animais , Plaquetas/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/fisiologia , Moléculas de Adesão Celular/análise , Linhagem Celular , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Plantas Tóxicas , Fumaça/efeitos adversos , Células Estromais/efeitos dos fármacos , Células Estromais/fisiologia , Nicotiana , Veias UmbilicaisRESUMO
The dispersed neuroendocrine system includes cells with different embryological derivations, sharing a common neuroendocrine (NE) program, as indicated by the expression of NE markers, some of which are shared antigenic determinants. We report here that the small cell lung carcinoma cells NCI-H69 express the two human melanoma-associated antigens (HMAA) NGA/LS62 an LS109. Incubation of NCI-H69 cells with maturational inducers, such as retinoic acid and bromodeoxyuridine (BrdU), upregulated the expression of both HMAA. Exposure to BrdU for 4 weeks induced the appearance of a different phenotype in subpopulations of NCI-H69 cells, which became epithelioid, substrate-adherent, grew in monolayer and continued to express NE-associated antigens in variable amount. The shift in phenotype was not reversible after BrdU withdrawal and was maintained for at least 6 months in continuous culture. The substrate adhesion of NCI-H69 cells was paralleled by a change in NGA glycosylation pattern, thus suggesting a possible functional role for NGA in cell substrate adhesion/recognition.
Assuntos
Antígenos de Neoplasias/análise , Bromodesoxiuridina/farmacologia , Carcinoma de Células Pequenas/imunologia , Neoplasias Pulmonares/imunologia , Melanoma/imunologia , Proteínas de Neoplasias/análise , Tretinoína/farmacologia , Carcinoma de Células Pequenas/patologia , Adesão Celular , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/patologia , Antígenos Específicos de Melanoma , Testes de Precipitina , Células Tumorais CultivadasRESUMO
We have described two human melanoma-associated antigens (HMAA), recognized by the murine monoclonal antibodies LS62 and LS109. LS62 recognizes the neuroglandular antigen (NGA), which is overexpressed in neoplastic melanocytes as well as in several tissues of neuroectodermal origin. These antibodies were used to screen six neuroblastoma cell lines and one neuroepithelioma cell line. A melanoma cell line, G361, known to express the two antigens, was used as the positive control. Variable expression of the two antigens was detected in neuroblastoma cells. The surface expression of NGA and of the LS109 antigen was modulated in parallel with the morphological differentiation induced by retinoic acid, 5-bromodeoxyuridine, or cyclic AMP analog/activators. The modulation of the expression of the two HMAA was detected in G361 melanoma cells and in one of the neuroblastoma cell lines, SK-N-SH. These results suggest altered expression of both antigens during melanoma and neuroblastoma cell differentiation in culture.
Assuntos
Antígenos de Neoplasias/imunologia , Melanoma/imunologia , Neuroblastoma/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Neoplasias/metabolismo , Bromodesoxiuridina/farmacologia , Diferenciação Celular/efeitos dos fármacos , AMP Cíclico/farmacologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Testes de Precipitina , Tretinoína/farmacologiaRESUMO
Neuroglandular antigen (NGA) was identified as a human melanoma-associated antigen by a panel of murine monoclonal antibodies of both IgG2a (LS62, LS76, LS159) and IgG1 (LS113, LS140, LS152) subclasses, developed in this laboratory (L. Sikora, A. Pinto, D. Demetrick, W. Dixon, S. Urbanski, and L. M. Jerry, Int. J. Cancer, 39: 138-145, 1987). Monoclonal antibody LS62 was used to immunoprecipitate NGA from radiolabeled cultured melanoma cells, and it behaved as a heterogeneous glycoprotein "smear" on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (Mr 29,000-70,000). Radioactive pulse-chase time course experiments using human melanoma cells cultured in the presence or absence of inhibitors of protein glycosylation showed that the antigen consisted of a core protein with a molecular weight of 22,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This molecule was modified by the addition of at least three N-linked oligosaccharide side chains (as revealed by limited N-glycanase digestion) to give a precursor form with a molecular weight of approximately 34,000. Subsequent processing steps yielded a heterogeneous family of glycoproteins with varying amounts of covalently attached carbohydrate. Much of this heterogeneity in both molecular weight and pI (as revealed by two-dimensional electrophoresis) could be removed by treatment of the antigen with neuraminidase, suggesting heavy sialylation of the glycoprotein. NGA could be detected on the surface of melanoma cells by fluorescence-activated cell sorter analysis, surface radioiodination, and, as previously shown, immunoperoxidase staining. However, there was a larger intracellular pool of the molecule and the antigen was rapidly released into the culture supernatant. The function of NGA remains unknown but its elevated expression in transformed melanocytes have prompted this characterization to understand its biochemical nature and relation to other melanoma-associated antigens.
Assuntos
Antígenos de Neoplasias/genética , Proteínas de Neoplasias/biossíntese , Processamento de Proteína Pós-Traducional , Anticorpos Monoclonais , Divisão Celular , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Glicosilação , Humanos , Melanoma/imunologia , Antígenos Específicos de Melanoma , Metionina/metabolismo , Peso Molecular , Monensin/farmacologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/isolamento & purificação , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/imunologia , Tunicamicina/farmacologiaRESUMO
Murine monoclonal antibody (MAb) LS109 was one of a series of MAbs produced by hyperimmunization of mice with detergent extracts of pooled melanoma cell lines and metastatic melanoma patient tumors. ELISA screening of extracts of individual cultured melanoma cell lines and single patient tumors with MAb LS109 gave an interesting pattern of reactivity. The antibody was strongly positive with some of these extracts, yet negative or weakly positive with others. In addition, there was strong reactivity with a restricted set of normal necropsy tissues and certain non-melanoma tumor extracts. Taken together, our data suggest that MAb LS109 recognizes a normal differentiation antigen which is perhaps aberrantly expressed or over-produced during certain stages of melanoma tumor progression. The antigen recognized by LS109 is a heterodimeric surface glycoprotein molecule, consisting of an 89-kDa "heavy" chain linked by disulfide bonds to an 83-kDa "light" chain. Under non-reducing SDS-PAGE conditions, the intact dimer migrates with an Mr of approximately 140kDa. The 89-kDa component appears to be heavily N-glycosylated whereas the 38-kDa component has little, if any, covalently attached carbohydrate. Our data show the biosynthesis, glycosylation and turnover of the LS109 antigen, as well as evidence of its surface localization. In addition, evidence is presented that the LS109 antigen is identical to the 4F2 cell activation/proliferation molecule previously described on a variety of normal and neoplastic cells.
Assuntos
Antígenos de Neoplasias/isolamento & purificação , Melanoma/imunologia , Neoplasias Cutâneas/imunologia , Animais , Anticorpos Monoclonais/isolamento & purificação , Antígenos de Neoplasias/análise , Antígenos de Superfície/análise , Antígenos de Superfície/isolamento & purificação , Divisão Celular , Linhagem Celular/imunologia , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Glicosilação , Humanos , Camundongos , Peso Molecular , Testes de Precipitina/métodos , Células Tumorais Cultivadas/imunologiaRESUMO
NGA is a human melanoma-associated antigen recognized by a panel of murine monoclonal antibodies developed in this laboratory. NGA consists of a 23.5 kDa core protein which is glycosylated in vivo to give a family of glycoproteins (30-60 kDa). Treatment of human melanoma G361 cells with the phorbol ester PMA resulted in apparent partial inhibition of NGA glycosylation. After PMA treatment, NGA appeared as 3 different bands of 24, 29 and 34 kDa on SDS-PAGE. The 29 kDa band is similar to the one obtained by treatment with the ionophore monensin, which inhibits NGA O-glycosylation. PMA can modulate plasma membrane ion exchange, most likely by activating protein kinase C. In G361 cells PMA may produce the same net effect as monensin, by impairing transport in the Golgi complex and consequently inhibiting protein O-glycosylation through an ionophore-like effect. Treatment of G361 cells with both PMA and protein kinase C inhibitors re-established the usual NGA glycosylation pattern. Thus the observed effect of PMA on NGA glycosylation is reversible and appears to be mediated by protein kinase C activation.
Assuntos
Antígenos de Neoplasias , Glicoproteínas/metabolismo , Melanoma/imunologia , Proteínas de Neoplasias , Acetato de Tetradecanoilforbol/farmacologia , Transporte Biológico , Membrana Celular/imunologia , Clomifeno/farmacologia , Eletroforese em Gel de Poliacrilamida , Glicosilação , Humanos , Técnicas de Imunoadsorção , Antígenos Específicos de Melanoma , Peso Molecular , Floretina/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Células Tumorais CultivadasRESUMO
Murine monoclonal antibodies (MAbs) were produced with reactivity to human malignant melanoma. Six MAbs, 3 of the IgGI (LS113, LS140, LS152) and 3 of the IgG2a (LS59, LS62, LS76) subclasses, were selected for their binding, with an identical pattern of reactivity, to a novel melanoma-associated antigen. As characterized by the enzyme-linked immunosorbent assay (ELISA), these MAbs were found to be positive on n-octyl-beta-D-glucopyranoside extracts of all 10 melanoma cell lines tested and on extracts of 22 metastatic melanoma tumors. The antibodies had minimal reaction with a panel of 14 normal adult tissue extracts. A degree of cross-reactivity was observed with 50% of 39 non-melanoma tumor extracts. The results obtained with the ELISA on cell line and tissue extracts were duplicated using the ABC method of peroxidase staining. The pattern of cross-reactivity, as demonstrated by the intense staining of paraffin-embedded and frozen tissue sections of normal, benign and malignant tissues, defines the recognized protein as a neuroglandular antigen (NGA). Immunoadsorbents made with the antibodies were used to purify the antigen shed from cultured melanomas. All 6 MAbs recognized this purified antigen while 5 other antimelanoma antibodies did not react with it. On gel electrophoresis this antigen is a highly glycosylated glycoprotein with a protein core of 21 kDa.
Assuntos
Antígenos de Neoplasias/análise , Melanoma/imunologia , Proteínas de Neoplasias/análise , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Linhagem Celular , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Humanos , Antígenos Específicos de Melanoma , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/isolamento & purificação , Tunicamicina/farmacologiaRESUMO
Four monoclonal antibodies specific for somatostatin have been produced and characterized. These antibodies were used to assess the anatomical relationship of somatostatin-containing cells in the pancreas and gastrointestinal tract of man, baboon and rat with ten other peptide-containing endocrine cells. The peptides investigated were gastrin, cholecystokinin, motilin, secretin, neurotensin, gastric inhibitory polypeptide, gut-glucagon, pancreatic glucagon, pancreatic polypeptide and insulin. The only regions in which somatostatin cells were seen in close contact with another endocrine cell were in the pancreas and the gastric antrum. In the pancreas somatostatin cells were commonly seen in close contact with insulin, glucagon and pancreatic polypeptide cells and infrequent contact was demonstrable with the gastrin-immunoreactive cells in the antrum of both rat and man. In all other cases no evidence was obtained for a close anatomical relationship between somatostatin cells and the other enteroendocrine cells.
Assuntos
Anticorpos Monoclonais/imunologia , Somatostatina/metabolismo , Animais , Especificidade de Anticorpos , Colecistocinina/metabolismo , Colo/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Mucosa Gástrica/metabolismo , Gastrinas/metabolismo , Glucagon/metabolismo , Humanos , Técnicas Imunológicas , Intestino Delgado/metabolismo , Motilina/metabolismo , Neurotensina/metabolismo , Pâncreas/metabolismo , Papio , Ratos , Secretina/metabolismo , Somatostatina/imunologiaRESUMO
Four monoclonal antibodies to VIP have been generated and shown to be N-terminal specific with high affinity for VIP. VIP-containing nerve fibers and cell bodies were visible in the upper small intestine from day 1 of neonatal life. Initially the immunoreactivity was mostly in the myenteric plexus but extended into the sub-mucous plexus by day 7. From day 1 to day 7 the VIP-innervation developed both orally and caudally at a similar rate. In the stomach, the antrum showed sub-mucosal cell bodies by day 14, while in the corpus the cell bodies remained confined to the myenteric plexus. The colon showed positive fibers in the myenteric plexus at day 7 and cell bodies and fibers in the sub-mucous plexus by day 14. The size (cross-sectional area) of the individual VIP-immunoreactive cell bodies increased significantly between day 1 and day 14 with no further increase with age. At no time were immunoreactive cell bodies shown to migrate from the myenteric to the sub-mucous plexus. VIP-immunoreactive epithelial cells were not detected in the present study.
Assuntos
Sistema Digestório/crescimento & desenvolvimento , Plexo Mientérico/crescimento & desenvolvimento , Neurônios/fisiologia , Peptídeo Intestinal Vasoativo/fisiologia , Envelhecimento , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais , Sistema Digestório/inervação , Ensaio de Imunoadsorção Enzimática , Especificidade de Órgãos , Fragmentos de Peptídeos/análise , Ratos , Ratos Endogâmicos , Peptídeo Intestinal Vasoativo/análiseRESUMO
The ferredoxin (Fd) molecule is a small non-mammalian immunogenic protein containing 55 amino acid residues with only two major antigenic determinants located with the NH2-terminal heptapeptide and the COOH-terminal pentapeptide. Selective enzyme cleavages of Fd with either trypsin or carboxypeptidase A result in the inactivation of the antigenic determinants by the removal of a tripeptide at the NH2-terminal and two amino acid residues at the COOH-terminal, effectively leaving 52 and 53 amino acid fragments respectively, each containing a single antigenic determinant. Fd digested with both enzymes yielded a 50 amino acid peptide with both determinants inactivated. Purity of these digests was assessed using monoclonal antibodies in standard and antigen-blocking ELISAs. The doubly digested peptide had virtually no reactivity with anti-Fd sera, reconfirming that the central cysteine-rich region is serologically silent. It was found that the sum of the reactivities of the N- and C-determinant-bearing peptides as equal to that of the native Fd and that the ratio of the reactivities could be used to assess determinant selectivity in the response to Fd in congenic recombinant strains of mice. This method was used in mapping the determinant selectivity in the antibody response to the MHC of mice to the left of the I-B subregion. Use of the B10.HTT strain indicated that separate genes mapping to the same subregion code for the magnitude of the antibody response and the determinant selectivity of the response.
Assuntos
Mapeamento Cromossômico , Epitopos/imunologia , Ferredoxinas/imunologia , Genes MHC da Classe II , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Feminino , Masculino , Camundongos , Fragmentos de Peptídeos/imunologiaRESUMO
Ferredoxin (Fd) is a low mol. wt protein (6000 d) isolated from Clostridium pasteurianum. This antigen possess two non-cross-reactive antigenic determinants and engenders a restricted antibody response in selected strains of mice. Immunochemical studies of Fd have shown that antibody responses are confined to two sequences of between five and seven amino acids in extent located at the NH2-and COOH-termini of the molecule. Serum antibodies from responder strains of mice bind these epitopes in proportions which are regulated by genes mapping in the Ir-region of the H-2 complex. A hybrid cell line secreting monoclonal Fd-binding antibody has been isolated from an immune mouse through fusion with the SP2/0 myeloma cell line. The resulting antibody binds to a single determinant located at the nH2-terminal of the molecule. An anti-idiotype antibody to this monoclonal antibody was raised in rabbits. After appropriate absorptions, its specificity for the paratopic regions of the hybridoma antibody was established by demonstrating its displacement from reaction with the idiotype by Fd. Analysis of the distribution of the hybridoma idiotype in serum antibodies from congenic mouse strains indicates that it is a major idiotype expressed in different inbred strains sharing identity at the Igh-1 locus.
Assuntos
Ferredoxinas/imunologia , Idiótipos de Imunoglobulinas , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Epitopos , Hibridomas/imunologia , Camundongos , Camundongos Endogâmicos/imunologia , Fragmentos de Peptídeos/imunologiaRESUMO
The solid phase enzyme linked immunosorbent assay (ELISA) has been used to quantify anti-keyhole limpet haemocyanin (anti-KLH) antibody in the serum of KLH-immune C57Bl/6 mice. When spleen cells from immune mice were cultured overnight in ELISA microtitre wells to which KLH had been adsorbed it was found that easily quantifiable amounts of anti-KLH antibody were synthesized and were detectable. It was found further that spleen cells from KLH-primed mice, when cultured in vitro in the presence of KLH, transferred to KLH-labelled ELISA plates, and cultured overnight, also produced detectable levels of antibody. Levels of antibody were detectable only after 4 and 5 days of in vitro stimulation. A comparison was made between detectable numbers of plaque forming cells to sheep red blood cells (SRBC) in SRBC primed CBA mice and levels of antibody detected by the ELISA procedure. It was found that the sensitivities of the two tests were comparable. The applications of this technique to the study of in vitro antibody synthesis using soluble antigens are discussed.