RESUMO
The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.
Assuntos
Sangue , Rejeição de Enxerto , Transplante das Ilhotas Pancreáticas , Transplante Heterólogo , Animais , Anticorpos/sangue , Bovinos , PapioRESUMO
Adenoviral (Adv) vectors are widely used in both experimental and clinical trials for vaccination and gene therapy. Recombinant Adv can evoke potent innate immune responses and adaptive immune responses to encoded antigens. However, how Adv infection affects the response to subsequently encountered antigens is poorly understood. We show that intravenously administered replication defective (E1 and E3 deleted) Adv educes functional changes in dendritic cells (DC) resulting in impaired priming of cytotoxic T lymphocytes (CTL) more than 7 days after Adv treatment. Generalized DC activation was indicated by transient upregulation of CD86 and reduced endocytosis of fluorescent beads. It is known that CD8+ DC are predominantly responsible for uptake and presentation (cross-presentation) of exogenous antigens to CD8+ CTL. Hence, impaired endocytosis in CD8+, but not CD8-, DC at 7 days after Adv administration provided an explanation for the impaired CTL response to antigen at this time. Shutdown of cross-presentation was confirmed using cytochrome c (cytc), an agent that selectively depletes cross-presenting DC. Adv-infection rendered CD8+ DC resistant to depletion by cytc. As the cross-presentation pathway underlies CD8 T-cell responses to many cancers and to vaccines or viruses that do not directly infect DC, systemic Adv administration may impair these responses.
Assuntos
Adenoviridae/genética , Adenoviridae/imunologia , Apresentação de Antígeno , Apresentação Cruzada , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Animais , Células Dendríticas/imunologia , Vetores Genéticos/administração & dosagem , Humanos , Terapia de Imunossupressão , Ativação Linfocitária , Contagem de Linfócitos , Camundongos , Linfócitos T Citotóxicos/imunologiaRESUMO
The aim of the present study was to define the point at which mesothelioma T-cell responses fail in order to design better immunotherapies. A murine model of mesothelioma was used which was established with asbestos. Inoculation of tumour cells into syngeneic mice results in progressing tumours with similar histopathology to human mesothelioma. The tumour cells secrete a marker tumour antigen similar to secreted tumour-associated products, such as mesothelin. The mesothelioma microenvironment contains stromal elements including dendritic cells, effector CD8(+) and CD4(+) T-cells, and CD4(+) T-regulatory (Tregs) cells, all of which are activated in situ, implying chronic inflammation. Tumour antigens are rapidly transported to draining lymph nodes wherein tumour-specific T-cell responses are generated. Despite the generation of potent CD8(+) cytotoxic lymphocyte in lymphoid organs, those that infiltrate tumours cannot restrain tumour growth suggesting local suppression. Splenic Tregs did not suppress protective responses in adoptive transfer experiments suggesting that systemic Tregs play little role in regulating anti-mesothelioma immune responses. Finally, removal of CD25(+) Tregs from the tumour site and lymphoid organs did not alter tumour growth with or without interleukin (IL)-2 or IL-21 immunotherapy. Tregs are not potent regulators of anti-mesothelioma immunity and targeting these cells may not improve results.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Mesotelioma/sangue , Linfócitos T Reguladores/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Proteínas Ligadas por GPI , Imunoterapia/métodos , Interleucina-2/metabolismo , Interleucinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Mesotelina , Mesotelioma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Linfócitos T Reguladores/imunologiaRESUMO
Plasmodium falciparum-infected erythrocytes (IRBCs) synthesize several histidine-rich proteins (HRPs) that accumulate high levels of [3H]histidine but very low levels of amino acids such as [3H]isoleucine or [35S]methionine. We prepared a monoclonal antibody which reacts specifically with one of these HRPs (Pf HRP II) and studied the location and synthesis of this protein during the parasite's intracellular growth. With the knob-positive Malayan Camp strain of P. falciparum, the monoclonal antibody identified a multiplet of protein bands with major species at Mr 72,000 and 69,000. Pf HRP II synthesis began with immature parasites (rings) and continued through the trophozoite stage. The Mr 72,000 band of Pf HRP II, but not the faster moving bands of the multiplet, was recovered as a water-soluble protein from the culture supernatant of intact IRBCs. Approximately 50% of the total [3H]histidine radioactivity incorporated into the Mr 72,000 band was extracellular between 2 and 24 h of culture. Immunofluorescence and cryothin-section immunoelectron microscopy localized Pf HRP II to several cell compartments including the parasite cytoplasm, as concentrated "packets" in the host erythrocyte cytoplasm and at the IRBC membrane. Our results provide evidence for an intracellular route of transport for a secreted malarial protein from the parasite through several membranes and the host cell cytoplasm.
Assuntos
Eritrócitos/metabolismo , Malária/parasitologia , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Transporte Biológico , Eritrócitos/parasitologia , Imunofluorescência , Interações Hospedeiro-Parasita , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium falciparum/imunologia , Plasmodium falciparum/metabolismo , Proteínas/imunologiaRESUMO
While much information exists about the structure and function of the clonally distributed T cell receptor (TCR) alpha beta heterodimer, little is known about the gamma protein, the product of a third rearranging TCR gene. An antiserum to a carboxyl-terminal peptide common to several of the murine gamma chain constant regions and a monoclonal antibody to the murine T3 complex were used to identify products of this TCR gene family in a subpopulation of Lyt2-, L3T4- thymocytes. This subpopulation does not express TCR alpha or full-length TCR beta messenger RNA. The gamma chain is a 35-kilodalton (kD) protein that is disulfide-bonded to a 45-kD partner and is associated with the T3 complex. Analysis of the glycosylation pattern of this thymic gamma chain revealed that the major variable region gamma (V gamma) gene transcribed in activated peripheral T cells is absent from this subpopulation. The cells that bear this second T cell receptor may therefore represent a distinct lineage differentiating within the thymus.
Assuntos
Receptores de Antígenos de Linfócitos T/biossíntese , Timo/metabolismo , Sequência de Aminoácidos , Animais , Dissulfetos/análise , Eletroforese em Gel de Poliacrilamida , Glicosilação , Substâncias Macromoleculares , Camundongos , Peso Molecular , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Relação Estrutura-AtividadeRESUMO
The polymerase chain reaction, and other methods for rapidly amplifying DNA or RNA are versatile tools that can be used in diverse applications that include HLA typing, analyzing the T-cell repertoire, constructing chimaeric antibodies and quantifying cytokine expression. The sensitivity of the polymerase chain reaction has allowed many cellular events to be investigated in vitro and in vivo. Many currently emerging adaptions of the basic technique are of interest to both the research and the clinical immunologist.
Assuntos
Reação em Cadeia da Polimerase/métodos , Animais , Quimera/imunologia , DNA/análise , Teste de Histocompatibilidade , RNA/análise , RNA Mensageiro/análise , Linfócitos T/imunologiaRESUMO
We selected lines of Plasmodium chabaudi that are resistant to high levels of the antifolate drug pyrimethamine and have shown that rearrangement and duplication of a portion of chromosome 7 has occurred in the resistant lines. This chromosomal duplication results in an increase in the chromosome number from 14 to 15: two derived chromosomes (450 kilobases and 1.1 megabases) were smaller than the original chromosome 7 (1.3 megabases), so that essentially only a 200-kilobase region was duplicated. This region contained the DHFR-TS gene and the closely linked Hsp70 gene. We have macrorestriction mapped chromosome 7 from the pyrimethamine-susceptible line (DS) and also the duplicated chromosome 7s in the resistant line. From these maps, we have proposed a process for the karyotype changes. Sequencing of the DHFR gene from the parent and derived chromosomes showed that there were no mutations in the coding sequence. As a result of the duplication of the DHFR-TS gene, there is at least a twofold increase in expression of the DHFR-TS gene, and this may explain the ability of the pyrimethamine-resistant lines to grow in increased amounts of the drug.
Assuntos
Cromossomos/efeitos dos fármacos , Rearranjo Gênico , Família Multigênica , Plasmodium/genética , Pirimetamina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Resistência a Medicamentos , Genes , Ligação Genética , Proteínas de Choque Térmico/genética , Cariotipagem , Dados de Sequência Molecular , Plasmodium/efeitos dos fármacos , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
IDDM in humans and in nonobese diabetic (NOD) mice is a T-cell-dependent autoimmune disease in which the beta-cells of the pancreatic islets are destroyed. Several putative beta-cell autoantigens have been identified, but insulin and its precursor, proinsulin, are the only ones that are beta-cell specific. (Pro)insulin may be a key autoantigen in IDDM. To address the role of proinsulin in the development of IDDM, we generated NOD mice transgenic for the mouse proinsulin II gene driven off a major histocompatibility complex (MHC) class II promoter to direct expression of the transgene to MHC class II bearing cells, including those in the thymus, with the aim of deleting proinsulin-reactive T-cells. The mononuclear cell infiltration of the islets (insulitis) is almost completely absent, and diabetes is prevented in these transgenic NOD mice. The mononuclear cell infiltration of the salivary glands (sialitis) and immune responses to ovalbumin (OVA) are not altered, indicating that the protective effect of the transgene is specific for islet pathology and not due to general immunosuppression. We conclude that autoimmunity to proinsulin plays a pivotal role in the development of IDDM.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Proinsulina/biossíntese , Precursores de Proteínas/biossíntese , Envelhecimento , Animais , Autoantígenos/imunologia , Ciclofosfamida , Primers do DNA , Diabetes Mellitus Experimental/epidemiologia , Diabetes Mellitus Experimental/prevenção & controle , Diabetes Mellitus Tipo 1/epidemiologia , Diabetes Mellitus Tipo 1/genética , Feminino , Genes MHC da Classe II , Terapia Genética , Glutamato Descarboxilase/imunologia , Humanos , Insulina , Ilhotas Pancreáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Proinsulina/genética , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , RNA Mensageiro/biossíntese , Transcrição GênicaRESUMO
Amino acid sequences encoded by exon 8 of the H-2K and H-2D/L genes appear to be locus specific. The majority of H-2Kb molecules contain 10 amino acids that are derived from exon 8. In contrast, the H-2Db, -Dd and -Ld molecules contain only one amino acid which is encoded by exon 8, even though the genetic information exists to encode 10 amino acids analogous to those encoded by the majority of H-2Kb transcripts. We have produced a rabbit anti-peptide serum reactive with the exon 8 encoded sequence of H-2Kb (alpha K-C) that specifically immunoprecipitates a molecule of 45 K mol. wt from spleen cell lysates of b, d, p, q and k haplotype mice. Further analysis by Western blots indicated that virtually all mouse strains express a 45 K protein reactive with alpha K-C. In sequential immunoprecipitations of spleen cell lysates from b, q, p and d haplotype mice using alpha K-C followed by H-2K or H-2D private specificity alloantisera, the anti-peptide serum removed nearly all of the molecules reactive with the anti-H-2K alloantisera (except in the k haplotype) and none of the molecules reactive with the anti-H-2D serum. In addition, no D-region molecules possessing a long C-terminal sequence were detected with an antiserum directed against a representative D-region long C-terminal peptide. We conclude that even though the genetic information for an extended exon 8 exists in K, D and L locus genes, apparently only K-region molecules are expressed with such a C-terminus. Furthermore, in most haplotypes the great majority of H-2K molecules are produced using long exon 8; however, H-2Kk is produced mostly from short exon 8. The absence or presence of key adenosine residues is predicted to be responsible for the variability in class I exon 8 splicing.
Assuntos
Antígenos H-2/genética , Animais , Fenômenos Químicos , Química , Eletroforese em Gel de Poliacrilamida , Antígenos H-2/imunologia , Antígeno de Histocompatibilidade H-2D , Soros Imunes/imunologia , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos , Fragmentos de Peptídeos/imunologiaRESUMO
Dendritic cells (DC) are potent antigen presenting cells that activate naive T cells. It is becoming increasingly clear that DC are not a homogeneous cell population, but comprise different subpopulations that differ in ontogeny and function. To further the molecular characterisation of DC, we screened for genes that were differentially expressed amongst DC subsets and could therefore give insight into their varying biological functions. Using Representational Difference Analysis (RDA) we identified a gene (CIRE) that is expressed at higher levels in the myeloid-related CD8alpha(-) DC than in the lymphoid-related CD8alpha(+) DC. CIRE is a 238 amino acid type II membrane protein, of approximately 33 kDa in size, whose extracellular region contains a C-type lectin domain. Northern blot analysis revealed that CIRE is almost exclusively expressed in DC and was not detected in organs such as heart, brain, kidney, liver, and thymus. T cells failed to express message for CIRE, whilst B cells expressed very low levels. These data here further substantiated by Northern blot analysis of 18 cell lines of various origins (myeloid, macrophage, B and T cell) where only one cell line, which was of myeloid origin and could give rise to DC, expressed mRNA for CIRE. Semi-quantitative RT-PCR suggested that CIRE is down-regulated upon activation. CIRE shares 57% identity with human DC-SIGN, a molecule that has been shown to be the ligand of ICAM-3 and that is also a receptor that binds HIV and facilitates trans-infection of T cells.
Assuntos
Antígenos CD8 , Moléculas de Adesão Celular , Células Dendríticas/metabolismo , Lectinas/genética , Proteínas de Membrana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , DNA Complementar , Regulação para Baixo , Expressão Gênica , Lectinas/classificação , Lectinas/metabolismo , Lectinas Tipo C , Proteínas de Membrana/classificação , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Receptores de Superfície Celular/classificação , Receptores de Superfície Celular/genética , Homologia de Sequência de Aminoácidos , Baço/citologia , Ativação TranscricionalRESUMO
Pit-1 is a transcription factor that has been shown to be critical for pituitary-specific activation of the GH and PRL genes. In rodents and humans, differentiation and/or maintenance of somatotroph, lactotroph, and thyrotroph phenotypes are dependent on expression of a functional pit-1 gene. In rodents, Pit-1 protein is detectable in only these three cell types; however, pit-1 mRNA transcripts appear to be present at comparable levels in all adenohypophysial cell types, suggesting that translational controls may dictate the pattern of Pit-1 expression. We examined the distribution of pit-1 transcripts in the human pituitary and pituitary adenomas. All tumors were characterized by immunocytochemistry, electron microscopy, and tissue culture for accurate classification. Northern blot analysis demonstrated abundant levels of pit-1 mRNA in somatotroph, mammosomatotroph, and lactotroph adenomas. Two clinically silent adenomas that expressed TSH as well as gonadotropins contained detectable levels of pit-1 mRNA. No pit-1 expression was otherwise detected in corticotroph, gonadotroph, null cell, or oncocytic adenomas. In situ hybridization localized pit-1 mRNA transcripts in adenomas that contained GH, PRL, or TSH, but not in adenomas composed of other cell types. Pit-1 mRNA was also localized to selected subpopulations of the human nontumorous adenohypophysis that contained immunoreactivity for GH, PRL, and/or TSH. Pit-1 protein immunoreactivity was detected in the nuclei of adenomas that expressed pit-1 mRNA, but not in those that were negative for pit-1 mRNA; it was also localized only in cells containing GH, PRL, or TSH beta in the nontumorous adenohypophysis. These data demonstrate selective expression of the human pit-1 gene in adenohypophysial cell types responsible for GH, PRL, and/or TSH synthesis and are consistent with a predominantly pretranslational regulatory mechanism for Pit-1 expression in the human.
Assuntos
Adenoma/metabolismo , Proteínas de Ligação a DNA/metabolismo , Adeno-Hipófise/metabolismo , Neoplasias Hipofisárias/metabolismo , Fatores de Transcrição/metabolismo , Northern Blotting , Técnicas de Cultura , Humanos , Imuno-Histoquímica , Hibridização In Situ , Fator de Transcrição Pit-1RESUMO
Consideration of the pathophysiology of insulin-dependent diabetes mellitus in the nonobese diabetic (NOD) mouse can be viewed from a temporal perspective. We argue that there are discontinuous phases and each phase may reflect a phenotype educed by a particular set of genetic and epigenetic events. Therefore, temporal dissection may be a useful platform for causal dissection and we have set out this article as follows: 1. Introduction. 2. "Pre-time." a. Genetics. b. Parental effects. 3. Development of insulitis. a. Development of autoimmunity vs waning of or failure to establish tolerance. b. Importance of beta cell mass. c. Homing. 4. Onset of beta cell destruction. 5. Further Discussion.
Assuntos
Autoimunidade/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Animais , Diabetes Mellitus Tipo 1/genética , Progressão da Doença , Humanos , Cinética , Camundongos , Camundongos Endogâmicos NOD , Fatores de TempoRESUMO
There are no adequate vaccines against some of the new or reemerged infectious scourges such as HIV and TB. They may require strong and enduring cell-mediated immunity to be elicited. This is quite a task, as the only known basis of protection by current commercial vaccines is antibody. As DNA or RNA vaccines may induce both cell-mediated and humoral immunity, great interest has been shown in them. However, doubt remains whether their efficacy will suffice for their clinical realization. We look at the various tactics to increase the potency of nucleic acid vaccines and divided them broadly under those affecting delivery and those affecting immune induction. For delivery, we have considered ways of improving uptake and the use of bacterial, replicon or viral vectors. For immune induction, we considered aspects of immunostimulatory CpG motifs, coinjection of cytokines or costimulators and alterations of the antigen, its cellular localization and its anatomical localization including the use of ligand-targeting to lymphoid tissue. We also thought that mucosal application of DNA deserved a separate section. In this review, we have taken the liberty to discuss these enhancement methods, whenever possible, in the context of the underlying mechanisms that might argue for or against these strategies.
Assuntos
Vacinas de DNA , Adjuvantes Imunológicos/administração & dosagem , Animais , Antígenos/genética , Bactérias/genética , Ilhas de CpG , Citocinas/administração & dosagem , Citocinas/genética , Vetores Genéticos , Humanos , Imunidade nas Mucosas , Plasmídeos/genética , Replicon , Linfócitos T Citotóxicos/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vírus/genéticaRESUMO
The antibody titre and the optical density values in an ELISA are influenced by the epitope density of the antigen and the affinity of the antibody tested. This has major implications in the interpretation of ELISA results.
Assuntos
Anticorpos Monoclonais/análise , Afinidade de Anticorpos , Epitopos/imunologia , Animais , Anticorpos Monoclonais/imunologia , Dinitrofenóis/imunologia , Ensaio de Imunoadsorção Enzimática , Cinética , Camundongos , Soroalbumina Bovina/imunologiaRESUMO
The IgG binding domains of staphylococcal protein A and streptococcal protein G were expressed as a chimaera using the pGEX vector which has been advocated because its fusion proteins tend to be soluble and easily isolated on immobilised glutathione. This chimaera was soluble and abundant (yield = 18 mg/l of bacterial culture) and was tested by double diffusion in agarose and by ELISA. It was found to bind IgG of all species that either parent could bind. It was superior to protein A or protein G in binding mouse Ig. A chimaera of protein G and alkaline phosphatase was also constructed and found to be soluble and abundant (yield = 20 mg/l of bacterial culture). This protein could be used as a secondary reagent in ELISA at 5 micrograms/ml for human, rabbit and mouse and at 25 micrograms/ml for sheep.
Assuntos
Fosfatase Alcalina/genética , Quimera/imunologia , Clonagem Molecular , Proteínas do Tecido Nervoso/genética , Proteína Estafilocócica A/genética , Fosfatase Alcalina/imunologia , Animais , Sequência de Bases , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Humanos , Imunodifusão , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/imunologia , Regiões Promotoras Genéticas , Proteínas Recombinantes , Proteína Estafilocócica A/imunologiaRESUMO
The affinities of 9 IgG1 monoclonal anti-dinitrophenol (DNP) antibodies for 3H-epsilon-DNP-L-lysine, 125I-HOP-DNP-L-lysine and 125I-DNP-human serum albumin (HSA) were determined. 3H-DNP-lysine was used in equilibrium dialysis and ammonium sulphate globulin precipitation assays; 125I-HOP-DNP-lysine was used in equilibrium dialysis and polyethylene glycol precipitation; and 125I-DNP5-HSA in the polyethylene glycol precipitation assay for affinity. The ranking order of the monoclonal antibodies in terms of affinity by the assays was significantly correlated. Of particular importance was the observation that the simple and widely applicable globulin precipitation assay utilizing a protein antigen produced affinity values which showed concordance with the least equivocal but cumbersome assay, equilibrium dialysis. Mixing of antibodies of high and low affinity demonstrated that even a low proportion of high affinity antibody had marked effect on measurements of the amount and affinity of a predominantly low affinity antibody preparation.
Assuntos
Anticorpos Monoclonais , Complexo Antígeno-Anticorpo/análise , Animais , Anticorpos Monoclonais/isolamento & purificação , Dinitrofenóis , Hibridomas/imunologia , Imunoglobulina G/imunologia , Radioisótopos do Iodo , Cinética , Camundongos , Camundongos Endogâmicos BALB C , TrítioRESUMO
The regions encoding the IgG-binding domains of protein A (PA) and protein G (PG) were cloned into the bacterial expression vector pGEX. Both proteins were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (PA-GST and PG-GST) and were found to be soluble, abundant and easily purified in one step from the bacterial lysate by affinity chromatography on immobilized glutathione. Yields of 50 mg/litre of cultures were obtained. Both purified fusion proteins were shown to be functional in a variety of immunochemical procedures. In radial diffusion tests, PA-GST precipitated IgG from human, squirrel monkey, rabbit, dog, cat and pig but not mouse, sheep, goat, cow, horse or chicken. PG-GST formed precipitin bands with IgG from human, rabbit, mouse, pig, sheep, goat, cow and horse but not squirrel monkey, dog, cat and chicken IgG. The fusion proteins were shown to function as effective detection reagents in ELISA and Western blotting. Glutathione agarose beads with bound fusion protein were shown to be useful for immunoprecipitation.
Assuntos
Escherichia coli/genética , Glutationa Transferase , Proteínas do Tecido Nervoso/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteína Estafilocócica A/biossíntese , Sequência de Bases , Western Blotting , Cromatografia de Afinidade , Clonagem Molecular , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/imunologia , Reação em Cadeia da Polimerase , Testes de Precipitina , Proteínas Recombinantes de Fusão/imunologia , Proteína Estafilocócica A/imunologia , Especificidade por SubstratoRESUMO
Isotyping and quantitation of murine IgG2a antibodies are widely performed with commercial monoclonal and polyclonal antisera raised against BALB/c IgG2a myeloma proteins. Recently it became evident that inbred mouse strains with the Igh1-b allele do not have the gene for IgG2a and instead express the IgG2c isotype. We show that commercial anti-IgG2a sera cross-react inadequately against IgG2c in immunoblot and ELISA and hence, are not suitable to detect and measure this subclass in mouse strains such as C57BL/6, C57BL/10 and NOD. We have used DNA immunization to generate polyclonal anti-IgG2c serum and demonstrated that it is essential to use IgG2c-specific antiserum to quantify accurately isotypic responses in mouse strains with the Igh1-b allele.
Assuntos
Especificidade de Anticorpos , Imunoensaio/métodos , Imunoglobulina G/sangue , Isotipos de Imunoglobulinas/sangue , Camundongos Endogâmicos/imunologia , Alelos , Animais , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Genes de Imunoglobulinas , Immunoblotting , Imunoglobulina G/genética , Isotipos de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL/imunologia , Camundongos Endogâmicos NOD/imunologia , Reprodutibilidade dos TestesRESUMO
The effect of antibody affinity on the performance of 5 commonly used assays was studied. The assays used were measurement of antigen binding capacity in a Farr type assay, haemagglutination, solid-phase radioimmunoassay (SP-RIA), solid-phase ELISA and precipitation. The first 4 assays were all more sensitive for high affinity antibodies. Precipitation was not related to affinity, suggesting that factors secondary to antigen-antibody binding may be more important in determining the level of precipitate formation. The effect of epitope density of the antigen was also investigated in the SP-RIA and ELISA. Affinity dependence was more marked when antigen of low epitope density was used and this dependence could be reduced in the ELISA by choosing a low OD endpoint. Thus, the most reliable way to estimate antibody content by the ELISA may be to determine a low OD endpoint titre against antigen of high epitope density. When epitope density per molecule cannot be increased, an alternative approach to the problem is to increase epitope density by covalent coupling of antigen to the solid-phase rather than by adsorption.