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1.
Bioanalysis ; 9(20): 1603-1615, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29072493

RESUMO

AIM: Development of drug-related surrogate positive controls for isotype anti-drug antibodies assay remains challenging. Efforts on antibody engineering or chemical crosslinking have been made. However, multiple epitope recognition, purity and stability are often of concern. To tackle these challenges, we used LC-SPDP/SMPB crosslinking to conjugate polyclonal anti-drug IgG and human immunoglobulin isotype (hIgE, hIgA, hIgM or hIgG4) with optimized conditions. RESULTS: The final product was a hybrid of anti-drug IgG cross-linked to human isotype immunoglobulin through stable thioether bond. The characteristics of the hybrids and their performance as assay positive controls were further evaluated. CONCLUSION: The results demonstrate that LC-SPDP/SMPB chemical crosslinking method is suitable for generating stable hybrids to be used as assay positive controls in isotype anti-drug antibodies assay.


Assuntos
Anticorpos/análise , Cromatografia em Gel , Isotipos de Imunoglobulinas/análise , Anticorpos/química , Anticorpos/metabolismo , Complexo Antígeno-Anticorpo/análise , Ensaio de Imunoadsorção Enzimática , Humanos , Isotipos de Imunoglobulinas/química , Maleimidas/química , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Sulfetos/química
2.
Microbiol Immunol ; 61(10): 452-458, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28877365

RESUMO

The primordial immunoglobulin class, IgD, was the first non-IgM isotype discovered in teleosts. The crucial roles of IgM and IgZ in imparting systemic and mucosal immunity, respectively, in various fish species have been widely established. However, the putative function of a unique IgD isotype during pathogenic invasions has not been well explored. The present study reports the existence of an IgD ortholog in freshwater carp, Catla catla, and further evaluates its differential expression profile in response to bacterial, parasitic and viral antigenic exposure and pathogen associated molecular patterns (PAMPs) stimulation. The IgD of C. catla (CcIgD) cDNA sequence was found to encode 226 amino acids and confirmed homology with heavy chain delta region of Cyprinidae family members. Phylogenetic analysis of CcIgD exhibited greatest similarity with Ctenopharyngodon idella. qRT-PCR analysis revealed significant upregulation (P < 0.001) of IgD gene expression in kidney with respect to other tissues at 24 hr post-Aeromonas hydrophila challenge. CcIgD gene expression in skin was enhanced following Streptococcus uberis infection and in blood following Argulus infection and inactivated rhabdoviral antigen stimulation. Further, the treatment of bacterial and viral products (PAMPs) also triggered significant (P < 0.05) increases in CcIgD mRNA expression in kidney. These findings indicate the functional importance of teleost IgD in orchestrating tissue specific neutralization of antigens on stimulation with different pathogens and PAMPs.


Assuntos
Carpas/genética , Carpas/imunologia , Clonagem Molecular , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica , Imunoglobulina D/química , Imunoglobulina D/genética , Padrões Moleculares Associados a Patógenos , Aeromonas hydrophila/imunologia , Aeromonas hydrophila/patogenicidade , Sequência de Aminoácidos , Animais , Arguloida/patogenicidade , Infecções Bacterianas/imunologia , Cyprinidae/imunologia , DNA Complementar/genética , Doenças dos Peixes/microbiologia , Doenças dos Peixes/parasitologia , Doenças dos Peixes/virologia , Água Doce , Expressão Gênica , Perfilação da Expressão Gênica/veterinária , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunoglobulina D/classificação , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/química , Isotipos de Imunoglobulinas/genética , Rim , Doenças Parasitárias/imunologia , Filogenia , Rhabdoviridae/patogenicidade , Análise de Sequência de Proteína , Pele/imunologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus/patogenicidade , Viroses/imunologia
3.
J Proteome Res ; 16(7): 2560-2570, 2017 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-28516782

RESUMO

The full potential of recombinant Immunoglobulin A as therapeutic antibody is not fully explored, owing to the fact that structure-function relationships of these extensively glycosylated proteins are not well understood. Here monomeric IgA1, IgA2m(1), and IgA2m(2) variants of the anti-HER2 antibody (IgG1) trastuzumab were expressed in glyco-engineered Nicotiana benthamiana plants and in human HEK293-6E cells. All three IgA isotypes were purified and subjected to biophysical and biochemical characterization. While no differences in assembly, antigen binding, and glycosylation occupancy were observed, both systems vary tremendously in terms of glycan structures and heterogeneity of glycosylation. Mass-spectrometric analysis of site-specific glycosylation revealed that plant-produced IgAs carry mainly complex-type biantennary N-glycans. HEK293-6E-produced IgAs, on the contrary, showed very heterogeneous N-glycans with high levels of sialylation, core-fucose, and the presence of branched structures. The site-specific analysis revealed major differences between the individual N-glycosylation sites of each IgA subtype. Moreover, the proline-rich hinge region from HEK293-6E cell-derived IgA1 was occupied with mucin-type O-glycans, whereas IgA1 from N. benthamiana displayed numerous plant-specific modifications. Interestingly, a shift in unfolding of the CH2 domain of plant-produced IgA toward lower temperatures can be observed with differential scanning calorimetry, suggesting that distinct glycoforms affect the thermal stability of IgAs.


Assuntos
Imunoglobulina A/metabolismo , Isotipos de Imunoglobulinas/metabolismo , Polissacarídeos/química , Receptor ErbB-2/metabolismo , Trastuzumab/metabolismo , Especificidade de Anticorpos , Sequência de Carboidratos , Expressão Gênica , Glicosilação , Células HEK293 , Humanos , Imunoglobulina A/química , Imunoglobulina A/classificação , Imunoglobulina A/genética , Isotipos de Imunoglobulinas/química , Isotipos de Imunoglobulinas/classificação , Isotipos de Imunoglobulinas/genética , Mucinas/química , Mucinas/metabolismo , Polissacarídeos/metabolismo , Ligação Proteica , Receptor ErbB-2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/classificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Tabaco/genética , Tabaco/metabolismo , Trastuzumab/química
5.
Methods Mol Biol ; 1503: 63-82, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27743359

RESUMO

In recent years, high-throughput glycomics approaches have been developed and applied to either complete biofluids, cell lysates or tissues, or proteins isolated thereof. However, during such analyses the N-glycan are released from the protein backbone and therefore site- and protein-specific information is lost. There exists a need for high-throughput methods that allow quantification of site- and protein-specific glycosylation patterns from complex biological mixtures. We here describe the use of a multiple reaction monitoring mass spectrometry based method for the generation of glycopeptide profiles of the nine high abundance glycoproteins IgG, IgA, IgM, haptoglobin, alpha-1-antitrypsin, alpha-2-macroglobulin, alpha-1-acid glycoprotein, transferrin, and complement C3. We show that the sample preparation can be performed at the 96-well level, and using a 17-min gradient on a RP-UPLC-QQQ instrument, 96 samples can be analyzed within 3 days.


Assuntos
Proteínas Sanguíneas/química , Glicopeptídeos/química , Glicoproteínas/química , Espectrometria de Massas/métodos , Proteínas Sanguíneas/análise , Glicopeptídeos/sangue , Glicoproteínas/sangue , Glicosilação , Humanos , Isotipos de Imunoglobulinas/sangue , Isotipos de Imunoglobulinas/química
6.
Comput Biol Chem ; 64: 210-216, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27433817

RESUMO

The human leukocyte antigen class II (HLA II) molecules are implicated in the immunopathogenesis of allergic rhinitis (AR). The HLA II contains three allelic isotypes HLA-DR, -DQ, and -QP that exhibit considerably different susceptibility to AR. Here, we investigated the structural basis and energetic landscape of the susceptibility difference between the three HLA II isotypes to AR by combining computational analysis and experimental assay. Multiple sequence alignment revealed a low conservation among the three subtypes with sequence identity of ∼10% between them, suggesting that the peptide repertoires presented by HLA-DR, -DP and -DQ are not overlapped to each other, and they may be involved in different immune functions and dysfunctions. Structural analysis imparted that the antigenic peptides are rooted on the peptide-binding groove of HLA molecules and hold in a PPII-like helical conformation. Subsequently, the interaction behavior of 17 AR allergen-derived peptides with HLA-DR, -DP and -DQ was investigated using a statistics-based quantitative structure-activity relationship (QSAR) predictor. It was found a significant difference between the binding capabilities of these antigenic peptides to HLA-DR and to HLA-DP/-DQ; the former showed a generally higher affinity than the latter with p-value of 0.02 obtained from 2-tailed Student's t-test. The computational findings were then confirmed by HLA II-peptide stability assay, which demonstrated that the AR allergen-derived peptides have a high in vitro selectivity for HLA-DR over HLA-DP/-DQ. Thus, the HLA-DR isotype, rather than HLA-DP and -DQ, is expected to associate with the pathological process of AR.


Assuntos
Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Isotipos de Imunoglobulinas/imunologia , Rinite Alérgica/imunologia , Sequência de Aminoácidos , Simulação por Computador , Cristalografia por Raios X , Suscetibilidade a Doenças , Antígenos HLA-DQ/química , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/imunologia , Antígenos HLA-DR/química , Antígenos HLA-DR/genética , Antígenos HLA-DR/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Isotipos de Imunoglobulinas/química , Isotipos de Imunoglobulinas/genética , Rinite Alérgica/genética
7.
MAbs ; 8(6): 1107-17, 2016 Aug-Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27310175

RESUMO

Nivolumab, an anti-programmed death (PD)1 IgG4 antibody, has shown notable success as a cancer treatment. Here, we report that nivolumab was susceptible to aggregation during manufacturing, particularly in routine purification steps. Our experimental results showed that exposure to low pH caused aggregation of nivolumab, and the Fc was primarily responsible for an acid-induced unfolding phenomenon. To compare the intrinsic propensity of acid-induced aggregation for other IgGs subclasses, tocilizumab (IgG1), panitumumab (IgG2) and atezolizumab (aglyco-IgG1) were also investigated. The accurate pH threshold of acid-induced aggregation for individual IgG Fc subclasses was identified and ranked as: IgG1 < aglyco-IgG1 < IgG2 < IgG4. This result was cross-validated by thermostability and conformation analysis. We also assessed the effect of several protein stabilizers on nivolumab, and found mannitol ameliorated the acid-induced aggregation of the molecule. Our results provide valuable insight into downstream manufacturing process development, especially for immune checkpoint modulating molecules with a human IgG4 backbone.


Assuntos
Anticorpos Monoclonais/química , Antineoplásicos/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Isotipos de Imunoglobulinas/química , Anticorpos Monoclonais Humanizados/química , Citotoxicidade Celular Dependente de Anticorpos , Varredura Diferencial de Calorimetria , Humanos , Concentração de Íons de Hidrogênio , Manitol/química , Nivolumabe , Panitumumabe , Pontuação de Propensão , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína
8.
Microbiol Immunol ; 60(8): 561-7, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27301776

RESUMO

Immunoglobulins serve as a crucial arm of the adaptive immune system against detrimental pathogenic threats in teleosts. However, whether the novel Ig isotype IgZ is present in the Indian major carp, Catla catla, has not yet been elucidated. The present study reports the presence of IgZ ortholog in C. catla (CcIgZ) and further demonstrates its comparative tissue specific expression with IgM (CcIgM) in response to bacterial and parasitic stimulation. The putative 139 amino acid sequence of IgZ heavy chain cDNA of C. catla showed homology with IgZ constant domains of other teleosts. Phylogenetic analysis of the predicted IgZ transcript sequence clustered with previously identified IgZ heavy chain sequences of Cyprinidae family members. The inductive expression profiles of IgZ and IgM genes were evaluated in immunologically relevant tissues at 24, 48 and 72 hr post infection with Aeromonas hydrophila, Streptococcus uberis and Argulus sp. Both CcIgZ and CcIgM were expressed most strongly in the kidneys of healthy fish. Basal expression of CcIgM transcript was higher than that of CcIgZ in all the examined tissues. Stimulation with bacteria triggered significant increase of IgZ in the intestine (P < 0.001) and spleen (P < 0.01), whereas IgM was relatively up-regulated in blood (P < 0.001) after stimulation with each of the three pathogens assessed. The study is the first to report identification of IgZ in C. catla. Further, it provides insights into the differential expression profiles of IgZ and IgM isotypes against various pathogenic infection in C. catla, which may facilitate better prophylaxis again such infections.


Assuntos
Clonagem Molecular , Doenças dos Peixes/genética , Peixes/genética , Expressão Gênica , Cadeias Pesadas de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/genética , Imunoglobulina M/genética , Sequência de Aminoácidos , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/parasitologia , Peixes/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Isotipos de Imunoglobulinas/química , Imunoglobulina M/química , Filogenia , Transcriptoma
9.
Proteomics ; 16(9): 1321-30, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26960168

RESUMO

With mice being the top model organism in immunology and with Fc glycosylation being increasingly recognized as important modulator of antibody function, the time has come to take a look at the glycosylation of mouse IgG isotypes. Tryptic glycopeptides of mouse IgG1, IgG2, and IgG3 differ in mass and so these three isoforms can be easily discriminated by MS. Commercial IgG contained a rare IgG1 variant but no IgG3, which, however, was found in sera of C57BL/6 and BALB/c mice. These strains deviated with regard to IgG2a and IgG2b alleles. The Ig2a B allele was not observed in any of the four samples investigated. All a/c isotypes contain the same glycopeptide sequence, which deviates from that of IgG2b by containing Leu instead of Ile. The Leu/Ile glycopeptide variants were separated by RP chromatography and the order of elution was determined. The major glycoforms on all isotypes were fucosylated with no and one galactose (GnGnF and GnAF) followed by fully galactosylated AAF and smaller amounts of mono- and disialylated N-glycans. In the commercial serum pool, the relative ratios of glycans differed between isotypes. Sialic acid exclusively occurred as N-glycolylneuraminic acid. Fucosylation was essentially complete. No bisected and no α1,3-galactosylated glycans were found.


Assuntos
Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Isotipos de Imunoglobulinas/química , Alelos , Sequência de Aminoácidos , Animais , Glicosilação , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Imunoglobulina G/sangue , Isotipos de Imunoglobulinas/sangue , Isotipos de Imunoglobulinas/classificação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/isolamento & purificação , Ácidos Neuramínicos/química , Ácidos Neuramínicos/isolamento & purificação , Mapeamento de Peptídeos , Peptídeos/química , Peptídeos/imunologia , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
10.
Immunol Rev ; 270(1): 51-64, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26864104

RESUMO

Since the late 1990s, the use of transgenic animal platforms has transformed the discovery of fully human therapeutic monoclonal antibodies. The first approved therapy derived from a transgenic platform--the epidermal growth factor receptor antagonist panitumumab to treat advanced colorectal cancer--was developed using XenoMouse(®) technology. Since its approval in 2006, the science of discovering and developing therapeutic monoclonal antibodies derived from the XenoMouse(®) platform has advanced considerably. The emerging array of antibody therapeutics developed using transgenic technologies is expected to include antibodies and antibody fragments with novel mechanisms of action and extreme potencies. In addition to these impressive functional properties, these antibodies will be designed to have superior biophysical properties that enable highly efficient large-scale manufacturing methods. Achieving these new heights in antibody drug discovery will ultimately bring better medicines to patients. Here, we review best practices for the discovery and bio-optimization of monoclonal antibodies that fit functional design goals and meet high manufacturing standards.


Assuntos
Anticorpos Monoclonais Humanizados/biossíntese , Anticorpos Monoclonais Humanizados/uso terapêutico , Biotecnologia , Descoberta de Drogas , Camundongos Transgênicos , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/genética , Formação de Anticorpos , Engenharia Genética , Humanos , Hibridomas/imunologia , Hibridomas/metabolismo , Isotipos de Imunoglobulinas/biossíntese , Isotipos de Imunoglobulinas/química , Isotipos de Imunoglobulinas/genética , Camundongos
11.
Artif Cells Nanomed Biotechnol ; 44(2): 532-9, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-25307269

RESUMO

Tuberculosis (TB) is one of the major devastating diseases in the world, mainly caused by Mycobacterium tuberculosis. Furthermore, multi-drug resistant TB and extremely drug resistant TB are becoming big problems globally. Bacillus Calmette-Guerin (BCG) is the only available vaccine which provides protection against TB. The BCG vaccine is effective in children but not recommended in adults and elderly patients due to an associated low risk of infection with Mycobacterium tuberculosis and variable effectiveness of the vaccine. The main aim of this research study is to develop such a vaccine which will provide a better and safer profile in children and adults, as well as in elderly patients. In this present study, we prepared pulmonary tubercular vaccine by using an Antigen 85 complex (Ag85)-loaded nanocarrier such as the immunostimulating complex (ISCOM). Immunological outcomes clearly indicated significant improvement in humoral as well as cellular immune responses after pulmonary immunization with ISCOMs containing Quil A in mice.


Assuntos
Aciltransferases/química , Aciltransferases/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Portadores de Fármacos/química , ISCOMs/química , Tuberculose Pulmonar/prevenção & controle , Vacinação , Animais , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Feminino , Hemólise/efeitos dos fármacos , Humanos , ISCOMs/imunologia , ISCOMs/toxicidade , Isotipos de Imunoglobulinas/sangue , Isotipos de Imunoglobulinas/química , Camundongos , Membrana Mucosa/imunologia , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/fisiologia , Nanoestruturas/química
12.
Immunol Rev ; 268(1): 328-39, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26497531

RESUMO

Antibodies are key molecules in the fight against infections. Although previously thought to mediate protection solely in the extracellular environment, recent research has revealed that antibody-mediated protection extends to the cytosolic compartment of cells. This postentry viral defense mechanism requires binding of the antibody to a cytosolic Fc receptor named tripartite motif containing 21 (TRIM21). In contrast to other Fc receptors, TRIM21 shows remarkably broad isotype specificity as it does not only bind IgG but also IgM and IgA. When viral pathogens coated with these antibody isotypes enter the cytosol, TRIM21 is rapidly recruited and efficient neutralization occurs before the virus has had the time to replicate. In addition, inflammatory signaling is induced. As such, TRIM21 acts as a cytosolic sensor that engages antibodies that have failed to protect against infection in the extracellular environment. Here, we summarize our current understanding of how TRIM21 orchestrates humoral immunity in the cytosolic environment.


Assuntos
Especificidade de Anticorpos/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Isotipos de Imunoglobulinas/imunologia , Isotipos de Imunoglobulinas/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Citosol/metabolismo , Ativação Enzimática , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Isotipos de Imunoglobulinas/química , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Ribonucleoproteínas/química , Ubiquitina-Proteína Ligases/metabolismo
13.
Methods Mol Biol ; 1318: 1-14, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26160559

RESUMO

Immunoglobulins (Ig) or antibodies are powerful molecular recognition tools that can be used to identify minute quantities of a given target analyte. Their antigen-binding properties define both the sensitivity and selectivity of an immunoassay. Understanding the biochemical properties of this class of protein will provide users with the knowledge necessary to select the appropriate antibody composition to maximize immunoassay results. Here we define the general biochemical properties of antibodies and their similarities and differences, explain how these properties influence their functional relationship to an antigen target, and describe a method for the enzymatic fragmentation of antibodies into smaller functional parts.


Assuntos
Anticorpos/isolamento & purificação , Cromatografia de Afinidade/métodos , Imunoensaio , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Fragmentos Fc das Imunoglobulinas/isolamento & purificação , Animais , Anticorpos/química , Afinidade de Anticorpos , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos/química , Epitopos/química , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , Isotipos de Imunoglobulinas/química , Isotipos de Imunoglobulinas/isolamento & purificação , Subunidades de Imunoglobulinas/química , Subunidades de Imunoglobulinas/isolamento & purificação , Modelos Moleculares , Papaína/química , Ligação Proteica , Sensibilidade e Especificidade , Proteína Estafilocócica A/química
14.
Methods Mol Biol ; 1318: 29-41, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26160561

RESUMO

Antibodies are provided in a variety of formats that include antiserum, hybridoma culture supernatant, or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facilitate assay reproducibility, economy, and reduced interference of nonspecific components as well as improved storage, stability, and bio-conjugation. Although not always necessary, the relative simplicity of antibody purification using commercially available protein-A, protein-G, or protein-L resins with basic chromatographic principles warrants purification when antibody source material is available in sufficient quantity. Here, we define three simple methods using immobilized (1) protein-A, (2) protein-G, and (3) protein-L agarose beads to yield highly purified antibody.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Proteínas de Bactérias/química , Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Proteína Estafilocócica A/química , Animais , Ascite/metabolismo , Cromatografia de Afinidade/instrumentação , Cromatografia Líquida/instrumentação , Meios de Cultivo Condicionados/química , Humanos , Hibridomas/metabolismo , Proteínas Imobilizadas/química , Soros Imunes/química , Isotipos de Imunoglobulinas/química , Camundongos , Ligação Proteica
15.
Blood Rev ; 29(6): 369-76, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26006097

RESUMO

Autoimmune hemolytic anemia (AIHA) is a rare autoimmune disease characterized by a hemolytic anemia caused by autoantibodies against red blood cells (RBCs). These autoantibodies are routinely detected via the direct antiglobulin test (DAT). As expected, the DAT score correlates with the presence of clinical symptoms, but this correlation is far from perfect. Regularly, strongly positive DAT scores are encountered with no sign of hemolysis, while severe hemolysis can be seen even in patients with a negative DAT score. Apparently, the mere amount of antibody is not the sole predictor of disease. In this paper, we review the current literature on aspects of both the autoantibodies and the patients' clearance system that together determine the clinical significance of an anti-RBC autoantibody, ranging from antibody isotype to antibody Fc-glycosylation to alternative clearance mechanisms. From this, the ensemble of tests is inferred that in our view best assesses the main determinants for pathogenicity of autoantibodies.


Assuntos
Anemia Hemolítica Autoimune/diagnóstico , Autoanticorpos/análise , Teste de Coombs/estatística & dados numéricos , Eritrócitos/imunologia , Isotipos de Imunoglobulinas/análise , Anemia Hemolítica Autoimune/sangue , Anemia Hemolítica Autoimune/patologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Autoanticorpos/biossíntese , Autoanticorpos/química , Eritrócitos/patologia , Hemólise/imunologia , Humanos , Isotipos de Imunoglobulinas/química , Fagocitose , Ligação Proteica
17.
Thromb Haemost ; 112(5): 972-80, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25104096

RESUMO

Replacement therapy is currently used to prevent and treat bleeding episodes in coagulation factor deficiencies. However, structural differences between the endogenous and therapeutic proteins might increase the risk for immune complications. This study was aimed at identifying factor (F)VII variants resistant to inhibitory antibodies developed after treatment with recombinant activated factor VII (rFVIIa) in a FVII-deficient patient homozygous for the p.A354V-p.P464Hfs mutation, which predicts trace levels of an elongated FVII variant in plasma. We performed fluorescent bead-based binding, ELISA-based competition as well as fluorogenic functional (activated FX and thrombin generation) assays in plasma and with recombinant proteins. We found that antibodies displayed higher affinity for the active than for the zymogen FVII (half-maximal binding at 0.54 ± 0.04 and 0.78 ± 0.07 BU/ml, respectively), and inhibited the coagulation initiation phase with a second-order kinetics. Isotypic analysis showed a polyclonal response with a large predominance of IgG1. We hypothesised that structural differences in the carboxyl-terminus between the inherited FVII and the therapeutic molecules contributed to the immune response. Intriguingly, a naturally-occurring, poorly secreted and 5-residue truncated FVII (FVII-462X) escaped inhibition. Among a series of truncated rFVII molecules, we identified a well-secreted and catalytically competent variant (rFVII-464X) with reduced binding to antibodies (half-maximal binding at 0.198 ± 0.003 BU/ml) as compared to the rFVII-wt (0.032 ± 0.002 BU/ml), which led to a 40-time reduced inhibition in activated FX generation assays. Taken together our results provide a paradigmatic example of mutation-related inhibitory antibodies, strongly support the FVII carboxyl-terminus as their main target and identify inhibitor-resistant FVII variants.


Assuntos
Fator VII/imunologia , Fator VIIa/imunologia , Isoanticorpos/imunologia , Sequência de Aminoácidos , Reações Antígeno-Anticorpo , Coagulação Sanguínea , Fator VII/antagonistas & inibidores , Fator VII/química , Fator VII/genética , Deficiência do Fator VII/tratamento farmacológico , Fator VIIa/química , Fator VIIa/uso terapêutico , Fator Xa/biossíntese , Mutação da Fase de Leitura , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Isotipos de Imunoglobulinas/química , Isotipos de Imunoglobulinas/imunologia , Isoanticorpos/química , Dados de Sequência Molecular , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , Deleção de Sequência , Relação Estrutura-Atividade , Trombina/biossíntese
18.
BMC Vet Res ; 10: 134, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24938323

RESUMO

BACKGROUND: Oral fluid collected by means of ropes has the potential to replace serum for monitoring and surveillance of important swine pathogens. Until now, the most commonly used method to collect oral fluid is by hanging a cotton rope in a pen. However, concerns about the influence of rope material on subsequent immunological assays have been raised. In this study, we evaluated six different rope materials for the collection of oral fluid and the subsequent detection of total and PRRSV-specific antibodies of different isotypes in oral fluid collected from PRRSV-vaccinated and infected pigs. RESULTS: An initial experiment showed that IgA is the predominant antibody isotype in porcine saliva. Moreover, it was found that synthetic ropes may yield higher amounts of IgA, whereas all rope types seemed to be equally suitable for IgG collection. Although IgA is the predominant antibody isotype in porcine oral fluid, the PRRSV-specific IgA-based IPMA and ELISA tests were clearly not ideal for sensitive detection of PRRSV-specific IgA antibodies. In contrast, PRRSV-specific IgG in oral fluids was readily detected in PRRSV-specific IgG-based IPMA and ELISA tests, indicating that IgG is a more reliable isotype for monitoring PRRSV-specific antibody immunity in vaccinated/infected animals via oral fluids with the currently available tests. CONCLUSIONS: Since PRRSV-specific IgG detection seems more reliable than PRRSV-specific IgA detection for monitoring PRRSV-specific antibody immunity via oral fluids, and since all rope types yield equal amounts of IgG, it seems that the currently used cotton ropes are an appropriate choice for sample collection in PRRSV monitoring.


Assuntos
Anticorpos Antivirais/química , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Saliva/química , Manejo de Espécimes/veterinária , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Cannabis , Fibra de Algodão , Feminino , Isotipos de Imunoglobulinas/sangue , Isotipos de Imunoglobulinas/química , Nylons , Poliésteres , Síndrome Respiratória e Reprodutiva Suína/sangue , Síndrome Respiratória e Reprodutiva Suína/virologia , Manejo de Espécimes/instrumentação , Suínos
19.
Immunogenetics ; 66(5): 335-51, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24676685

RESUMO

Lungfish (Dipnoi) are the closest living relatives to tetrapods, and they represent the transition from water to land during vertebrate evolution. Lungfish are armed with immunoglobulins (Igs), one of the hallmarks of the adaptive immune system of jawed vertebrates, but only three Ig forms have been characterized in Dipnoi to date. We report here a new diversity of Ig molecules in two African lungfish species (Protopterus dolloi and Protopterus annectens). The African lungfish Igs consist of three IgMs, two IgWs, three IgNs, and an IgQ, where both IgN and IgQ originated evidently from the IgW lineage. Our data also suggest that the IgH genes in the lungfish are organized in a transiting form from clusters (IgH loci in cartilaginous fish) to a translocon configuration (IgH locus in tetrapods). We propose that the intraclass diversification of the two primordial gnathostome Ig classes (IgM and IgW) as well as acquisition of new isotypes (IgN and IgQ) has allowed lungfish to acquire a complex and functionally diverse Ig repertoire to fight a variety of microorganisms. Furthermore, our results support the idea that "tetrapod-specific" Ig classes did not evolve until the vertebrate adaptation to land was completed ~360 million years ago.


Assuntos
Peixes/classificação , Peixes/genética , Variação Genética , Cadeias Pesadas de Imunoglobulinas/genética , Sequência de Aminoácidos , Animais , Enterobacteriaceae , Infecções por Enterobacteriaceae/genética , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Cadeias Pesadas de Imunoglobulinas/química , Isotipos de Imunoglobulinas/química , Isotipos de Imunoglobulinas/genética , Imunoglobulina M/química , Imunoglobulina M/genética , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Filogenia , Alinhamento de Sequência , Transcriptoma
20.
Mol Immunol ; 56(4): 588-98, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23911417

RESUMO

Until relatively recently the immunoglobulin molecule was viewed as composed of two independent domains comprised of the variable (V) and constant (C) regions. However, recent work has established that the C region mediates allosteric changes in the V region that can influence specificity and affinity. To further explore cross-domain interrelationship in murine IgG structure we carried out solution small angle X-ray scattering (SAXS) measurements for four V region identical IgG isotypes. SAXS analysis revealed elongated Y-shaped structures in solution with significantly different, isotype-dependent domain orientations. To further explore local C region effects on the V region, the IgG3 Fab crystal structure from the same family was determined to 2.45 Å resolution. The IgG3 Fab crystal structure differs from a closely related previously solved IgG1 Fab revealing significant structural differences, which may account for isotype-related specificity differences in V region identical Abs. Among the four murine isotypes, IgG3 was the most different in solution with regards to overall structure as well as aggregate formation in solution suggesting that the greater apparent affinity of this isotype resulted from polyvalent complexes with enhanced avidity. Our results provide additional evidence that Ig V and C domains influence each other structurally and suggest that V region structure can have significant effects on overall Ig structure.


Assuntos
Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Isotipos de Imunoglobulinas/química , Região Variável de Imunoglobulina/química , Animais , Cristalografia por Raios X , Regiões Constantes de Imunoglobulina/química , Camundongos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X
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