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1.
J Immunol ; 178(11): 7467-72, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17513798

RESUMO

Evaluation of the immunogenicity of panitumumab, a fully human anti-epidermal growth factor receptor mAb approved for use in colorectal cancer patients, led to the development of two separate immunoassays for the detection of anti-panitumumab Abs. The first immunoassay used a bridging ELISA capable of detecting 10 ng/ml positive control anti-panitumumab Ab. The ELISA incorporated an acid dissociation step to reduce drug interference and tolerated the presence of approximately 100-fold molar excess of drug. During eight clinical trials, the ELISA detected developing Ab responses in 2 of 612 (0.3%) subjects. In one of the ELISA positive subjects, neutralizing Abs were detected using an epidermal growth factor receptor phosphorylation bioassay. The second immunoassay used a Biacore biosensor immunoassay format capable of detecting 1 mug/ml positive control Ab while tolerating the presence of equal molar amounts of drug. Although less sensitive and less tolerant to competing drug in the assay, the Biacore assay detected developing Ab responses in 25 of the 604 (4.1%) subjects. Additionally, the Biacore assay identified eight subjects who developed neutralizing Abs. Mouse mAbs with affinities ranging from 1.1 x 10(-6) to 8.4 x 10(-10) M were used to characterize both assay types. The ELISA was more sensitive for the detection of higher affinity mAbs and detected high-affinity mAbs in the presence of higher molar ratio of drug to mAb. The Biacore assay was more sensitive for detection of lower affinity mAbs and detected low affinity Abs in the presence of higher molar ratios of drug to mAb.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Ressonância de Plasmônio de Superfície/métodos , Animais , Anticorpos Anti-Idiotípicos/biossíntese , Anticorpos Anti-Idiotípicos/sangue , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Linhagem Celular Tumoral , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática/normas , Mapeamento de Epitopos , Receptores ErbB/imunologia , Feminino , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Panitumumabe , Sensibilidade e Especificidade , Ressonância de Plasmônio de Superfície/normas
2.
J Immunol Methods ; 308(1-2): 101-8, 2006 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-16356511

RESUMO

Neutralizing antibodies against therapeutic proteins can be potentially harmful if the antibody blocks not only the therapeutic activities of the therapeutic protein but also the normal functions of the endogenous counterpart. Detection of the neutralizing anti-therapeutic protein antibodies generally relies on bioassays measuring changes in the biologic activity of the therapeutic protein triggered by the presence of the antibody. Most of the bioassays, particularly the cell-based in vitro assays, fail to detect neutralizing anti-therapeutic protein antibodies when the remaining therapeutic protein level in the assay samples is high. The remaining therapeutic protein, either a free molecule or an immune complex with anti-therapeutic protein antibodies, can inhibit the neutralizing activity of the antibody and prevent detection. We describe the development of a procedure that uses acid dissociation and affinity adsorption to remove therapeutic protein from assay samples. With this procedure, we can detect the presence of neutralizing anti-therapeutic protein antibodies from samples containing high levels of therapeutic protein.


Assuntos
Anticorpos/sangue , Testes de Neutralização/métodos , Proteínas Recombinantes/sangue , Proteínas Recombinantes/imunologia , Anticorpos Monoclonais/sangue , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/isolamento & purificação , Linhagem Celular , Humanos , Concentração de Íons de Hidrogênio , Técnicas de Imunoadsorção , Técnicas In Vitro , Interleucina-8/biossíntese , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/imunologia , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/uso terapêutico
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