RESUMO
Bifunctional antibodies were prepared using the principle of solid-phase synthesis. The two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')2 fragments from intact IgG were prepared using an immobilized pepsin column. Goat, mouse and human antibodies were digested completely within 4 h. The F(ab')2 fragments thus produced did not contain any IgG impurities. The Fab' fragments were produced by reducing the inter-heavy chain disulfide bonds using 2-mercaptoethylamine. The use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of the bifunctional antibody preparation and the rapid optimization of reaction conditions.
Assuntos
Anticorpos Biespecíficos/biossíntese , Imunoglobulina G/biossíntese , Animais , Anticorpos Biespecíficos/química , Cromatografia em Gel , Reagentes de Ligações Cruzadas , Enzimas Imobilizadas , Cabras , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/química , Imunoglobulina G/química , Mercaptoetilaminas , Camundongos , Oxirredução , Pepsina ARESUMO
This review summarizes our recent in vitro studies of the factors affecting the tumor penetration of immunoconjugates. The studies were designed to probe the mechanisms of diffusion and convection, using a cultured layer of mouse melanoma cells as a model tumor cell layer and an antibody to the murine transferrin receptor as a model ligand. Transport of the binding antibody was observed to be slower than that of a non-binding control, a result that is consistent with the "binding site barrier" hypothesis (Fujimori et al., J. Nucl. Med., 31: 1191-1198, 1990). Internalization of the antibody/receptor complex was necessary for this effect to be observed, implying that intracellular trafficking is a determinant of net tumor transport rates. Convective fluid flow exhibited a dependence on cell density that is consistent with a Poiseuille flow model, suggesting that convective transport occurs as laminar flow in tortuous channels. Implications for immunoconjugate therapy, limitations of the approach, and future directions of the research program are discussed.
Assuntos
Anticorpos/administração & dosagem , Animais , Anticorpos/imunologia , Sítios de Ligação de Anticorpos , Difusão , Relação Dose-Resposta Imunológica , Endocitose , Camundongos , Receptores da Transferrina/imunologia , Células Tumorais CultivadasRESUMO
This paper describes a project for the establishment of a cancer pain clinic in a developing country. The project was conducted according to guidelines from the World Health Organization and utilized a link with an existing cancer pain clinic in the UK. The principal methods used for establishing the new pain clinic included: an assessment of barriers to effective cancer pain control, teaching programs for nurses and trainee doctors, educational links with a UK cancer pain clinic, and analgesic guidelines and introduction of a pain assessment tool. As a result of these interventions, a new cancer pain clinic was founded. The methods used serve as one possible model for establishing cancer pain treatment facilities in developing countries.
Assuntos
Países em Desenvolvimento , Neoplasias/complicações , Clínicas de Dor/organização & administração , Dor/etiologia , Comportamento Cooperativo , Humanos , Clínicas de Dor/normas , Sri Lanka , Reino UnidoRESUMO
The convective transport of fluid and of a binding antibody through a cultured tumor cell layer was investigated with a mouse melanoma cell line (B16F10) grown on a microporous polycarbonate filter (Snapwell inserts). The inserts were precoated with Matrigel or collagen, or were uncoated. The cell layers were exposed to nominal pressure gradients from 5 to 25 cm H2O, and the volume flux was measured by collecting the effluent volume over time. The rate of convective transport of a binding monoclonal antibody that recognizes the murina transferrin receptor (a-TfR) was investigated at a nominal pressure gradient of 15 cm H2O and compared with that of an isotype matched, nonbinding control. The resistance, R, of the cell layer to fluid flow was quantified as the hydraulic conductivity, Lp (= 1/R); the ability of the cell layer to retard antibody transport was quantified as the reflection coefficient, sigma. The resulting Lp values decreased with increasing cell density, in a manner consistent with Poiseuille flow. Collagen or Matrigel precoating also decreased Lp values, with cells grown on Matrigel providing the greatest resistance. The sigma values were 0.67 (+/-0.08) for the a-TfR antibody and 0.51 (+/-0.06) for the control, indicating that the cell layer acts as a semipermeable barrier to convective transport of antibody that is less permeable to the binding antibody.
Assuntos
Anticorpos Monoclonais/metabolismo , Técnicas de Cultura de Células/métodos , Animais , Transporte Biológico , Contagem de Células , Melanoma Experimental/imunologia , Camundongos , Microscopia Eletrônica , Pressão , Receptores da Transferrina/imunologia , Células Tumorais CultivadasRESUMO
Immunoassays are bioanalytical methods in which quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. Although applicable to the analysis of both low molecular weight xenobiotic and macromolecular drugs, these procedures currently find most consistent application in the pharmaceutical industry to the quantitation of protein molecules. Immunoassays are also frequently applied in such important areas as the quantitation of biomarker molecules which indicate disease progression or regression, and antibodies elicited in response to treatment with macromolecular therapeutic drug candidates. Currently available guidance documents dealing with the validation of bioanalytical methods address immunoassays in only a limited way. This review highlights some of the differences between immunoassays and chromatographic assays, and presents some recommendations for specific aspects of immunoassay validation. Immunoassay calibration curves are inherently nonlinear, and require nonlinear curve fitting algorithms for best description of experimental data. Demonstration of specificity of the immunoassay for the analyte of interest is critical because most immunoassays are not preceded by extraction of the analyte from the matrix of interest. Since the core of the assay is an antigen-antibody reaction, immunoassays may be less precise than chromatographic assays; thus, criteria for accuracy (mean bias) and precision, both in pre-study validation experiments and in the analysis of in-study quality control samples, should be more lenient than for chromatographic assays. Application of the SFSTP (Societe Francaise Sciences et Techniques Pharmaceutiques) confidence interval approach for evaluating the total error (including both accuracy and precision) of results from validation samples is recommended in considering the acceptance/rejection of an immunoassay procedure resulting from validation experiments. These recommendations for immunoassay validation are presented in the hope that their consideration may result in the production of consistently higher quality data from the application of these methods.
Assuntos
Imunoensaio/normas , Algoritmos , Calibragem , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Success in generating catalytic antibodies as enzyme mimics lies in the strategic design of the transition-state analog (TSA) for the reaction of interest, and careful development of screening processes for the selection of antibodies that are catalysts. Typically, the choice of TSA structure is straightforward, and the criterion for selection in screening is often binding of the TSA to the antibody in a microtiter-plate assay. This article emphasizes the problems of TSA design in complex reactions and the importance of selecting antibodies on the basis of catalysis as well as binding to the TSA. The target reaction is the derivatization of primary amines with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide ion. The desired outcome is selective catalysis of formation of the fluorescent derivative in preference to nonfluorescent side-products. In the study, TSA design was directed toward the reaction branch leading to the fluorescent product. Here, we describe a microtiter plate-based assay that is capable of detecting antibodies showing catalytic activity at an early stage. Of the antibodies selected, 36% showed no appreciable binding to any of the substrates tested, but did show catalytic activity in derivatizing one or more of the amino acids screened. In contrast, only two out of 77 clones that showed binding did not show catalysis. Thus, in this complex system, observation of binding is a good predictor of the presence of catalytic activity, and failure to observe binding is a poor predictor of the absence of catalytic activity.
Assuntos
Anticorpos Catalíticos/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Catálise , Cianetos/metabolismo , Corantes Fluorescentes/metabolismo , Haptenos/química , Hibridomas/imunologia , Técnicas In Vitro , Camundongos , Naftalenos/imunologia , Naftalenos/metabolismoRESUMO
The synthesis of bifunctional antibodies using the principle of solid-phase synthesis is described. Two Fab' fragments were chemically linked together via a bismaleimide crosslinking reagent. The F(ab')(2) fragments from intact immunoglobulin G (IgG) were prepared using an immobilized pepsin column. Goat, mouse, and human antibodies were digested completely within 4 h. The F(ab')(2) fragments thus produced did not contain any IgG impurities. Fab' fragments were produced by reducing the heavy interchain disulfide bonds using 2-mercaptoethylamine. Use of the solid-phase reactor in the preparation of the bifunctional antibodies eliminated many of the time-consuming separation steps between the fragmentation and conjugation steps. This procedure facilitates the automation of bifunctional antibody preparation and the rapid optimization of reaction conditions.