RESUMO
The ability of small interfering RNA (siRNA) to efficiently silence the expression of specific genes provides the basis for exciting new therapies based on RNA interference (RNAi). The efficient intracellular delivery of siRNA from cell uptake through the endosomal trafficking pathways into the cytoplasm remains a significant challenge. Previously we described the synthesis of a new family of diblock copolymer siRNA carriers using controlled reversible addition-fragmentation chain transfer (RAFT) polymerization. The carriers were composed of a positively charged block of dimethylaminoethyl methacrylate (DMAEMA) to mediate siRNA binding and a second pH-responsive endosome releasing block composed of DMAEMA and propylacrylic acid (PAA) in roughly equimolar ratios and butyl methacylate (BMA). Here we describe the development of a new generation of siRNA delivery polymers based on this design that exhibit enhanced transfection efficiency and low cytotoxicity. This design incorporates a longer endosomolytic block with increased hydrophobic content to induce micelle formation. These polymers spontaneously form spherical micelles in the size range of 40 nm with CMC (critical micelle concentration) values of approximately 2 µg/mL based on dynamic light scattering (DLS), (1)H NMR, electron microscopy, and selective partitioning of the small molecule pyrene into the hydrophobic micelle core. The siRNA binding to the cationic shell block did not perturb micelle stability or significantly increase particle size. The self-assembly of the diblock copolymers into particles was shown to provide a significant enhancement in mRNA knockdown at siRNA concentrations as low as 12.5 nM. Under these conditions, the micelle-based systems showed an 89% reduction in GAPDH mRNA levels as compared to only 23% (10 nM siRNA) for the nonmicelle system. The reduction in mRNA levels becomes nearly quantitative as the siRNA concentration is increased to 25 nM and higher. Flow cytometry analysis of fluorescent-labeled siRNA showed uptake in 90% of cells and a 3-fold increase in siRNA per cell compared to a standard lipid transfection agent. These results demonstrate the potential utility of this carrier design for siRNA drug delivery.
Assuntos
Portadores de Fármacos/química , Polímeros/química , RNA Interferente Pequeno/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Portadores de Fármacos/síntese química , Portadores de Fármacos/toxicidade , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Micelas , Tamanho da Partícula , Polímeros/síntese química , Polímeros/toxicidade , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/toxicidade , Relação Estrutura-Atividade , Propriedades de SuperfícieRESUMO
X-ray absorption spectroscopy is an element-specific technique for probing the local atomic-scale environment around an absorber atom. It is widely used to investigate the structures of liquids and solids, being especially valuable for characterization of solid-supported catalysts. Reported cell designs are limited in capabilities-to fluorescence or transmission and to static or flowing atmospheres, or to vacuum. Our goal was to design a robust and widely applicable cell for catalyst characterizations under all these conditions-to allow tracking of changes during genesis and during operation, both under vacuum and in reactive atmospheres. Herein, we report the design of such a cell and a demonstration of its operation both with a sample under dynamic vacuum and in the presence of gases flowing at temperatures up to 300 °C, showing data obtained with both fluorescence and transmission detection. The cell allows more flexibility in catalyst characterization than any reported.
RESUMO
This study comprehensively investigates the changing biodistribution of fluorescent-labelled polystyrene latex bead nanoparticles in a mouse model of inflammation. Since inflammation alters systemic circulatory properties, increases vessel permeability and modulates the immune system, we theorised that systemic inflammation would alter nanoparticle distribution within the body. This has implications for prospective nanocarrier-based therapies targeting inflammatory diseases. Low dose lipopolysaccharide (LPS), a bacterial endotoxin, was used to induce an inflammatory response, and 20 nm, 100 nm or 500 nm polystyrene nanoparticles were administered after 16 hours. HPLC analysis was used to accurately quantify nanoparticle retention by each vital organ, and tissue sections revealed the precise locations of nanoparticle deposition within key tissues. During inflammation, nanoparticles of all sizes redistributed, particularly to the marginal zones of the spleen. We found that LPS-induced inflammation induces splenic macrophage polarisation and alters leukocyte uptake of nanoparticles, with size-dependent effects. In addition, spleen vasculature becomes significantly more permeable following LPS treatment. We conclude that systemic inflammation affects nanoparticle distribution by multiple mechanisms, in a size dependent manner.
Assuntos
Corantes Fluorescentes , Inflamação/metabolismo , Nanopartículas , Animais , Cromatografia Líquida de Alta Pressão , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Masculino , Camundongos , Nanopartículas/química , Nanopartículas/metabolismo , Tamanho da Partícula , Baço/química , Baço/metabolismo , Distribuição TecidualRESUMO
The outgrowth of corneal epithelial cells onto a polymeric substrate is expected to be the primary event in the epithelialization of a synthetic corneal graft. To study the effects of polymer surface properties on corneal epithelial cell outgrowth, a quantitative in vitro cell outgrowth assay was done. Polymers with systematic variations in hydroxyl content were used as outgrowth substrates. These polymers were characterized by electron spectroscopy for chemical analysis for elemental surface composition and by captive air-bubble contact angle for surface wettability. Circular corneal buttons were punched from excised rabbit corneas, placed onto these substrates, and incubated in a hormonally enriched culture medium. Outgrowth of the epithelial cells was allowed to proceed onto the substrates for 4 days. The outgrowth areas were measured, and an outgrowth index was calculated for each substrate by comparing it with tissue culture polystyrene substrate. The highest outgrowth generally occurred on substrates with intermediate wettabilities (captive air-bubble contact angles of approximately 45-75 degree); it was less on substrates of lower or higher wettabilities. Protein coatings of albumin, immunoglobulin G (IgG), fibronectin, and culture medium were found to lower the wettabilities of native substrates. Albumin and IgG precoating were shown to reduce epithelial cell outgrowth; fibronectin precoating was shown to improve outgrowth on most substrates. These results suggest that epithelial cell outgrowth is influenced by both substrate and protein interactions.
Assuntos
Córnea/citologia , Polímeros , Actinas/metabolismo , Adsorção , Albuminas/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Córnea/efeitos dos fármacos , Células Epiteliais , Epitélio/efeitos dos fármacos , Fator VIII/metabolismo , Fibronectinas/farmacologia , Imunofluorescência , Imunoglobulina G/farmacologia , Queratinas/metabolismo , Cinética , Metacrilatos , Metilmetacrilatos , Propriedades de Superfície , Vimentina/metabolismoRESUMO
The conjugate of protein A with poly(N-isopropylacrylamide) was synthesized and utilized in the separation of human immunogammaglobulin. In the separation process, poly(N-isopropylacrylamide)-protein A conjugate binds to the immunoglobulin with high specificity to form the poly(N-isopropylacrylamide)-protein A/immunoglobulin complex. The complex can be conveniently separated by precipitation upon heating above the lower critical solution temperature of the poly(N-isopropylacrylamide)-protein A/immunogammaglobulin complex. The separation capacity of poly(N-isopropylacrylamide)-protein A conjugate for human immunogammaglobulin was studied and it was demonstrated that approximately one out of every four protein A molecules binds to human immunogammaglobulin with a dissociation constant (Ks) of 3 x 10(-6) M. The affinity precipitation separation of human immunogammaglobulin is a rapid process which avoids the need for chromatographic columns. It can also be designed to run in a continuous mode.
Assuntos
Resinas Acrílicas/química , Imunoglobulina G/química , Proteína Estafilocócica A/química , Precipitação Química , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/metabolismo , Proteína Estafilocócica A/metabolismoRESUMO
Poly (N-isopropylacrylamide) (p(NIPAAm] exhibits a cloud point around 32 degrees C. In this study we have conjugated several proteins to p(NIPAAm), and the effects of protein molecular weight, pH and ionic strength on the cloud point of the p(NIPAAm)-protein conjugates have been studied. The cloud points of p(NIPAAm)-protein conjugates appear to be unaffected by the molecular weight or isoelectric point (pl) of the proteins, the pH or ionic strength.
Assuntos
Resinas Acrílicas/química , Preparações de Ação Retardada/química , Polímeros/química , Proteínas/química , Fenômenos Químicos , Físico-Química , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Peso MolecularRESUMO
The high affinity recognition of biotin and biotinylated molecules has made streptavidin one of the most important components in diagnostics and laboratory kits. While it is extremely useful as the native protein, there are many applications where its function can be improved re-engineering the subunits. We review here our efforts to construct streptavidin tetramers that have 'smart' recognition capabilities, and which display functional peptide sequences. These smart and biofunctional streptavidin derivatives can 'talk' to cells, and 'listen' to external signals which control capture and release of biotinylated molecules.
Assuntos
Estreptavidina/química , Estreptavidina/metabolismo , Marcadores de Afinidade , Sítios de Ligação , Biotecnologia , Biotina/metabolismo , Modelos Moleculares , Conformação Proteica , Engenharia de Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Estreptavidina/genéticaRESUMO
This paper reviews the composition and synthesis of hydrogels, the character of their absorbed water, and permeation of solutes within their swollen matrices. The most important properties of hydrogels relevant to their biomedical applications are also identified, in particular for use of hydrogels as drug and cell carriers, and as tissue engineering matrices.
Assuntos
Materiais Biocompatíveis , Hidrogéis , ÁguaRESUMO
We have designed a new mucoadhesive drug delivery formulation based on H-bonded complexes of poly(acrylic acid) (PAA) or poly(methacrylic acid) (PMAA) with the poly(ethylene glycol) (PEG), of a (PEG)-drug conjugate. The PEGylated prodrugs are synthesized with degradable PEG-anhydride-drug bonds for eventual delivery of free drug from the formulation. In this work we have used indomethacin as the model drug which is PEGylated via anhydride bonds to the PEG. The complexes are designed first to dissociate as the formulation swells in contact with mucosal surfaces at pH 7.4, releasing PEG-indomethacin, which then hydrolyses to release free drug and free PEG. We found that as MW of PAA increases, the dissociation rate of the complex decreases, which results in decreased rate of release of the drug. On the other hand, the drug release from PEG-indomethacin alone and from solid mixture of PEG-indomethacin+PAA was much faster than that from the H-bonded complexes. Due to the differences in the thermal stability, PMAA complex exhibited slightly faster drug release than that of the PAA complex of comparable MW. These H-bonded complexes of degradable PEGylated drugs with bioadhesive polymers should be useful for mucosal drug delivery.
Assuntos
Resinas Acrílicas/química , Portadores de Fármacos/química , Polietilenoglicóis/química , Ácidos Polimetacrílicos/química , Adesivos , Anidridos/química , Concentração de Íons de Hidrogênio , Hidrólise , Indometacina/química , Espectroscopia de Ressonância Magnética , Mucosa , Espectrofotometria Infravermelho , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
An AB block copolymer of oligo(methyl methacrylate) (oMMA) and poly(acrylic acid) (PAAc) has been synthesized. The block copolymer forms micelles in an aqueous medium, as confirmed by a fluorescence probe technique using pyrene. Doxorubicin hydrochloride was incorporated into the micelle and the release profile of doxorubicin hydrochloride was investigated. Slow and prolonged release of doxorubicin hydrochloride from the micelle was observed. The AB block copolymer micelle can be useful for prolonged mucosal drug delivery of hydrophobic drugs.
Assuntos
Resinas Acrílicas/química , Sistemas de Liberação de Medicamentos , Polimetil Metacrilato/química , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Preparações de Ação Retardada , Doxorrubicina/química , Doxorrubicina/metabolismo , Corantes Fluorescentes/análise , Micelas , Pirenos/análise , Espectrometria de FluorescênciaRESUMO
The intracellular trafficking of drugs is critical to the efficacy of drugs that are susceptible to attack by lysosomal enzymes. It is therefore an important goal to design and synthesize molecules which can enhance the transport of endocytosed drugs from the endosomal compartments to the cytoplasm. The pH of an endosome is lower than that of the cytosol by one to two pH units, depending on the stage of endosomal development. This pH gradient is a key factor in the design of membrane-disruptive polymers which could enhance the endosomal release of drugs. Such polymers should disrupt lipid bilayer membranes at pH 6.5 and below, but should be non-lytic at pH 7.4. We have designed and synthesized pH-sensitive synthetic polymers which efficiently disrupt red blood cells within a sharply defined pH range. One of these polymers, poly(ethyl acrylic acid) (PEAAc) has been previously shown to disrupt synthetic vesicles in a pH-dependent fashion [6]. PEAAc hemolyzes red blood cells with an activity of 10(7) molecules per red blood cell, which is as efficient on a molar basis as the peptide melittin. The mechanism of RBC hemolysis by PEAAc is consistent with the colloid osmotic mechanism. PEAAc's hemolytic activity rises rapidly as the pH decreases from 6.3 to 5.0, and there is no hemolytic activity at pH 7.4. A related polymer, poly(propyl acrylic acid) (PPAAc), was synthesized to test whether making the pendant alkyl group more hydrophobic by adding one methylene group would increase the hemolytic activity. PPAAc was found to disrupt red blood cells 15 times more efficiently than PEAAc at pH 6.1. PPAAc was also not active at pH 7.4 and displayed a pH-dependent hemolysis that was shifted toward higher pH's. Random 1:1 copolymers of ethyl acrylate (EA) and acrylic acid (AAc) (which contain random -COOH and -C(2)H(5) groups that are present and regularly repeat in PEAAc) also displayed significant hemolytic activity, with an efficiency close to PEAAc. These results demonstrate that pH-sensitive synthetic polymers can be molecularly engineered to efficiently disrupt eukaryotic membranes within defined and narrow pH ranges. Thus, these polymers might serve as endosomal disruptive agents with specificities for early or late endosomes.
Assuntos
Membrana Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Polímeros/farmacologia , Hemólise , Humanos , Concentração de Íons de Hidrogênio , Meliteno/farmacologia , Polímeros/síntese químicaRESUMO
There are many protein and DNA based therapeutics under development in the biotechnology and pharmaceutical industries. Key delivery challenges remain before many of these biomolecular therapeutics reach the clinic. Two important barriers are the effective targeting of drugs to specific tissues and cells and the subsequent intracellular delivery to appropriate cellular compartments. In this review, we summarize protein engineering work aimed at improving the stability and refolding efficiency of antibody fragments used in targeting, and at constructing new streptavidin variants which may offer improved performance in pre-targeting delivery strategies. In addition, we review recent work with pH-responsive polymers that mimic the membrane disruptive properties of viruses and toxins. These polymers could serve as alternatives to fusogenic peptides in gene therapy formulations and to enhance the intracellular delivery of protein therapeutics that function in the cytoplasm.
Assuntos
Sistemas de Liberação de Medicamentos , Polímeros/química , Engenharia de Proteínas , Anticorpos/química , Terapia Genética , Indicadores e Reagentes , Polímeros/síntese química , Conformação Proteica , Estreptavidina/administração & dosagemRESUMO
We have prepared a new formulation for mucosal delivery of GM-CSF or PEGylated GM-CSF based on a chitosan carrier plus added glycerol to control the rate of release of the protein. Thin dry films comprised of various weight ratios of chitosan to glycerol and containing either granulocyte-macrophage colony-stimulating factor (GM-CSF) or PEGylated GM-CSF, PEG-(GM-CSF), were prepared. The amount of GM-CSF or PEG-(GM-CSF) released from the chitosan/glycerol films was determined using size exclusion high performance liquid chromatography (HPLC-SEC). The amount of PEG-(GM-CSF) released from the films decreased with an increase in the amount of glycerol present in the film. In parallel with this, films with higher glycerol content exhibited a lower degree of equilibrium swelling when immersed in release media. pH measurements of the release media and analysis of the dried films by Fourier-transform infrared spectroscopy (FTIR) suggested that the amount of residual acetic acid in the dry films decreased as the glycerol content increased. This indicates that glycerol may act by displacing and releasing bound acetic acid from the chitosan molecules, resulting in chitosan--glycerol hydrogen bond formation as the film dries. Further, it was found that the release rate and the amount of PEG-(GM-CSF) released decreased with increasing molecular weight of the conjugated PEG. This effect was not observed with films containing physical mixtures of PEG and GM-CSF. The decrease in the fraction of PEG-(GM-CSF) released with increasing PEG molecular weight is believed to be due to the increased steric hindrance of the PEGylated protein molecule during its diffusion out of the swollen chitosan/glycerol film.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Materiais Biocompatíveis , Soluções Tampão , Quitina/análogos & derivados , Quitosana , Glicerol , Fator Estimulador de Colônias de Granulócitos e Macrófagos/química , Concentração de Íons de Hidrogênio , Membranas Artificiais , Veículos Farmacêuticos , Polietilenoglicóis/química , Proteínas Recombinantes , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
We conducted a prospective study of a smoking ban on a general inpatient psychiatry service in response to staff concerns about the feasibility of a proposed hospital-wide ban. Demographic information, smoking history, and DSM III-R diagnoses were obtained for consecutively admitted patients during two study conditions: smoking and nonsmoking. A log of p.r.n. medication, seclusion, restraint, elopement, incident reports, and smoking-related discharges was kept for each patient. Chi-square analysis of 232 patients for whom demographic, smoking, diagnostic, and log data were complete showed no significant differences between study conditions for demographic or diagnostic variables. Two-tailed t-test analysis of the log data for these 232 patients showed no significant difference in disruptive incidents during smoking and nonsmoking conditions (p = 0.183). Fifty staff members answered pre- and post-ban questionnaires. Paired t-test analysis demonstrated a significant change in staff attitude toward supporting the ban. These data indicate that smoking can be stopped on inpatient psychiatry units without increases in unit disruption or adverse effects on staff morale.
Assuntos
Atitude do Pessoal de Saúde , Atitude Frente a Saúde , Transtornos Mentais/psicologia , Política Organizacional , Recursos Humanos em Hospital/psicologia , Unidade Hospitalar de Psiquiatria/organização & administração , Abandono do Hábito de Fumar , Adulto , Estudos de Viabilidade , Feminino , Hospitais de Ensino , Humanos , Masculino , Transtornos Mentais/epidemiologia , Pessoa de Meia-Idade , Cidade de Nova Iorque/epidemiologia , Estudos Prospectivos , Fumar/epidemiologia , Fumar/psicologia , Prevenção do Hábito de Fumar , Inquéritos e QuestionáriosRESUMO
The pore sizes of various temperature-sensitive hydrogel beads have been estimated at different temperatures by a gel filtration method using several probe molecules of known molecular weight. The hydrogel beads were prepared by inverse suspension copolymerizations of a series of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm) monomers with a small amount of cross-linker. These hydrogel beads exhibit LCST (lower critical solution temperature) behavior in aqueous solution, that is, expanding and swelling when cooled below the LCST and shrinking and collapsing when heated above the LCST. The pore sizes of these temperature-sensitive hydrogel beads are affected significantly by both the temperature and the gel composition, but not much by cross-linker concentration.
Assuntos
Resinas Acrílicas , Polietilenoglicóis , Hidrogel de Polietilenoglicol-Dimetacrilato , TemperaturaRESUMO
Arthrobacter simplex cells have been immobilized in a series of thermally reversible hydrogels having different gel hydrophobicities. Steroid conversion from hydrocortisone to prednisolone via the delta 1-dehydrogenase system was greatly affected by the relative hydrophobicities of the gel matrices, which were prepared by copolymerizing varying ratios of N-isopropylacrylamide to acrylamide. The characteristics of the immobilized cells, such as optimal temperatures, Km values, and the effects of an added artificial electron acceptor, were largely influenced by the gel matrices and their different lower critical solution temperatures (LCST). The data indicate that the microenvironment of the dehydrogenation system is quite different within the different hydrophilic/hydrophobic gel matrices. The high partitioning of water-insoluble steroids into the hydrophobic regions and the reduced possibility of product inhibition within the more hydrophobic gel matrices may cause the observed higher steroid conversion in these gels. A possible model for immobilized A. simplex cells in such different gel matrices is proposed.
Assuntos
Arthrobacter/enzimologia , Géis/química , Temperatura Alta , Hidrocortisona/metabolismo , Prednisolona/metabolismo , Acrilamidas , Fenômenos Químicos , Físico-Química , Cinética , Metilfenazônio Metossulfato/farmacologia , ÁguaRESUMO
Previous studies have shown that certain glow discharge treated polymers strongly retain adsorbed albumin and fibrinogen. On the basis of this phenomenon, we have investigated the possibility of immobilizing antibodies on glow discharge treated surfaces for diagnostic immunoassay applications. As a model for antibody immobilization, bovine IgG was immobilized on the following polymers: polyethylene (PE), tetrafluoroethylene glow discharge treated PE (TFE/PE), poly(ethylene terephthalate) (PET), TFE/PET, poly(tetrafluoroethylene) (PTFE), ethylene glow discharge treated PET (E/PET) and hexamethyldisiloxane glow discharge treated PET (HMDS/PET). IgG was radiolabeled with 125I and immobilized by either of the following two methods: (a) physical adsorption of IgG on untreated and glow discharge treated polymers or (b) physical adsorption of albumin followed by chemical coupling of IgG to albumin by glutaraldehyde. IgG concentration as well as adsorption times were varied in order both to optimize the immobilization conditions and to investigate the adsorption and retention mechanisms. To evaluate the efficiency of the immobilization techniques, blood plasma, Tween-20, and sodium dodecyl sulfate (SDS) were used to elute the adsorbed IgG layer. We found that IgG was successfully immobilized on the fluorocarbon glow discharge treated surfaces by using either the physical adsorption or the glutaraldehyde coupling method, although the former is more efficient than the latter method.
Assuntos
Anticorpos/química , Imunoadsorventes/química , Polímeros/química , Albuminas/química , Glutaral/química , Imunoglobulina G/química , Propriedades de SuperfícieRESUMO
The enzyme beta-galactosidase was immobilized in thermally reversible hydrogel beads that exhibit a reversible expansion and collapse of the gel volume in response to temperature. The kinetic performances of immobilized enzyme bead reactors were studied during thermal cycling operation. A periodic cycling of temperature for the packed-bed reactor induced a cyclic swelling and deswelling of the hydrogel beads. A temperature cycling operation around the phase transition temperature of the gel matrix enhanced the overall enzymatic conversion of the substrate, compared to its upper and lower isothermal operations. The effects on the overall conversion of various thermal cycling operational conditions, such as cycling range and heating and cooling rates, were investigated.
Assuntos
Enzimas Imobilizadas/metabolismo , beta-Galactosidase/metabolismo , Biotecnologia/métodos , Hidrogel de Polietilenoglicol-Dimetacrilato , Cinética , Polietilenoglicóis , Temperatura , TermodinâmicaRESUMO
The enzyme beta-galactosidase has been immobilized within thermally reversible hydrogel beads that exhibit LCST (lower critical solution temperature) behavior. The hydrogel beads containing the immobilized enzymes swell and expand below the LCST and deswell and shrink above the LCST. This behavior is reversible. The enzyme was physically entrapped in a crosslinked hydrogel of a copolymer of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm), and formed as beads in an inverse suspension polymerization. The beads were placed in a packed bed column reactor which was operated in a continuous, single pass mode, either isothermally at 30 or 35 degrees C, or with temperature cycling between 30 and 35 degrees C. The thermal cycling significantly enhanced overall reactor enzyme activity relative to isothermal operation at either the higher or lower temperature. It is postulated that mass transfer rates within the hydrogel beads are greatly enhanced by the movement of water in and out of the beads during the expansion or collapse of the polymer chain network as temperature is cycled.
Assuntos
Enzimas Imobilizadas/metabolismo , Galactosidases/metabolismo , Temperatura , beta-Galactosidase/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato , PolietilenoglicóisRESUMO
Poly-N-isopropylacrylamide (polyNIPAAm), a water-soluble, thermally precipitating synthetic polymer, has been conjugated together with a monoclonal antibody (MAb) and utilized in a novel separation method for an immunoassay. The PolyNIPAAm precipitates out of water above a critical temperature of 31 degres C, enabling a polymer-bound immune complex to be separated from the solution. The principal advantages of this method are that it utilizes a homogeneous incubation for the antigen-antibody reaction, plus, it has the ability to assay large-molecular-weight antigens with sensitivities equivalent to other nonisotopic heterogeneous immunoassays. In addition, since the polymer-immune complex may be reversibly redissolved by cooling, the method may be used both to concentrate the signal and isolate the analyte. This general technique may also be used for a wide variety of separation processes in addition to immunoassays, in which a specific component in a biological fluid, industrial process stream, or body of water is to be isolated for analysis, recovery, or disposal. Thus, product recovery and/or toxin or pollutant removal processes are possible with this methodology.