Abstract:
Disclosed herein is a methodology for analyzing isoenzymes using capillary zone electrophoresis ('CZE') techniques. Briefly, an isoenzyme-containing sample and a substrate capable of being catalyzed by said isoenzyme into a reaction product are introduced into a capillary column comprising a buffer. Most preferably, the buffer contains the substrate prior to introduction of the sample into such substrate-buffer. CZE separation techniques are applied to the column such that the isoenzymes are separated from each other into discrete zones. The separation techniques are terminated such that product is rapidly generated by the catalytic conversion of substrate by the isoenzymes, and accumulated, within each discrete zone, followed by detection of product. Information regarding the relative distribution of the isoenzymes can be derived from the relative distribution of the product.
Abstract:
Specific binding assays for measuring the concentration of an analyte in a sample involve forming a complex comprising a solid support, the analyte or an analogue of the analyte, and an enzyme. The enzyme is used to cleave non-isotopic labels that are directly detectable from a substrate/label conjugate to provide a plurality of released labels in the free state, which are then separated from conjugate containing unreleased label. The released labels are detected for measuring the concentration of the analyte in the sample. Preferably the analyte or analogue is connected to the solid support by a cleavable bond, so that the enzyme can be separated from the solid support for use in the enzymatic cleaving of labels from the conjugate. This preferred version reduces the adverse effect of non-specific binding on the sensitivity and accuracy of the assay.
Abstract:
Double stranded nucleic acid duplexes serve as universal harvestable and cleavable link systems in a variety of different types of immnuoassays (e.g., sandwich, competitive, etc.). Depending upon the type of assay, at least one specific component involved in the assay system is attached to a first member of a pair of sequences forming a double stranded nucleic acid (i.e., two oligonucleotides comprising substantially complementary sequences). The assay is carried out in the presence of a support to which is attached an oligonucleotide which is the other member of the pair of sequences forming a double-stranded nucleic acid duplex under hybridization conditions. Upon the hybridization of the two complementary oligonucleotides to form a duplex, the component of the assay system to which the first member of the pair of oligonucleotides is attached may thereby be effectively removed from the solution phase and harvested onto the support. Oligonucleotides bound to a support are reusable in multiple successive assays. Moreover, any given support-bound oligonucleotide can be used in accordance with the present invention for the analysis of a variety of different analytes. In many cases, the assay system includes a label to facilitate quantifying the amount of analyte; in others, the amount of analyte may be determined without the use of any extraneous label.
Abstract:
The present invention provides processes for isoelectric focusing ("IEF") and associated detection, which incorporates a dynamic means of electroosmotic flow ("EOF") control during IEF and/or after IEF to effect solute mobilization. In accordance with the present invention, the EOF control during IEF and/or solute mobilization after IEF are accomplished by applying an external electric field, relative to an internal electric field, to modify the electroosmotic flow in the capillary. This can be done by disposing a conductive member at one or more locations outside and along the buffer column in the capillary. The conductive member may be statically charged or caused to conduct a current to create the external required electric field. The applied external electric field may be adjusted, relative to the external electric field, during IEF necessary to reduce or completely suppress EOF to prevent flow of the buffer. Upon the completion of IEF (irrespective of the method of reducing or removing EOF during IEF), the external electric field is adjusted, relative to the internal electric field, such that the buffer carrying the focused solutes are moved electroosmotically through the capillary past a detection point. The present invention is applicable to internally coated capillary which suppresses EOF even in the absence of the external electric field.
Abstract:
A tridentate conjugate has three chemical moieties, or tridentate members, attached through an appropriate spacer moiety. At least two of the tridentate members are relatively small molecules, usually less than about 7,000 Daltons in size. The particular appropriate spacer moiety selected for a tridentate imparts certain steric properties to the tridentate conjugate. In one embodiment, the binding of a macromolecular specific binding partner to one of the tridentate members sterically inhibits the binding of a different macromolecule to another tridentate member. In another embodiment, the binding of a first tridentate member to a macromolecule restricts the subsequent binding of a second tridentate member to a proximate location on the same macromolecule.
Abstract:
The present invention provides processes for isoelectric focusing ('IEF') and associated detection, which incorporates a dynamic means of electroosmotic flow ('EOF') control during IEF and/or after IEF to effect solute mobilization. In accordance with the present invention, the EOF control during IEF and/or solute mobilization after IEF are accomplished by applying an external electric field, relative to an internal electric field, to modify the electroosmotic flow in the capillary. This can be done by disposing a conductive member at one or more locations outside and along the buffer column in the capillary. The conductive member may be statically charged or caused to conduct a current to create the external required electric field. The applied external electric field may be adjusted, relative to the external electric field, during IEF necessary to reduce or completely suppress EOF to prevent flow of the buffer. Upon the completion of IEF (irrespective of the method of reducing or removing EOF during IEF), the external electric field is adjusted, relative to the internal electric field, such that the buffer carrying the focused solutes are moved electroosmotically through the capillary past a detection point. The present invention is applicable to internally coated capillary which suppresses EOF even in the absence of the external electric field.
Abstract:
Des méthodes de titrages par liaison spécifique, permettant de mesurer la concentration d'un analyte dans un échantillon, consistent à former un complexe comprenant un support solide, l'analyte ou un analogue de l'analyte, et une enzyme. On utilise l'enzyme pour cliver des marques non isotropiques directement détectables à partir d'un conjugué substrat/marque, afin de produire une pluralité de marques libérées dans l'état libre, que l'on sépare ensuite du conjugué contenant la marque non libérée. On détecte les marques libérées pour mesurer la concentration de l'analyte dans l'échantillon. L'analyte ou analogue est de préférence fixé au support solide par une liaison clivable, de sorte que l'on peut séparer l'enzyme du support solide pour l'utiliser dans le clivage enzymatique de marques à partir du conjugué. Cette version préférée réduit l'effet désavantageux d'une liaison non spécifique sur la sensibilité et la précision du titrage.
Abstract:
Double stranded nucleic acid duplexes serve as universal harvestable and cleavable link systems in a variety of different types of immnuoassays (e.g., sandwich, competitive, etc.). Depending upon the type of assay, at least one specific component involved in the assay system is attached to a first member of a pair of sequences forming a double stranded nucleic acid (i.e., two oligonucleotides comprising substantially complementary sequences). The assay is carried out in the presence of a support to which is attached an oligonucleotide which is the other member of the pair of sequences forming a double-stranded nucleic acid duplex under hybridization conditions. Upon the hybridization of the two complementary oligonucleotides to form a duplex, the component of the assay system to which the first member of the pair of oligonucleotides is attached may thereby be effectively removed from the solution phase and harvested onto the support. Oligonucleotides bound to a support are reusable in multiple successive assays. Moreover, any given support-bound oligonucleotide can be used in accordance with the present invention for the analysis of a variety of different analytes. In many cases, the assay system includes a label to facilitate quantifying the amount of analyte; in others, the amount of analyte may be determined without the use of any extraneous label.
Abstract:
Procédé d'analyse d'isoenzymes utilisant des techniques d'électrophorèse à zone capillaire ("CZE"). Un échantillon contenant une isoenzyme et un substrat pouvant être catalysé par ladite isoenzyme en un produit de réaction sont introduits dans une colonne capillaire comprenant un tampon. Idéalement, le tampon contient le substrat avant l'introduction de l'échantillon. On applique à la colonne des techniques de séparation CZE, afin de séparer les isoenzymes en des zones discrètes. A la fin de la séparation, le produit est rapidement généré par la conversion catalytique du substrat par les isoenzymes, et accumulé dans chaque zone discrète. Le produit est ensuite détecté. On peut dériver les informations sur la répartition relative des isoenzymes à partir de la distribution relative du produit.
Abstract:
Disclosed herein is a methodology for analyzing isoenzymes using capillary zone electrophoresis ('CZE') techniques. Briefly, an isoenzyme-containing sample and a substrate capable of being catalyzed by said isoenzyme into a reaction product are introduced into a capillary column comprising a buffer. Most preferably, the buffer contains the substrate prior to introduction of the sample into such substrate-buffer. CZE separation techniques are applied to the column such that the isoenzymes are separated from each other into discrete zones. The separation techniques are terminated such that product is rapidly generated by the catalytic conversion of substrate by the isoenzymes, and accumulated, within each discrete zone, followed by detection of product. Information regarding the relative distribution of the isoenzymes can be derived from the relative distribution of the product.