Beckman Instruments, Inc.

James C Sternberg

Fu-Tai A Chen

Chan S Oh

Gerald L Klein

Cheng-Ming Liu

M Parameswara Reddy

Edmund E Buzza

John E Lillig

Hann-Ping Wang

You-Zung Hsieh

Steven S Herrick

You-Zung Hseih

Iqbal W Siddigi

A trifunctional conjugate is provided having three chemical moieties attached through a spacer moeity. At least two of the chemical moieties are relatively small molecules, usually less than about 7,000 Daltons in size. The spacer moiety is selected to impart certain steric properties to the conjugate. In one embodiment, the binding of a macromolecular specific binding partner to one of the chemical moieties sterically inhibits the binding of a different macromolecule to another chemical moiety. In another embodiment, the binding of a first chemical moiety to a macromolecule restricts the subsequent binding of a second chemical moiety to a proximate location on the same macromolecule. The three chemical moieties are preferably a nitrophenylazido residue, a phenyl boronic acid residue, and a solid support or a label such as biotin. The spacer is preferably cysteine, lysine, glutamic acid, pyroglutamic acid, S-acetylmercaptosuccinic anhydride or .omega.-carbobenzoxylysine. The conjugate is useful in immunoassays and for targeted labeling of proteins. In an assay, a sample is contacted with the conjugate, a limited quantity of analyte binding partner and an excess of small molecule binding partner. Presence of the analyte is determined by detecting the amount of analyte binding partner diverted away from analyte attached to the spacer of the conjugate.

Analyte assay using a trifunctional conjugate

A trifunctional conjugate is providing having three chemical moieties attached through a spacer moiety. At least two of the chemical moieties are relatively small molecules, usually less than about 7,000 Daltons in size. The spacer moiety is selected to impart certain steric properties to the conjugate. In one embodiment, the binding of a macromolecular specific binding partner to one of the chemical moieties sterically inhibits the binding of a different macromolecule to another chemical moieties. In another embodiment, the binding of a first chemical moiety to a macromolecule restricts the subsequent binding of a second tridentate member to a proximate location on the same macromolecule. The three chemical moieties are preferably a nitrophenylazido residue, a phenyl boronic acid residue, and a solid support or a label such as biotin. The spacer is preferably cysteine, lysine, glutamic acid, pyroglutamic acid, S-acetylmercaptosuccinic anhydride or .omega.-carbobenzoxylysine. The conjugate is useful in immunoassays and for targeted labeling of proteins.

Trifunctional conjugates

Double stranded nucleic acid duplexes serve as universal harvestable and cleavable link systems in a variety of different types of immunoassays (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.

Compositions and methods for use in detection of analytes

A method for detecting an analyte, in an aqueous solution at a physiological pH, by reductive or oxidative/reductive electrochemical luminescence methodologies is disclosed. The method proceeds by labelling the analyte with a transition metal complex, followed by inducing the transition metal label to luminescence by application of a suitable electrical potential to a solution containing the label and the analyte. The transition metal complex can be a tris-ruthenium(bipyridine) complex. A hydroxylamine and/or a halogen-containing moiety can be used to enhance both reductive and/or oxidative electrochemical luminescence of the transition metal complex. The transition metal chelate can be used as a label for the detection of picomolar concentrations of an analyte of interest, such as an analyte present in a sample of a physiological fluid.

Method for detecting an analyte using an electrochemical luminescent

A method for correcting for light scattering affects obtained from a sample to which a fluorophore has been added. In accordance with the invention, a sample to which a fluorophore has been added is irradiated with light in the adsorption band of the fluorophore such that the fluorophore emits light at a different intensity. By manipulating the pH of the sample, and obtaining both pre- and post-manipulation emission light intensity readings, the value of the reading attributed to 'light scattering' can be determined, such that correction of an erroneous fluorescence reading can be obtained.

Use of pH dependence for scatter correction in fluorescent methods

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 internal electric field, during IEF as 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 is 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.

Methods and apparatus for separating and mobilizing solutes in a solute

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.

Capillary zone electrophoretic analysis of isoenzymes

Disclosed herein are methodologies for the Capillary Electrophoretic Immunosubtraction ('CEI') of samples. In a preferred embodiment, CEI of a sample comprising constituent parts to be separated comprises the steps of: (1) separating a first aliquot of the sample into constituent parts by capillary electrophoretic techniques, and detecting said parts; (2) admixing a second aliquot of said sample with at least one specific binding partner to a constituent of said sample, said specific binding partner capable of being substantially removed from said aliquot; (3) separating said second aliquot into constituent parts by capillary electrophoretic techniques and detecting said parts; and (4) comparing the separated constituent parts of step (3) with the separated constituent parts of step (1).

Analysis of samples by capillary electrophoretic immunosubtraction

A trifunctional conjugate is provided having three chemical moieties, attached through a spacer moiety. At least two of the chemical moieties are relatively small molecules, usually less than about 7,000 Daltons in size. The spacer moiety is selected to impart certain steric properties to the conjugate. In one embodiment, the binding of a macromolecular specific binding partner to one of the chemical mouths sterically inhibits the binding of a different macromolecule to another chemical moiety. In another embodiment, the binding of a first chemical moiety to a macromolecule restricts the subsequent binding of a second chemical moiety to a proximate location on the same macromolecule.

Method and electrolyte buffer useful in the analysis of samples comprising glycoproteins by capillary zone electrophoresis. The buffer comprises at least one agent capable of complexing with sugar moieties of glycoproteins. The buffer preferably further comprises at least one pH modifier. An embodiment of the buffer comprises boric acid as the complexing agent and sodium hydroxide as the pH modifier.

Analysis of samples utilzing capillary electrophoresis

Photometric analysis apparatus having a cylindrical glass sample container which is rotated about its axis during optical measurement of sample in the container. Rotation of the 5 container window areas past an optical detector compensates for optical variations and imperfections in the container wall which would otherwise render the measured photometric signal sensitive to each particular rotational orientation of the container.

Photometer with rotating sample container

A method of determining the concentration of antigen in a rate nephelometric immunochemical analysis wherein an observed maximum rate of change of a nephelometric signal is used to determine the antigen concentration by employing a mathematical expression derived on a semi-empirical basis.

Method determining concentration in rate nephelometric immunochemical

A method of determining the need for post-addition of antigen or antibody to an antigen-antibody reaction to ascertain whether the reaction is in an antigen excess or an antibody excess condition. The time rate of change of a nephelometric signal developed from the reaction is monitored to generate a rate signal having a peak value providing a measure of antigen concentration. The post-addition step is performed only if the peak rate value is ambiguous--, i.e. if it indicates one antigen concentration value for a reaction in antigen excess but another value for a reaction in antibody excess. The need for the post-addition step is determined from measured qualities of the rate signal including one or more of (1) the peak rate value, (2) the elapsed time from the start of the reaction to the peak rate, and (3) the product of the peak rate and the elapsed time. When required, the post-addition step may be performed during progress of the antigen-antibody reaction after measurement of the peak rate value.

Method of rate immunonephelometric analysis

A portable, automated chemical analyzer having a polarographic oxygen electrode submerged in a batch of stirred solution. The signal from the electrode is directly differentiated and amplified to produce a signal proportional to the time rate of change of oxygen concentration. The membrane of the polarographic sensor is stretched very tightly over the cathode surface to provide a high signal-to-noise ratio. Methods for analyzing cholesterol-cholesterol oxidase and various other enzyme systems by the polarographic electrode oxygen sensing apparatus include the steps of converting the sensed signal into time rate of change of oxygen concentration and recording the maximum rate of change of oxygen concentration.

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Fullerton, CA, United States of America

James C Sternberg