JP4753945B2 - Methods for analyzing the presence or amount of silver-labeled species - Google Patents

Description

  The present invention relates to a method and apparatus for analyzing the presence or amount of metal-labeled chemical species. In particular, but not exclusively, it relates to a method and apparatus for detecting a chemical species comprising an analyte of interest or a chemical species that specifically binds to the analyte.

  A compound in which a chemical species having a specific binding ability such as an antibody is complexed with a label is routinely used as a reagent for rapid assay in detection of an analyte. One such assay is described in European Patent Publication No. 0291194. In this assay, a laterally-flowing porous carrier and a mobile antibody that specifically binds to the target analyte and labeled with microparticles are used. It also uses a second antibody that is also specific for this analyte, but immobilized in the detection zone. The analyte binds to the labeled mobile antibody to form a first complex, which is subsequently captured by the second antibody immobilized in the detection zone. The amount of label present in the detection zone is an indicator of the amount of analyte present in the sample. The label may be a fine particle species such as gold, and its presence in the detection zone may be detected visually or optically.

  Another method for detecting labeled specific binding species is disclosed in US Patent Application No. 20030186274. In this method, the amount or presence of a metal-labeled binding species is detected by solubilizing the label in the presence of an oxidizing agent such as a strong acid to produce a metal ion. When an appropriate negative potential is applied, the generated ions are electrochemically reduced and deposited on the electrode surface. Then, when the potential is changed, the deposited metal is reoxidized and returned to the solution, producing a current response. . The intensity of this response is an indicator of the amount of metal ions deposited on the electrode surface and the amount of label initially present. This type of electrochemical analysis method is known as stripping voltammetry and can be used to measure trace amounts of metal ions (eg, parts per million or less). This method can be used to detect the presence or to analyze the amount of an analyte that has binding specificity for a labeled binding species.

  The method described in U.S. Patent No. 2003018627 is exemplified using gold as a label. However, in many respects, silver is considered a better label for use in electrochemical detection. This is mainly because it is easier to oxidize silver to produce silver ions than gold. For example, the oxidant required to produce silver ions from silver has a lower oxidizing power than that needed to produce gold ions from gold and is therefore more suitable for use in diagnostic kits.

  US 2003018627 discloses silver as a detectable metal particle, but the method described herein cannot be used for electrochemical analysis of silver or similar metals in biological samples. . This is because these metals in an oxidized state react with chemical species present in biological samples such as blood, plasma and urine to form insoluble electrochemically inert complexes. For example, since the chloride ion concentration in urine is usually 150 mM, all silver ions generated by oxidation are immediately precipitated as AgCl, and the oxidation on the electrode surface is inhibited. Similarly, sulfur-containing species such as bromide ions and cysteine that are also present in biological samples form insoluble electrochemically inactive complexes with silver ions.

In a first aspect, the present invention provides:
A metal-labeled species in a sample is dissolved in a soluble electrochemically active complex [the complex is or may be present in the sample (the component is Insoluble and / or electrochemically inactive complexes)). And measuring the formed complex electrochemically to provide an indication of the presence or amount of the metal-labeled chemical species, in the sample A method for measuring the presence or amount of a chemical species is provided.

  In the present invention, the electrochemical measurement of a metal is performed through the formation of a soluble electrochemically active complex from the metal. By this method, a metal is present in the sample and reacts with a substance or component that should form an insoluble and / or electrochemically inactive complex with the metal if the complexation does not occur. It is suppressed. Therefore, the electrochemical measurement of the generated complex can be easily performed without the need to remove the component that makes the metal into a chemical form that cannot be electrochemically measured. U.S. Pat. No. 2003018627 discloses that metal ions produced by the method disclosed therein can be complexed, the purpose of which is to analyze electrochemically inert metal ions, Converting to a chemically active compound and forming a more hydrophobic substructure that can be adsorbed to the electrode. In contrast, in the present invention, in order to prevent electrochemically active metal ions from forming insoluble and / or electrochemically inactive complexes with components present in the sample, Is converted to

  In one embodiment, the electrochemically active complex is produced by oxidizing a metal and reacting the resulting metal ion with a suitable complexing agent. The metal may be oxidized in the presence of a suitable complexing agent.

  The metal label used in the present invention may be silver, but other metals such as lead, cadmium, copper, thallium, mercury and zinc can also be used. Compared to gold, which is the preferred metal for fine particles in the past, silver more readily generates an electrochemical response that can be measured on a platinum or carbon electrode. For this reason, the carbon electrode obtained conveniently and cheaply can be used.

  A further advantage is that the combination of oxidant and carbon electrode used provides a stable reference potential that can be used as a potential reference. For this reason, it is not necessary to separately provide an electrode such as a commonly used silver / silver chloride electrode.

In one embodiment, the metal label is a particulate label such as a silver sol. The advantage of using fine particles label, many very By single label is oxidized, for example, by the size of the sol generates 10 ⁶ or even more metal ions is that Therefore high sensitivity can be obtained . A commercially available metal sol is available, and a complex with a binding reagent can be prepared by a known method as described in European Patent Publication No. 0291194.

Silver is also more convenient to use than gold because the oxidation conditions required to produce silver ions are less harsh than less susceptible metals such as gold. For example, oxidation of metallic gold to ionic gold species (AuCl ₄ ) requires a mixture of concentrated nitric acid and concentrated hydrochloric acid, or sodium hypochlorite and hydrochloric acid. In contrast, to oxidize silver particles to silver ions, peroxides, ferric ions and ferricyanides can be used as oxidizing agents. It can also be oxidized at a more preferred pH, eg 3 to 7.

  In the present invention, an electrochemically active complex capable of electrochemical detection is formed on the metal label. As used herein, an “electrochemically active” species is an ion, neutral compound, or complex that can accept or emit one or more electrons.

  The role of the electrochemically active complex is to prevent the metal from reacting with the chemical species in the sample to form an electrochemically undetectable insoluble and / or electrochemically inactive complex. It is. For example, silver ions react with halide ions such as chloride ions to form insoluble salts, or contain sulfur such as cysteine, glutathione, cysteinyl glycine or other sulfhydryl group (mercapto group) containing compounds. Reacts with chemical species to form electrochemically inert silver ion complexes. Therefore, electrochemically active complexes must be stable even in the presence of such chemical species. As is well known to those skilled in the art, the electrochemically active complex is in dynamic equilibrium between the metal (or metal ion) and the complexing agent. In addition, an equilibrium is established between a metal or a metal ion and a component that is present in the sample and can form an insoluble and / or electrochemically inactive complex with the metal. Therefore, in principle, all of the above chemical species will be present in some amount. However, these equilibrium positions are present in the sample in the amount of complexing agent capable of forming a soluble electrochemically active complex, insoluble and / or electrochemically present with the metal or metal ion. It depends at least in part on the amount of chemical species that can form an inert complex and the amount of the metal or metal ion present. For this reason, the term “stable” as used herein is a situation where an electrochemically active complex is preferentially formed, and the concentration of the electrochemically inactive and / or insoluble complex is electrochemically determined. A state that can be ignored with respect to the concentration of the active complex. It is sufficient that the concentration of the electrochemically inactive and / or insoluble complex does not substantially affect the measurement result. The higher the amount of complexing agent initially present, the more the equilibrium moves in the direction of the formation of a soluble electrochemically active complex, and thus the less metal or metal ion present. For this reason, electrochemically active complexes are preferentially produced regardless of the concentration of components present in the sample that can form insoluble and / or electrochemically inactive complexes with the metal. In order to ensure that the complexing agent is used in excess.

  The present invention is suitably used for biological samples containing all fluids that can be obtained from the mammalian body such as blood, plasma, urine, lymph, gastric fluid, bile, serum, saliva, sweat and cerebrospinal fluid. Furthermore, these body fluids may be treated (eg, serum) or untreated. Methods for obtaining a sample from a subject are known to those skilled in the art. The biological sample may be a solid sample, for example, a liquid sample obtained by processing a cell tissue. The present invention also provides a liquid industrial sample or liquid environment, such as wastewater or seawater, that may contain a large amount of chemical species that can form insoluble and / or electrochemically inactive complexes with the metal. It is also suitable for measuring other liquids including samples. The sample may be of plant origin. When the test sample contains a protein component, iodinated acetamide may be added to react with a thiol group in the protein to form a thioester. This prevents the metal ions from binding to the protein via the thiol group and failing to form a soluble electrochemically active complex.

As described above, an electrochemically active complex can be produced by reacting a metal ion produced by oxidizing a metal with an appropriate complexing agent. Any convenient oxidizing agent capable of oxidizing the metal can be used, for example, permanganic acid salts, chromate, peroxides, persulfates, ferric ions and ferricyanide compounds can be used. In one specific embodiment, the oxidant is ferric ion, which is a particularly mild oxidant. According to the present invention, the oxidizing agent may be used in a reduced form and oxidized only when necessary to be activated. The oxidation at this time may be performed chemically or electrochemically. In certain embodiments, oxidation can be induced by contacting the reduced form of the oxidant with the test sample.

In one embodiment, silver is oxidized using iron in the oxidation state + III as shown in the following reaction scheme:
Fe ³⁺ + Ag → Ag ⁺ + Fe ²⁺
Iron in the oxidation state + III can be used in the form of a suitable soluble ferric salt such as nitrate, acetate, citrate, ferric ammonium sulfate and the like.

Nitric Alternatives to ferric, using potassium ferricyanide in accordance with the following reaction formula, and then may be reacted with thiocyanate or thiosulfate:
4K ₃ Fe (CN) _{6 (aq)} + 4Ag _(s) → 3K ₄ Fe (CN) _{6 (aq)} + Ag ₄ Fe (CN) _{6 (aq)}

  Ferricyanide ions (FeIII) may be generated by oxidizing ferrocyanide ions (FeII) on an electrode for oxidizing a metal such as silver sol particles. This method has several advantages. First, ferricyanide ions can be generated at the point of use at the required time and in a controlled manner and amount. As a result, loss due to diffusion of the metal from the measurement zone can be minimized. The same electrode may be used for oxidant generation and metal ion concentration analysis. Second, since ferrocyanide is more stable than ferricyanide in the dry state, it can be stored in the apparatus or kit of the present invention until the sample is added. Third, since ferrocyanide ions form weaker complexes with silver than ferricyanide ions, it is beneficial to generate ferricyanide ions only when needed.

  The fact that ferrocyanide ions are relatively insensitive to silver and can only be reacted when oxidized is the fact that the silver-labeled binding partner binds to the analyte to produce a silver-labeled species. Is important in embodiments of the invention that use (see below). In these embodiments, the unbound silver-labeled binding partner is soluble in an electrochemically active complex even though the analyte and unbound silver-labeled binding partner are in contact with the ferrocyanide ion. There is no danger of distorting the results by acting as silver ions that produce. After removing all analyte and unbound silver-labeled binding partners, ferrocyanide ions are oxidized to generate ferricyanide ions that are soluble only in the bound silver-labeled species of silver. Electrochemically active complexes can be formed.

  As a result, it is possible to provide a one-step test / kit in which a necessary reagent is prepared in a dry state in the apparatus and a user can perform a test simply by adding a liquid sample. become. In such a test / kit, the sample itself can act as a wash solution, and unbound silver-labeled binding partners can be removed from the zone / portion that detects soluble electrochemically active complexes. The ferrocyanide may be supplied upstream or in the detection zone. Potassium ferrocyanide can be used because it is highly water-soluble, relatively difficult to react with silver, and stable in the dry state.

An excess of ammonium thiocyanate may be used after production of silver ions to produce a soluble silver ion complex that can be detected electrochemically as shown below.
Ag ⁺ + m (NH ₄ ) ⁺ + n (SCN) ⁻ → [Ag (NH ₄ ⁺ ) m (SCN ⁻ ) n] ^{m−n + 1}

  Instead of thiocyanate, an excess of ammonium thiosulfate may be used to form a soluble silver ion complex. This silver ion complex is formed preferentially over the insoluble silver salt, and therefore remains in the form of a soluble complex even if there are chemical species that can form an insoluble salt with the silver ion.

  Reagents used to oxidize silver (or other metals) to form a stable complex may be in a dry state, which is advantageous when incorporating these reagents into an immunoassay device. In use, these reagents are lysed by the biological sample to be analyzed.

  A further advantage of using silver as the metal label is that the reduction potential of the silver species stripped from the electrode is a negative potential less than that of silver ions. This can suppress interference and background effects from electrochemically active compounds such as ascorbate ions that may be present and other metal ions such as copper, cadmium and mercury.

  For the measurement of the electrochemically active complex, a convenient electrochemical method can be used as appropriate. Anodic stripping voltammetry (or polarography) with potential scanning, which can be linear, cyclic, square wave, normal pulse or differential pulse, or superimposed sinusoidal voltage Can be used. Alternatively, anodic stripping chronopotentiometry may be used. Other measurement methods that can be used include ion exchange voltammetry; linear, cyclic, square wave, normal or differential pulsed scanning, or superimposed sinusoidal voltage adsorption cathodes Stripping voltammetry (or polarography); chronoamperometry; chronoculometry; voltammetry (or polarography) of linear, cyclic, square wave, normal pulse or differential pulse; or superimpose Examples include voltammetry (or polarography) of dosinusoidal voltage.

  The metal-labeled species can be generated by the binding of the analyte to be detected and the metal-labeled binding partner. Thus, the metal-labeled species and hence the soluble electrochemically active complex produced from the metal-labeled species is an indicator of the presence or amount of the analyte in a biological sample. The metal-labeled binding partner may be any as long as it can bind to the analyte to be detected. Thus, depending on the analyte to be detected, an antibody or antigen-binding fragment thereof, protein receptor, T cell receptor, antigen, hapten, protein, peptide, oligonucleotide, boronic acid derivative, polymeric acid or base, carbohydrate, lectin , Complementary nucleotide or peptide sequences, specific protein binding substances, nucleic acids (such as DNA or RNA), and analyte conjugates.

In a further aspect, the invention provides:
Contacting a sample that may contain an analyte with a metal-labeled binding partner to form a metal-labeled species;
Oxidizing the metal labeled chemical species, the resulting metal ions are
Soluble electrochemically active complex [the complex is a component that is or may be present in the sample (the component is an insoluble and / or electrochemically inactive complex with the metal It is more stable than And the presence of an analyte in the sample, or electrochemically measuring the formed complex to provide an indication of the presence or amount of the metal-labeled species. Provide a way to analyze quantities.

The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active regions or fragments in immunoglobulin molecules, ie, molecules that contain an antigen binding site that specifically binds an antigen. The immunoglobulin molecules used in the present invention may be any class (eg, IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecules. Antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies, bispecific antibodies, humanized and chimeric antibodies, single chain antibodies, Fab fragments and F (ab ′) ₂ fragments, Fab expression libraries Produced fragments, anti-idiotype (anti-Id) antibodies and epitope binding fragments thereof. An antibody, or generally any molecule, will preferentially bind to the antigen, eg, less than about 30%, preferably 20%, 10% or 1% cross-reactivity with other molecules. If so, it is said to “specifically bind”. Antibody regions include Fv and Fv ′ regions.

  The analyte to be detected may be measured by immunoassay. Such an assay may be a competitive immunoassay or a non-competitive (sandwich) immunoassay. Such assays are well known in the art, both homogeneous and heterogeneous.

  In one embodiment, the immunoassay involves contacting a sample from a test subject with an appropriate metal-labeled antibody to generate a metal-labeled species, and an electrochemically active complex for the species. Treatment (which may be oxidation of the metal and complexation of the resulting metal ions), followed by electrochemical measurement of the electrochemically active complex, This is done by analyzing the amount or presence (which corresponds to the amount or presence of the analyte).

  Analytes in biological samples can be detected by a two-step sandwich assay. In the first step, an analyte is captured using a capture reagent (eg, an anti-analyte antibody). If necessary, the capture reagent may be immobilized on a solid phase. In the second step, the analyte is detected using a metal labeled detection reagent. Alternatively, the capture reagent may be metal-labeled and the detection reagent immobilized. In this case, the analyte can be detected at a specific portion where the detection reagent is immobilized.

  In one embodiment, the lateral flow immunoassay device is configured in a “sandwich” fashion in which a sufficient amount of analyte marker in the sample causes a “sandwich” interaction in the capture zone of the lateral flow assay. May be used. As used herein, a capture zone contains capture reagents such as antibody molecules, antigens, nucleic acids, lectins, and enzymes suitable for capturing the analyte of interest. Other assays used in the methods of the present invention include, but are not limited to, flow-through devices.

The present invention also provides
A metal-labeled binding partner that can bind to an analyte believed to be present in the sample to form a metal-labeled species,
From a metal of the metal-labeled species, a soluble electrochemically active complex [the complex is or may be present in the sample (the component is insoluble in the metal And / or form an electrochemically inactive complex). One or more reagents for forming
A volume space in which a metal-labeled binding reagent can bind to an analyte, and the metal-labeled species for allowing the one or more reagents to form a soluble electrochemically active complex A kit is provided that includes a capture zone for immobilizing.

  The kit may be provided in combination with a reader for electrochemically measuring soluble electrochemically active complexes. In one embodiment, the kit is disposable and the reader can be reused and assembled, such as by engaging each part together before use. Alternatively, these may be provided as an integrated system.

  The kit or reader may be provided with at least two electrodes for electrochemical measurement. If provided in the kit, the electrode may be connectable via a terminal to an electrode terminal on the inner or outer surface of the reader. When provided in a reader, the kit is positioned so that the electrodes are located at appropriate locations in the kit when the kit and reader are engaged.

  The reader may include signal conversion and signal processing means for applying signals to the electrodes and for converting and processing specific signals obtained from the electrodes. The reader may include display means for displaying information; kit installation means, power supply or external power capture means, storage means, calibration means, and data input / output means if necessary.

  In one embodiment, the volume space of the kit is formed by a multiwell plate or the like. The sample is placed in a well where a metal labeled binding partner is added. At this time, the analyte present produces a metal-labeled species. The species is immobilized in a capture zone (eg, a filter or immobilized antibody) and one or more reagents to form an electrochemically active complex that is soluble in the metal of the metal-labeled species. Is added there. Alternatively, the volume space may be formed or provided by a porous carrier or microfluidic device that may be a lateral flow porous carrier.

  Since the present invention involves the generation of metal ions and subsequent complexation, in order to obtain accurate results, electrochemical measurements must be limited to complexed metal ions only. In other words, non-complexed metal-labeled binding partners should not be oxidized or, if oxidized, should not affect the measurement results. This forms a complex between the immobilized metal-labeled binding partner and the analyte, so that the sample is first contacted with the binding partner and then unbound metal-labeled binding partner is washed from the vicinity of the capture zone. This can be achieved by removing them. Then immobilize one or more reagents to form an electrochemically active complex that is soluble in the metal of the metal-labeled species to form an electrochemically stable metal ion complex The contact zone may be brought into contact with the crystallization zone.

  The electrode can be obtained by many methods such as various printing methods such as screen printing, inkjet printing, offset lithography printing, and rotary printing. Alternatively, the electrode may be deposited by vacuum deposition or sputtering. The electrode may be provided on the inner surface of the substrate. In a two electrode configuration, one electrode is the working electrode and the other is the counter / reference electrode. A third electrode that can function as a reference electrode may be provided. The same electrode or even additional electrodes may be used as an electrode to indicate the presence of the sample in the chamber and / or to verify that the chamber is properly filled. The electrode may be a part of a structure composed of three screen-printed flat carbon electrodes. This construction prints a low-temperature carbon pattern on the substrate and overlays a low-temperature dielectric pattern on it to define the area of the electrode and coat other carbon parts in contact with water. It can be manufactured by printing. The substrate may be a polymer although alumina can also be used. Lamination of the electrode material and the dielectric can be performed by various methods such as screen printing.

  In one embodiment, the immobilization or capture of the metal labeled species (binding partner-analyte complex) is performed by using a filter structure. In the absence of analyte, the metal-labeled binding partner will pass through the filter, and in the presence of analyte, a complex will be formed and immobilized or captured on the filter.

  Preferred features of each aspect of the invention are the same for other aspects, each with the necessary changes. The prior literature mentioned here is incorporated to the maximum extent permitted by law.

  The invention is described in more detail in the following examples with reference to the accompanying drawings.

Electrochemical determination of silver sol in serum Ammonium thiocyanate (0.4 g) was added to a 1 ml serum sample. A 1% EDTA (100 μl) solution containing ferric nitrate (0.01 g) as an oxidant was used with a pH 4 buffer (citric acid / sodium citrate). This pH has long been known to give an optimal silver ion analytical signal in biological samples and pure buffers (see FIG. 1). From FIG. 1 it can be seen that pH 4 is optimal because it gives the highest current reading, ie the highest sensitivity. It can also be seen that the oxidation reaction of the silver ion complex is pH dependent. However, when measuring in whole blood, a higher pH such as pH 7 may be selected to prevent coagulation of the blood sample. When 10 μl of silver particles of known concentration were added to a serum sample, a rapidly dissolving and optically clear solution was obtained.

  The generated silver ions were measured on a screen-printed three-plate carbon electrode structure (see FIG. 2). This electrode was manufactured by printing a low-temperature carbon pattern (D2, Gwent Electronic Materials, Inc.) on a 25 μ alumina substrate. Next, an electrode region was defined thereon, and a low temperature dielectric pattern (Gwent Electronic Materials, Inc.) was overprinted to cover other carbon regions so as not to come into contact with water (see FIG. 2).

  The volume of the sample added to the electrode surface in each measurement was 10 μl. Carbon / ammonium thiocyanate / ferric ion was used as a reference electrode. This has a potential difference of about -450 mV for an Ag / AgCl / 3.5M KCl half-cell. Here, high-speed rectangular wave anode stripping voltammetry (FQWASV) is used as the electroanalytical method, but other standard electroanalytical methods may be used.

  The measurement was performed in the stripping rectangular wave voltammetry mode using Easyscan (Ezescan (trade name), Whistonbrook Technologies, Inc.). Pre-concentration was performed for 120 seconds at an accumulated potential of -900 mV (no stirring). After this preconcentration time (a 5 second pause may be provided if necessary), stripping from -900 mV to -100 mV was started. The operating conditions at this time were as follows: potential step: 1 mV; peak width: 5 ms; half cycle amplitude: 25 mV; frequency: 100 Hz. The peak intensity obtained during the scan was adopted as an index of the silver ion concentration in the sample. Alternatively, the area surrounded by the peak may be measured and used as an indicator of charge.

  The obtained results are shown in FIGS. An increase in response current is observed as the amount of silver particles initially present increases.

Analysis of serum hCG levels Using the electrochemical method of Example 1 with human chorionic gonadotropin (hCG) as an analyte, standard ELISA type treatment was used to analyze serum levels. Briefly, samples containing analyte hCG at various concentrations were mixed with silver particulate labeled antibody (anti-αhCG) to form a first antibody-analyte complex. This complex was then added to an ELISA plate containing an immobilized antibody reagent (anti-βhCG) to form a second immobilized antibody-analyte-antibody “sandwich” complex. Finally, excess unbound silver microparticles-labeled first antibody was removed from the plate by washing. This is schematically shown in FIG.

  Subsequently, an aqueous sample solution containing the reagent described in Example 1 is added to oxidize the silver-antibody complex to form a soluble silver ion complex, followed by electrochemical measurement of silver ions and the existing analyte hCG. The amount of was analyzed.

  Preparation of the silver labeled antibody reagent and the reagent immobilized on the ELISA plate was carried out by the standard procedure shown below.

  S-acetylmercaptosuccinic acid (SAMSA) (Sigma Sigma) was reacted with anti-αhCG monoclonal antibody (Mabs). A PD-10 size exclusion chromatography column (Pharmacia Pharmacia) was equilibrated with 25 ml of pH 6.0 phosphate buffer, 2.5 ml of MAb stock solution was added to the column, and 3.5 ml of pH 6.0 phosphate buffer. It eluted with. 100 μl of the eluted MAb solution was taken, 900 μl of phosphate buffer was added thereto, the absorbance at 280 nm was measured, and the concentration was calculated (Abs = εcl, ε = 1.4). The total mass of the protein in the solution (including the remaining amount of 3.4 ml) was calculated and the number of moles of MAb was calculated (MAb MW = 150,000). When calculating the number of moles of SAMSA required for an excess of 35 equivalents (SAMSA MW = 174.2) and adding this required amount of SAMSA as a dimethylformamide (DMF) solution containing SAMSA at a concentration of 50 mg / ml Was converted into the volume (μl unit) necessary for the above.

  SAMSA was dissolved in dry DMF to a concentration of 50 mg / ml and an appropriate amount of this solution was added to the MAb solution with stirring. The resulting solution was incubated at room temperature overnight using a mixer.

  The complexing of the silver colloid and the anti-αhCG monoclonal antibody (MAb) bound to SAMSA obtained above was performed as follows.

  A known amount of silver colloid was incubated with a nonionic surfactant (Pluronic F108-PMPI) to suppress nonspecific binding. 5 mg of F108-PMPI was dissolved in deionized water and placed in an Eppendorf tube to which about 2 mg of silver sol was added. This was incubated at room temperature for 1 hour using a rotary mixer. The resulting solution was then centrifuged at 17000 rpm for 15 minutes at 5 ° C. The supernatant was removed and the pellet was resuspended in 50 mM HEPES buffer (pH 7.5).

  In order to enhance the binding property to silver colloid, the SAMSA-MAb was deprotected and converted to a monoclonal antibody thiol (MAb-SH). To 1 ml of the SAMSA-MAb solution, 40 μl of 0.1 M tris (hydroxymethylamino) ethane (Tris) solution was added and mixed for 5 minutes. 20 μl of 0.1 M ethylenediaminetetraacetic acid (EDTA) solution was added and mixed for 5 minutes. 40 μl of 1M hydroxylamine solution was added and mixed for 5 minutes. A NAP-10 packed column (Pharmacia) was equilibrated with 20 ml of 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.5) and 1 ml deprotected. The MAb solution was loaded onto the column and eluted with 1.5 ml HEPES buffer.

  This deprotected SAMSA-Mab was reacted with the silver colloid. The silver colloidal suspension was divided in half and 500 μl of deprotected SAMSAAb (MAb-SH) solution was added to each sample with mixing. The sample was incubated overnight, centrifuged at 17000 rpm for 30 minutes at 5 ° C., the supernatant was removed, and each sample pellet was resuspended in 500 μl borate buffer (pH 8.6). The two samples were recombined and placed in one Eppendorf tube and centrifuged at 17000 rpm for 30 minutes at 5 ° C. The supernatant was removed and the solid was resuspended in 1 ml borate buffer. The obtained silver fine particle labeled antibody was stored at 4 ° C.

  A monoclonal anti-βhCG antibody reagent was immobilized on an ELISA plate (Greiner high binding plates) as follows.

  200 μl of 50 mM borate buffer (pH 8.6) in which anti-βhCG was dissolved was added to each well. The plate was capped and incubated for 1 hour at 37 ° C. with shaking and then washed. DBS / BSA buffer (Dulbecco, phosphate buffered saline—1 tablet per 100 ml R.O. water, 1% BSA) was added to the wells and allowed to stand overnight before buffering. Was removed. 200 μl of DBS / BSA buffer containing hCG at the desired concentration (0, 0.2, 1, 5, 10, 20 and 50 mlU) was added to each well in two samples, incubated at 37 ° C. for 1 hour, and washed. did. 100 μl (excessive amount) of borate buffer (pH 8.6) containing silver colloid bound to SAMSA-MAb was added to each well, incubated at 37 ° C. for 1 hour, and then washed.

  To each well was added 100 μl of a solution containing 5 M ammonium thiocyanate, 0.05 M citrate buffer (pH 4), 1 M ferric nitrate, 1% EDTA, and then incubated at 37 ° C. for 30 seconds to be immobilized. The silver-antibody complex was oxidized. The solution was pipetted and placed on the three-electrode assembly described in Example 1 and the amount of silver ions was measured electrochemically according to Example 1 except that a conventional Ag / AgCl reference electrode was used. . Therefore, the accumulated potential was −300 mV, and the stripping potential was changed between −300 mV and +400 mV. The results are shown in FIG. From this figure, it can be seen that the current response increases as the concentration of the analyte increases.

NT-proBNP immunoassay based on utilization of magnetic capture particles and silver sol signal particles In this example, anti-NT-proBNP antibodies labeled with silver-labeled anti-NT-proBNP antibodies and latex-coated magnetic particles are labeled with NT- Mixed with proBNP to form a sandwich complex. The resulting sample was concentrated under a magnetic field, which has the effect of separating unbound silver labeled antibody from the complex (and antibody labeled with unbound magnetic particles). The supernatant was removed and the sample was resuspended in PBS and placed on the electrode surface. Ferrocyanide was added to the electrode surface and oxidized to ferricyanide ions as an active reagent. The amount of silver ions produced was measured by reducing on the electrode surface followed by electrostripping, and the charge was also measured. This experiment was repeated using various concentrations of NT-proBNP, and the results obtained are shown in FIG. 7 as the relationship between charge and concentration (pg / ml). Here, the concentration (pg / ml) is the final concentration, not the concentration at the time of adding the solution.

In this example, and in the following example, the working electrode area is 0.8 mm × 4 mm = 3.2 mm ² and the charge is approximately −0.4 after subtracting the background (due to charging current, interference, etc.). It was determined by integrating the region under the peak of ~ 0.0V. The potential (unit V) may be relative to (carbon / K ₄ Fe (CN) ₆ , K ₃ Fe (CN) ₆ , NH ₄ SCN).

  More specifically, the silver sol complex was prepared by British Biocell international using anti-NT-ProBNP antibody and 80 nM diameter silver particles (OD 10). The magnetic particle composite used is a latex-coated paramagnetic particle (diameter 1.5 μm) on which Hytest 15F11 anti-NT-proBNP antibody is adsorbed and blocked with BSA to obtain a solution having a solid content of 10%. It was prepared by.

The assay was performed by incubating a sample (5 μl) supplemented with silver sol complex (10 μl), magnetic particle complex (5 μl) and NT-proBNP for 5 minutes. The sample was then concentrated under a magnetic field and the supernatant was removed. The sample was resuspended in PBS (10 μl) and concentrated using a bar magnet to remove the supernatant. After repeating this operation once more, the particles were resuspended and placed on the electrode surface. At this time, all the magnetic particles moved to the working electrode. A solution (5 μl) of 0.106 g K ₄ Fe (CN) ₆ dissolved in 1 ml of 4M SCN solution was added onto the electrode. Electrochemical measurements were performed using a constant voltage autolab, essentially as described in Example 1, using three screen printed carbon electrodes as working, counter and reference electrodes, respectively. The carbon ink used is D2 carbon ink manufactured by Gwent Electronic Materials.

The parameters in the electrochemical analysis of silver by anodic stripping voltammetry are as follows:
1. 1. 0.35 V for 10 seconds for conversion from Fe ²⁺ to Fe ³⁺ -1.6 V for 5 seconds Scan from -1.2V to 0.1V at a scan speed of 4.1Vs ^-1 for 55 seconds at -1.2V

  In step 1, ferrocyanide ions are oxidized on the working electrode to produce ferricyanide ions, which ferricyanide ions oxidize and solubilize silver. In steps 2 and 3, silver is deposited on the electrode and in step 4 the silver is stripped from the electrode. The strip charge Q was determined by subtracting the background (due to charge current, interference, etc.) and then integrating the area under the strip peak at about −0.4 to 0.0V.

  FIG. 7 shows the response of stripping charge with increasing analyte concentration.

NT-proBNP immunoassay based on trapping by silver sol signal particles of 20um diameter latex capture particles using a filter in the channel In this example, latex-labeled NT-proBNP antibody and silver-labeled NT-proBNP antibody Was mixed with NT-proBNP. The obtained mixture was injected into the channel and allowed to flow into the filter region having a pore size of 20 μm or less by a capillary effect. The filter serves to capture latex labeled antibody-analyte-silver labeled antibody complexes (and unbound latex antibodies as well). Unbound silver-labeled antibody passes through the filter. The captured species were then washed to remove any remaining unbound silver-labeled antibody and ferrocyanide solution was added. Both the oxidation of the reagent and the detection of silver ions are performed by an electrode provided in the vicinity of the filter region and upstream. Therefore, the complex of anti-NT-proBNP and 20 μm latex capturing particles is utilized to capture the complex of silver sol capturing NT-proBNP and anti-NT-proBNP.

  Latex particles were captured by a filter adjacent to the electrode in the channel, and the unbound silver complex was washed away by passing the solution through a bed packed with captured particles. The bound silver particles were solubilized and subsequently analyzed by stripping voltammetry on the electrode. This experiment was repeated several times to determine the charge (corresponding to the amount of silver ions detected at the electrode). FIG. 8 shows a plot of measured charge versus amount of NT-proBNP added.

  More specifically, the apparatus (see FIG. 9) comprises a working electrode, a counter electrode, and a reference electrode obtained by screen printing D2 carbon ink, manufactured by Gwent Electronic Materials (Gwent Electronic Materials), on a mylar sheet. This was affixed to a polystyrene molded product having a straight channel and a filter having a depth of 50 μm and subjected to a hydrophilic treatment using a U-shaped double-sided adhesive tape. The working electrode (WE) is the leftmost electrode next to the filter and was tested by adding the sample to the right side of the channel. The channel width used was 4 mm. The dimensions of the filter were as follows: length 5 mm, width 4 mm, channel width 30 μm, wall thickness 50 μm. The number of channels (openings) in the filter was 49.

The solution was prepared as follows. A standard solution (1.953 × 10 ⁶ pgml ⁻¹ ) obtained by dissolving NT-proBNP in PBSA (phosphate buffered saline, containing 0.05% azide) was diluted with 40 mgml ⁻¹ BSA. Thus, standard solutions having other concentrations were prepared.

In a 1.5 ml Eppendorf tube, 176 μl of PBSA solution containing 40 mg ml ⁻¹ BSA and 30 μl of standard solution were placed. After mixing this, 40 μl of silver sol (this sol was prepared by British Biocell International using anti-NT-ProBNP antibody and 80 nM diameter silver particles, blocked with β-casein, 0 (Suspended in 10 mM borate buffer (pH 8.5) containing 5 mgml ⁻¹ β-casein) and mixed for another 60 seconds. To this was added 33 μl nominal 5% solids latex (20 μm in diameter and sensitized with 15FI1 antibody PBSA) and the solution was mixed for 3 minutes.

  0.5% Tween 20 solution was added to fill the apparatus, followed by the latex, NTproBNP standard solution, and silver sol solution to the bead bed. To remove unbound silver, it was washed with 15 μl PBSA, and another 5 μl was added to fill the capillary. The filled bed was visually observed to estimate the covered electrode area. The capillary was sucked and dried by gel blotting, but liquid remained in the bead bed filled at this time. Immediately before the electrochemical measurement, 2.5 μl of PBSA solution containing 0.8 M SCN and 50 mM ferrocyanide was added to the bead bed.

For electrochemical measurements:
1. 1. Preconditioning for 5 seconds at 0V 2. Bias for 40 seconds at 0.5V Hold at -1.0V for 60 seconds 4. Scan from potential -1.0V to final potential 0.5V at scan speed 0.5Vs- ¹ .

  In step 2, ferrocyanide ions are oxidized on the working electrode to produce ferricyanide ions, and the ferricyanide ions oxidize and solubilize silver. In step 3, silver is deposited on the electrode, and then in step 4, the silver is stripped from the electrode. The strip charge Q was obtained by subtracting the background and then integrating the area under the peak during stripping.

  The results are shown in FIG. Charge measurements are plotted on a logarithmic scale against NT-proBNP concentration. Error bars represent the standard deviation of the signal in 3 or 4 measurements. FIG. 8 shows the stripping charge response with increasing analyte concentration.

Use of iodinated acetamide in the determination of silver in plasma This example shows the effect of adding iodinated acetamide. A possible problem when using silver in the presence of a protein-containing solution such as blood or plasma is that the silver binds tightly to the protein through the thiol group and becomes electrochemically inactive or electrochemically active. Is to decrease. Therefore, iodinated acetamide, which strongly binds to protein, was added to function as a protein masking agent, and the ability of protein to bind to silver was removed. This reagent can be provided dry in the apparatus.

  The principle of using iodoacetamide is that it reacts with thiol groups in the protein to create thioesters. The structure of iodoacetamide and its reaction with thiols is shown below:

Iodinated acetamide was added to plasma samples at a concentration of 80% from a 500 mM stock solution in methanol to a concentration of 5 mM. 20 μl of plasma (with or without iodoacetamide) was mixed with 5 μl of a solution containing ₄ M NH ₄ SCN, 250 mM potassium ferricyanide, 25 mM potassium ferrocyanide, and various concentrations of AgNO ₃ . The obtained solution containing ₄ mM iodoacetamide, 0.8 M NH ₄ SCN, 50 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and various concentrations of AgNO ₃ was placed on the electrode. The potential was scanned to −1.6 V at 1 V / s, held at −1.6 V for 5 seconds, then switched to −1.2 V and held for 115 seconds. Subsequently, the potential was scanned to 0.1 V at 1 V / s. The strip charge Q was obtained by subtracting the background and then integrating the area under the strip peak.

  The results are shown in FIG. Without the addition of iodoacetamide, the low concentration silver signal is very small. Addition of iodoacetamide restores some of the silver signal and allows detection of lower concentrations of silver.

It is a graph of electric current vs potential for showing the optimal pH at the time of silver forming a silver ion complex by mixing with the aqueous solution containing ammonium thiocyanate and ferric nitrate. It is a schematic diagram of the electrode used for an electrochemical measurement in an Example. 2 is a graph showing the current response for various amounts of silver particles present in a liquid sample measured according to the method of Example 1. FIG. 4 is an enlarged view of FIG. 3 obtained for a specific silver particle amount range. It is a schematic diagram of the immunoassay of the present invention. FIG. 6 shows the current response measured according to the method of Example 2 as a function of the concentration of human chorionic gonadotropin (hCG) used as the analyte. FIG. 7 is a graph showing the relationship between the measured charge value and the concentration of NTproBNP. Error bars represent the standard deviation of the signal determined by two or three measurements. FIG. 8 is a graph showing the relationship between the measured charge value and the concentration of added NTproBNP. FIG. 9 illustrates the microfluidic device used in Example 5. FIG. 10 is a graph showing the results of measuring silver ions in a plasma sample in the presence or absence of 5 mM iodoacetamide.

Claims (15)

Silver is oxidized to form silver ions, and the silver ions are reacted with thiocyanate and / or thiosulfate to form a soluble electrochemically active complex, where the complex is a sample. Is stable compared to components present or possibly present therein that form insoluble and / or electrochemically inactive complexes with the silver ions,
A method for measuring the presence or amount of a silver-labeled chemical species in a sample comprising electrochemically measuring the formed complex to provide an indication of the presence or amount of a silver-labeled chemical species .   The silver is oxidized using one or more oxidizing agents selected from permanganate, chromate, peroxide, persulfate, ferric ion and ferricyanide. The method described in 1. The method according to claim 1 or 2, wherein the soluble electrochemically active complex is formed in the presence of ammonium thiocyanate by the following reaction.
Ag ⁺ + m (NH ₄ ) ⁺ + n (SCN) ⁻ → [Ag (NH ₄ ⁺ ) m (SCN ⁻ ) n] ^{m−n + 1}   The method according to any one of claims 1 to 3, wherein the silver label is a fine particle.   The method according to claim 1, wherein the measurement of the electrochemically active complex is carried out by anodic stripping voltammetry.   6. A method according to any one of claims 1 to 5, wherein the silver labeled chemical species is formed by binding an analyte to be detected and a silver labeled binding partner. 7. The method of claim 6 , wherein the silver labeled binding partner is an antibody or antigen binding fragment thereof.   The method according to any one of claims 1 to 7, wherein the sample is a biological sample.   9. The method of claim 8, further comprising adding iodoacetamide as a protein masking agent. A silver-labeled binding partner that can bind to an analyte believed to be present in the sample to form a silver-labeled species,
A reagent for oxidizing silver to form silver ions, and a thiocyanate and / or thiosulfate for reacting with the silver ions to form a soluble electrochemically active complex Reagent, where said complex is stable compared to components present or possibly present in the sample that form insoluble and / or electrochemically inactive complexes with said silver ions And
A volume space in which the silver-labeled binding reagent can bind to the analyte, and capture to immobilize the silver-labeled chemical species so that the reagent can form a soluble electrochemically active complex Kit containing zones.   11. The kit of claim 10, wherein the silver labeled binding partner is an antibody or antigen binding fragment thereof.   The said reagent for oxidizing silver is one or more oxidizing agents selected from permanganate, chromate, peroxide, persulfate, ferric ion and ferricyanide. The kit according to 10 or 11.   The kit according to any one of claims 10 to 12, wherein the silver label is a fine particle.   14. A kit according to any one of claims 10 to 13 provided in combination with a reader for electrochemical measurement of the soluble electrochemically active complex. The kit according to any one of claims 10 to 14, further comprising iodoacetamide as a protein masking agent.

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