JP7546084B2 - Immunoassay reagent using insoluble carrier - Google Patents

Description

The present invention relates to an immunoassay reagent that uses an insoluble carrier.

Automated analyzers that analyze components in biological samples such as blood and urine are widely used in hospitals, testing institutions, etc., because they can simultaneously perform large-scale component analysis and investigation. Among them, immunoassays using insoluble carriers are widely used because they are suitable for measurement using automated analyzers. Examples include latex agglutination immunoassays that utilize the agglutination reaction of latex particles, and CLEIA (chemiluminescent enzyme immunoassay) that uses magnetic particles.
In large-scale automatic analyzers, in order to save the effort and time required to set up measurement reagents in the device, each reagent container is stored in the device's reagent storage after the measurement of the test sample is completed, and can be used for the next measurement as is. Therefore, the reagent storage generally has a cooling function to prevent the deterioration of the reagents due to temperature changes.
Furthermore, as in Patent Document 1, a lid that automatically opens and closes when dispensing reagent is attached, or as in Patent Document 2, the humidity inside the reagent chamber is increased to suppress evaporation of reagent inside the reagent chamber, and measures are taken to prevent concentration of reagent due to evaporation and contamination of other reagents with volatilized reagent.
Nevertheless, some measurement reagents have a problem in that their reactivity decreases over time when stored in the reagent storage of an automatic analyzer (poor on-board stability).

JP 2006-030170 A JP 2011-191117 A

Therefore, the object of the present invention is to provide a method for suppressing the on-board decrease in reactivity and improving the on-board stability of an immunoassay reagent that uses an insoluble carrier, such as a latex agglutination immunoassay reagent that contains sensitized latex particles, and to provide an immunoassay reagent with improved on-board stability.

As a result of intensive research aimed at solving the above problems, the inventors discovered that adding an oxygen radical absorber to an immunoassay reagent that uses an insoluble carrier can suppress the decrease in reactivity and improve on-board stability, leading to the present invention.

The present invention relates to the following inventions:
[1] An immunoassay reagent using an insoluble carrier containing an oxygen radical absorber.
[2] The measurement reagent according to [1], wherein the immunoassay is a latex agglutination immunoassay or an immunoassay using magnetic particles.
[3] The measuring reagent according to [1] or [2], wherein the oxygen radical absorber is contained together with an insoluble carrier having an antigen or antibody for the analyte immobilized thereon.
[4] A measurement reagent according to any one of [1] to [3], which is a reagent for an automatic analyzer.
[5] The measurement reagent according to any one of [1] to [4], wherein the oxygen radical absorber is a compound selected from the group consisting of methionine, cystine, glutathione (oxidized form), hydroxymethyl EDOT, and sodium nitrite.
[6] The measurement reagent according to any one of [1] to [5], wherein the concentration of the oxygen radical absorber is 1.0×10 ⁻⁶ % by weight to 2.0% by weight.
[7] A method for improving the on-board stability of an immunoassay reagent, comprising adding an oxygen radical absorber to the immunoassay reagent, the method comprising the steps of: immobilizing an antigen or antibody against a substance to be analyzed on an insoluble carrier; and

The immunoassay reagent of the present invention can suppress the decrease in reactivity on-board and improve the on-board stability of the assay reagent.

1 is a graph showing the inhibitory effect of various substances on the decrease in reactivity of an anti-soluble fibrin (SF) antibody-sensitized latex solution caused by the presence of sodium hypochlorite. 1 is a graph showing the suppressive effect of various oxygen radical absorbents on the decrease in reactivity of an anti-soluble fibrin (SF) antibody-sensitized latex solution caused by the presence of sodium hypochlorite. 1 is a graph showing the inhibitory effect of L-methionine at various concentrations on the decrease in reactivity of an anti-soluble fibrin (SF) antibody-sensitized latex solution caused by the presence of sodium hypochlorite. 1 is a graph showing the effect of L-(-)-cystine at various concentrations in suppressing the decrease in reactivity of an anti-soluble fibrin (SF) antibody-sensitized latex solution caused by the presence of sodium hypochlorite. 1 is a graph showing the inhibitory effect of various concentrations of glutathione (oxidized type) on the decrease in reactivity of an anti-soluble fibrin (SF) antibody-sensitized latex solution caused by the presence of sodium hypochlorite. 1 is a graph showing the inhibitory effect of various concentrations of sodium nitrite on the decrease in reactivity of an anti-soluble fibrin (SF) antibody-sensitized latex solution caused by the presence of sodium hypochlorite. 1 is a graph showing the inhibitory effect of L-methionine on the decrease in reactivity of an anti-tissue plasminogen activator/plasminogen activator inhibitor-1 complex (t-PA/PAI-1 complex) antibody-sensitized latex solution caused by the presence of sodium hypochlorite.

The measurement reagent of the present invention is an immunological measurement reagent that uses an insoluble carrier and contains an oxygen radical absorber.

In this specification, oxygen radical means a free radical having an oxygen atom, which is an atom (or molecule) having an unpaired electron. Examples include superoxide anion radical (.O ₂ ⁻ ) and hydroxyl radical (.OH). Molecules having unpaired electrons are very unstable and attempt to become stable by depriving electrons from their counterparts in order to form a pair, so they are highly reactive and can attack lipids, proteins, nucleic acids, carbohydrates, etc., and damage their functions. For example, sodium hypochlorite is relatively unstable and easily decomposed at room temperature, and releases oxygen and generates oxygen radicals through natural decomposition, photolysis, thermal decomposition, acid decomposition, and catalytic decomposition by heavy metals. It is known that it is easily decomposed at high storage temperatures, but is relatively stable at low temperatures. However, it was unexpectedly discovered in the course of trying to solve this problem that sodium hypochlorite can be decomposed even in the reagent storage of an automatic analyzer, which is kept at about 2 to 10°C in a dark place, and can affect the measurement reagent.

The oxygen radical absorbent used in the present invention is not particularly limited as long as it absorbs oxygen radicals, but low molecular weight compounds having the structure -S-, -N-, or -O- are preferred. In addition, since they are prone to problems such as precipitation, it is desirable to use compounds that have a water solubility of 0.1 g/L (25°C) or more, preferably a water solubility of 1 g/L (25°C) or more, and more preferably a water solubility of 10 g/L (25°C) or more, and that have a pH in the range of 5 to 10 when made into a solution.

Specific examples of amino acids or compounds having the -S- structure include methionine, cystine, cysteine, glutathione (oxidized form), N-acetylcysteine, which is a precursor of glutathione, the synthetic compound (2,3-dihydrothieno[3,4-b][1,4]dioxin-2-yl)methanol (also known as hydroxymethylEDOT), and sulfites (sodium sulfite, ammonium sulfite, potassium sulfite, sodium hydrogen sulfite). Examples of compounds having the -N- structure include nitrites (sodium nitrite, potassium nitrite, calcium nitrite). Compounds with the -O- structure include ascorbic acid (vitamin C), uric acid, tocopherols (alpha-tocopherol (vitamin E)), polyphenols (catechin, tannin, rutin, isoflavone, lebiretin, chlorogenic acid, ellagic acid, lignan, sesamin, curcumin, coumarin, oleocanthal, quercetin), and the synthetic compound butylhydroxyanisole (BHA). Two or more of these compounds may be used in combination.

The concentration of the oxygen radical absorber is not particularly limited as long as it is a concentration that can absorb the effects of oxygen radicals on-board. Those skilled in the art can determine the optimal concentration to be added for each measurement reagent, taking into consideration the solubility, pH, and effect on the antigen-antibody reaction of the oxygen radical absorber. Usually, an effect is obtained at 1.0×10 ⁻⁶ wt % or more, an excellent effect is obtained at 1.0×10 ⁻⁴ wt % or more, and a particularly excellent effect is obtained at 1.0×10 ⁻² wt % or more. In particular, compounds having -S- tend to be effective even at low concentrations, and methionine is effective over a wide concentration range from 1.0×10 ⁻⁷ wt % to 2.0 wt %. The upper limit of the amount to be added depends on the solubility of the oxygen radical absorber, etc., but is preferably 2.0 wt % or less, more preferably 1.0 wt % or less, and even more preferably 0.3 wt % or less.

The reagent of the present invention can be used for measurement by manual methods, but is particularly suitable for use as a reagent to be installed in an automatic analyzer. In the present invention, an automatic analyzer refers to one manufactured and sold by various companies for use mainly in clinical testing. In particular, the effects of the present invention are easily obtained in an automatic analyzer having a reagent storage for storing each reagent container used in the measurement within the device until the next measurement. Specific examples include so-called multi-item (general-purpose reagent type) automatic analyzers such as the Hitachi High-Technologies automatic analyzer series, Toshiba Medical Systems' TBA series, JEOL's BM series, Beckman Coulter Biomedical, Roche, and Ortho Clinical Diagnostics, as well as so-called dedicated reagent type automatic analyzers such as the near-infrared measuring device LPIA (registered trademark) (LSI Medience) and the scattered light intensity measuring device (Dade Behring), as well as blood coagulation measuring devices and luminescence immunoassay devices capable of optical measurement, such as the Sysmex CS-CA series, the Sekisui Medical CP series, the IL ACLTOP series, the LSI Medience STACIA, the Abbott ARCHITECT analyzer series, and the Siemens ADVIA Centaur series.

The reagent storage of the automatic analyzer has a cooling function to store the measurement reagents at a predetermined temperature range according to the measurement reagents to be stored, and preferably has measures to prevent condensation and evaporation of the reagents. Depending on the automatic analyzer, cleaning solutions and rinse solutions for cleaning the nozzles and cuvettes used in the measurement are also stored in the reagent storage together with the measurement reagents. Cleaning solutions include alkaline solutions, acidic solutions, neutral detergents (surfactants), and hypochlorite agents, and when stored together with hypochlorite-based cleaning solutions, the measurement reagents are more susceptible to the effects of oxygen radicals. The measurement reagent of the present invention can suppress a decrease in reactivity and maintain on-board stability even when stored together with reagents and cleaning solutions that can generate oxygen radicals in the reagent storage.

The measurement reagent of the present invention may be in the form of a single reagent, or in the form of a multiple reagent such as a two-reagent system consisting of a first reagent and a second reagent (i.e., a kit). For example, in the case of latex agglutination immunoassay, a two-reagent system consisting of a first reagent and a second reagent is generally used, in which the first reagent is a buffer solution for preparing the conditions during the antigen-antibody reaction, and the second reagent is latex particles on which an antigen or antibody for the analyte is immobilized. In the case of a multiple reagent reagent, it is sufficient that at least the latex particles on which the antigen or antibody is immobilized contain an oxygen radical absorbent, and each reagent may contain an oxygen radical absorbent. In the case of an immunoassay using magnetic particles for B/F separation, it is often composed of a two to three liquid system, but as above, it is sufficient that at least the magnetic particles on which the antigen or antibody is immobilized contain an oxygen radical absorbent.

The substance to be analyzed is not particularly limited as long as it is a substance (particularly a physiologically active substance) that can generally be analyzed using an antigen-antibody reaction. Representative examples of substances to be analyzed include proteins and lipids, and more specifically, various antigens, antibodies, receptors, enzymes, etc. Specific examples include C-reactive protein (CRP), rheumatoid factor, ferritin, β-2 microglobulin, α-fetoprotein (AFP), anti-streptolysin O antibody, IgE, Treponema pallidum antibody, antibody against syphilis lipid antigen, hepatitis B virus (HBS antibody, HBS antigen, HBc antibody, HBe antibody), D-dimer, fibrin-fibrinogen degradation products (FDP), soluble fibrin (SF), plasmin-α2-plasmin inhibitor complex (PPI), prostate-specific antigen (PSA), elastase 1, elastase XDP, thrombomodulin, thrombin-antithrombin complex (TAT), tissue plasminogen activator-plasminogen activator inhibitor 1 complex (t-PA-PAI-1 complex), hyaluronic acid, presepsin, IL-2R, and anti-DNA antibodies.

The insoluble carrier according to the present invention is not particularly limited as long as it is one that is usually used in this field, and examples thereof include insoluble carriers of homopolymers (e.g., polystyrene) obtained by polymerizing vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylic acid esters, and methacrylic acid esters. In addition, examples thereof include insoluble carriers made of copolymers (e.g., styrene-styrene sulfonate copolymers, methacrylic acid polymers, acrylic acid polymers, vinyl chloride-acrylic acid ester copolymers, etc.), and insoluble carriers made of butadiene copolymers such as styrene-butadiene copolymers, methyl methacrylate-butadiene copolymers, and acrylonitrile-butadiene copolymers. Examples of insoluble carriers include insoluble carriers having functional groups such as carboxyl groups, primary amino groups, carbamoyl groups (-CONH ₂ ), hydroxyl groups, and aldehyde groups, and having a base made of the above-mentioned organic fine particles.

In the present invention, magnetic particles can be used as the insoluble carrier (see Japanese Patent Application Laid-Open No. 7-151755). The magnetic particles can _be easily magnetized by magnetic induction, and examples of such magnetic particles include magnetic particles made of hydrophobic polymers such as polystyrene, polyacrylonitrile, polymethacrylonitrile, polymethyl methacrylate, polycapramide, and polyethylene terephthalate, crosslinked hydrophilic _polymers such as polyacrylamide, polymethacrylamide, polyvinylpyrrolidone, polyvinyl alcohol, poly( ₂ -oxyethyl acrylate), poly( ₂ -oxyethyl methacrylate), poly(2,3-dioxypropyl acrylate), poly(2,3-dioxypropyl methacrylate), and polyethylene glycol methacrylate, or latex, gelatin, and liposomes containing metals such as iron tetroxide (Fe 3 O 4 ), iron sesquioxide (γ-Fe 2 O 3 ), various ferrites, iron, manganese, nickel, cobalt, and chromium, or alloys of cobalt, nickel, and manganese inside.

The antigen or antibody immobilized on the insoluble carrier is not particularly limited as long as it is an antigen or antibody that undergoes an antigen-antibody reaction with the analyte, and the antibody (including both monoclonal and polyclonal antibodies) may be an immunoglobulin molecule itself or an antibody fragment [e.g., Fab, Fab', F(ab') ₂ , or Fv]. When using a monoclonal antibody, there are cases where two or more types of monoclonal antibodies that bind to the antigen, which is the analyte, at different sites are used, and cases where one type of monoclonal antibody is used when there are two or more recognition sites for the antigen.

The method of immobilizing an antigen or antibody on an insoluble carrier may be carried out in accordance with a known method. For example, the antibody and insoluble carrier may be suspended in a buffer solution, reacted at 25°C for 1 hour, and then centrifuged, blocked, or otherwise treated as typically performed in this field. Alternatively, the antigen or antibody may be immobilized on the insoluble carrier by chemical bonding or by a biotin-avidin reaction.

The components used in blocking the insoluble carrier can be appropriately selected from known blocking agents, such as blocking agents made of protein components such as bovine serum albumin (BSA), human serum albumin, casein, skim milk, and sericin, as well as blocking agents made of synthetic polymers consisting of hydrophilic and hydrophobic blocks.

The measurement reagent of the present invention may further contain additives that can be added to immunological measurement reagents, such as buffers, non-specific reaction inhibitors, sensitizers, etc.

As the buffer solution, various buffer solutions appropriate for the type of the analyte can be selected. This buffer solution may be any buffer solution that does not inactivate the analyte and has an ion concentration and pH that does not inhibit the antigen-antibody reaction. For example, Good's buffer solution, glycine buffer solution, or Tris buffer solution can be used.

Examples of non-specific reaction inhibitors that can be added to the measurement reagent of the present invention include antibodies or receptors against substances that cause non-specific reactions; buffers such as Tris buffer, phosphate buffer, glycine buffer, borate buffer, citrate buffer, acetate buffer, and Good's buffer; chelating agents such as EDTA, CyDTA, DTPA, EGTA, NTA, and NTP; salts such as sodium chloride, potassium chloride, sodium sulfate, calcium sulfate, magnesium sulfate, calcium carbonate, and sodium carbonate; and non-ionic surfactants such as fatty acid diethanolamide, polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, fatty acid sorbitan ester, alkyl polyglucoside, alkyl monoglyceryl ether, polyoxyethylene sorbitan fatty acid ester, fatty acid alkanolamide, and alkyl glycoside.

In addition, water-soluble polymers and proteins are preferably used as sensitizers that can be added to the measurement reagent of the present invention. Examples include water-soluble polymers such as sodium alginate, propylene glycol alginate, dextran, dextran sulfate, polyvinyl alcohol, polyethylene glycol, and polyvinylpyrrolidone, albumins such as bovine serum albumin, and globulins such as gamma-globulin.

The test sample is not particularly limited as long as it is a sample that may contain the antigen or antibody that is the substance to be analyzed, and examples include biological fluids commonly used in clinical diagnosis, such as blood, serum, plasma, or urine, or experimental samples.

The present invention also includes a method for suppressing a decrease in the on-board reactivity of an immunoassay reagent and improving its on-board stability. In the present invention, on-board stability means storage stability of the assay reagent in a state in which it is placed in a reagent storage of an automatic analyzer. Improved on-board stability means that fluctuations in the reactivity of the reagent are suppressed after a certain period of time has elapsed with the reagent placed in a reagent storage of an automatic analyzer.

The method for evaluating on-board stability involves loading the reagent into the reagent storage of an automated analyzer in accordance with the device's operating instructions and the reagent's accompanying documentation, and evaluating the stability over a certain period of time. There are no limitations on the time the reagent is opened or the period it is left in the reagent storage, but typically the stability is evaluated over a period of 5 to 105 days (15 weeks) with the reagent open for 5 to 24 hours per day. In terms of stability, it is desirable for the reactivity of the reagent after it has been loaded into the device for a certain period of time to be within 100±20%, and preferably 100±15%, of the reagent immediately after opening.

The present invention will be explained in detail below with reference to examples, but these are not intended to limit the scope of the present invention.

Example 1: Examination of substances that suppress reactivity decrease
For the study, Iatro SF II (manufactured by LSI Medience Corporation), a general-purpose reagent for automatic analyzers, was used.
The substance that suppresses the decrease in reactivity was examined using activated carbon (As One Corporation), 5 mol/L sodium hydroxide solution (Wako), and Ageless (registered trademark) (Mitsubishi Gas Chemical). In order to reproduce the conditions in the reagent storehouse, 50 mL of sodium hypochlorite-based cleaning solution used in automatic analyzers was prepared in a polyethylene container (50 mL, Eye Boy). 9 mL of the second reagent of Iatro SF II (anti-SF (soluble fibrin) antibody-sensitized latex solution) was placed in a polyethylene container (11 mL, Shoei Resin), and placed in a zippered plastic bag (Seinichi) with the lid open. The cleaning solution and each substance were placed in the bag, and the bag was closed and stored at 2 to 10°C for 21 days.
As a cleaning solution control, 50 mL of RO water was similarly stored with the latex solution and each test substance.

Using the reagents left for 21 days, the reactivity was evaluated using a Hitachi 7170S automatic analyzer. 3 μL of the SF standard and the first reagent of Iatro SF II were mixed and kept at 37° C. for about 5 minutes, and then the second reagent stored with each test substance was added, and the change in absorbance after about 5 minutes was measured at a wavelength of 800 nm.
Measurements were carried out for the reagent coexisting with the cleaning solution and the reagent coexisting with RO water, and the percent change in absorbance was determined for the reagent coexisting with RO water.

The results are shown in Figure 1. As a result, it was found that sodium hydroxide, which has a hydroxyl radical absorbing effect, and Ageless, an oxygen scavenger that creates an oxygen-free state in a sealed container, were effective in suppressing the decline in reactivity, and it was found that the measurement reagents in the reagent storage of the automatic analyzer are affected by oxygen and oxygen radicals from the air conditioning.

Example 2: Examination of oxygen radical absorbents
We searched for oxygen radical absorbers that can be added to the measurement reagent. As candidate compounds for oxygen radical absorbers, 0.1% each of L-(-)-cystine (Nacalai Tesque), hydroxymethyl EDOT (Sigma-ALDRICH), sodium nitrite (Wako), oxidized glutathione (Wako), and L-methionine (Wako) were added to the second reagent of Iatro SF II (anti-SF (soluble fibrin) antibody-sensitized latex solution). Instead of a cleaning solution, 50 mL of sodium hypochlorite solution (Wako) was prepared in a polyethylene container (50 mL, Aiboy). 4 mL of the latex solution to which each oxygen radical absorber was added was placed in a polyethylene container (11 mL, Shoei Resin), and the solution was placed in a zipper-type plastic bag (Seinichi) with the lid open together with the sodium hypochlorite solution, and the zipper was closed and stored at 2 to 10°C for 14 days. Five bottles of latex solution were placed together in 50 mL of sodium hypochlorite solution.
As a control for the sodium hypochlorite solution, 50 mL of RO water was stored together with the latex solutions to which each absorbent had been similarly added.

Using the reagents left for 14 days, the reactivity was evaluated using a Hitachi 7170S automatic analyzer. 3 μL of the SF standard and the first reagent of Iatro SF II were mixed and kept at 37° C. for about 5 minutes, and then the second reagent containing various oxygen radical absorbents was added, and the change in absorbance after about 5 minutes was measured at a wavelength of 800 nm.
Measurements were carried out for the reagent coexisting with the sodium hypochlorite solution and the reagent coexisting with RO water, and the percentage change in absorbance was determined for the reagent coexisting with RO water.

The results are shown in Figure 2. Reagents that were coexistent with RO water and showed 85% or more reactivity were judged to have a reactivity decrease inhibitory effect. As a result, all candidate compounds were found to have a reactivity decrease inhibitory effect.

Example 3: Investigation into the cause of decreased reactivity
To investigate the cause of the decrease in reactivity, the antibody-sensitized latex solution after storage was analyzed by Western blotting.
2 mL of each of the latex solutions obtained in Example 2 (Sample 1: 0% methionine packaged with RO water, Sample 2: 0.1% methionine packaged with RO water, Sample 3: 0% methionine packaged with sodium hypochlorite solution, Sample 4: 0.1% methionine packaged with sodium hypochlorite solution) was centrifuged at 14,000 rpm at 10° C. for 30 minutes to separate the antibody-sensitized latex particles and the supernatant. The supernatant was concentrated from 1.8 mL to 70 μL using an Amicon Ultra-0.5 mL Centrifugal Filters Ultracel-10k; Merck) to prepare a supernatant sample.
Anti-SF antibodies in the above samples were detected by WB Western blotting. The NuPAGE electrophoresis system and X-Cell II blot module (both from Invitrogen) were used. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on non-reduced samples. Rabbit anti-mouse IgG (DAKO) and alkaline phosphatase-labeled anti-rabbit antibody (anti-rabbit antibody-ALP; from Invitrogen) were used to detect the antibodies.

As a result, IgG-derived fragments were detected in the supernatant samples of samples 2 and 4 to the same extent as in sample 1. However, a larger amount of IgG-derived fragments was detected in the supernatant sample of sample 3 compared to sample 1, confirming that the antibody had peeled off from the antibody-sensitized latex particles in the supernatant due to the influence of the sodium hypochlorite solution. Furthermore, a sample stored for an extended period of one week was examined, and it was confirmed that the antibody that had peeled off in the supernatant had decomposed over time due to the influence of the sodium hypochlorite solution.

Example 4: Examination of effective concentration of oxygen radical absorber
(1) Effective concentration of L-methionine The effective concentration of methionine, which is inexpensive and easily available, was examined. L-methionine (Wako) was added to the second reagent of Iatro SF II at the following concentrations: 0.0 wt%, 1.0× ^10-6 wt%, 1.0× ^10-5 wt%, 3.0× ^10-5 wt%, 6.0× ^10-6 wt%, 1.0× ^10-4 wt%, 3.0× ^10-4 wt%, 6.0× ^10-4 wt%, 1.0× ^10-3 wt%, 1.0× ^10-1 wt%, 3.0× ^10-1 wt%, 1.0 wt%, and 2.0 wt% to prepare latex solutions.
50 mL of sodium hypochlorite solution (Wako) was placed in a polyethylene container (50 mL, Aiboy), and 4 mL of each latex container was placed in a polyethylene container (11 mL, Shoei Resin), and both were placed in a checked plastic bag (Seinichi) with the lid open, and the bag was closed and stored at 2 to 10°C for 14 days. Five bottles of latex solution were placed in one 50 mL bottle of sodium hypochlorite solution.
As a control for the sodium hypochlorite solution, 50 mL of RO water was stored with the latex solution in the same manner.

Using the reagent that had been left for 14 days, the reactivity was evaluated using a Hitachi 7170S automatic analyzer. 3 μL of SF standard and 150 μL of the first reagent of Iatro SF II were mixed and kept at 37° C. for about 5 minutes, and then the above-mentioned latex solution was added as the second reagent, and the absorbance was measured at a wavelength of 800 nm for about 5 minutes. Measurements were carried out for the following L-methionine (Wako)-added latex reagents: 0.0 wt%, 1.0 x 10 ^-6 wt%, 1.0 x 10 ^-5 wt%, 3.0 x 10 ^-5 wt%, 6.0 x 10 ^-6 wt%, 1.0 x 10 ^-4 wt%, 3.0 x 10 ^-4 wt%, 6.0 x 10 ^-4 wt%, 1.0 x 10 ^-3 wt%, 1.0 x 10 ^-1 wt%, 3.0 x 10 ^-1 wt%, 1.0 wt%, and 2.0 wt%, in the presence of sodium hypochlorite solution and in the presence of RO water, and the change in absorbance for the reagent in the presence of RO water was determined.

The results are shown in Figure 3. Reagents that were coexistent with RO water and showed 85% or higher reactivity were judged to be effective. As a result, it was confirmed that L-methionine concentrations between 1.0 x 10 ^-6 % by weight and 2.0% by weight were effective in suppressing the decrease in reactivity caused by sodium hypochlorite, and on-board stability was improved.

Example 5: Examination of effective concentrations of other oxygen radical absorbents
To the second reagent of Iatro SF II, 0.0 wt%, 1.0× ^10-4 wt%, 1.0× ^10-3 wt%, 1.0×10-1 wt%, and 3.0× ^10-1 wt% of L-(-)-cystine (Nacalai Tesque), 0.0 wt%, 1.0× ^10-4 wt%, 1.0× ^10-3 wt%, 1.0× ^10-1 wt%, and 1.0 wt% of glutathione (oxidized type) (Wako), and 0.0 wt%, 1.0× ^10-4 wt%, 1.0× ^{10-3 wt} %, 1.0× ^10-1 wt%, and 1.0 wt% of sodium nitrite (Wako) were added to prepare latex solutions.
As in Example 4, the reagent was stored at 2 to 10°C for 16 days in the presence of sodium hypochlorite solution and RO water, respectively, and measurements were performed using a Hitachi 7170S automatic analyzer to determine the change in absorbance for the reagent in the presence of RO water.

The results are shown in Figures 4 to 6. The inhibitory effect of sodium hypochlorite on the decrease in reactivity was observed at ^1.0x10-4wt % to 3.0x10-1wt% for cystine, ^10-4wt % to ^1.0wt % for glutathione (oxidized form), and ^1.0x10-4wt % to ^1.0x10-1wt % for sodium nitrite, and on-board stability was improved. Note that the latex solution to which 1.0wt% sodium nitrite was added coagulated during storage and could not be measured.

Example 6: Confirmation of the effect of suppressing the decrease in reactivity in another reagent
The effect of sodium hypochlorite solution was confirmed using a commercially available tissue plasminogen activator-plasminogen activator inhibitor 1 complex (t-PA-PAI-1 complex) measurement kit. This reagent is a two-reagent system, with the first reagent containing a buffer solution and the second reagent containing antibody-sensitized latex particles. L-methionine (Wako) was added to the latex solution, which is the second reagent, at 0.0% by weight and 0.1% by weight, respectively, and the latex solution was stored at 2 to 10°C for 21 days in the presence of sodium hypochlorite solution and RO water, respectively, as in Example 2, and measurement was performed using a Hitachi 7170S automatic analyzer to determine the absorbance change for the reagent in the presence of RO water.

The results are shown in Figure 7. As a result, even in the t-PA/PAI-1 complex measurement kit, a decrease in reactivity was observed when L-methionine was 0.0% by weight (additive-free product) in the presence of sodium hypochlorite solution, but the addition of 0.1% by weight of L-methionine suppressed the decrease in reactivity and improved on-board stability.

The measurement reagent of the present invention can be suitably used as a measurement reagent for an automatic analyzer that analyzes components in biological samples. Furthermore, the method of improving on-board stability of the present invention can be used to manufacture measurement reagents for the automatic analyzer.

Claims (7)

A method for preventing the antibody from peeling off from an insoluble carrier, comprising adding an oxygen radical absorber to an immunoassay reagent containing an insoluble carrier having an antibody against an analyte immobilized thereon, the antibody being used in combination with sodium hypochlorite . A method for improving the on-board stability of an immunoassay reagent, which includes an insoluble carrier having an antibody against an analyte immobilized thereon and is used in combination with sodium hypochlorite, by adding an oxygen radical absorber to the immunoassay reagent, thereby preventing the antibody from peeling off from the insoluble carrier. 3. The method according to claim 1 or 2 , wherein the immunoassay is a latex agglutination immunoassay or an immunoassay using magnetic particles. The method according to any one of claims 1 to 3 , wherein the oxygen radical absorber is contained together with an insoluble carrier having an antibody against the analyte immobilized thereon. The method according to any one of claims 1 to 4 , which is a reagent for an automatic analyzer. The method according to any one of claims 1 to 5 , wherein the oxygen radical absorber is a compound selected from the group consisting of methionine, cystine, glutathione (oxidized form), hydroxymethyl EDOT, and sodium nitrite. The method according to any one of claims 1 to 6 , wherein the concentration of the oxygen radical absorber is 1.0 x 10 ^-6% by weight to 2.0% by weight.