CN115023611A - Immunological analysis method and immunological analysis kit - Google Patents
Immunological analysis method and immunological analysis kit Download PDFInfo
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- CN115023611A CN115023611A CN202180011768.7A CN202180011768A CN115023611A CN 115023611 A CN115023611 A CN 115023611A CN 202180011768 A CN202180011768 A CN 202180011768A CN 115023611 A CN115023611 A CN 115023611A
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- antibody
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- polyethylene glycol
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
Abstract
The present invention addresses the problem of providing a technique for improving the sensitivity of an immunological analysis method and for accurately analyzing a substance to be detected. The aforementioned object can be achieved by an immunological method for analyzing a test substance, which comprises bringing a biological sample that may contain the test substance into contact with a conjugate of a polyethylene glycol-modified antibody and a labeling substance.
Description
Technical Field
The present invention relates to an immunoassay method and an immunoassay kit for detecting a substance to be detected. The present invention also relates to a method for reducing measurement error in an immunological assay method for detecting a substance to be detected. The invention also relates to a preparation method of the conjugate of the polyethylene glycol modified antibody and the ruthenium complex.
Background
When the amount of a substance to be detected in a biological sample is analyzed, an immunological analysis method using an antibody against the substance to be detected may be used. In the immunological analysis method, since simple and highly sensitive analysis is possible, automation of an analysis device such as an immunoassay reader is possible, and a method of measuring luminescence or fluorescence to measure a substance to be detected in a sample is utilized in many fields including clinical examination.
In the method of measuring luminescence or fluorescence, when obtaining a measurement value of the amount of a substance to be detected, it is necessary to subtract a value of a blank to which a biological sample is not added. However, when a highly hydrophobic labeling substance binds to an antibody, the labeled antibody may be attached to the carrier, and the amount of the substance to be detected may not be accurately measured. In addition, it has been observed that the amount of the substance to be detected cannot be accurately measured because the labeled antibodies are aggregated with each other.
Patent document 1 teaches a chemiluminescent immunoassay reagent characterized by binding a conjugate of an antibody and a hydrophilic compound having a dissociative group or a polar group and having a substituent reactive with an amino group of the antibody, a water-soluble polymer compound and a chemiluminescent substance. In patent document 1, in the case of using a labeling substance having low solubility under neutral pH conditions, the isoelectric point of the labeled antibody is lowered by adding a linker having a low isoelectric point to the antibody, thereby improving the solubility under neutral pH conditions. However, in order to improve the sensitivity, a technique for accurately measuring the amount of a substance to be detected by applying an antibody labeled with a larger amount of a labeling substance is required to further improve the sensitivity.
Documents of the prior art
Patent document
Patent document 1: japanese patent laid-open publication No. 58-77662
Summary of The Invention
The present invention aims to provide a technique for improving the sensitivity of an immunological analysis method and accurately analyzing the amount and/or presence of a substance to be detected.
The present inventors have found that, in a method for immunologically analyzing a substance to be detected, when a biological sample having a possibility of containing the substance to be detected, a carrier to which a capture antibody is immobilized, and a labeled antibody in which a labeled substance is conjugated to a polyethylene glycol-modified antibody are brought into contact with each other, the substance to be detected can be accurately analyzed by preventing aggregation of the labeled antibody, nonspecific adsorption of the labeled antibody to the carrier, and the like, and have completed the present invention.
Specifically, the present invention is as follows.
<1> a method for immunological analysis of a substance to be detected, which comprises contacting a biological sample having a possibility of containing the substance to be detected with a conjugate, wherein the conjugate is a conjugate of a polyethylene glycol-modified antibody and a labeling substance.
The immunological assay method according to <2> <1>, wherein the biological sample having a possibility of containing the substance to be detected is at least one selected from the group consisting of blood, plasma, serum and urine.
The immunoassay method according to any one of the items <3> <1> and <2>, wherein the polyethylene glycol has a weight average Molecular Weight (MW) of 250 to 15000.
The immunological analysis method according to any one of <4> to <1> to <3>, wherein the labeling substance is an electrochemiluminescent substance, and the immunological analysis method is an electrochemiluminescence method (ECL method).
The immunological assay method according to any one of <5> to <1> to <4>, wherein the polyethylene glycol-modified antibody is a polyethylene glycol-modified IgM antibody or a polyethylene glycol-modified IgG antibody.
The immunoassay method according to any one of <6> <1> to <5>, wherein the number of binding molecules of the labeling substance in 1-molecule antibody is 10 to 200.
<7> a kit for immunological analysis of a substance to be detected, which comprises a conjugate of a polyethylene glycol-modified antibody and a labeling substance.
<8> <7> the kit for immunological analysis, which is used for at least one biological sample selected from the group consisting of blood, plasma, and serum.
The kit for an immunological assay according to any one of <9> <7> and <8>, wherein the polyethylene glycol has a weight average Molecular Weight (MW) of 250 to 15000.
The kit for an immunological analysis according to any one of <10> <7> to <9>, wherein the labeling substance is an electrochemiluminescent substance, and the immunological analysis is an electrochemiluminescence method (ECL method).
The immunoassay kit according to any one of <11> <7> to <10>, wherein the polyethylene glycol-modified antibody is a polyethylene glycol-modified IgM antibody or a polyethylene glycol-modified IgG antibody.
The immunoassay kit according to any one of <12> <7> to <11>, wherein the number of binding molecules of the labeling substance in 1-molecule antibody is 10 to 200.
<13> a method for reducing measurement error in immunological analysis of a test substance, which comprises contacting a biological sample having a possibility of containing the test substance with a conjugate, wherein the conjugate is a conjugate of a polyethylene glycol-modified antibody and a labeling substance.
<14> the method for reducing measurement error of <13>, wherein the biological sample having a possibility of containing the test substance is one or more selected from the group consisting of blood, plasma, and serum. The method for reducing measurement error of <15> <13> or <14>, wherein the polyethylene glycol has a weight average Molecular Weight (MW) of 250 to 15000.
<16> a method for producing a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex, which comprises the following steps (1) to (3):
(1) a first complex forming step of bringing the antibody into contact with the nail complex to form a first complex,
(2) a conjugate-forming step of bringing polyethylene glycol into contact with the first complex, forming a conjugate of a polyethylene glycol-modified antibody and a peg complex, and
(3) a free ruthenium complex removal step of removing free ruthenium complexes which are not bound to the antibody.
<17> and <16>, wherein the conjugate formation step (2) is performed after the free ruthenium complex removal step (3).
The present invention also includes the following embodiments.
(a) The immunological assay method according to any one of <1> to <6>, wherein the antibody is a monoclonal antibody.
(b) The immunoassay kit according to any one of <7> to <12>, wherein the antibody is a monoclonal antibody.
(c) The method for reducing measurement error according to any one of <13> to <15>, wherein the antibody is a monoclonal antibody.
(d) <16> or <17> of the method for producing a conjugate between a polyethylene glycol-modified antibody and a ruthenium complex, wherein the antibody is a monoclonal antibody.
According to the present invention, the deviation of the blank value can be reduced. Further in detail, according to the present invention, even if the number of binding molecules of the labeled substance per 1 molecule of the antibody is increased, it is possible to prevent the deviation of the blank value, the increase of the blank value due to the non-specific adsorption of the labeled antibody to the carrier, and the aggregation of the labeled antibodies with each other via the labeled substance. Therefore, according to the present invention, the amount of the substance to be detected can be accurately measured with high sensitivity.
Brief Description of Drawings
FIG. 1 is a schematic diagram showing an example of a conjugate of a polyethylene glycol-modified antibody and a labeling substance used in the immunological analysis method of the present invention.
FIG. 2 is a schematic diagram showing the sequence and principle of an ECL method, which is one embodiment of the immunological analysis method of the present invention.
Means for carrying out the invention (substance to be detected)
The substance to be detected in the immunological analysis method of the present invention is a substance that can be measured by an antigen-antibody reaction and can serve as an antigen. Examples of the substance to be detected in the immunological analysis method of the present invention include the following substances.
Myocardial markers such as CK-MB, HFABP, troponin (troponin I, troponin T), and myoglobin; thrombin/fibrinolysis markers such as a fibrin degradation product (e.g., D dimer), soluble fibrin, TAT (thrombin-antithrombin complex), PIC (plasmin-plasmin inhibitor complex); circulation-related markers such as oxidized LDL and BNP (brain natriuretic peptide); metabolism-related markers such as adiponectin; CEA (carcinoembryonic antigen), AFP (alpha fetoprotein), PSA (prostate specific antigen), mucin-type glycoprotein and other tumor markers; inflammation-related markers such as CRP (C-reactive protein), LRG (leucine-rich alpha glycoprotein); influenza, HIV (human immunodeficiency virus), HBV (hepatitis B virus), HCV (hepatitis C virus), toxoplasma, chlamydia, syphilis, staphylococcus aureus, escherichia coli, procalcitonin and other infection related markers.
(biological samples)
In the present invention, as a biological sample having a possibility of containing a substance to be detected, a substance derived from a living body (organism), an extract solution from which the substance to be detected is extracted, and the like are mainly mentioned. Specific examples of the substance derived from a living body (organism) include blood (whole blood), serum, plasma, lymph fluid, urine, feces, ascites, pleural effusion, and cerebrospinal fluid. In the present invention, as the biological sample, a body fluid, in particular, one or more selected from the group consisting of blood (whole blood), serum, plasma, and urine is preferably used. The biological sample having a possibility of containing a substance to be detected includes a component separated and fractionated from the whole blood or the like by means of centrifugation, filtration, purification, or the like, a component extracted with an organic solvent or the like, a component dissolved with a surfactant or the like, a component diluted with a buffer or the like, a component modified or changed by a chemical reaction or the like, and the like.
(immunological analysis method)
Examples of the immunological analysis method of the present invention include, but are not limited to, electrochemiluminescence immunoassay (ECL method), enzyme immunoassay (ELISA method), latex agglutination immunoassay (LTIA method), chemiluminescence immunoassay, and fluorescent antibody method. The immunological assay method of the present invention is preferably an electrochemiluminescence immunoassay (ECL method).
Furthermore, in the present specification, "analysis" includes the proof and/or quantification of the presence of the substance to be detected. In addition, a conjugate of a polyethylene glycol-modified antibody and a labeling substance is sometimes referred to as a PEG-modified labeled antibody.
The electrochemiluminescence immunoassay (ECL method) is a method of measuring the amount of a substance to be detected by causing a labeled substance to emit light by energization and detecting the amount of the light. In the electrochemiluminescence immunoassay (ECL method), a ruthenium complex is used as a labeling substance. An electrode is provided on a solid phase (microplate or the like) and radicals are generated on the electrode, whereby the ruthenium complex is excited and emits light. Then, the luminescence amount of the ruthenium complex can be detected.
An electrochemiluminescence immunoassay (ECL method) can be performed by using a first antibody as a solid-phase antibody and a second antibody recognizing an epitope different from that of the first antibody as a detection antibody (labeled antibody). As the labeled antibody, a polyethylene glycol-modified antibody (PEG-modified labeled antibody) to which a labeling substance is bound is used. In the case where a plurality of the same epitopes are present on the substance to be detected, the same antibody can be used as the solid phase antibody and the PEG-modified labeled antibody. The measurement principle when magnetic particles are used as insoluble carrier particles and ruthenium complexes are used as PEG-modified labeling substances is as follows. The following shows the principle of the assay in one embodiment of the present invention, without limiting the scope of the invention in any way.
1. When the magnetic particles to which the antibody is fixed are brought into contact with the biological sample, the substance to be detected in the biological sample is bound to the solid-phase antibody.
2. Thereafter, when the PEG-modified labeled antibody is brought into contact with the sample, the PEG-modified labeled antibody binds to the sample bound to the magnetic particles.
3. After removing the unbound PEG-modified labeled antibody, the amount of the PEG-modified labeled antibody bound to the substance to be detected emits light when the electric current is applied. By measuring the amount of luminescence, the amount of the substance to be detected in the sample is accurately measured.
In the immunological analysis method, an enzyme immunoassay method (ELISA method) using an enzyme label is also preferable because the target can be simply and rapidly measured. In the case of sandwich ELISA, an insoluble carrier to which a first antibody (solid phase antibody) recognizing a substance to be detected is immobilized and a second antibody (labeled antibody) labeled with a labeling substance are used. As the labeled antibody, a polyethylene glycol-modified antibody (PEG-modified labeled antibody) to which a labeling substance is bound is used. In the case where the first antibody used recognizes a plurality of epitopes on the substance to be detected, the first antibody can be used as a solid phase antibody and a labeled antibody. The insoluble carrier is preferably a plate (immunoplate).
The solid phase antibody immobilized on the insoluble carrier captures a substance to be detected in a biological sample, and an antibody-substance to be detected complex is formed on the insoluble carrier. The PEG-modified labeled antibody is bound to the captured analyte and forms a sandwich with the antibody-analyte complex. The substance to be detected in the sample can be determined by measuring the amount of the labeled substance in accordance with the method corresponding to the labeled substance. Specific methods such as a method of immobilizing an antibody on an insoluble carrier and a method of binding an antibody to a labeling substance may be any known method by those skilled in the art without any particular limitation.
In the immunoassay method, a signal may be detected even in the absence of an antigen due to nonspecific adsorption of a magnetic particle (or a carrier to which an antibody is bound) to which a labeled antibody is bound. For example, in the case of the ECL method, a complex of an antibody bound to a magnetic particle and an antibody labeled with an electrochemiluminescent substance is generated at a certain ratio even in the absence of an antigen, and a certain amount of light emission is detected even in the absence of an antigen. In particular, in the case where the labeling substance is a ruthenium complex having a high hydrophobicity, erroneous detection is likely to occur due to non-specific adsorption of the labeled antibody to the magnetic particles or the carrier. In addition, in the case of IgM antibodies, since the IgM antibodies themselves have high hydrophobicity and thus nonspecific adsorption of the bound magnetic particles (or antibody-bound carrier) by the antibodies, there is a high possibility that signals are detected even in the absence of antigens, as compared with IgG antibodies which are generally used in many immunoassay systems. To avoid this, the hydrophilicity of the labeled antibody is increased by modification with polyethylene glycol. This can suppress nonspecific adsorption of the labeled antibody to the magnetic particles or the carrier, suppress the value of the blank, and improve the reproducibility of the measured value when measuring solutions having the same antigen concentration.
Further, it is possible to increase the detection sensitivity by increasing the amount of the labeling substance bound to each 1 molecule of the antibody. However, when a labeling substance having a high degree of hydrophobicity is labeled on an antibody, the degree of hydrophobicity of the labeled antibody increases as the amount of the labeling substance bound to 1 molecule of the antibody increases. That is, when the amount of the labeling substance bound to 1 molecule of the antibody is increased, the detection sensitivity is improved, but there are disadvantages that the blank value is improved and the reproducibility is deteriorated. By modifying the labeled antibody with polyethylene glycol, even if the amount of a highly hydrophobic labeling substance (e.g., ruthenium complex or the like) bound to 1 molecule of the antibody is increased, the increase in the value of the blank and the deterioration in reproducibility can be suppressed, and therefore, the improvement in sensitivity, the increase in the value of the blank and the suppression of the deterioration in reproducibility can be achieved.
Any of the monoclonal antibody and the polyclonal antibody can be produced by a known method, and the monoclonal antibody is preferred as the antibody used in the immunological analysis method of the present invention. Monoclonal antibodies can be obtained by, for example, isolating spleen cells or lymph node cells as antibody-producing cells from a non-human mammal immunized with a test substance or a fragment thereof, fusing the cells with a myeloma-derived cell line or the like having a high proliferation ability to prepare hybridomas, and purifying antibodies produced by the hybridomas. In addition, the polyclonal antibody can be obtained from the serum of an animal immunized with the substance to be detected or a fragment thereof. The fragment is a partial fragment of the substance to be detected, and the antibody bound to the fragment recognizes the substance to be detected.
As the antibody, a fragment of an antibody having antigen-antibody reactivity may be used in addition to the whole antibody molecule, and a fragment obtained by using a gene recombination technique and a chimeric antibody may be used in addition to the one obtained by the immunization step to an animal as described above. As the fragment of the antibody, a functional fragment is preferable, and for example, F (ab') 2 Fab', scFv and the like, which can be prepared by reactingThe obtained antibody is treated with a protease (e.g., pepsin or papain), or a DNA of the antibody is cloned and expressed in a culture system using escherichia coli or yeast.
The antibody used in the immunological analysis method of the present invention is preferably an antibody that specifically reacts with a substance to be detected. "specifically react with a substance to be detected" means to react with the substance to be detected but not substantially react with other substances. The meaning of "substantially non-reactive" is described later.
In the present specification, the terms "reacting with", "recognizing" a substance to be detected, and "binding with" a substance to be detected are used synonymously, but are not limited to these examples, and need to be interpreted in the broadest sense. The "reaction" of the antibody with the antigen (compound) can be confirmed by, for example, an antigen immobilization ELISA method, a competition ELISA method, or a sandwich ELISA method, and also by a method utilizing the principle of surface plasmon resonance (SPR method). The SPR method can be performed using a device, a sensor, and reagents commercially available under the name of Biacore (registered trademark).
The term "non-reactive" with respect to an antibody used in the present invention means that the antibody used in the present invention does not substantially react with a compound, and the term "substantially non-reactive" means that, for example, when the antibody used in the present invention is immobilized and measured by using Biacore (registered trademark) T100 or T200 based on the SPR method described above, the reactivity of the antibody used in the present invention is not enhanced. In detail, the reactivity of the antibody with the compound means that there is no significant difference in reactivity compared to the control (compound not added). Needless to say, the "substantially non-reactive" state can be confirmed by a method or means known to those skilled in the art other than the SPR method.
In the present specification, the "carrier" refers to a substance that specifically recognizes a substance to be detected, or fixes an antibody or the like that specifically recognizes the substance to be detected. Examples include, but are not limited to, insoluble substances such as immunoplates, membranes, latex particles, and magnetic particles.
(polyethylene glycol)
Used in the immunological analysis method of the present inventionThe polyethylene glycol (polyethylene glycol may be abbreviated as PEG) is not particularly limited as long as the effect of the present invention is obtained, and is preferably a polyethylene glycol having a polymerization degree of 2 to 300, more preferably 2 to 200, further preferably 2 to 150, further preferably 2 to 100, further preferably 2 to 50, further preferably 2 to 20, further preferably 2 to 10, and most preferably 4 to 10. In addition, the degree of polymerization means the structural formula "HO- (CH) of PEG 2 -CH 2 -O) n-H' n. In the case of branched PEG, the degree of polymerization represents (CH) 2 -CH 2 -O) total number of repeating units. That is, the branched PEG contains "(CH) 2 -CH 2 -O)1”、“(CH 2 -CH 2 -O) m ', and' (CH) 2 -CH 2 In the case of-O) n', the degree of polymerization means the total value of l + m + n.
The PEG used in the immunological analysis method of the present invention preferably has a weight average Molecular Weight (MW) of 250 to 15000, more preferably 250 to 10000, further preferably 250 to 5000, further preferably 250 to 2000, and further preferably 250 to 1000. In the present specification, the term "2 to 200" includes values at both ends thereof, and the range includes 2 and 200. The PEG may be either branched or linear, or both. The weight average Molecular Weight (MW) can be determined using Size Exclusion Chromatography (SEC).
(polyethylene glycol modified antibody)
The term "polyethylene glycol-modified antibody" as used herein means an antibody which is chemically reacted with PEG by contacting with PEG. Binding may be generated at any position of the antibody as long as the effect of the present invention is obtained, and for example, the binding may be generated by reaction between an amino group and PEG. Preferably, PEG having an NHS group at one end is used to react with the amino group of the antibody for conjugation.
The antibody used in the immunological analysis method of the present invention is a polyethylene glycol-modified antibody, preferably a polyethylene glycol-modified IgM antibody or a polyethylene glycol-modified IgG antibody, and more preferably a polyethylene glycol-modified IgM antibody. The IgM antibody has higher hydrophobicity than the IgG antibody, and is labeled with a labeling substance having higher hydrophobicity, thereby further increasing hydrophobicity. By modifying the IgM antibodies with polyethylene glycol, agglutination and the like of the IgM antibodies can be effectively prevented due to strong hydrophobicity. Modification of the antibody by polyethylene glycol may be carried out immediately before analysis of the substance to be detected, or a previously prepared antibody may be used. By modifying the antibody with polyethylene glycol, adsorption of a complex of the antibody and the labeling substance to a carrier or the like, or aggregation of the complex can be prevented.
(conjugate of polyethylene glycol-modified antibody and labeling substance)
The PEG-modified labeled antibody, which is a conjugate of the polyethylene glycol-modified antibody and the labeling substance that can be used in the immunological analysis method of the present invention, is not limited as long as the effect of the present invention is obtained, and a conjugate of the polyethylene glycol-modified antibody and the ruthenium complex is preferable. The conjugate of the polyethylene glycol-modified antibody and the ruthenium complex that can be used in the immunological analysis method of the present invention is not limited as long as the effect of the present invention is obtained, and can be prepared, for example, by a method including the following procedures (1) to (3).
(1) A first complex formation step of bringing the antibody into contact with a ruthenium complex to form a first complex,
(2) a conjugate-forming step of bringing polyethylene glycol into contact with the first complex, forming a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex, and
(3) and a free ruthenium complex removing step of removing free ruthenium complexes which are not bound to the antibody.
In the foregoing production method, in order to reduce the blank value, the foregoing conjugate formation step (2) is preferably performed after the foregoing free ruthenium complex removal step (3). In the case of using other labeling substances, conjugates can also be prepared in the same manner.
In the first complex formation step (1), the ratio of the ruthenium complex to the antibody is preferably 1: 1 to 1: 200, more preferably 1: 5 to 1: 200, further preferably 1: 10 to 1: 200, further preferably 1: 20 to 1: 200, further preferably 1: 50 to 1: 150, based on the number of molecules of the ruthenium complex and the antibody.
In the conjugate forming step (2), the ratio of the first complex to the polyethylene glycol may be 1: 1 to 1: 1000, 1: 1 to 1: 500, 1: 5 to 1: 500, 1: 10 to 1: 500, or 1: 30 to 1: 300, based on the number of molecules of the first complex to the polyethylene glycol.
The detection sensitivity becomes high when the number of ruthenium complexes bound to 1 molecule of antibody is large. On the other hand, in this case, in a case where the labeled substance exhibits hydrophobicity, the amount of the substance to be detected may not be accurately measured due to adsorption of a complex of the antibody and the labeled substance to a carrier or the like, or aggregation of the complex. However, even in such a case, by using the polyethylene glycol-modified antibody, the substance to be detected can be analyzed with good sensitivity and accurately.
In the conjugate of a polyethylene glycol-modified antibody and a labeling substance of the present invention, the number of molecules of the labeling substance, preferably the ruthenium complex, to be bound to 1 molecule of the antibody is preferably 1 to 200, more preferably 10 to 200, further preferably 15 to 200, and further preferably 20 to 100. The binding of the labeling substance to the antibody may be direct binding or via a linker or the like.
(labeling substance)
The "labeling substance" in the present specification means a substance that binds directly or indirectly to a substance to be detected and then generates a signal for detecting the substance to be detected. The signal from the labeling substance may be measured by a known method, and for example, the absorbance or the intensity of the reflected light may be measured. The signal can be confirmed visually or by using a specific measuring instrument.
Examples of the labeling substance used for producing the labeled antibody include an enzyme, a fluorescent substance, an electrochemiluminescent substance, biotin, avidin, a radioisotope, a colloidal gold particle, and colored latex. The immunological analysis method can be appropriately selected by those skilled in the art depending on the antibody and the labeling substance used. The electrochemical luminescence material is a material which emits light by excitation with electric energy, and ruthenium complex and osmium complex are listed. As the ruthenium complex, a ruthenium pyridine complex is exemplified.
It is preferable to use an electrochemiluminescence immunoassay (ECL method) as the immunological analysis method and an electrochemiluminescence, particularly a ruthenium complex, as the labeling substance. The electrochemically luminescent substances, in particular the ruthenium complexes, are very hydrophobic. Therefore, the ECL method can greatly enjoy the effect of the immunological analysis method of the present invention.
The immunological assay method of the present invention may comprise a step of measuring a signal from the labeled substance. This step may include subtracting the blank value.
(measurement error reduction)
In the immunological measurement method, it is known that a positive or negative measurement error of the measurement value occurs due to, for example, an increase in the value of the blank or adhesion of the labeled antibody to the carrier. In the present specification, "reduction of measurement error" means that the positive or negative measurement error of the measurement value is closer to the original measurement value (true value).
(reagent kit for immunological analysis)
The kit for immunological analysis of a substance to be detected of the present invention may be any kit as long as it contains a reagent containing a conjugate of a polyethylene glycol-modified antibody and a labeling substance (PEG-modified labeled antibody). In the reagent containing the PEG-modified labeled antibody (hereinafter, sometimes referred to as a labeled reagent), the concentration of the PEG-modified labeled antibody in the labeled reagent may be 0.01 to 1000. mu.g/mL, 0.05 to 200. mu.g/mL, 0.1 to 100. mu.g/mL, or 0.1 to 20. mu.g/mL. Among the labeled reagents, the PEG-modified labeled antibody is preferably contained in a form adjusted to 0.1 to 100. mu.g/mL, particularly 0.1 to 20. mu.g/mL, in a mixed state of all reagents to be added to the measurement system and the sample.
The kit for immunological analysis of a substance to be detected of the present invention preferably further comprises a reagent (hereinafter, sometimes referred to as a capture reagent) comprising a carrier to which an antibody against the substance to be detected (hereinafter, sometimes referred to as a "capture antibody") that is not labeled with a labeling substance is bound. The carrier to which the capture antibody is bound may have a concentration of 0.01 to 1000mg/mL, 0.05 to 200mg/mL, 0.1 to 100mg/mL, or 0.1 to 2mg/mL in the capture reagent. In the capture reagent, the carrier to which the capture antibody is bound is preferably contained in a form adjusted to 0.1 to 10mg/mL, particularly 0.1 to 2mg/mL, in a mixed state of all reagents and samples to be added to the measurement system.
The kit for immunological analysis of a substance to be detected of the present invention may further contain other detection reagents such as a standard antigen substance and an antigen sample for quality control, a sample diluent, and/or instructions for use.
In the kit for immunological analysis of a substance to be detected of the present invention, the PEG-modified labeled antibody and the capture antibody may recognize different epitopes, and in the case where the PEG-modified labeled antibody recognizes a plurality of epitopes on an antigen, the same (i.e., an antibody recognizing the same epitope) antibody may be used as the PEG-modified labeled antibody and the capture antibody.
In the case of using the ECL method, the kit for immunological analysis of a substance to be detected of the present invention may contain the following (a) and (B).
(A) A labeling agent comprising a conjugate of a polyethylene glycol-modified antibody and an electrochemically luminescent substance (e.g., ruthenium complex, etc.), and
(B) a solid phase on which a capture antibody bound to a substance to be detected is immobilized.
For example, in a kit using magnetic particles as a solid phase, a biological sample is added to the magnetic particles on which a capture antibody that binds to a substance to be detected is immobilized and reacted, and then the sample is removed and washed. Then, a conjugate of the polyethylene glycol-modified antibody and an electrochemiluminescent substance (e.g., ruthenium complex or the like) is added to react. After washing the magnetic particles, the amount of luminescence of the labeled substance is measured by applying electric energy to the magnetic particles, and the substance to be detected can be analyzed.
In the case of using the sandwich ELISA method, the kit for immunological analysis of a substance to be detected of the present invention may contain the following (A) and (B).
(A) Labeling reagent containing PEG modified labeled antibody
(B) An insoluble carrier to which the capture antibody is immobilized.
In such a kit, first, a biological sample is added to an insoluble carrier on which a capture antibody that binds to a substance to be detected is immobilized, followed by incubation, removal of the sample and washing. Then, the mixture is incubated after adding a labeling agent, and a substrate is added to develop color. The substance to be detected can be analyzed by measuring the color development using a plate reader or the like.
The present invention will be specifically described with reference to examples, which are not intended to limit the scope of the present invention. In addition,% means mass% unless otherwise specified. The "Ru — Ab mixing ratio" in the table indicates the ratio of the number of molecules of the ruthenium complex when the number of antibody molecules is 1.
Examples
[ preparation example 1 [ preparation of antibody to be used
anti-Dupan-2 antibody and anti-procalcitonin antibody were used as antibodies. The anti-Dupan-2 antibody is an IgM antibody and the anti-procalcitonin antibody is an IgG antibody. Furthermore, they are all monoclonal antibodies.
For the anti-Dupan-2 antibody, the cell of clone No. S19201R was cultured, and the resulting culture supernatant containing the anti-Dupan-2 antibody was purified by anion exchange and gel filtration chromatography, dialyzed against 150mM potassium phosphate/150 mM NaCL (pH7.8), and used. Refrigerating and storing until use.
For anti-procalcitonin antibodies, clone numbers S02211 and S02203R cells were transplanted into BALB/c mice, respectively, to generate ascites. With respect to each ascites containing the obtained antibody, ascites obtained from the S02211 clone was purified by anion exchange chromatography. Ascites fluid obtained from clone S02203R was purified by protein G chromatography. The purified respective antibodies were dialyzed against 150mM potassium phosphate/150 mM NaCL (pH7.8), and stored under refrigeration until use.
[ preparation example 2-1 binding of ruthenium Complex to anti-Dupan-2 antibody
Purified antibody 1.73mg/mL (anti-Dupan-2 antibody, dissolved in 150mM potassium phosphate/150 mM NaCL (pH7.8)) was added to a 2.0mL capacity Eppendorf tube. Ruthenium complex 10mg/mL (dissolved in DMSO) was added to the Eppendorf tube such that the ratio of the number of antibody molecules to the number of ruthenium complex molecules was 1: 5, 1: 20, 1: 50, or 1: 100, respectively. Thereafter, the mixture was stirred at room temperature for 30 minutes. As ruthenium complex, ruthenium (II) tris (bipyridyl) -NHS Ester (Ruthenium (II) Tris (bispyridyl) -NHS Ester) was used.
For the purpose of blocking the NHS group (N-hydroxysuccinimide group) of the free ruthenium complex with an excess of the amino group of glycine, 2M glycine (dissolved in distilled water) was added to the solution after the reaction so that the final concentration of glycine became 100 mM. Thereafter, the mixture was stirred at room temperature for 20 minutes.
The reacted solution was applied to a SephadexG25 column (gel volume 20mL) equilibrated with 10mM potassium phosphate/150 mM NaCL (pH6.0) to remove the free ruthenium complex.
The absorbance of the eluate at A280nm was measured, and a fraction in which the absorbance at the absorption wavelength of protein, A280nm, was observed was separated. The Protein concentration of the separated fractions was quantified using the Micro BCA Protein Assay Kit (Thermo Fisher Co.), and the number of antibody molecules was calculated from the quantified values. Further, the absorbance of a455nm having the absorbance of the ruthenium complex was measured, and the number of ruthenium complex molecules in the separated fraction was calculated from the absorbance of a455 nm. From the calculated number of antibody molecules and ruthenium complex molecules, the number of ruthenium complex molecules labeled in 1-molecule antibody was calculated for the ruthenium-labeled antibody in the separation fraction (table 1). The prepared ruthenium-labeled antibody is stored in a refrigerated state until the use.
[ Table 1]
(Table 1) binding ratio of anti-Dupan-2 antibody to ruthenium Complex
[ preparation example 2-2 [ binding of ruthenium Complex to anti-Procalcitonin antibody
The same procedure as for the anti-Dupan-2 antibody was carried out except that an antibody purified from ascites fluid using the S02211 clone was used as a labeled antibody and the ratio of the number of antibody molecules to the number of ruthenium complex molecules was added at a mixing ratio of 1: 20. The number of bound ruthenium complex molecules per 1 molecule of antibody was 10.
Preparation example 3-1 PEG modification of ruthenium-labeled antibody
As PEG with one end NHS, MS (PEG)4Methyl-PEG-NHS-Ester Regent with a degree of polymerization of 4 (manufactured by Thermo Fisher, MW: about 330), MS (PEG)24Methyl-PEG-NHS-Ester Regent with a degree of polymerization of 24 (manufactured by Thermo Fisher, MW: about 1210), and Acrylate-PEG7500-NHS with a degree of polymerization of about 170 (manufactured by Merck, average Mn: about 7500) were used. Immediately before use, each PEG was dissolved in distilled water to 0.1 mM. Each of the ruthenium-labeled antibodies prepared in preparation examples 2-1 and 2-2 was diluted 2-fold with 150mM potassium phosphate/150 mM NaCl (pH7.8) in order to make the pH of the solution weakly basic, and then the ruthenium-labeled antibody molecules diluted 2-fold were mixed with each of the PEG molecules at an arbitrary ratio and stirred at room temperature for 16 hours. To block the NHS group (N-hydroxysuccinimide group) of the free ruthenium complex with an excess of the amino group of glycine, 2M glycine (dissolved in distilled water) was added to the stirred solution so that the final concentration of glycine became 100mM, and the mixture was stirred at room temperature for 20 minutes. The prepared PEG modified ruthenium labeled antibody is refrigerated and stored until use.
Details of the PEG-modified ruthenium-labeled antibody prepared are shown in table 2 below.
[ Table 2]
(Table 2) details of the PEG-modified ruthenium-labeled antibody prepared
[ preparation example 3-2 [ binding of ruthenium Complex to antibody and PEG modification of ruthenium-labeled antibody
To examine the effect of the case where the removal of the free ruthenium complex was performed after PEG modification of the antibody, a PEG-modified ruthenium-labeled antibody was prepared in the following order.
For the purified antibody 1.73mg/mL (anti-Dupan-2 purified IgM antibody, dissolved in 150mM potassium phosphate/150 mM NaCl (pH7.8)), 2.0mL capacity Eppendorf tubes (3 in total) were added at 1.84mg parts, 0.92mg parts, 1.5mg parts, respectively. To this, 10mg/mL (m.w1057, dissolved in DMSO) of ruthenium complex was added at 4.8 μ L (1.84 mg against antibody), 6.0 μ L (0.92 mg against antibody), and 19.6 μ L (1.5 mg against antibody) so that the ratio of the number of antibody molecules to the number of ruthenium complex molecules became 1: 20, 1: 50, and 1: 100, respectively, and the mixture was stirred and mixed at room temperature for 30 minutes.
Subsequently, MS (PEG)4Methyl-PEG-NHS-Ester Regent (manufactured by Thermo Fisher Co., Ltd.) in which PEG4 molecules each having an NHS-modified end were linked was dissolved with 150mM potassium phosphate/150 mM NaCl (pH7.8) so as to be 1mM immediately before use, mixed so that the ratio of the number of ruthenium-labeled antibody molecules to the number of PEG molecules became 1: 30, and stirred at room temperature for 1 hour. For the purpose of blocking the NHS group of free ruthenium complex and free PEG with an excess of the amino group of glycine, 2M glycine (dissolved in distilled water) was added to the reacted solution so that the final concentration of glycine became 100mM, and the mixture was stirred at room temperature for 20 minutes.
The reacted solution was applied to a SephadexG25 column (gel volume 20mL) equilibrated with 10mM potassium phosphate/150 mM NaCL (pH6.0) to remove free ruthenium complex and free PEG.
The absorbance of the eluate at A280nm was measured, and a fraction in which the absorbance at the absorption wavelength of protein, A280nm, was observed was separated. The separated fractions were quantified for Protein concentration using Micro BCA Protein Assay Kit (Thermo Fisher Co.), and the number of antibody molecules was calculated from the quantified values. Further, the absorbance of a455nm having the absorbance of the ruthenium complex was measured, and the number of ruthenium molecules in the separated fraction was calculated from the absorbance of a455 nm. From the calculated number of antibody molecules and ruthenium complex molecules, the number of ruthenium complex molecules labeled in 1-molecule antibody was calculated for the ruthenium-labeled antibody in the separation fraction (table 3). The prepared ruthenium-labeled antibody is stored in a refrigerated state until the use.
[ Table 3]
(Table 3) binding ratio of anti-Dupan-2 antibody prepared in preparation example 3-2 to ruthenium Complex
Details of the prepared PEG-modified ruthenium-labeled antibody are shown in table 4 below.
[ Table 4]
(Table 4) details of PEG-modified ruthenium-labeled antibody prepared in preparation example 3-2
Preparation example 4 preparation of antibody-bound magnetic particles
1mL of 30mg/mL magnetic particles having epoxy groups on the surface and a diameter of 3 μm was separated, and the solvent was removed by collecting the magnetic particles with a magnet. In order to wash the magnetic particles, 1mL of 150mM potassium phosphate/150 mM NaCl (pH7.8) was mixed with the magnetic particles and stirred, and then the solvent was collected with a magnet again to remove the solvent. To the magnetic particles from which the solvent was removed, 1mL of each purified antibody (anti-Dupan-2 antibody, anti-procalcitonin antibody dissolved in 150mM potassium phosphate/150 mM NaCl (pH7.8)) was added at 0.6mg/mL, and the antibodies were bound to the magnetic particles by stirring with an inverted stirrer at 25 ℃ for 17 hours. The antibody-bound magnetic particles are washed and stored refrigerated until use.
Example 1-1 analysis of Dupan-2 by anti-Dupan-2 antibody
(1-1) preparation of ruthenium-labeled antibody solution
The diluted PEG-unmodified ruthenium-labeled anti-Dupan-2 antibody and the antibodies of Nos. 2 to 11 prepared in preparation example 3-1 were each used in the measurement so as to be 5. mu.g/mL.
(1-2) preparation of antibody-bound magnetic particle solution
The antibody prepared in preparation example 4 was diluted so that the binding amount to the magnetic particles became 2mg/mL, and used in the measurement.
(1-3) measuring apparatus
The measurement was carried out using picolium III (manufactured by hydropsy medical corporation) as an electrochemiluminescence immunoassay device.
(1-4) measurement
A magnetic particle solution and a ruthenium-labeled antibody solution or a PEG-modified ruthenium-labeled antibody are placed in a predetermined position of a measurement device in a predetermined container. To the reaction vessel designated in the measurement apparatus, 60. mu.L of the reaction solution was dispensed, followed by addition of 30. mu.L of the measurement sample. The reaction vessel containing the measurement sample is set at a predetermined position of the measurement apparatus, and the preparation for measurement is completed. When the apparatus was started, 25. mu.L of the magnetic particle solution was added to the reaction vessel containing the measurement sample, and the antigen-antibody reaction (first reaction) was performed at 28 ℃ for about 5 minutes. Thereafter, BF separation was carried out using a BF separating liquid specified by the apparatus. Subsequently, 100. mu.L of the ruthenium-labeled antibody or the PEG-modified ruthenium-labeled antibody was added, and the antigen-antibody reaction (second reaction) was performed at 28 ℃ for about 3 minutes. Thereafter, BF separation was performed using a BF separation solution designated by the apparatus to remove unreacted ruthenium-labeled antibody or PEG-modified ruthenium-labeled antibody. An antigen-antibody sandwich complex generated by the reaction is caused to chemiluminescence (ECL luminescence) by applying electricity to a solution of a luminescent substrate (tripropylamine) prescribed by the device at a predetermined field of the device.
The intensity of ECL luminescence differs depending on the number of antigen-antibody sandwich complexes (magnetic particle-bound antibody-antigenic substance in sample-ruthenium-labeled antibody (or PEG-modified ruthenium-labeled antibody)) generated by the reaction, and the antigen concentration can be quantified based on the difference in ECL luminescence intensity corresponding to the antigen concentration in the measurement sample. The results are shown in Table 5 below.
[ Table 5]
(Table 5) results of measurement based on blank of anti-Dupan-2 antibody
As a result, in the measurement system using the PEG-unmodified ruthenium-labeled anti-Dupan-2 antibody, it was found that the blank simultaneous reproducibility (variation: CV) was significantly deteriorated and the blank value was improved as the ruthenium binding number to the antibody was increased. The antibody having a CV of 10% or less is a ruthenium-labeled antibody in which ruthenium is labeled only 5 molecules with respect to 1 molecule of the antibody. On the other hand, in the assay system using various PEG-modified ruthenium-labeled anti-Dupan-2 antibodies, the reproducibility was greatly improved at the same time. In the ruthenium-labeled antibodies (antibody Nos. 8 to 10) in which ruthenium was labeled with respect to 1-molecule antibody 67, CV was also suppressed to 10% or less. In addition, the blank ECL luminescence intensity also gave inhibition results for PEG unmodified labeled antibodies. In addition, due to the difference in the degree of polymerization of the conjugated PEG, no clear difference was observed in the blank simultaneous reproducibility and ECL luminescence intensity.
Example 1-2 confirmation test results of the degree of effect due to the difference in PEG conjugation method it was confirmed that the difference in performance of the PEG-modified ruthenium-labeled anti-Dupan-2 antibody was observed due to the difference in PEG conjugation method. The PEG-modified ruthenium-labeled anti-Dupan-2 antibody prepared in the method of preparation example 3-1 or preparation example 3-2 was used, and a blank was measured with N ═ 10 to confirm the reproducibility of the antibody at the same time. In any of the PEG coupling methods, the PEG coupling of the antibody is carried out in a mixing ratio of ruthenium-labeled antibody molecules to PEG molecules of 1: 30. The results are shown in Table 6.
[ Table 6]
(Table 6) measurement results by blank of anti-Dupan-2 antibody prepared by preparation method 3-1 or 3-2
As a result, the PEG-modified ruthenium-labeled anti-Dupan-2 antibody prepared by either of the binding methods showed improvement in blank simultaneous reproducibility (variation: CV) as compared with the PEG-unmodified ruthenium-labeled anti-Dupan-2 antibody. However, the PEG-modified ruthenium-labeled anti-Dupan-2 antibody prepared by preparation method 3-1 was more improved in reproducibility (deviation: CV) than preparation method 3-2.
Example 2-1 [ confirmation test of effect due to difference in mixing ratio of PEG to ruthenium-labeled anti-Dupan-2 antibody ] the difference in performance of PEG-modified ruthenium-labeled anti-Dupan-2 antibody due to difference in mixing ratio of PEG to antibody was confirmed. The mixing ratio of PEG was adjusted so that the ratio of the number of ruthenium-labeled antibody to the number of PEG molecules was 1: 30 or 1: 300. The antibodies prepared in preparation example 3-1 were used in the study in the numbers 2 and 3. The simultaneous reproducibility and detection sensitivity in each assay system were confirmed using the respective antibodies, a blank (N ═ 10) and Dupan-2 antigen (STD: 0.05, 0.1, 0.5, 1, 2 μ g/mL in table 7) prepared using synthetic sugar chains mimicking the sequence of Dupan-2 sugar chains. The results are shown in Table 7.
[ Table 7]
(Table 7) confirmation of Effect due to difference in PEG mixing ratio
As a result, in the measurement system using any PEG-modified ruthenium-labeled anti-Dupan-2 antibody, improvement (CV 3.7% and 7.7%) was observed with respect to the blank N ═ 10 simultaneous reproducibility, relative to the measurement system using PEG-unmodified ruthenium-labeled anti-Dupan-2 antibody (CV 19.1%). On the other hand, when comparing the ECL luminescence intensity of STD, the PEG-modified ruthenium-labeled anti-Dupan-2 antibody prepared with a mixing ratio of ruthenium-labeled antibody to PEG of 1: 30 gave a strong ECL luminescence intensity compared to that of 1: 300.
Example 2-2 sensitivity test results of Dupan-2 measurement System Using PEG-modified ruthenium-labeled anti-Dupan-2 antibody
With respect to the Dupan-2 assay system using various PEG-modified ruthenium-labeled anti-Dupan-2 antibodies and the Dupan-2 assay system using PEG-unmodified ruthenium-labeled anti-Dupan-2 antibodies, assays were carried out with respect to 4 serum samples of pancreatic cancer patients and Dupan-2 antigen (STD: 0.025, 0.05, 0.1. mu.g/mL in tables 8 and 9) prepared using synthetic sugar chains mimicking the sequence of Dupan-2 sugar chains, and confirmation was made with respect to the detection sensitivity of each assay system. The results are shown in tables 8 and 9.
[ Table 8]
(Table 8) results of measurement of serum samples and synthetic sugar chains of pancreatic cancer patients
[ Table 9]
(Table 9) measurement results of serum samples and synthetic sugar chains (ECL luminescence intensity ratio) of pancreatic cancer patients
As a result, higher emission intensities were obtained in the measurement system using the PEG-modified ruthenium-labeled antibody (ruthenium 12, 22, and 67 molecules/antibody) in which CV suppression with simultaneous reproducibility was 10% or less and more ruthenium could be labeled to the antibody, compared to the emission intensities in the measurement system using the ruthenium-labeled antibody (ruthenium 5 molecule/antibody) in which the blank simultaneous reproducibility was CV 10% or less. In addition, as the number of ruthenium-labeled molecules against the antibody increased, the ECL luminescence intensity also gradually increased. In the relationship between the difference in the kind of bound PEG and ECL luminescence intensity, the polymerization degree of labeled PEG was decreased (short PEG), and ECL luminescence intensity of the sample tended to be increased. In the measurement system using the PEG 4-modified ruthenium-labeled antibody having the strongest emission intensity in this test, the average emission intensity of 13 times and the average emission intensity of 29 times in the synthesized sugar chains in the serum samples of pancreatic cancer patients were obtained compared to the measurement system using the PEG unmodified ruthenium-labeled antibody.
Example 3 analysis of Procalcitonin
(3-1) preparation of ruthenium-labeled antibody solution the ruthenium-labeled anti-procalcitonin antibody and the PEG-modified ruthenium-labeled anti-procalcitonin antibody prepared in preparation examples 1, 2-2 and 3-1 were diluted so as to be 20. mu.g/mL and used in the measurement.
(3-2) preparation of antibody-bound magnetic particle solution the antibody-bound magnetic particles prepared in preparation example 4 were diluted so that they became 1mg/mL and used in the measurement.
(3-3) measurement device the measurement was carried out using PICOLUMI III (manufactured by HYPERPOMER MEDICAL CO., LTD.) as an electrochemiluminescence immunoassay device.
(3-4) measurement of the magnetic particle solution and the ruthenium-labeled antibody solution or the PEG-modified ruthenium-labeled antibody in a predetermined place of the measurement device in a predetermined container. The reaction solution was dispensed to a reaction vessel designated in the measurement apparatus, followed by addition of 20. mu.L of a measurement sample (physiological saline as a blank). The reaction vessel containing the measurement sample is set at a predetermined position of the measurement apparatus, and the preparation for measurement is completed. When the apparatus was started for measurement, 50. mu.L of the magnetic particle solution was added to the reaction vessel containing the measurement sample, and an antigen-antibody reaction (first reaction) was performed at 28 ℃ for about 5 minutes. Subsequently, 50. mu.L of ruthenium-labeled antibody solution or PEG-modified ruthenium-labeled antibody was added, and antigen-antibody reaction (second reaction) was performed at 28 ℃ for about 3 minutes. Thereafter, BF separation was performed using a BF separating medium specified in the apparatus to remove unreacted ruthenium-labeled antibody solution or PEG-modified ruthenium-labeled antibody. An antigen-antibody sandwich complex generated by the reaction is caused to chemiluminescence (ECL luminescence) by applying electricity to a solution of a luminescent substrate (tripropylamine) prescribed by the device at a predetermined field of the device. The results are shown in Table 10.
[ Table 10]
(Table 10) analysis results of Procalcitonin
In the PEG-unmodified ruthenium-labeled anti-procalcitonin antibody, an abnormally high value (jump value) of ECL luminescence was generated, which is considered to be caused by nonspecific binding of the ruthenium-labeled antibody to antibody-bound magnetic particles at a certain ratio, but when the PEG-modified ruthenium-labeled anti-procalcitonin antibody was used, it was suppressed (the respective data are not described). As a result, the PEG-modified ruthenium-labeled anti-procalcitonin antibody showed improved reproducibility (deviation: CV) as compared with the PEG-unmodified ruthenium-labeled anti-procalcitonin antibody (14%).
The present invention aims to provide a technique for improving sensitivity in an immunological analysis method and accurately analyzing a substance to be detected.
Claims (17)
1. A method for immunological analysis of a substance to be detected, which comprises contacting a biological sample having a possibility of containing the substance to be detected with a conjugate, wherein the conjugate is a conjugate of a polyethylene glycol-modified antibody and a labeling substance.
2. The immunological analysis method according to claim 1, wherein the biological sample having a possibility of containing the test substance is at least one selected from the group consisting of blood, plasma, serum and urine.
3. The immunological assay method of claim 1 or 2, wherein the polyethylene glycol has a weight average Molecular Weight (MW) of 250 to 15000.
4. The immunological analysis method as claimed in any one of claims 1 to 3, wherein the labeling substance is an electrochemiluminescent substance, and the immunological analysis method is an electrochemiluminescence method (ECL method).
5. The immunological assay of any one of claims 1 to 4, wherein the polyethylene glycol-modified antibody is a polyethylene glycol-modified IgM antibody or a polyethylene glycol-modified IgG antibody.
6. The immunoassay method according to any one of claims 1 to 5, wherein the number of binding molecules of the labeling substance in 1-molecule antibody is 10 to 200.
7. A kit for immunological analysis of a substance to be detected, comprising a conjugate of a polyethylene glycol-modified antibody and a labeling substance.
8. The immunoassay kit according to claim 7, which is used for at least one biological sample selected from the group consisting of blood, plasma and serum.
9. The immunoassay kit according to claim 7 or 8, wherein the polyethylene glycol has a weight average Molecular Weight (MW) of 250 to 15000.
10. The kit for an immunological analysis as claimed in any one of claims 7 to 9, wherein the labeling substance is an electrochemiluminescent substance and the immunological analysis is an electrochemiluminescence method (ECL method).
11. The immunoassay kit according to any one of claims 7 to 10, wherein the polyethylene glycol-modified antibody is a polyethylene glycol-modified IgM antibody or a polyethylene glycol-modified IgG antibody.
12. The immunoassay kit according to any one of claims 7 to 11, wherein the number of binding molecules of the labeling substance in 1-molecule antibody is 10 to 200.
13. A method for reducing measurement error in immunological analysis of a substance to be detected, which comprises contacting a biological sample having a possibility of containing the substance to be detected with a conjugate of a polyethylene glycol-modified antibody and a labeling substance.
14. The method for reducing measurement error according to claim 13, wherein the biological sample having a possibility of containing the test substance is one or more selected from the group consisting of blood, plasma, and serum.
15. The method for reducing measurement error according to claim 13 or 14, wherein the polyethylene glycol has a weight average Molecular Weight (MW) of 250 to 15000.
16. A method for producing a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex, which comprises the following steps (1) to (3):
(1) a first complex formation step of bringing the antibody into contact with a ruthenium complex to form a first complex,
(2) a conjugate-forming step of bringing polyethylene glycol into contact with the first complex, forming a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex, and
(3) a free ruthenium complex removal step of removing free ruthenium complexes which are not bound to the antibody.
17. The method for preparing a conjugate of a polyethylene glycol-modified antibody and a ruthenium complex according to claim 16, wherein the conjugate formation step (2) is performed after the free ruthenium complex removal step (3).
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