WO2016052690A1 - Blocking method for immunoassay, and immunoassay instrument - Google Patents

Blocking method for immunoassay, and immunoassay instrument Download PDF

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Publication number
WO2016052690A1
WO2016052690A1 PCT/JP2015/077904 JP2015077904W WO2016052690A1 WO 2016052690 A1 WO2016052690 A1 WO 2016052690A1 JP 2015077904 W JP2015077904 W JP 2015077904W WO 2016052690 A1 WO2016052690 A1 WO 2016052690A1
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blocking
blocking agent
protein
proteins
coating
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PCT/JP2015/077904
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French (fr)
Japanese (ja)
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洋一 青木
鶴紀 田村
真紀子 大谷
松本　大輔
麻衣 片岡
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コニカミノルタ株式会社
アークレイ株式会社
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Priority to JP2016552159A priority Critical patent/JP6731347B2/en
Publication of WO2016052690A1 publication Critical patent/WO2016052690A1/en

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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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a blocking method for immunoassay and an instrument used for immunoassay. More specifically, the present invention relates to a method for blocking non-specific adsorption between two or more kinds of substances to be measured (proteins) contained in a blood sample and a measuring instrument, and two or more kinds of substances to be measured contained in a blood sample. The present invention relates to a measurement instrument in which non-specific adsorption with (protein) is blocked.
  • Patent Document 1 provides a system for detecting and quantifying troponin I and troponin T in blood, which is a myocardial examination item, by immunoassay.
  • various measurement substances are contained in a blood sample, and two or more kinds of measurement objects contained in the blood sample using the same measuring instrument (blood collection tube, filter, plate, etc.). Measuring a substance (protein) is useful as a method for improving the efficiency of measurement. In this case, in order to perform an accurate measurement, it is necessary to block non-specific adsorption to the same measurement instrument as described above for each of two or more kinds of measurement target substances (proteins).
  • the blocking agent of the cited reference 1 prevents nonspecific adsorption
  • the effect of preventing non-specific adsorption to the measuring instrument will be different, and these blocking agents do not guarantee the same blocking effect for proteins other than troponin I and T. Therefore, when two or more types of measurement target substances (proteins) contained in a blood sample are immunoassay using the same measurement instrument, all of the two or more types of measurement target substances (proteins) A method for effectively blocking non-specific adsorption is desired.
  • the measurement instruments for all of the two or more kinds of measurement target substances (proteins). It is an object of the present invention to provide a method for effectively blocking non-specific adsorption.
  • an object of the present invention is to provide an instrument for immunoassay in which nonspecific adsorption is effectively blocked for all of two or more kinds of substances to be measured (proteins).
  • the present inventors have prevented the measurement target substance (proteins) from nonspecifically adsorbing to the measuring instrument.
  • the optimal blocking agent to be used may be different for each measurement target substance (protein), and effectively prevents non-specific adsorption of two or more measurement target substances (proteins) to the same measurement instrument.
  • the present inventors have found that it is effective to coat a measuring instrument with two or more types of blocking agents that are optimal for each substance to be measured (protein), and have completed the present invention.
  • the coating with two or more kinds of blocking agents described above is applied to a measurement target substance (protein) having a high molecular weight from a blocking agent for a measurement target substance (protein) having a low molecular weight among two or more kinds of measurement target substances (proteins).
  • the inventors have found that it is preferable to coat in the order of blocking agents, and have completed the present invention.
  • the present invention is a blocking method for immunoassay in which two or more proteins contained in a blood sample are measured using the same measuring instrument, Provided is a blocking method in which at least a part of the surface of the device that comes into contact with the blood sample is coated with two or more blocking agents that prevent each non-specific adsorption.
  • the present invention provides an instrument used for immunoassay that measures two or more proteins contained in a blood sample, wherein at least a part of the surface of the instrument that comes into contact with the blood sample is the 2
  • a device having a coating layer of two or more blocking agents that prevent non-specific adsorption of each of the two or more proteins.
  • the present invention when two or more kinds of measurement target substances (proteins) contained in a blood sample are subjected to immunoassay using the same measuring instrument, all of the two or more kinds of measurement target substances (proteins) are used for measurement.
  • a method is provided for effectively blocking non-specific adsorption with an instrument.
  • an immunoassay instrument in which nonspecific adsorption is effectively blocked for all of two or more kinds of substances to be measured (proteins).
  • a measuring instrument When coating a measuring instrument with two or more blocking agents, the effect of preventing non-specific binding between each measurement target substance (protein) and the measuring instrument when all the blocking agents have been coated is It depends on the coating order.
  • the most effective non-specific adsorption preventing effect is achieved by coating sequentially from a blocking agent to a measurement target substance (protein) having a smaller molecular weight among two or more measurement target substances (proteins). (See Examples below).
  • the blocking method or the instrument for immunoassay of the present invention is used, even when two or more kinds of measurement target substances contained in the same blood sample are immunoassayed using the same measurement instrument, Non-specific adsorption to the measuring instrument is prevented, and accurate measurement can be performed for each measurement target substance.
  • One aspect of the blocking method of the present invention is: “A blocking method for immunoassay in which two or more proteins contained in a blood sample are measured using the same measuring instrument, and two types of preventing non-specific adsorption of each of the two or more proteins A blocking method in which at least a part of the surface of the instrument that comes into contact with the blood sample is coated with the above blocking agent " It is.
  • the blocking method of the present invention is a blocking method for immunoassay of two or more proteins contained in a blood sample.
  • the blood sample in this case is a blood-derived sample collected from a human or an animal.
  • whole blood, plasma, serum, blood cells, and other samples prepared according to the purpose can be used.
  • the protein as the measurement target substance is not particularly limited as long as it functions as an antigen in immunoassay, and two or more kinds may be appropriately selected according to the purpose of immunoassay. Examples thereof include two or more selected from the cardiac markers troponin I, troponin T, CK-MB, myoglobin, H-FABP, BNP, NT-proBNP, and the like.
  • the substance to be measured refers to protein.
  • blocking means that the substance to be measured is non-specifically adsorbed on the surface of a measuring instrument (such as a blood collection tube, a filter, a well, or a plate with a channel).
  • a measuring instrument such as a blood collection tube, a filter, a well, or a plate with a channel.
  • the blocking agent refers to a substance that prevents such adsorption.
  • Examples of the material of the instrument used in the immunoassay include, for example, a blood collection tube such as polyester, polyethylene terephthalate, polypropylene, and glass, and a filter such as polyester, polypropylene, polyamide, polyethylene, polycarbonate, polyethylene terephthalate, polyethersulfone, glass fiber, There are cellulose, cellulose acetate and the like, and the plate includes polyethylene, polypropylene, polystyrene and the like. Proteins adsorb nonspecifically to instruments made of these materials.
  • non-specific adsorption between a protein and an instrument made of such a material is caused by electrostatic adsorption or adsorption by hydrophobic interaction, and it is considered that the two are actually related to each other.
  • the action of the blocking agent is not clearly understood, by previously coating the contact surface of the measuring instrument with the protein with the blocking agent, the protein contained in the blood sample to be added later can be absorbed on the surface of the measuring instrument. This is thought to be due to the fact that contact with the above material is hindered.
  • non-specificity of a low molecular weight protein of two or more proteins Coating with a blocking agent that prevents general adsorption can provide a high non-specific adsorption preventing effect for each of two or more proteins.
  • the coating amount of the blocking agent is smaller as the coating of the upper layer. This is a blocking agent that prevents nonspecific adsorption of proteins having a small molecular weight. It is speculated that this leads to an improvement in non-specific adsorption prevention efficiency when coating is performed.
  • Blocking agent What is necessary is just to select the blocking agent used by this invention from the blocking agents currently used for protein blocking to the thing suitable for the blocking of the protein which is a measuring object substance.
  • blocking agents used for protein blocking include skim milk, fish gelatin, bovine serum albumin (BSA), surfactants, casein, protamine, polyethylene glycol, trehalose, dextran and the like.
  • Examples of the method for selecting a blocking agent in the present invention are as follows. That is, after the candidate blocking agent is coated on the measuring instrument, the standard solution of the protein as the measurement target substance is brought into contact with the measuring instrument, and then the standard solution is recovered, and is usually used for ELISA and other immunoassays.
  • the recovery rate of the protein (the concentration of the protein in the recovered solution after contact with the measuring instrument (weight / mL) / the concentration of the protein standard solution before contacting the measuring instrument (weight / mL) (%)) is determined by Of 80% or more, preferably 85% or more is selected as the blocking agent for the substance to be measured (see Examples below). In this case, when one blocking agent shows a recovery rate of 80% or more, preferably 85% or more with respect to two or more kinds of proteins, the blocking agent is used as a blocking agent for any of these proteins. You can also.
  • two or more kinds of blocking agents are selected for two or more kinds of substances to be measured.
  • the selected blocking agents may be two or more kinds.
  • the same blocking agent is selected for two types of measurement target substances, and a different blocking agent is selected for the other one type of measurement target substance.
  • Three kinds of substances to be measured may be blocked with a kind of blocking agent.
  • one type of blocking agent shows a recovery rate of 80% or more, preferably 85% or more in common with respect to all of two or more kinds of substances to be measured, only one type of blocking agent is used. It is not included in the present invention.
  • the present invention uses two or more blocking agents in combination. Examples of combinations of substances to be measured and blocking agents will be described later.
  • the blocking method of the present invention uses the same measuring instrument (such as a blood collection tube, a filter, a well or a plate with a flow path), and two or more types of measurement objects contained in the same blood sample. Used for immunoassay of substances. For example, this is a case where a blood sample collected in one blood collection tube is filtered with one filter, and two or more kinds of substances to be measured are immunoassayed with one plate having wells or flow paths. Even in such a case, the above-described effects can be obtained by the blocking method of the present invention.
  • the measuring instrument is not particularly limited, and examples of the material of each measuring instrument are as described above.
  • the blocking agent selected as described above at least a part, preferably the entire surface, of the surface (for example, the inner surface of the blood collection tube, the inside of the filter, the flow path and well of the plate, etc.) in contact with the blood sample of the measuring instrument Coating.
  • the coating method in this case may be a commonly used method.
  • the blocking agent in a blocking state, the blocking agent is covered in addition to the surface with which the blood sample of the measuring instrument comes into contact. What is necessary is just to remove a blocking agent.
  • Examples of the coating processing conditions include holding for 1 hour at room temperature in a state of being covered with a blocking agent solution, washing with pure water, and drying (see Examples below).
  • a blocking agent that prevents non-specific adsorption of a protein having a large molecular weight from a blocking agent that prevents non-specific adsorption of a protein having a low molecular weight among two or more proteins as a measurement target substance.
  • the nonspecific adsorption preventing effect of each blocking agent on each protein is larger.
  • both blocking agent protamine (BNP recovery rate 85%) for BNP (protein with a molecular weight of about 3,500) and blocking agent casein (troponin recovery rate 85%) for troponin I (a protein with a molecular weight of about 23,500) are used.
  • BNP recovery rate 85% for BNP
  • troponin I a protein with a molecular weight of about 23,500
  • the BNP recovery is 82%, but the troponin I recovery is Remains 56%.
  • casein and protamine are mixed and the filter is coated simultaneously, the troponin I recovery rate is 85%, but the BNP recovery rate is only 56%.
  • the order of coating the measuring instrument with the blocking agent is the non-specific adsorption of the protein with a large molecular weight from the blocking agent that prevents the non-specific adsorption of the protein with a small molecular weight among the two or more kinds of proteins to be measured. It is preferable to perform the coating in order on the blocking agent to be prevented.
  • the coating with the next blocking agent after the coating layer of the previous blocking agent dries the coating of the instrument for a measurement by 2 or more types of blocking agents.
  • An example of the combination of the substance to be measured and the blocking agent is as follows.
  • Immunoassay After coating a measuring instrument with a blocking agent for two or more kinds of substances to be measured contained in a blood sample as described above, the same coated measuring instrument (blood collection tube, filter, well or A target immunoassay is performed on the two or more kinds of substances to be measured contained in one blood sample using a plate having a flow path or the like.
  • the immunoassay method is not particularly limited, and detection, quantification, and the like may be performed by a commonly used ELISA or other methods.
  • a measuring device having a well or a channel for example, a surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) measuring device, etc.
  • SPFS surface plasmon excitation enhanced fluorescence spectroscopy
  • mode of the instrument for immunoassay of this invention is, “An instrument used for immunoassay that measures two or more proteins contained in a blood sample, wherein at least a part of the surface of the instrument that contacts the blood sample is not coated with each of the two or more proteins. Instrument having two or more blocking agent coating layers to prevent specific adsorption " It is.
  • the instrument for immunoassay of this invention has a coating layer of 2 or more types of blocking agents.
  • the coating method to 2 or more types of blocking agents and the instrument for immunoassay it is as above-mentioned.
  • the immunoassay instrument of the present invention has two or more layers coated with a blocking agent.
  • the form of the coating layer of the blocking agent (layer thickness, layer thickness uniformity, etc.) is not particularly limited, and the target blocking effect (non-specific adsorption of the protein as the detection target substance to the instrument)
  • the target blocking effect non-specific adsorption of the protein as the detection target substance to the instrument
  • a layer with a uniform thickness is rarely formed on the surface of the device, and the thickness of the coating layer of the blocking agent is not uniform.
  • the layer to be coated later tends to have less adhesion of the blocking agent than the layer to be coated first. Even in the coating layer in such a state, the above-described effects of the present invention can be obtained and included in the scope of the immunoassay device of the present invention.
  • the blocking agent coating is performed sequentially from the blocking agent that prevents non-specific adsorption of proteins having a low molecular weight among these proteins.
  • the immunoassay device of the present invention also has a measurement on a coating layer of a blocking agent that prevents nonspecific adsorption of a protein having a low molecular weight among these proteins (measurement is also performed).
  • a device having a coating layer of a blocking agent in order to prevent nonspecific adsorption of a protein having a large molecular weight (in a direction further away from the device surface) is preferable.
  • the instrument for immunoassay of the present invention is an instrument used for immunoassay, and is not particularly limited as long as it is in contact with a blood sample. Examples thereof include a blood collection tube, a filter, a well, or a plate having a flow path. .
  • the blocking effect was examined using a sample (a glass filter (Advantech, GA-100, GA-100) was used) with a sample (troponin I (hereinafter cTnI), BNP or NT-proBNP as a substance to be measured).
  • a sample a glass filter (Advantech, GA-100, GA-100) was used
  • a sample troponin I (hereinafter cTnI), BNP or NT-proBNP as a substance to be measured).
  • cTnI troponin I
  • BNP NT-proBNP
  • the recovery rate of the substance to be measured was calculated from the ratio of the concentration measurement values after contact. The higher the recovery rate, the higher the blocking ability.
  • the instrument was immersed in a blocking solution at room temperature for 1 hour, washed with pure water, and dried in a thermostatic bath.
  • the relationship between combinations of substances to be measured (multiple staining myocardial markers) and blocking agents is shown in Table 1 below. Further, the results of comparing the recovery rates in the respective comparative examples and examples are shown in Tables 3 to 4 and FIGS.
  • Example 1 After blocking Protamine (Wako Pure Chemicals: Protamine sulfate / salmon derived) with blocking effect confirmed against BNP, blocking casein (Thermo Fisher: Casein in PBS) with blocking effect confirmed against cTnI, The recovery rates were as high as 80% for BNP and 85% for cTnI (see Table 3 and FIG. 1).
  • Example 2 After blocking casein (Thermo Fisher: Casein in PBS) whose blocking effect was confirmed against cTnI and blocking protamine whose blocking effect was confirmed against BNP, the recovery rate was 82% for BNP and 56% for cTnI. The cTnI recovery rate was lower than that in Example 1 (see Table 3 and FIG. 1).
  • Casein manufactured by Thermo Fisher: Casein in PBS
  • protamine Wired Chemicals: Protamine sulfate / salmon derived
  • the recovery rate was 56% for BNP and 85% for cTnI.
  • the recovery rate of BNP was lower than that in Example 1 (see Table 3 and FIG. 1).
  • Example 4 After blocking BSA (Thermo Fischer: BSA in PBS) whose blocking effect was confirmed against NT-proBNP, casein (Thermo Fisher: Casein in PBS) whose blocking effect was confirmed against cTnI was recovered. The rates were as high as 82% for NT-proBNP and 84% for cTnI (see Table 4 and FIG. 2).
  • Example 5 After blocking casein (Thermo Fischer: Casein in PBS) whose blocking effect was confirmed against cTnI, BSA (Thermo Fisher: BSA in PBS) whose blocking effect was confirmed against NT-proBNP was recovered. The rates were 81% for NT-proBNP and 52% for cTnI. The recovery rate of cTnI was lower than that of Example 4 (see Table 4 and FIG. 2).

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Abstract

The present invention addresses the problem of providing a method or the like for effectively blocking non-specific adsorption, on a measuring instrument, of all of at least two substances (proteins) to be measured included in a blood sample, in cases when the same measuring instrument is used to perform immunoassay on the at least two substances (proteins) to be measured. A blocking method according to the present invention is for immunoassay in which the same measuring instrument is used to measure at least two proteins included in a blood sample. In the blocking method, at least a portion of the surface of the instrument which is brought into contact with the blood sample is coated with at least two blocking agents which inhibit non-specific adsorption of each of the at least two proteins.

Description

免疫測定用ブロッキング方法及び免疫測定用器具Immunoassay blocking method and immunoassay instrument
　本発明は、免疫測定のためのブロッキング方法及び免疫測定に用いる器具に関する。更に詳しくは、本発明は、血液試料に含まれる２種以上の測定対象物質（タンパク質）と測定用器具との非特異吸着をブロッキングする方法及び血液試料中に含まれる２種以上の測定対象物質（タンパク質）との非特異吸着がブロッキングされた測定用器具に関する。 The present invention relates to a blocking method for immunoassay and an instrument used for immunoassay. More specifically, the present invention relates to a method for blocking non-specific adsorption between two or more kinds of substances to be measured (proteins) contained in a blood sample and a measuring instrument, and two or more kinds of substances to be measured contained in a blood sample. The present invention relates to a measurement instrument in which non-specific adsorption with (protein) is blocked.
　医療分野における診断や生物、生化学の分野における研究において、ヒトや動物の血液を試料として、抗原抗体反応を利用した免疫測定が広く行われている。血液試料を用いた免疫測定においては、例えば、全血そのものを流す場合は測定キットの採血管やプレート等の測定用器具に全血が接触し、又、血球と血漿を分離する場合はフィルター等の測定用器具に全血、血球、血漿等が接触する。このように測定試料の血液（全血、血液成分）が測定用器具に接触することによって、血液試料中の測定対象物質であるタンパク質（抗原）がこれらの測定用器具に非特異的に吸着し、本来得られるはずの定量値よりも低い定量値を算出してしまうという問題が知られている。 In the field of diagnosis in the medical field, research in the field of biology, and biochemistry, immunoassay using antigen-antibody reaction is widely performed using human or animal blood as a sample. In an immunoassay using a blood sample, for example, when whole blood is allowed to flow, the whole blood contacts a measuring instrument such as a blood collection tube or a plate of a measurement kit, and when blood cells and plasma are separated, a filter or the like. Whole blood, blood cells, plasma, etc. are in contact with the measuring instrument. Thus, when blood (whole blood, blood components) of a measurement sample comes into contact with a measurement instrument, a protein (antigen) that is a measurement target substance in the blood sample is adsorbed nonspecifically on these measurement instruments. There is a known problem that a quantitative value lower than the quantitative value that should originally be obtained is calculated.
　この問題に対する対策として、事前に測定用器具に他のタンパク質を吸着させ、測定対象物質（タンパク質）の非特異吸着をブロッキングするという技術が知られている。この場合、従来は、１種の測定対象物質に対し、１種のブロッキング剤でブロッキングを行うことが通常であった。また、２種の測定対象物質に対するブロッキングに関しては、例えば、特許文献１は、免疫測定によって心筋検査項目である血液中のトロポニンＩ及びトロポニンＴの検出と定量のシステムを提供するものであるが、その中で、イムノアッセイ法においてトロポニンＩ及びＴの回収率を改良するために、強塩基性のペプチド又はタンパク質をアッセイ法に用いる膜若しくはラテックス粒子又は装置の表面上に添加することが紹介されている。そして、このような用途に用いる好ましいブロッキング剤として、ｐＩ値が約８よりも大きいポリマー、タンパク質及びペプチドが紹介され、サルミン、リゾチーム、シトクロムその他が例示されている（特許文献１の５１～５２ページ）。 As a countermeasure against this problem, a technique is known in which a non-specific adsorption of a substance to be measured (protein) is blocked by previously adsorbing another protein to a measuring instrument. In this case, conventionally, it has been usual to perform blocking with one type of blocking agent for one type of substance to be measured. Regarding blocking against two kinds of substances to be measured, for example, Patent Document 1 provides a system for detecting and quantifying troponin I and troponin T in blood, which is a myocardial examination item, by immunoassay. Among them, in order to improve the recovery rate of troponin I and T in an immunoassay method, it is introduced that a strongly basic peptide or protein is added on the membrane or latex particle used in the assay method or on the surface of the apparatus. . As preferable blocking agents used in such applications, polymers, proteins and peptides having a pI value greater than about 8 have been introduced, and salmine, lysozyme, cytochrome, etc. are exemplified (Patent Document 1, pages 51 to 52). ).
　ところで、血液試料には種々の測定対象物質（タンパク質）が含まれており、同じ測定用器具（採血管、フィルター、プレート等）を用いて、その血液試料中に含まれる２種以上の測定対象物質（タンパク質）を測定することは、測定の効率化を図る方法として有用である。この場合、正確な測定を行うためには、２種以上の測定対象物質（タンパク質）のそれぞれについて、前記したように、同じ測定用器具への非特異吸着をブロッキングすることが必要である。引用文献１のブロッキング剤の例は、トロポニンＩ及びＴの非特異吸着を防止するものであるが、これらのブロッキング剤の中から一つを選択した場合、トロポニンＩに対してとトロポニンＴに対してとでは、測定用器具への非特異吸着防止効果が異なるであろうし、又、これらのブロッキング剤は、トロポニンＩ及びＴ以外のタンパク質に対しても同様なブロッキング効果を保証するものではない。従って、同じ測定用器具を用いて血液試料中に含まれる２種以上の測定対象物質（タンパク質）を免疫測定する場合に、２種以上の測定対象物質（タンパク質）の全てについて測定用器具との非特異吸着を効果的にブロッキングする方法が望まれている。 By the way, various measurement substances (proteins) are contained in a blood sample, and two or more kinds of measurement objects contained in the blood sample using the same measuring instrument (blood collection tube, filter, plate, etc.). Measuring a substance (protein) is useful as a method for improving the efficiency of measurement. In this case, in order to perform an accurate measurement, it is necessary to block non-specific adsorption to the same measurement instrument as described above for each of two or more kinds of measurement target substances (proteins). Although the example of the blocking agent of the cited reference 1 prevents nonspecific adsorption | suction of troponin I and T, when one is selected from these blocking agents, with respect to troponin I and troponin T In the past, the effect of preventing non-specific adsorption to the measuring instrument will be different, and these blocking agents do not guarantee the same blocking effect for proteins other than troponin I and T. Therefore, when two or more types of measurement target substances (proteins) contained in a blood sample are immunoassay using the same measurement instrument, all of the two or more types of measurement target substances (proteins) A method for effectively blocking non-specific adsorption is desired.
特表平11-505605号公報Japanese National Patent Publication No. 11-505605
　本発明は、同じ測定用器具を用いて血液試料中に含まれる２種以上の測定対象物質（タンパク質）を免疫測定する場合に、２種以上の測定対象物質（タンパク質）の全てについて測定用器具との非特異吸着を効果的にブロッキングする方法を提供することを課題とする。 In the present invention, when two or more kinds of measurement target substances (proteins) contained in a blood sample are subjected to immunoassay using the same measurement instrument, the measurement instruments for all of the two or more kinds of measurement target substances (proteins). It is an object of the present invention to provide a method for effectively blocking non-specific adsorption.
　更に、本発明は、このように２種以上の測定対象物質（タンパク質）の全てについて非特異吸着が効果的にブロッキングされた免疫測定用器具を提供することを課題とする。 Furthermore, an object of the present invention is to provide an instrument for immunoassay in which nonspecific adsorption is effectively blocked for all of two or more kinds of substances to be measured (proteins).
　本発明者らは、血液中の種々の測定対象物質（タンパク質）について各種ブロッキング剤の非特異吸着防止効果を調べた結果、測定対象物質（タンパク質）が測定用器具と非特異吸着することを防止するための最適なブロッキング剤は、それぞれの測定対象物質（タンパク質）で異なる場合があり、２種以上の測定対象物質（タンパク質）が同じ測定用器具へ非特異吸着することを効果的に防止するためには、それぞれの測定対象物質（タンパク質）に最適な２種以上のブロッキング剤で測定用器具をコーティングすることが有効であることを見出し、本発明を完成させた。 As a result of examining the nonspecific adsorption preventing effect of various blocking agents on various measurement target substances (proteins) in blood, the present inventors have prevented the measurement target substance (proteins) from nonspecifically adsorbing to the measuring instrument. The optimal blocking agent to be used may be different for each measurement target substance (protein), and effectively prevents non-specific adsorption of two or more measurement target substances (proteins) to the same measurement instrument. In order to achieve this, the present inventors have found that it is effective to coat a measuring instrument with two or more types of blocking agents that are optimal for each substance to be measured (protein), and have completed the present invention.
　更に、上記の２種以上のブロッキング剤によるコーティングは、２種以上の測定対象物質（タンパク質）のうち、分子量の小さな測定対象物質（タンパク質）に対するブロッキング剤から分子量の大きな測定対象物質（タンパク質）に対するブロッキング剤の順にコーティングすることが好ましいことを見出し、本発明を完成させた。 Furthermore, the coating with two or more kinds of blocking agents described above is applied to a measurement target substance (protein) having a high molecular weight from a blocking agent for a measurement target substance (protein) having a low molecular weight among two or more kinds of measurement target substances (proteins). The inventors have found that it is preferable to coat in the order of blocking agents, and have completed the present invention.
　すなわち、本発明は、一つの側面において、血液試料中に含まれる２種以上のタンパク質を同じ測定用器具を用いて測定する免疫測定のためのブロッキング方法であって、前記２種以上のタンパク質のそれぞれの非特異吸着を防止する２種以上のブロッキング剤によって、前記血液試料と接触する前記器具の表面の少なくとも一部をコーティングするブロッキング方法を提供する。
　また、本発明はさらなる側面において、血液試料中に含まれる２種以上のタンパク質を測定する免疫測定に用いる器具であって、前記器具の前記血液試料と接触する面の少なくとも一部に、前記２種以上のタンパク質のそれぞれの非特異吸着を防止する２種以上のブロッキング剤のコーティング層を有する器具を提供する。 That is, in one aspect, the present invention is a blocking method for immunoassay in which two or more proteins contained in a blood sample are measured using the same measuring instrument, Provided is a blocking method in which at least a part of the surface of the device that comes into contact with the blood sample is coated with two or more blocking agents that prevent each non-specific adsorption.
In addition, in a further aspect, the present invention provides an instrument used for immunoassay that measures two or more proteins contained in a blood sample, wherein at least a part of the surface of the instrument that comes into contact with the blood sample is the 2 Provided is a device having a coating layer of two or more blocking agents that prevent non-specific adsorption of each of the two or more proteins.
　本発明によれば、同じ測定器具を用いて血液試料中に含まれる２種以上の測定対象物質（タンパク質）を免疫測定する場合に、２種以上の測定対象物質（タンパク質）の全てについて測定用器具との非特異吸着を効果的にブロッキングする方法が提供される。 According to the present invention, when two or more kinds of measurement target substances (proteins) contained in a blood sample are subjected to immunoassay using the same measuring instrument, all of the two or more kinds of measurement target substances (proteins) are used for measurement. A method is provided for effectively blocking non-specific adsorption with an instrument.
　更に、本発明によれば、２種以上の測定対象物質（タンパク質）の全てについて非特異吸着が効果的にブロッキングされた免疫測定用器具が提供される。
　２種以上のブロッキング剤で測定用器具をコーティングする場合、全てのブロッキング剤のコーティングが終わった時の各測定対象物質（タンパク質）と測定用器具との非特異結合の防止効果は、各ブロッキング剤のコーティングの順序によって異なってくる。本発明の好ましい態様では、２種以上の測定対象物質（タンパク質）のうち、分子量の小さい方の測定対象物質（タンパク質）に対するブロッキング剤から順にコーティングすることにより、最も効果的な非特異吸着防止効果が得られる（後記実施例参照）。 Furthermore, according to the present invention, there is provided an immunoassay instrument in which nonspecific adsorption is effectively blocked for all of two or more kinds of substances to be measured (proteins).
When coating a measuring instrument with two or more blocking agents, the effect of preventing non-specific binding between each measurement target substance (protein) and the measuring instrument when all the blocking agents have been coated is It depends on the coating order. In a preferred embodiment of the present invention, the most effective non-specific adsorption preventing effect is achieved by coating sequentially from a blocking agent to a measurement target substance (protein) having a smaller molecular weight among two or more measurement target substances (proteins). (See Examples below).
　従って、本発明のブロッキング方法又は免疫測定用器具を用いれば、同じ測定器具を用いて、同じ血液試料に含まれる２種以上の測定対象物質を免疫測定する場合であっても、測定対象物質の測定器具への非特異吸着が防止され、それぞれの測定対象物質について正確な測定を行うことができる。 Therefore, if the blocking method or the instrument for immunoassay of the present invention is used, even when two or more kinds of measurement target substances contained in the same blood sample are immunoassayed using the same measurement instrument, Non-specific adsorption to the measuring instrument is prevented, and accurate measurement can be performed for each measurement target substance.
図１は、比較例１及び２、実施例１～３におけるｃＴｎＩ及びＢＮＰの各回収率を示したグラフである。FIG. 1 is a graph showing the recovery rates of cTnI and BNP in Comparative Examples 1 and 2 and Examples 1 to 3. 図２は、比較例３及び４、実施例４及び５におけるｃＴｎＩ及びＮＴ－ｐｒｏＢＮＰの各回収率を示したグラフである。FIG. 2 is a graph showing the recovery rates of cTnI and NT-proBNP in Comparative Examples 3 and 4 and Examples 4 and 5.
　以下、本発明を実施するための形態について詳細に説明するが、本発明はこれらに限定されるものではない。
　１．本発明のブロッキング方法について
　本発明のブロッキング方法の一態様は、
「血液試料中に含まれる２種以上のタンパク質を同じ測定用器具を用いて測定する免疫測定のためのブロッキング方法であって、前記２種以上のタンパク質のそれぞれの非特異吸着を防止する２種以上のブロッキング剤によって、前記血液試料と接触する器具の表面の少なくとも一部をコーティングするブロッキング方法」
である。 Hereinafter, although the form for implementing this invention is demonstrated in detail, this invention is not limited to these.
1. About the blocking method of the present invention One aspect of the blocking method of the present invention is:
“A blocking method for immunoassay in which two or more proteins contained in a blood sample are measured using the same measuring instrument, and two types of preventing non-specific adsorption of each of the two or more proteins A blocking method in which at least a part of the surface of the instrument that comes into contact with the blood sample is coated with the above blocking agent "
It is.
　（１）血液試料、タンパク質
　本発明のブロッキング方法は、血液試料に含まれる２種以上のタンパク質を免疫測定するためのブロッキング方法である。この場合の血液試料は、ヒトや動物から採取された血液由来の試料であり、例えば、全血、血漿、血清、血球、その他目的に応じた調製を行ったものを試料とすることができる。 (1) Blood sample, protein The blocking method of the present invention is a blocking method for immunoassay of two or more proteins contained in a blood sample. The blood sample in this case is a blood-derived sample collected from a human or an animal. For example, whole blood, plasma, serum, blood cells, and other samples prepared according to the purpose can be used.
　測定対象物質としてのタンパク質は、免疫測定で抗原として機能するものであれば特に制限はなく、免疫測定の目的に応じて適宜２種以上を選定すればよい。例えば、心筋マーカーであるトロポニンＩ、トロポニンＴ、ＣＫ－ＭＢ、ミオグロビン、Ｈ－ＦＡＢＰ，ＢＮＰ、ＮＴ－ｐｒｏＢＮＰ等から選定した２種以上が挙げられる。以下、本明細書では、単に測定対象物質といえばタンパク質をいう。 The protein as the measurement target substance is not particularly limited as long as it functions as an antigen in immunoassay, and two or more kinds may be appropriately selected according to the purpose of immunoassay. Examples thereof include two or more selected from the cardiac markers troponin I, troponin T, CK-MB, myoglobin, H-FABP, BNP, NT-proBNP, and the like. Hereinafter, in this specification, simply speaking, the substance to be measured refers to protein.
　（２）非特異吸着及びブロッキング
　本発明でブロッキングとは、測定対象物質が測定用器具（採血管、フィルター、ウエル又は流路を備えたプレート等）の表面に非特異的に吸着することを防止することをいい、本発明でブロッキング剤とは、このような吸着を防止する物質をいう。 (2) Non-specific adsorption and blocking In the present invention, blocking means that the substance to be measured is non-specifically adsorbed on the surface of a measuring instrument (such as a blood collection tube, a filter, a well, or a plate with a channel). In the present invention, the blocking agent refers to a substance that prevents such adsorption.
　免疫測定で用いられる器具の材質としては、例えば、採血管はポリエステル、ポリエチレンテレフタレート、ポリプロピレン、ガラス等があり、フィルターはポリエステル、ポリプロピレン、ポリアミド、ポリエチレン、ポリカーボネート、ポリエチレンテレフタレート、ポリエーテルスルホン、ガラス繊維、セルロース、セルロースアセテート等があり、プレートはポリエチレン、ポリプロピレン、ポリスチレン等がある。タンパク質は、これらの材質からなる器具に非特異的に吸着する。 Examples of the material of the instrument used in the immunoassay include, for example, a blood collection tube such as polyester, polyethylene terephthalate, polypropylene, and glass, and a filter such as polyester, polypropylene, polyamide, polyethylene, polycarbonate, polyethylene terephthalate, polyethersulfone, glass fiber, There are cellulose, cellulose acetate and the like, and the plate includes polyethylene, polypropylene, polystyrene and the like. Proteins adsorb nonspecifically to instruments made of these materials.
　タンパク質とこのような材質からなる器具との非特異的吸着には、静電的吸着や疎水的相互作用による吸着があるといわれており、実際は、両者が関連して生じていると考えられる。ブロッキング剤の作用は明確には分かっていないが、予めブロッキング剤で測定器具のタンパク質との接触面をコーティングしておくことによって、後から加える血液試料に含まれるタンパク質が、測定用器具の表面上で上記の材質と接触することが妨げられることによると考えられる。後述するように、２種以上のブロッキング剤で測定用器具をコーティングして２種以上のタンパク質の非特異吸着を防止するためには、２種以上のタンパク質のうち、分子量の小さいタンパク質の非特異的吸着を防止するブロッキング剤からコーティングする方が、２種以上のそれぞれのタンパク質について高い非特異吸着防止効果が得られる。この理由は明確ではないが、上層のコーティングほどブロッキング剤のコーティング量が少なくなることが発明者らの検討によって分かっており、このことが、分子量の小さいタンパク質の非特異的吸着を防止するブロッキング剤からコーティングした場合の非特異吸着防止効率の向上に繋がっていると推測される。 It is said that non-specific adsorption between a protein and an instrument made of such a material is caused by electrostatic adsorption or adsorption by hydrophobic interaction, and it is considered that the two are actually related to each other. Although the action of the blocking agent is not clearly understood, by previously coating the contact surface of the measuring instrument with the protein with the blocking agent, the protein contained in the blood sample to be added later can be absorbed on the surface of the measuring instrument. This is thought to be due to the fact that contact with the above material is hindered. As will be described later, in order to prevent non-specific adsorption of two or more proteins by coating a measuring instrument with two or more blocking agents, non-specificity of a low molecular weight protein of two or more proteins Coating with a blocking agent that prevents general adsorption can provide a high non-specific adsorption preventing effect for each of two or more proteins. The reason for this is not clear, but it has been found by the inventors that the coating amount of the blocking agent is smaller as the coating of the upper layer. This is a blocking agent that prevents nonspecific adsorption of proteins having a small molecular weight. It is speculated that this leads to an improvement in non-specific adsorption prevention efficiency when coating is performed.
　（３）ブロッキング剤
　本発明で用いるブロッキング剤は、通常、タンパク質のブロッキングに用いられているブロッキング剤の中から、測定対象物質であるタンパク質のブロッキングに適したものを選定すればよい。タンパク質のブロッキングに用いられているブロッキング剤の例としては、スキムミルク、フィッシュゼラチン、ウシ血清アルブミン（ＢＳＡ）、界面活性剤、カゼイン、プロタミン、ポリエチレングリコール、トレハロース、デキストラン等がある。 (3) Blocking agent What is necessary is just to select the blocking agent used by this invention from the blocking agents currently used for protein blocking to the thing suitable for the blocking of the protein which is a measuring object substance. Examples of blocking agents used for protein blocking include skim milk, fish gelatin, bovine serum albumin (BSA), surfactants, casein, protamine, polyethylene glycol, trehalose, dextran and the like.
　本発明におけるブロッキング剤の選定方法の例は、次の通りである。すなわち、候補とするブロッキング剤を測定用器具にコーティングした後、測定対象物質としてのタンパク質の標準液を測定器具に接触させ、その後、標準液を回収して、ＥＬＩＳＡその他の免疫測定等、通常用いられている方法により当該タンパク質の回収率（測定器具接触後の回収液のタンパク質濃度（重量／ｍＬ）／測定器具接触前のタンパク質標準液濃度（重量／ｍＬ）（％））を求め、回収率が８０％以上、好ましくは８５％以上のブロッキング剤をその測定対象物質に対するブロッキング剤として選定する（後記実施例参照）。この場合、１つのブロッキング剤が２種以上のタンパク質に対して、８０％以上、好ましくは８５％以上の回収率を示す場合は、そのブロッキング剤はこれらのいずれのタンパク質に対するブロッキング剤としても使用することもできる。 Examples of the method for selecting a blocking agent in the present invention are as follows. That is, after the candidate blocking agent is coated on the measuring instrument, the standard solution of the protein as the measurement target substance is brought into contact with the measuring instrument, and then the standard solution is recovered, and is usually used for ELISA and other immunoassays. The recovery rate of the protein (the concentration of the protein in the recovered solution after contact with the measuring instrument (weight / mL) / the concentration of the protein standard solution before contacting the measuring instrument (weight / mL) (%)) is determined by Of 80% or more, preferably 85% or more is selected as the blocking agent for the substance to be measured (see Examples below). In this case, when one blocking agent shows a recovery rate of 80% or more, preferably 85% or more with respect to two or more kinds of proteins, the blocking agent is used as a blocking agent for any of these proteins. You can also.
　本発明では、２種以上の測定対象物質に対して、２種以上のブロッキング剤を選定する。この場合、測定対象物質が３種以上の場合は、選定したブロッキング剤が２種以上であればよい。例えば、測定対象物質が３種の場合、２種の測定対象物質に対して同じブロッキング剤を選定し、他の１種の測定対象物質に対してはそれとは異なるブロッキング剤を選定して、２種のブロッキング剤で３種の測定対象物質をブロッキングしてもよい。しかし、２種以上の測定対象物質の全てに対して１種のブロッキング剤が共通に８０％以上、好ましくは８５％以上の回収率を示す場合に、１種のブロッキング剤のみを使用する態様は本発明には含まれない。本発明は、２種以上のブロッキング剤を組合せて使用するものである。測定対象物質とブロッキング剤の組合せの例については、後記に記す。 In the present invention, two or more kinds of blocking agents are selected for two or more kinds of substances to be measured. In this case, when there are three or more kinds of substances to be measured, the selected blocking agents may be two or more kinds. For example, when there are three types of measurement target substances, the same blocking agent is selected for two types of measurement target substances, and a different blocking agent is selected for the other one type of measurement target substance. Three kinds of substances to be measured may be blocked with a kind of blocking agent. However, when one type of blocking agent shows a recovery rate of 80% or more, preferably 85% or more in common with respect to all of two or more kinds of substances to be measured, only one type of blocking agent is used. It is not included in the present invention. The present invention uses two or more blocking agents in combination. Examples of combinations of substances to be measured and blocking agents will be described later.
　（４）測定用器具
　本発明のブロッキング方法は、同じ測定用器具（採血管、フィルター、ウエル又は流路を備えたプレート等）を用いて、同一の血液試料に含まれる２種以上の測定対象物質を免疫測定する場合に用いられる。例えば、１本の採血管に採取した血液試料を、１個のフィルターでろ過し、ウエルまたは流路を有する１枚のプレートで、２種以上の測定対象物質を免疫測定する場合である。このような場合であっても、本発明のブロッキング方法により、前述の効果が得られる。この場合、例えば、１枚のプレートを用いて２種以上の測定対象物質を免疫測定する場合、プレート上には２種以上の測定対象物質に対応した２種以上の１次抗体が、それぞれ異なるウエルまたは流路中の所定の領域（測定領域）に固定化されているが、このような２種以上のウエルまたは領域で免疫測定をする実施形態であっても「同じ測定用器具を用いて測定する」ことに含まれる。測定器具の材質は特に制限はなく、個々の測定器具の材質の例は前述の通りである。 (4) Measuring instrument The blocking method of the present invention uses the same measuring instrument (such as a blood collection tube, a filter, a well or a plate with a flow path), and two or more types of measurement objects contained in the same blood sample. Used for immunoassay of substances. For example, this is a case where a blood sample collected in one blood collection tube is filtered with one filter, and two or more kinds of substances to be measured are immunoassayed with one plate having wells or flow paths. Even in such a case, the above-described effects can be obtained by the blocking method of the present invention. In this case, for example, when two or more kinds of measurement target substances are immunoassay using one plate, two or more kinds of primary antibodies corresponding to two or more kinds of measurement target substances are different on the plate. Although it is fixed to a predetermined region (measurement region) in a well or a flow path, even in an embodiment in which immunoassay is performed in such two or more wells or regions, “using the same measuring instrument” It is included in “measuring”. The material of the measuring instrument is not particularly limited, and examples of the material of each measuring instrument are as described above.
　（５）コーティング
　前述の通り選定したブロッキング剤によって、測定用器具の血液試料が接触する表面（例えば、採血管内面、フィルター内部、プレートの流路とウエル等）の少なくとも一部、好ましくは全面をコーティングする。この場合のコーティング方法は通常用いられている方法によればよく、例えば、ブロッキング剤を溶液状態として、測定用器具の血液試料が接触する表面に加えて覆い、所定時間保持後、測定用器具からブロッキング剤を除去すればよい。コーティングの処理条件は、例えば、ブロッキング剤溶液で覆った状態で室温で１時間保持し、純水で洗浄後、乾燥させることが挙げられる（後記実施例参照）。 (5) Coating By the blocking agent selected as described above, at least a part, preferably the entire surface, of the surface (for example, the inner surface of the blood collection tube, the inside of the filter, the flow path and well of the plate, etc.) in contact with the blood sample of the measuring instrument Coating. The coating method in this case may be a commonly used method. For example, in a blocking state, the blocking agent is covered in addition to the surface with which the blood sample of the measuring instrument comes into contact. What is necessary is just to remove a blocking agent. Examples of the coating processing conditions include holding for 1 hour at room temperature in a state of being covered with a blocking agent solution, washing with pure water, and drying (see Examples below).
　本発明者らの検討によれば、測定対象物質としての２種以上のタンパク質のうち、分子量の小さいタンパク質の非特異吸着を防止するブロッキング剤から分子量の大きいタンパク質の非特異吸着を防止するブロッキング剤へ順にコーティングを実施する方が、各タンパク質に対する各ブロッキング剤の非特異吸着防止効果が大きい。 According to the study by the present inventors, a blocking agent that prevents non-specific adsorption of a protein having a large molecular weight from a blocking agent that prevents non-specific adsorption of a protein having a low molecular weight among two or more proteins as a measurement target substance. When the coating is performed in order, the nonspecific adsorption preventing effect of each blocking agent on each protein is larger.
　例えば、ＢＮＰ（分子量約３，５００のタンパク質）に対するブロッキング剤プロタミン（ＢＮＰ回収率８５％）及びトロポニンＩ（分子量約２３，５００のタンパク質）に対するブロッキング剤カゼイン（トロポニン回収率８５％）の両者を用いてフィルターをブロッキングする場合、分子量の大きい方のタンパク質に対するブロッキング剤から順にコーティングすると、すなわち、先にカゼインでコーティングし次にプロタミンでコーティングすると、ＢＮＰ回収率は８２％であるが、トロポニンＩ回収率は５６％に留まる。また、カゼインとプロタミンを混合して同時にフィルターをコーティングすると、トロポニンＩ回収率８５％であるが、ＢＮＰ回収率は５６％に留まる。しかし、分子量の小さい方のタンパク質に対するブロッキング剤から順にコーティングすると、すなわち、先にプロタミンでコーティングし次にカゼインでコーティングすると、両者のコーティング後のフィルターは、ＢＮＰ回収率８０％、トロポニンＩ回収率８５％と、各ブロッキング剤を単独で使用した場合の各タンパク質に対する回収率とほぼ同じ回収率が得られる。（後記実施例、比較例参照）。 For example, both blocking agent protamine (BNP recovery rate 85%) for BNP (protein with a molecular weight of about 3,500) and blocking agent casein (troponin recovery rate 85%) for troponin I (a protein with a molecular weight of about 23,500) are used. When blocking the filter, coating with the blocking agent for the protein with the higher molecular weight, ie, coating with casein first and then with protamine, the BNP recovery is 82%, but the troponin I recovery is Remains 56%. When casein and protamine are mixed and the filter is coated simultaneously, the troponin I recovery rate is 85%, but the BNP recovery rate is only 56%. However, when coating is performed in order from the blocking agent for the protein having the smaller molecular weight, that is, when coated with protamine first and then with casein, both coated filters have a BNP recovery rate of 80% and a troponin I recovery rate of 85. %, And almost the same recovery rate as the recovery rate for each protein when each blocking agent is used alone. (See Examples and Comparative Examples below).
　従って、ブロッキング剤で測定用器具をコーティングする順序は、２種以上の測定対象物質としてのタンパク質のうち、分子量の小さいタンパク質の非特異吸着を防止するブロッキング剤から分子量の大きいタンパク質の非特異吸着を防止するブロッキング剤へ順にコーティングを実施する方が、好ましい。 Therefore, the order of coating the measuring instrument with the blocking agent is the non-specific adsorption of the protein with a large molecular weight from the blocking agent that prevents the non-specific adsorption of the protein with a small molecular weight among the two or more kinds of proteins to be measured. It is preferable to perform the coating in order on the blocking agent to be prevented.
　なお、この場合のタンパク質の分子量の測定は、通常行われている方法、例えば、ＳＤＳ－ＰＡＧＥ、ゲルろ過カラムクロマトグラフィー、質量分析、密度勾配遠心分離等で行えばよい。 In this case, the molecular weight of the protein may be measured by a commonly used method such as SDS-PAGE, gel filtration column chromatography, mass spectrometry, density gradient centrifugation and the like.
　また、２種以上のブロッキング剤による測定用器具のコーティングは、前のブロッキング剤のコーティング層が乾燥した後、次のブロッキング剤によるコーティングを実施することが好ましい。
　測定対象物質とブロッキング剤の組合せの一例は次の通りである。 Moreover, it is preferable to perform the coating with the next blocking agent after the coating layer of the previous blocking agent dries the coating of the instrument for a measurement by 2 or more types of blocking agents.
An example of the combination of the substance to be measured and the blocking agent is as follows.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　（６）免疫測定
　上記のようにして、血液試料に含まれる２種以上の測定対象物質に対するブロッキング剤で測定用器具をコーティングした後は、コーティングした同じ測定用器具（採血管、フィルター、ウエル又は流路を備えたプレート等）を用いて、一つの血液試料に含まれる当該２種以上の測定対象物質について目的とする免疫測定を行う。免疫測定の方法は特に制限はなく、通常用いられているＥＬＩＳＡその他の方法で検出、定量等を行えばよい。 (6) Immunoassay After coating a measuring instrument with a blocking agent for two or more kinds of substances to be measured contained in a blood sample as described above, the same coated measuring instrument (blood collection tube, filter, well or A target immunoassay is performed on the two or more kinds of substances to be measured contained in one blood sample using a plate having a flow path or the like. The immunoassay method is not particularly limited, and detection, quantification, and the like may be performed by a commonly used ELISA or other methods.
　例えば、ウエルまたは流路を備えた測定装置（例えば、表面プラズモン励起増強蛍光分光法（ＳＰＦＳ）測定装置等）を用いる場合、２種以上のウエルまたは流路中の測定領域に、それぞれ異なる検出対象物質を特異的に捕捉する１次抗体を固定化しておき、サンドイッチ法で２次抗体を結合させ標識して、測定対象物質を検出、定量する方法等がある。 For example, when using a measuring device having a well or a channel (for example, a surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) measuring device, etc.), two or more types of wells or different measurement targets in the channel are detected. There is a method in which a primary antibody that specifically captures a substance is immobilized, a secondary antibody is bound and labeled by a sandwich method, and a substance to be measured is detected and quantified.
　２．本発明の免疫測定用器具について
　本発明の免疫測定用器具の一態様は、
「血液試料中に含まれる２種以上のタンパク質を測定する免疫測定に用いる器具であって、前記器具の前記血液試料と接触する面の少なくとも一部に、前記２種以上のタンパク質のそれぞれの非特異吸着を防止する２種以上のブロッキング剤のコーティング層を有する器具」
である。 2. About the instrument for immunoassay of this invention One aspect | mode of the instrument for immunoassay of this invention is,
“An instrument used for immunoassay that measures two or more proteins contained in a blood sample, wherein at least a part of the surface of the instrument that contacts the blood sample is not coated with each of the two or more proteins. Instrument having two or more blocking agent coating layers to prevent specific adsorption "
It is.
　（１）コーティング層
　本発明の免疫測定用器具は、２種以上のブロッキング剤のコーティング層を有する。ここで、２種以上のブロッキング剤や免疫測定用器具へのコーティング方法については、前述の通りである。 (1) Coating layer The instrument for immunoassay of this invention has a coating layer of 2 or more types of blocking agents. Here, about the coating method to 2 or more types of blocking agents and the instrument for immunoassay, it is as above-mentioned.
　本発明の免疫測定用器具は、ブロッキング剤がコーティングされた二重以上の層を有する。この場合、ブロッキング剤のコーティング層の形態（層の厚さ、層の厚さの均一性等）は、特に制限はなく、目的とするブロッキング効果（検出対象物質としてのタンパク質が器具へ非特異吸着することを防止する効果、すなわち前記の回収率）が得られれば、特に制限はない。本発明者らが検討した結果によると、器具の表面に均一な厚さで層が形成されることは少なく、ブロッキング剤のコーティング層の厚さは不均一で、場所によっては、ブロッキング剤が付いていない部分がある場合もある。特に、先にコーティングする層に比べ、後にコーティングする層の方が、ブロッキング剤の付着が少なくなる傾向にある。このような状態のコーティング層であっても、上記の本発明の効果が得られ、本発明の免疫測定用器具の範囲に含まれる。 The immunoassay instrument of the present invention has two or more layers coated with a blocking agent. In this case, the form of the coating layer of the blocking agent (layer thickness, layer thickness uniformity, etc.) is not particularly limited, and the target blocking effect (non-specific adsorption of the protein as the detection target substance to the instrument) There is no particular limitation as long as the effect of preventing this, ie, the above-described recovery rate, is obtained. According to the results of the study by the present inventors, a layer with a uniform thickness is rarely formed on the surface of the device, and the thickness of the coating layer of the blocking agent is not uniform. There may be parts that are not. In particular, the layer to be coated later tends to have less adhesion of the blocking agent than the layer to be coated first. Even in the coating layer in such a state, the above-described effects of the present invention can be obtained and included in the scope of the immunoassay device of the present invention.
　また、２種以上の測定対象物質としてのタンパク質それぞれに対するブロッキング効果を十分得るために、ブロッキング剤のコーティングは、これらタンパク質のうち、分子量の小さいタンパク質の非特異吸着を防止するブロッキング剤から順に実施することが好ましいことは前述の通りであり、本発明の免疫測定用器具においても、同様に、これらタンパク質のうち、分子量の小さいタンパク質の非特異吸着を防止するブロッキング剤のコーティング層の上に（測定用器具表面からより離れた方向に）、分子量の大きいタンパク質の非特異吸着を防止するブロッキング剤のコーティング層をそれぞれ順に有する器具が好ましい。 In addition, in order to obtain a sufficient blocking effect on each of the two or more types of substances to be measured, the blocking agent coating is performed sequentially from the blocking agent that prevents non-specific adsorption of proteins having a low molecular weight among these proteins. As described above, it is preferable that the immunoassay device of the present invention also has a measurement on a coating layer of a blocking agent that prevents nonspecific adsorption of a protein having a low molecular weight among these proteins (measurement is also performed). A device having a coating layer of a blocking agent in order to prevent nonspecific adsorption of a protein having a large molecular weight (in a direction further away from the device surface) is preferable.
　本発明の免疫測定用器具は、免疫測定に用いられる器具であり、血液試料が接触するものであれば特に制限はなく、例えば、採血管、フィルター、ウエル又は流路を有するプレート等が挙げられる。 The instrument for immunoassay of the present invention is an instrument used for immunoassay, and is not particularly limited as long as it is in contact with a blood sample. Examples thereof include a blood collection tube, a filter, a well, or a plate having a flow path. .
　（２）その他
　本発明の免疫測定用器具についてのその他の説明は、前述の「１．本発明のブロッキング方法について」の説明の通りである。 (2) Others Other descriptions of the immunoassay device of the present invention are as described in the above-mentioned “1. About the blocking method of the present invention”.
　ブロッキング効果の検討は、ブロッキングされた器具（ガラスフィルター（アドバンテック社製、ＧＡ－１００）を使用した）に対して、サンプル（測定対象物質としてトロポニンＩ（以下、ｃＴｎＩ）、ＢＮＰ又はＮＴ－ｐｒｏＢＮＰを添加した血液を使用した）を接触させる前後において（すなわち、上記サンプルを上記ガラスフィルターでろ過する前とろ過した後において）、サンプル中の測定対象物質の濃度測定をＥＬＩＳＡ法によって行い、接触前に対する接触後の濃度測定値の割合から、測定対象物質の回収率を算出した。回収率が高い程、ブロッキング能力が高いと判断する。 The blocking effect was examined using a sample (a glass filter (Advantech, GA-100, GA-100) was used) with a sample (troponin I (hereinafter cTnI), BNP or NT-proBNP as a substance to be measured). Before and after contact (using the added blood) (ie, before and after filtering the sample through the glass filter), the concentration of the substance to be measured in the sample is measured by the ELISA method. The recovery rate of the substance to be measured was calculated from the ratio of the concentration measurement values after contact. The higher the recovery rate, the higher the blocking ability.
　ブロッキングは、器具をブロッキング溶液に室温で１時間浸水後、純水にて洗浄を行い、恒温槽にて乾燥させた。
　測定対象物質（多重染色する心筋マーカー）の組み合わせとブロッキング剤の関係は後記の表1に示す。また、各比較例、実施例における回収率を比較した結果は、後記の表３～４及び図１～２に示す。 For blocking, the instrument was immersed in a blocking solution at room temperature for 1 hour, washed with pure water, and dried in a thermostatic bath.
The relationship between combinations of substances to be measured (multiple staining myocardial markers) and blocking agents is shown in Table 1 below. Further, the results of comparing the recovery rates in the respective comparative examples and examples are shown in Tables 3 to 4 and FIGS.
　[比較例１]
　ｃＴｎＩ（トロポニンＩ、分子量：約23500）に対しブロッキング効果（回収率８５％）が確認されたカゼイン（サーモフィッシャー製:Casein in PBS)をガラスフィルターにブロッキングした系において、ＢＮＰ(分子量：約3500)に対するブロッキング効果を確認したところ、回収率は１％と低値となった（表３、図１参照）。 [Comparative Example 1]
In a system in which casein (manufactured by Thermo Fisher: Casein in PBS), whose blocking effect (recovery rate: 85%) was confirmed against cTnI (troponin I, molecular weight: about 23500), was blocked in a glass filter, BNP (molecular weight: about 3500) As a result of confirming the blocking effect against the above, the recovery rate was as low as 1% (see Table 3 and FIG. 1).
　[比較例２]
　ＢＮＰに対しブロッキング効果（回収率８５％）が確認されたプロタミン(和光純薬: プロタミン硫酸塩・サケ由来)をガラスフィルターにブロッキングした系において、ｃＴｎＩに対するブロッキング効果を確認したところ、回収率は２０％と低値となった（表３、図１参照）。 [Comparative Example 2]
When blocking effect against cTnI was confirmed in a system in which protamine (Wako Pure Chemical: protamine sulfate / salmon derived) with blocking effect (recovery rate 85%) confirmed against BNP was blocked on a glass filter, the recovery rate was 20 % (See Table 3 and FIG. 1).
　[比較例３]
　ｃＴｎＩに対しブロッキング効果（回収率８５％）が確認されたカゼイン(サーモフィッシャー製:Casein in PBS)をガラスフィルターにブロッキングした系において、ＮＴ－ｐｒｏＢＮＰ(分子量：約8500)に対するブロッキング効果を確認したところ、回収率は５３％と低値となった（表４、図２参照）。 [Comparative Example 3]
The blocking effect on NT-proBNP (molecular weight: about 8500) was confirmed in a system in which casein (Casein in PBS), which was confirmed to have a blocking effect (recovery rate of 85%) against cTnI, was blocked on a glass filter. The recovery rate was a low 53% (see Table 4 and FIG. 2).
　[比較例４]
　ＮＴ－ｐｒｏＢＮＰに対しブロッキング効果（回収率８３％）が確認されたＢＳＡ(サーモフィッシャー製:BSA in PBS)をガラスフィルターにブロッキングした系において、ｃＴｎＩに対するブロッキング効果を確認したところ、回収率は５５％と低値となった（表４、図２参照）。 [Comparative Example 4]
When blocking effect against cTnI was confirmed in a system where BSA (manufactured by Thermo Fisher: BSA in PBS), which was confirmed to have a blocking effect (recovery rate 83%) against NT-proBNP, was blocked on a glass filter, the recovery rate was 55%. (See Table 4 and FIG. 2).
　[実施例１]
　ＢＮＰに対しブロッキング効果が確認されたプロタミン(和光純薬: プロタミン硫酸塩・サケ由来)をブロッキング後、ｃＴｎＩに対しブロッキング効果が確認されたカゼイン(サーモフィッシャー製:Casein in PBS)をブロッキングしたところ、回収率はＢＮＰが８０％、ｃＴｎＩが８５％と、いずれも高回収率となった（表３、図１参照）。 [Example 1]
After blocking Protamine (Wako Pure Chemicals: Protamine sulfate / salmon derived) with blocking effect confirmed against BNP, blocking casein (Thermo Fisher: Casein in PBS) with blocking effect confirmed against cTnI, The recovery rates were as high as 80% for BNP and 85% for cTnI (see Table 3 and FIG. 1).
　[実施例２]
　ｃＴｎＩに対しブロッキング効果が確認されたカゼイン(サーモフィッシャー製:Casein in PBS)をブロッキング後、ＢＮＰに対しブロッキング効果が確認されたプロタミンをブロッキングしたところ、回収率はＢＮＰが８２％、ｃＴｎＩが５６％と、ｃＴｎＩの回収率は実施例１に比べ低値となった（表３、図１参照）。 [Example 2]
After blocking casein (Thermo Fisher: Casein in PBS) whose blocking effect was confirmed against cTnI and blocking protamine whose blocking effect was confirmed against BNP, the recovery rate was 82% for BNP and 56% for cTnI. The cTnI recovery rate was lower than that in Example 1 (see Table 3 and FIG. 1).
　[実施例３]
　カゼイン(サーモフィッシャー製:Casein in PBS)中にプロタミン(和光純薬: プロタミン硫酸塩・サケ由来)を1：1で混ぜ、ブロッキングを行ったところ、回収率はＢＮＰが５６％、ｃＴｎＩが８５％となり、ＢＮＰの回収率は実施例１に比べて低値となった（表３、図１参照）。 [Example 3]
Casein (manufactured by Thermo Fisher: Casein in PBS) was mixed with protamine (Wako Pure Chemicals: Protamine sulfate / salmon derived) 1: 1 and blocked. The recovery rate was 56% for BNP and 85% for cTnI. Thus, the recovery rate of BNP was lower than that in Example 1 (see Table 3 and FIG. 1).
　[実施例４]
　ＮＴ－ｐｒｏＢＮＰに対しブロッキング効果が確認されたＢＳＡ(サーモフィッシャー製:BSA in PBS)をブロッキング後、ｃＴｎＩに対しブロッキング効果が確認されたカゼイン(サーモフィッシャー製:Casein in PBS)をブロッキングしたところ、回収率はＮＴ－ｐｒｏＢＮＰが８２％、ｃＴｎＩが８４％と、いずれも高回収率となった（表４、図２参照）。 [Example 4]
After blocking BSA (Thermo Fischer: BSA in PBS) whose blocking effect was confirmed against NT-proBNP, casein (Thermo Fisher: Casein in PBS) whose blocking effect was confirmed against cTnI was recovered. The rates were as high as 82% for NT-proBNP and 84% for cTnI (see Table 4 and FIG. 2).
　[実施例５]
　ｃＴｎＩに対しブロッキング効果が確認されたカゼイン(サーモフィッシャー製:Casein in PBS)をブロッキング後、ＮＴ－ｐｒｏＢＮＰに対しブロッキング効果が確認されたＢＳＡ(サーモフィッシャー製:BSA in PBS)をブロッキングしたところ、回収率はＮＴ－ｐｒｏＢＮＰが８１％、ｃＴｎＩが５２％と、ｃＴｎＩの回収率は実施例４に比べ低値となった（表４、図２参照）。 [Example 5]
After blocking casein (Thermo Fischer: Casein in PBS) whose blocking effect was confirmed against cTnI, BSA (Thermo Fisher: BSA in PBS) whose blocking effect was confirmed against NT-proBNP was recovered. The rates were 81% for NT-proBNP and 52% for cTnI. The recovery rate of cTnI was lower than that of Example 4 (see Table 4 and FIG. 2).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004

Claims (8)

  1. 　血液試料中に含まれる２種以上のタンパク質を同じ測定用器具を用いて測定する免疫測定のためのブロッキング方法であって、前記２種以上のタンパク質のそれぞれの非特異吸着を防止する２種以上のブロッキング剤によって、前記血液試料と接触する前記器具の表面の少なくとも一部をコーティングするブロッキング方法。 A blocking method for immunoassay in which two or more kinds of proteins contained in a blood sample are measured using the same measuring instrument, wherein the two or more kinds prevent nonspecific adsorption of each of the two or more kinds of proteins. A blocking method of coating at least a part of the surface of the device that comes into contact with the blood sample with the blocking agent.
  2. 　前記２種以上のタンパク質のうち、分子量の小さいタンパク質の非特異吸着を防止するブロッキング剤から分子量の大きいタンパク質の非特異吸着を防止するブロッキング剤へ順にコーティングを実施する、請求項１に記載の方法。 The method according to claim 1, wherein coating is performed in order from a blocking agent that prevents nonspecific adsorption of a low molecular weight protein to a blocking agent that prevents nonspecific adsorption of a high molecular weight protein among the two or more proteins. .
  3. 　前のブロッキング剤が乾燥した後に次のブロッキング剤のコーティングを実施する、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the next blocking agent is coated after the previous blocking agent is dried.
  4. 　前記タンパク質、前記ブロッキング剤、及びコーティングを実施する順序が次のいずれかである、請求項２又は３に記載の方法。
    （１）前記タンパク質がＢＮＰ及びトロポニンＩであり、前記ブロッキング剤がプロタミン及びカゼインであり、プロタミンのコーティングの次にカゼインのコーティングを実施する。
    （２）前記タンパク質がＮＴ－ｐｒｏＢＮＰ及びトロポニンＩであり、前記ブロッキング剤がＢＳＡ及びカゼインであり、ＢＳＡのコーティングの次にカゼインのコーティングを実施する。     The method according to claim 2 or 3, wherein the order of performing the protein, the blocking agent, and coating is any of the following.
    (1) The protein is BNP and troponin I, the blocking agent is protamine and casein, and the coating of casein is performed after the coating of protamine.
    (2) The protein is NT-proBNP and troponin I, the blocking agent is BSA and casein, and the coating of casein is performed after the coating of BSA.
  5. 　血液試料中に含まれる２種以上のタンパク質を測定する免疫測定に用いる器具であって、前記器具の前記血液試料と接触する面の少なくとも一部に、前記２種以上のタンパク質のそれぞれの非特異吸着を防止する２種以上のブロッキング剤のコーティング層を有する器具。 A device used for immunoassay for measuring two or more proteins contained in a blood sample, wherein each of the two or more proteins is non-specific on at least a part of a surface of the device that contacts the blood sample. An instrument having a coating layer of two or more blocking agents for preventing adsorption.
  6. 　前記２種以上のタンパク質のうち、分子量の小さいタンパク質の非特異吸着を防止するブロッキング剤のコーティング層の上に、分子量の大きいタンパク質の非特異吸着を防止するブロッキング剤のコーティング層をそれぞれ順に有する請求項５に記載の器具。 A blocking agent coating layer for preventing nonspecific adsorption of a protein having a large molecular weight is sequentially formed on the coating layer of a blocking agent for preventing nonspecific adsorption of a protein having a low molecular weight among the two or more proteins. Item 6. The device according to Item 5.
  7. 　採血管、フィルター、又はウエル若しくは流路を備えたプレートである、請求項５又は６に記載の器具。 The instrument according to claim 5 or 6, which is a plate having a blood collection tube, a filter, or a well or a flow path.
  8. 　前記タンパク質、前記ブロッキング剤、及びコーティング層を有する順序が次のいずれかである、請求項５～７のいずれか一項に記載の器具。
    （１）前記タンパク質がＢＮＰ及びトロポニンＩであり、前記ブロッキング剤がプロタミン及びカゼインであり、プロタミンのコーティング層の上にカゼインのコーティング層を有する。
    （２）前記タンパク質がＮＴ－ｐｒｏＢＮＰ及びトロポニンＩであり、前記ブロッキング剤がＢＳＡ及びカゼインであり、ＢＳＡのコーティング層の上にカゼインのコーティング層を有する。     The device according to any one of claims 5 to 7, wherein the order of having the protein, the blocking agent, and the coating layer is any of the following.
    (1) The protein is BNP and troponin I, the blocking agent is protamine and casein, and has a casein coating layer on the protamine coating layer.
    (2) The protein is NT-proBNP and troponin I, the blocking agent is BSA and casein, and has a casein coating layer on the BSA coating layer.
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CN112730824A (en) * 2020-12-02 2021-04-30 广州迪澳生物科技有限公司 Sealing stabilizer for mechanized preparation of pre-coated plate
CN115184620A (en) * 2022-09-14 2022-10-14 山东子峰生物技术有限公司 Quantum dot fluorescence detection test strip and kit for PLA2R antibody and application of quantum dot fluorescence detection test strip and kit

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CN111965349A (en) * 2019-11-28 2020-11-20 上海荣盛生物药业有限公司 Composition for fluorescence immunochromatography detection and application of composition in HIV detection
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