JP2007225603A - Method and reagent for immunoassay of measured object in sample - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a reagent for immunoassay of a measured object in a sample where influence of hemoglobin is suppressed. <P>SOLUTION: In the method for immunoassay of the measured object, the measured object in the sample is made to react with an antibody to be bonded to the measured object under coexistence of cytolysate which does not originate from the sample. The reagent for immunoassay of the measured object contains the cytolysate. The influence of hemoglobin on the immuno-reaction of the measured object is suppressed by making the measured object in the sample react with the antibody to be bonded to the measured object under coexistence of cytolysate which does not originate from the sample. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an immunoassay method and immunoassay reagent for a measurement object, a method for suppressing the influence of hemoglobin on the immune reaction of the measurement object, and a reagent for suppressing the influence of hemoglobin on the immune reaction of the measurement object About.

  When measuring blood components immunologically, serum or plasma obtained by removing blood cells from whole blood is generally used as a sample. However, in order to remove blood cells, a dedicated device such as a centrifuge is required, which takes time, and a method for directly measuring a whole blood sample has been proposed (see Patent Document 1). When whole blood is used as a sample, blood cell components such as hemoglobin and blood cell components mixed into the specimen due to hemolysis affect the optical detection system, inhibit the immune reaction, and adsorb the measurement target substance. Therefore, there is a problem that affects the measurement. In order to avoid such a problem, an immunoassay method using whole blood that is measured without hemolysis as a sample has been reported (see Patent Documents 2, 3, 4, and 5).

When measuring blood cell components such as intracellular proteins, it is necessary to lyse the blood cells, and thus the above-described measurement method without hemolysis cannot be used. In this case, a method of measuring the target blood cell component by separating the target blood cells by flow cytometry and then lysing the separated cells has been used. Is necessary and time-consuming. On the other hand, as an example of a simple method for measuring intracellular protein, an immunoassay method for MxA protein using a sample obtained by dissolving blood cells in whole blood with a surfactant has been reported (Non-patent Document 1). reference). In this report, no measures are taken to suppress the influence of hemoglobin or the like. Therefore, it has not been established that a measurement method that dissolves blood cells in a sample containing hemoglobin such as whole blood, is simple and highly sensitive, and suppresses the influence of hemoglobin or the like.
JP-A-10-48214 JP-A-6-265554 International Publication No. 96/04558 Pamphlet International Publication No. 02/73203 Pamphlet JP 2004-45395 A Journal of Interferon Research, (USA), 1992, Vol. 12, No. 2, p. 67-74 Pediatric Research, (USA), 1997, Vol. 41, No. 5, p. 647-650

  An object of the present invention is to provide an immunoassay method and an immunoassay reagent for a measurement object in a sample, in which the influence of hemoglobin is suppressed.

The present inventors have found that the influence of hemoglobin can be suppressed by reacting a measurement object and an antibody that binds to the measurement object in the presence of a cell lysate not derived from the sample, and completed the present invention. It was. That is, the present invention relates to the following [1] to [23].
[1] A method for immunoassay of a measurement target, comprising reacting a measurement target in a sample with an antibody that binds to the measurement target in the presence of a cell lysate not derived from the sample.
[2] The method according to [1], wherein the immunoassay method is a sandwich method or a competitive method.
[3] An immunoassay method for an object to be measured, comprising reacting the object to be measured in the sample with any one of (i) to (iii) in the presence of a cell lysate not derived from the sample.
(I) a labeled antibody in which a label is bound to a first antibody that binds to the measurement object, and a second antibody that binds to the measurement object;
(Ii) a labeled competitor with a label bound to the competitor, and an antibody that binds to the analyte and competitor,
(Iii) A competitive substance, and a labeled antibody that binds to the measurement target and the competitive substance and has a label bound thereto.
[4] The influence of hemoglobin on the immune reaction of a measurement object, wherein the measurement object in a sample and an antibody that binds to the measurement object are reacted in the presence of a cell lysate not derived from the sample. How to suppress.

[5] The method according to any one of [1] to [4], wherein the sample is whole blood.
[6] The method according to [5], wherein the whole blood is whole blood obtained by dissolving blood cells.
[7] The method according to [6], wherein the whole blood in which blood cells are dissolved is whole blood in which blood cells are dissolved with a surfactant.
[8] The method according to any one of [1] to [7], wherein the cell lysate is a cell lysate of animal cells.
[9] The method according to [8], wherein the animal cell is an animal cell line.
[10] The method according to [9], wherein the animal cell line is Raji cell or T98G cell.
[11] The method according to any one of [1] to [10], wherein the object to be measured is MxA protein or Pancreatitis associated protein.

[12] A reagent for suppressing the influence of hemoglobin on the reaction between a measurement object and an antibody that binds to the measurement object, comprising a cell lysate.
[13] An immunoassay reagent for an object to be measured, comprising a cell lysate.
[14] The immunoassay reagent according to [13], further comprising any one of (i) to (iii).
(I) a labeled antibody in which a label is bound to a first antibody that binds to a measurement object, and a second antibody that binds to the measurement object;
(Ii) a labeled competitor having a label bound to the competitor, and an antibody that binds to the analyte and the competitor,
(Iii) A labeled antibody in which a label is bound to a competitive substance, and an antibody that binds to an analyte and a competitive substance.

[15] The immunoassay reagent according to [13] or [14], further comprising a reagent for dissolving blood cells in the sample.
[16] The immunoassay reagent according to [15], wherein the reagent for dissolving blood cells is a surfactant.
[17] The immunoassay reagent according to [16], wherein the surfactant is a nonionic surfactant.
[18] The immunoassay reagent according to [17], wherein the nonionic surfactant is a polyoxyethylene surfactant.
[19] The immunoassay reagent according to [18], wherein the polyoxyethylene-based surfactant is polyoxyethylene alkylphenyl ether.

[20] The reagent according to any one of [12] to [19], wherein the cell lysate is a cell lysate of animal cells.
[21] The reagent according to [20], wherein the animal cell is an animal cell line.
[22] The reagent according to [21], wherein the animal cell line is a Raji cell or a T98G cell.
[23] The reagent according to any one of [12] to [22], wherein the object to be measured is MxA protein or Pancreatitis associated protein.

  According to the present invention, there are provided an immunoassay method and an immunoassay reagent for a measurement object in a sample, in which the influence of hemoglobin is suppressed.

(1) Sample As a sample used as a sample in the immunoassay method of the present invention, for example, whole blood, a blood cell fraction containing red blood cells prepared from whole blood, plasma or serum suspected of hemolysis, red blood cells, arbitrary sample Examples include a sample containing hemoglobin or a sample suspected of containing hemoglobin, such as a sample added with hemoglobin. The whole blood may be the blood itself collected from the subject, or may be the processed blood, and the processed blood is preferred. Examples of the treatment include anticoagulation treatment and hemolysis treatment, and these treatments may be combined. When the measurement target is an intracellular component of a blood cell, the whole blood is preferably hemolyzed blood, and particularly preferably blood subjected to both anticoagulation treatment and hemolysis treatment. Examples of the anticoagulation treatment include a treatment of adding EDTA, heparin or the like to the collected blood. Examples of the hemolysis treatment include addition of a surfactant or a saponin solution, mixing with a hypotonic solution, freezing and thawing, ultrasonic treatment, and the like. As the surfactant, any surfactant can be used as long as it can lyse cells without affecting the measurement system. In the present invention, a nonionic surfactant is preferable. Examples of the nonionic surfactant include the nonionic surfactant described below. When using a blood cell fraction or erythrocyte as a sample, those subjected to anticoagulation treatment, hemolysis treatment and the like as described above are preferable.

(2) Measurement object The measurement object to be measured by the immunoassay method of the present invention is not particularly limited as long as it is a substance contained in the sample mentioned in (1). For example, it is contained in blood cell components or plasma. And proteins such as antibodies, interleukins and hormones, bioactive substances, drugs administered from the outside, and the like. Specific examples of measurement objects include MxA protein (Mol. Cell. Biol., 9 , 5062-5072, 1989; J. Virol. 64 , 1171-1181, 1990), which is a component of leukocytes, in plasma. Pancreatitis Associated Protein (hereinafter abbreviated as PAP) (Digestion, 29 , 242-249, 1984) and the like.

(3) Cell lysate The immunoassay method of the present invention is based on the measurement object and the measurement object in the presence of a cell lysate not derived from the sample, that is, a cell lysate different from the cell lysate contained in the sample. It is characterized in that it reacts with an antibody that binds to. The cell lysate coexisting in the method of the present invention does not include the cell lysate contained in the sample when the immunoassay is performed by lysing cells in the sample. For example, when whole blood is used as a sample and blood cells in whole blood are lysed for measurement, a lysate of blood cells will coexist. Perform an antibody reaction.

  The cell lysate means a liquid containing a cytoplasm obtained by lysing cells, that is, by destroying a cell membrane. For example, the cells can be lysed and then centrifuged to obtain a supernatant. Cell lysis can be performed, for example, by the above-described hemolysis treatment, but hemolysis with a surfactant is preferable. The surfactant is not particularly limited as long as it does not affect immunoassay and can lyse cells, but a nonionic surfactant is preferable.

  Examples of the nonionic surfactant used for the hemolysis treatment and cell lysis described above include polyoxyethylene surfactants, sorbitan fatty acid esters, glycerin fatty acid esters, and the like. Polyoxyethylene surfactants Is preferred. Examples of the polyoxyethylene surfactant include polyoxyethylene alkylphenyl ether, polyoxyethylene alkyl ether, polyoxyethylene sorbitan fatty acid ester, and the like, and polyoxyethylene alkylphenyl ether is preferable. Examples of the alkyl in the polyoxyethylene alkylphenyl ether include octyl and nonyl. Specific examples (commercially available products) of polyoxyethylene alkylphenyl ether include Nonidet P-40 (polyoxyethylene nonylphenyl ether) and the like.

  The cell used as a raw material of the cell lysate may be any cell such as an animal cell, a plant cell, or a microorganism, but an animal cell is preferable, and an animal cell line is more preferable. Animal cells include not only non-human animal cells but also human cells. Examples of animal cell lines include Raji, Namalwa, HL60 established from human blood cells, T98G established from human neurons, CCF-STTG1, H4, HeLa established from human uterine cancer cells, monkey kidney cells COS-7 established from Vero, CHO established from hamster ovary cells, BHK21 established from hamster kidney cells, NIH / 3T3 established from mouse fibroblasts, NS0 established from rat myeloma cells, dog MDCK established from kidney cells.

  The cell lysate coexists in the antigen-antibody reaction involving the measurement object in the sample in the immunoassay method of the present invention. That is, the cell lysate coexists in the primary reaction in the sandwich method (reaction between the first antibody that binds to the measurement object and the measurement object in the sample) and the competitive reaction in the competition method. The cell lysate may be added to the sample in advance, or may be added to the reaction solution. For example, a sample diluent containing a cell lysate can be prepared, and a sample diluted with a specimen diluent can be used as a sample. The sample diluent containing cell lysate may contain the aforementioned surfactant for dissolving blood cells in the sample.

The amount of cell lysate added is not particularly limited as long as it is a concentration that suppresses the influence of hemoglobin on the immune reaction of the measurement object. For example, the protein concentration of the cell lysate in the reaction solution of the measurement object is 1 μg. / ML to 2 mg / mL, more preferably 5 to 50 μg / mL. The volume is preferably 0.1 to 10% of the sample.
The cell lysate preferably does not contain the above-mentioned measurement object, and when it contains the measurement object, it is preferable to determine the concentration of the measurement object in advance.

(4) Antibody The antibody used in the measurement method of the present invention is not particularly limited as long as it is an antibody that specifically binds to a measurement target, and both a polyclonal antibody and a monoclonal antibody can be used. preferable. In the present invention, not only antibodies but also Fc portions such as Fab obtained by papain treatment, F (ab ′) ₂ obtained by pepsin treatment, Fab ′ obtained by pepsin treatment-reduction treatment, etc. were removed from the antibody. Antibody fragments can also be used. As the antibody fragment, F (ab ′) ₂ is particularly preferable.

  The antibody used in the present invention can be obtained by an ordinary method using a peptide to be measured or a peptide corresponding to its epitope as an antigen, but is also commercially available.

  When the measurement target is MxA protein, examples of antibodies that specifically bind to MxA protein include hybridoma cell lines KM1122, KM1123, KM1124 (FERM BP-4729), KM1125, described in WO 96/05230, KM1126, KM1127, KM1128, KM1129, KM1130, KM1131, KM1132 (FERM BP-4730), KM1133, KM1134, KM1135 (FERM BP-4731) produced by anti-human MxA protein monoclonal antibodies KM1122, KM1123, KM1124, KM1124 , KM1127, KM1128, KM1129, KM1130, KM1131, KM1132, KM1133, KM1134 KM1135 and the like. When the measurement target is PAP, examples of antibodies that specifically bind to PAP include 6F3E4 (WO94 / 15218), 16F4B8 (WO94 / 15218), and the like.

(5) Competing substance In the present invention, a “competitive substance” is a substance that can bind to an “antibody that binds to a measurement target” used in the immunoassay of the present invention, and the binding competes with the measurement target. It means a substance that is the target, and includes the measurement object itself. The “competitive substance” is used when measuring an object to be measured in a sample by a competitive method. Therefore, the antibody that binds to the measurement target used in the competition method is an antibody that binds to the measurement target and the competitive substance, and binds to the measurement target to generate an immune complex, and also binds to the competitive substance to immunize. Create a complex. The competitive substance is preferably a substance having the same structure as the epitope recognized by the antibody that binds to the measurement target, and the strength of the binding to the antibody that binds to the measurement target is determined by the measurement target for the antibody. Those having the same strength as the bond of the object are preferred. The measurement object itself is suitable as a competitive substance.

(6) Immunoassay method The immunoassay method of the present invention is characterized in that a measurement object in a sample and an antibody that binds to the measurement object are reacted in the presence of a cell lysate not derived from the sample. Is the method. Examples of the immunoassay method of the present invention include a sandwich method and a competition method. For example, an immunoassay method is characterized in that a measurement object in a sample is reacted with any of the following (i) to (iii) in the presence of a cell lysate not derived from the sample. (I) is a sandwich method and (ii) and (iii) are competitive methods.
(I) a labeled antibody in which a label is bound to a first antibody that binds to the measurement object, and a second antibody that binds to the measurement object;
(Ii) a labeled competitor with a label bound to the competitor, and an antibody that binds to the analyte and competitor,
(Iii) A competitive substance, and a labeled antibody that binds to the measurement target and the competitive substance and has a label bound thereto.

  Specifically, the method of the following aspect is mention | raise | lifted, for example. Measurement methods 1 and 2 are sandwich methods, measurement methods 3 to 6 are competition methods in which a measurement object in a sample and a competitive substance compete with each other, and measurement methods 7 are labeled antibodies that are not included in immune complexes and immune complexes or This is a homogeneous method that does not separate the labeled competitor (B / F separation).

Measurement method 1
A measurement method for sequentially performing the following steps (a) to (e).
(A) A measurement object in a sample and a first antibody that binds to the measurement object are reacted in the presence of a cell lysate that does not originate from the sample, and consists of the first antibody and the measurement object. Generating an immune complex;
(B) The immune complex generated in the step (a) is reacted with the labeled antibody having the label bound to the second antibody that binds to the measurement target, and the first antibody, the measurement target, and the labeling Generating an immune complex comprising an antibody;
(C) a step of separating the immune complex produced in the step (b) and a labeled antibody not contained in the immune complex;
(D) measuring the amount of the label in the immune complex produced in the step (b); and
(E) A step of determining the concentration of the measurement object in the sample from the amount of the label in the immune complex generated in the step (b).

  The first antibody is preferably immobilized on an insoluble carrier. Step (a) and step (b) may be performed sequentially or simultaneously. If the second antibody can bind to the measurement object bound to the first antibody, the site in the measurement object recognized by the first antibody and the site in the measurement object recognized by the second antibody May be the same or different, but are preferably different.

Measurement method 2
A measurement method in which the following steps (a) to (f) are sequentially performed.
(A) A measurement object in a sample and a first antibody that binds to the measurement object are reacted in the presence of a cell lysate that does not originate from the sample, and consists of the first antibody and the measurement object. Generating an immune complex;
(B) reacting the immune complex generated in the step (a) with the second antibody that binds to the measurement target, and preparing an immune complex comprising the first antibody, the measurement target, and the second antibody. Generating step;
(C) The immune complex produced in the step (b) is reacted with a labeled antibody having a label attached to a third antibody that binds to the second antibody to produce an immune complex containing the labeled antibody. The step of causing;
(D) a step of separating the immune complex produced in the step (c) and a labeled antibody not contained in the immune complex;
(E) a step of measuring the amount of the label in the immune complex produced in the step (c); and (f) measurement in the sample from the amount of the label in the immune complex produced in the step (c). Determining the amount of the object.

  The first antibody is preferably immobilized on an insoluble carrier. Steps (a) to (c) may be performed sequentially or simultaneously. If the second antibody can bind to the measurement object bound to the first antibody, the site in the measurement object recognized by the first antibody and the site in the measurement object recognized by the second antibody May be the same or different, but are preferably different.

  When the measurement target in the sample is an antibody against a specific antigen, the measurement target is measured by using the antigen instead of the first antibody in measurement method 1 or 2. You can also.

Measurement method 3
A measurement method for sequentially performing the following steps (a) to (d).
(A) A measurement object in a sample, a labeled competitor, and an antibody that binds to the measurement object and the competitor are reacted in the presence of cell lysate not derived from the sample, and labeled with the antibody. Producing an immune complex comprising a competitor and an immune complex comprising the antibody and a measurement object;
(B) separating the immune complex comprising the antibody and the labeled competitor from unreacted labeled competitor;
(C) measuring the amount of the label in the immune complex comprising the antibody and the labeled competitor; and
(D) A step of determining the concentration of the measurement object in the sample from the amount of the label in the immune complex comprising the antibody and the labeled competitor.
The antibody is preferably immobilized on an insoluble carrier.

Measurement method 4
A measurement method for sequentially performing the following steps (a) to (d).
(A) A measurement object in a sample, a competitive substance, and a labeled antibody in which a label is bound to an antibody that binds to the measurement object and the competitive substance are reacted in the presence of a cell lysate not derived from the sample. A step of generating an immune complex comprising a labeled antibody and a competitor and an immune complex comprising a labeled antibody and a measurement object;
(B) separating the immune complex composed of the labeled antibody and the competitor from the unreacted labeled antibody and the immune complex composed of the labeled antibody and the measurement target;
(C) measuring the amount of the label in the immune complex comprising the labeled antibody and the competitor; and
(D) A step of determining the concentration of the analyte in the sample from the amount of the label in the immune complex consisting of the labeled antibody and the competitor.

  The competitor is preferably immobilized on an insoluble carrier. When the competitive substance has the same structure as the measurement object, the competitive substance immobilized on the insoluble carrier in the step (a) is used.

Measurement method 5
A measurement method in which the following steps (a) to (f) are sequentially performed.
(A) A measurement object in a sample, a competitive substance, and a first antibody that binds to the measurement object and the competitive substance are reacted in the presence of a cell lysate not derived from the sample, and the first antibody Generating an immune complex composed of the first antibody and the competitor, and an immune complex composed of the first antibody and the measurement target;
(B) separating the immune complex composed of the first antibody and the competitor from the unreacted first antibody and the immune complex composed of the first antibody and the measurement target;
(C) reacting an immune complex comprising a first antibody and a competitor with a labeled antibody having a label bound to a second antibody that binds to the first antibody, and reacting the first antibody, the competitor and Generating an immune complex comprising a labeled antibody;
(D) separating the immune complex composed of the first antibody, the competitor and the labeled antibody from the unreacted labeled antibody;
(E) measuring the amount of label in the immune complex consisting of the first antibody, competitor and labeled antibody; and
(F) A step of determining the concentration of the measurement object in the sample from the amount of the label in the immune complex composed of the first antibody, the competitor and the labeled antibody.

  The competitor is preferably immobilized on an insoluble carrier. When the competitive substance has the same structure as the measurement object, the competitive substance immobilized on the insoluble carrier in the step (a) is used.

Measurement method 6
A measurement method for sequentially performing the following steps (a) to (e).
(A) A measurement object in a sample, a competitive substance, and a first antibody that binds to the measurement object and the competitive substance are reacted in the presence of a cell lysate not derived from the sample, and the first antibody Generating an immune complex composed of the first antibody and the competitor, and an immune complex composed of the first antibody and the measurement target;
(B) A label is bound to the immune complex composed of the first antibody and the competitor, the immune complex composed of the first antibody and the measurement object, and the second antibody that binds to the first antibody. Reacting the labeled antibody to produce an immune complex composed of the first antibody, the competitor and the labeled antibody, and an immune complex composed of the first antibody, the measurement object and the labeled antibody. ;
(C) Separating the immune complex composed of the first antibody, the competitor and the labeled antibody from the immune complex composed of the first antibody, the measurement object and the labeled antibody, and the unreacted labeled antibody The step of:
(D) measuring the amount of label in the immune complex consisting of the first antibody, the competitor and the labeled antibody; and
(E) A step of determining the concentration of the analyte in the sample from the amount of the label in the immune complex consisting of the first antibody, the competitor and the labeled antibody.

  The competitor is preferably immobilized on an insoluble carrier. When the competitive substance has the same structure as the measurement object, the competitive substance immobilized on the insoluble carrier in the step (a) is used.

Measurement method 7
The measuring method which has the process of the following (a)-(c).
(A) The measurement object in the sample, the labeled antibody 1 obtained by labeling the first antibody that specifically binds to the measurement object with the labeling substance 1, and the second that specifically binds to the measurement object The antibody is reacted with a labeled antibody 2 labeled with a labeling substance 2 different from the labeling substance 1 in the presence of a cell lysate not derived from the sample, and consists of a labeled antibody 1, a measurement object, and a labeled antibody 2. Generating an immune complex;
(B) a step of measuring the amount of change in the interaction between the labeling substance 1 and the labeling substance 2;

  If the second antibody can bind to the measurement object bound to the first antibody, the site in the measurement object recognized by the first antibody and the site in the measurement object recognized by the second antibody May be the same or different, but are preferably different.

  As described above, measurement methods 1 to 6 are measurement methods (heterogeneous methods) involving B / F separation. Step (c) of measurement method 1, step (d) of measurement method 2, step (b) of measurement methods 3 and 4, steps (b) and (d) of measurement method 5, step (c) of measurement method 6 Is the B / F separation step. This B / F separation can be easily performed by washing the insoluble carrier after removing the reaction solution when the antibody or the competitor is immobilized on the insoluble carrier. In other words, the reaction solution is removed after the antigen-antibody reaction, and the insoluble carrier is washed with a washing solution to separate the immune complex produced on the insoluble carrier from the unreacted label (labeled antibody, labeled competitor). can do. The washing solution contains phosphate buffered saline (10 mmol / L phosphate buffer containing 0.15 mol / L sodium chloride, pH 7.2, hereinafter referred to as PBS), and a surfactant. PBS, the aqueous medium mentioned later, etc. can be mention | raise | lifted. Examples of the surfactant include nonionic surfactants such as Tween 20 and the like. In measurement method 1 or 2, when the first antibody is immobilized on an insoluble carrier, a step of washing the insoluble carrier may be inserted between step (a) and step (b). it can. In measurement method 2, when the first antibody is immobilized on an insoluble carrier, it is preferable to have a step of washing the insoluble carrier between step (b) and step (c).

  In measurement method 3, when the antibody that binds to the measurement target is not immobilized on an insoluble carrier, the binding substance that binds to the antibody is immobilized without binding to the labeled competitor in step (c). The insoluble carrier is added to bind the immune complex to the insoluble carrier, and then the reaction solution is removed, and the insoluble carrier is washed, so that the immune complex and the labeled competition not included in the immune complex can be obtained. The substance can be separated. In addition, in the presence of an insoluble carrier immobilized with a binding substance that binds to the antibody without binding to the labeled competitor, a reaction for generating an immune complex in the step (a) is performed, After simultaneously generating and immobilizing the immune complex on an insoluble carrier, the reaction solution is removed, and the insoluble carrier is washed to remove the immune complex and the labeled competitor that is not included in the immune complex. Can be separated. Examples of the binding substance that does not bind to the labeled competitor but binds to the antibody include an antibody that binds to the constant region of the antibody. If the measurement object is not a protein, a protein precipitating agent such as ammonium sulfate or polyethylene glycol is added in the step (c) to precipitate only the immune complex, and then centrifuged to obtain the immune complex. And labeled competitors not contained in the immune complex can be separated.

In the method of measurement method 1 or 2, when the first antibody is not immobilized on an insoluble carrier, the binding ability that binds to the first antibody is not bound to the labeled antibody in the B / F separation step. The step of adding an insoluble carrier on which a substance is immobilized or generating an immune complex is performed in the presence of an insoluble carrier on which a binding substance that binds to the first antibody is immobilized without binding to the labeled antibody. After removing each reaction solution and washing the insoluble carrier, the labeled antibody and the labeled antibody not contained in the immune complex can be separated. As the binding substance that does not bind to the labeled antibody but binds to the first antibody, for example, when the animal species used for the production of the labeled antibody are different from those of the first antibody, the production of the first antibody Examples include antibodies against immunoglobulins of animal species used in the above, wherein the first antibody is an antibody having a constant region, and the labeled antibody is an antibody having no constant region such as Fab, F (ab ′) ₂ , Fab ′ In the case of a fragment, an antibody that specifically binds to the constant region of the first antibody can be used.

  In the methods of measurement methods 4 to 6, when the competitive substance is not immobilized on the insoluble carrier, if the measurement target and the competitive substance have different structures, they do not bind to the measurement target and An insoluble carrier in which a binding substance to be bound is immobilized is added to the reaction solution, or the step of generating an immune complex is not bound to the measurement target, and the binding substance that binds to the competitor is immobilized. The reaction is carried out in the presence of an insoluble carrier, the reaction solution is removed, and the insoluble carrier is washed. Thus, an immune complex containing a competitor (an immune complex composed of a competitor and a labeled antibody, a competitor and It is possible to separate only the immune complex consisting of one antibody or the immune complex consisting of a competitor, the first antibody and a labeled antibody). Examples of the binding substance that does not bind to the measurement target but binds to the competitive substance include an antibody that does not exist in the measurement target and binds to a partial structure that only the competitive substance has.

  In these measurement methods, by performing the step (a) in the presence of the cell lysate described in (3), the influence of hemoglobin can be suppressed, and more accurate measurement can be performed. .

(7) Immobilization of insoluble carrier and antibody or competitive substance to insoluble carrier Insoluble carrier for immobilizing antibody or competitive substance includes any substance that can stably hold antibody or competitive substance. Is done. Preferred materials for the insoluble carrier include polystyrene, polycarbonate, polyvinyltoluene, polypropylene, polyethylene, polyvinyl chloride, nylon, polymethacrylate, gelatin, agarose, cellulose, polyethylene terephthalate, and other polymer materials, glass, ceramics, magnetic particles, metals, etc. Can be given. Preferable shapes of the insoluble carrier include tubes, beads, plates, fine particles such as latex, sticks and the like. For example, a polystyrene microtiter plate having 96 wells per sheet is preferable.

  As a method for immobilizing an antibody or a competitive substance on an insoluble carrier, a known method such as a method using physical binding, a method using chemical binding, or a combination thereof is used. Examples of physical bonds include electrostatic bonds, hydrogen bonds, and hydrophobic bonds. Examples of the chemical bond include a covalent bond and a coordination bond. For example, when a polystyrene microterplate for immunoassay is used as an insoluble carrier, an antibody or competitor solution is added to the wells in the plate and incubated at 4 ° C. to 30 ° C. for 1 hour to 1 day. Thus, a method of physical adsorption and immobilization can be mentioned.

  The antibody or competitor may be directly immobilized on an insoluble carrier or indirectly immobilized on an insoluble carrier. As an indirect immobilization method, for example, a biotinylated antibody or competitor is added to an insoluble carrier on which avidin is immobilized, and the antibody or competitor is made insoluble on the insoluble carrier through specific binding between biotin and avidin. A method of immobilization is mentioned. Alternatively, an antibody that specifically binds to an antibody or an antibody that specifically binds to a competitor may be immobilized on an insoluble carrier, and the antibody or competitor may be immobilized to the insoluble carrier via this antibody. Alternatively, the antibody or competitor may be immobilized on an insoluble carrier by covalent bonding via a linker. The linker may be any molecule that can be covalently bonded to both the functional group of the antibody or the measurement target and the functional group of the side chain of the insoluble carrier. A preferred embodiment includes, for example, a first reactive group capable of reacting with a functional group possessed by an antibody or a measurement object, and a second reactive group capable of reacting with a functional group on the side chain of the insoluble carrier. It is a molecule having at the same time, and it is preferable that the first reaction active group and the second reaction active group are different groups. Examples of the functional group of the antibody or competitor and the functional group held on the surface of the insoluble carrier include a carboxy group, amino group, glycidyl group, sulfhydryl group, hydroxyl group, amide group, imino group, N-hydroxysuccinyl group, And maleimide groups. Examples of the active reactive group in the linker include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide (NHS) ester, pentafluorophenyl (PFP) ester, psoralen, And groups such as pyridyl disulfide and vinyl sulfone.

(8) Labeling of antibody or measurement object Examples of the labeling substance for labeling the antibody or the measurement object include enzymes, fluorescent substances, luminescent substances, radioisotopes, biotin, digoxigenin, polypeptides containing tag sequences, and the like.

Examples of the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like.
Examples of the fluorescent substance include FITC (fluorescein isothiocyanate), RITC (rhodamine B-isothiocyanate) and the like. As other fluorescent substances, for example, quantum dot (Science, 281 , 2016-2018, 1998), phycobiliproteins such as phycoerythrin, GFP (Green fluorescent Protein), RFP (Red fluorescent Protein), YFP (Yellow fluorescent Protein), Examples of the fluorescent protein include BFP (Blue fluorescent Protein).

Examples of the luminescent substance include acridinium and its derivatives, ruthenium complex compounds, and lophine. Further, as the ruthenium complex compound, those shown in Clin. Chem. 37 , 9, 1534-1539, 1991 that emit light electrochemically together with an electron donor are preferable.
Examples of the radioisotope include ³ H, ¹⁴ C, ³⁵ S, ³² P, ¹²⁵ I, ¹³¹ I, and the like.

  Examples of the polypeptide containing the tag sequence include FLAG peptide (FLAG tag, Asp Tyr Lys Asp Asp Asp Asp Asp Lys), polyhistidine (His tag, His His His His His), myc epitope peptide (myc tag, Glu Gln Lys Leu). Ile Ser Glu Glu Asp Leu), hemagglutinin epitope peptide (HA tag, Tyr Pro Tyr Asp Val Pro Asp Tyr Ala) and the like.

  The labeling of the antibody or the measurement target can be performed by a reaction that causes a covalent bond between the functional group of the antibody or the measurement target and the labeling substance through or without a linker. Examples of the functional group include a carboxy group, amino group, glycidyl group, sulfhydryl group, hydroxyl group, amide group, imino group, hydroxysuccinyl ester group, maleimide group, and isothiocyanate group. It is possible to cause a condensation reaction between these functional groups.

Examples of the bonding method without using a linker include a method using a carbodiimide compound such as EDC. In this case, it is also possible to use an active ester such as NHS or a derivative thereof. The condensation reaction between the isothiocyanate group and the amino group is preferable because it does not require other reagents and proceeds only by mixing under neutral to weakly alkaline conditions.
The linker may be any molecule as long as it can bind a labeling substance and an antibody via respective functional groups. A preferable embodiment has, for example, a first functional group capable of reacting with an amino acid residue of an antibody and a second functional group capable of reacting with a functional group of a side chain of a labeling substance in the same molecule. It is a molecule, and the first functional group and the second functional group are preferably different groups. Examples of the functional group of the linker include the functional groups described above.

As a method for chemically combining radioisotopes, there is a method described in literature (Antibody Immunoconj. Radiopharm., 3 , 60, 1990).

  When the labeling substance is a polypeptide such as an enzyme, avidin, a fluorescent protein, a phycobiliprotein, or a polypeptide containing a tag sequence, a known genetic recombination technique (Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring) According to Harbor Laboratory Press, 2001), an expression vector containing a DNA encoding a fusion protein of a labeling substance and an antibody can be produced, introduced into an appropriate host, and cultured by culturing the host. . The DNA encoding the fusion protein can be obtained by cloning the DNA encoding the antibody and the labeling substance by PCR or the like and ligating each DNA by a ligase reaction.

Examples of the labeling substances 1 and 2 used in the homogeneous method described in the measurement method 7 of (6) described above include labeling substances that interact with each other by binding to and approaching one measurement object. Examples of such a labeling substance include a fluorescent substance that causes fluorescence resonance energy transfer (FRET). FRET is a phenomenon in which the fluorescence energy generated when the first fluorescent material receives excitation light is used as the fluorescence energy of the adjacent second fluorescent material. Caused by close proximity. Examples of combinations of fluorescent substances that cause FRET include combinations in which there is an overlap between the spectrum of one fluorescence wavelength and the spectrum of the other excitation wavelength. Examples of the fluorescent substance include fluorescent proteins, low molecular organic fluorescent dyes, inorganic compounds, and the like. Examples of a combination of fluorescent proteins that cause FRET include a combination of CFP [yellow mutant of green fluorescent protein (GFP)] and YFP [cyan mutant of green fluorescent protein (GFP)]. Examples of the combination of the low-molecular organic fluorescent dyes include a combination of Cy3 and Cy5. Examples of the inorganic compound include quantum dot (Science, 281 , 2016-2018, 1998).

  In addition, as a combination of the labeling substances in the homogeneous method, a combination of an enzyme that generates bioluminescence resonance energy transfer (BRET) and a chemiluminescence and a fluorescent substance can also be mentioned. Examples of the combination of the enzyme that causes BRET and the fluorescent substance include a combination in which the spectrum of the emission wavelength generated by decomposing the substrate by the enzyme and the spectrum of the excitation wavelength of the fluorescent substance overlap. For example, a combination using Renilla luciferase (Rluc) as an enzyme, Deep Blue C (Deep Blue C, manufactured by Packard BioScience) as a substrate, GFP as a fluorescent substance, and the like can be mentioned. In this case, light having a wavelength of 395 nm is generated by decomposition of the substrate by Rluc, and when GFP comes close to Rluc, it is possible to detect fluorescence having a wavelength of 510 nm emitted by GFP by receiving the energy of this light.

  In addition, as a combination of the labeling substances in the homogeneous method, a combination of substances that cause enzyme activity when the labeling substance 1 and the labeling substance 2 are close to each other and bonded with a certain orientation can be mentioned. For example, a combination of labeling substances includes a combination using a Δα subunit of β-galactosidase as the labeling substance 1, a Δω subunit of β-galactosidase as the labeling substance 2, an N-terminal domain of Rluc as the labeling substance 1, and a labeling substance 2 And combinations using the C-terminal domain of Rluc.

(9) Reaction conditions The antigen-antibody reaction is preferably performed in an aqueous medium. As reaction temperature, 0-50 degreeC is mention | raise | lifted, for example, and 4-40 degreeC is preferable. The reaction time is preferably 5 minutes to 20 hours.

(10) Measurement of the amount of label As a method for measuring the amount of label of the immune complex, an appropriate one can be selected according to the labeling substance. That is, when the labeling substance is a coloring substance, that is, a substance that absorbs light of a certain wavelength, a spectrophotometer, a multiwell plate reader, or the like can be used. When the labeling substance is a fluorescent substance, a fluorometer, a fluorescent multiwell plate reader, or the like can be used. When the labeling substance is a luminescent substance, a luminescence photometer, a luminescent multiwell plate reader, or the like can be used. When the labeling substance is a radioisotope, the amount of radioisotope can be measured by measuring the radioactivity with a scintillation counter, a γ-well counter, or the like.

When the label is an enzyme, the amount of the label can be measured by reacting the enzyme substrate with the enzyme and measuring the produced substance.
When the enzyme is peroxidase, the amount of peroxidase can be measured by, for example, an absorbance method or a fluorescence method. As a method for measuring the amount of peroxidase by the absorbance method, for example, a peroxidase is reacted with a combination of its substrate hydrogen peroxide and an oxidative coloring chromogen, and the absorbance of the reaction solution is measured by a spectrophotometer, a multiwell plate reader, etc. The measurement method etc. are mention | raise | lifted. Examples of the oxidative coloring chromogen include a leuco chromogen and an oxidative coupling coloring chromogen.

  A leuco chromogen is a substance that is converted into a pigment by itself in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Specifically, tetramethylbenzidine, o-phenylenediamine, 10-N-carboxymethylcarbamoyl-3,7-bis (dimethylamino) -10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7- Bis (dimethylamino) -10H-phenothiazine (MCDP), N- (carboxymethylaminocarbonyl) -4,4′-bis (dimethylamino) diphenylamine sodium salt (DA-64), 4,4′-bis (dimethylamino) ) Diphenylamine, bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine (BCMA), and the like.

  The oxidative coupling chromogen is a substance that forms a dye by oxidative coupling of two compounds in the presence of a peroxide active substance such as hydrogen peroxide and peroxidase. Examples of the combination of the two compounds include a combination of a coupler and an aniline (Trinder reagent), a combination of a coupler and a phenol. Examples of the coupler include 4-aminoantipyrine (4-AA) and 3-methyl-2-benzothiazolinone hydrazine. Examples of anilines include N- (3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline (TOOS), N-ethyl-N- (2-hydroxy). -3-sulfopropyl) -3,5-dimethylaniline (MAOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS), N-ethyl-N- (3-sulfopropyl) -3-methylaniline (TOPS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N, N-dimethyl-3-methylaniline, N , N-di (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline, N-ethyl- -(3-sulfopropyl) aniline, N-ethyl-N- (3-sulfopropyl) -3,5-dimethoxyaniline, N- (3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N -(3-sulfopropyl) -3,5-dimethylaniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methoxyaniline, N-ethyl-N- (2-hydroxy-3- Sulfopropyl) aniline, N-ethyl-N- (3-methylphenyl) -N′-succinylethylenediamine (EMSE), N-ethyl-N- (3-methylphenyl) -N′-acetylethylenediamine, N-ethyl- N- (2-hydroxy-3-sulfopropyl) -4-fluoro-3,5-dimethoxyaniline (F-DAOS) and the like. Examples of phenols include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.

  As a method of measuring the amount of peroxidase by the fluorescence method, for example, the peroxidase is reacted with a combination of hydrogen peroxide and a fluorescent substance as a substrate thereof, and the intensity of fluorescence generated by a fluorometer, a fluorescent multiwell plate reader, or the like is measured. And the like. Examples of the fluorescent substance include 4-hydroxyphenylacetic acid, 3- (4-hydroxyphenyl) propionic acid, and coumarin.

As a method of measuring the amount of peroxidase by the luminescence method, for example, the peroxidase is reacted with a combination of hydrogen peroxide and a luminescent substance as a substrate, and the intensity of luminescence generated by a luminescence intensity meter or a luminescence multiwell plate reader is measured. And the like. Examples of the luminescent substance include a luminol compound and a lucigenin compound.
When the enzyme is alkaline phosphatase, the amount of alkaline phosphatase can be measured by, for example, a luminescence method. Examples of the method for measuring the amount of alkaline phosphatase by the luminescence method include a method in which alkaline phosphatase and its substrate are reacted and the luminescence intensity of the generated luminescence is measured with a luminescence intensity meter, a luminescence multiwell plate reader, or the like. Examples of alkaline phosphatase substrates include 3- (2′-spiroadamantane) -4-methoxy-4- (3′-phosphoryloxy) phenyl-1,2-dioxetane disodium salt (AMPPD), 2-chloro- 5- {4-methoxyspiro [1,2-dioxetane-3,2 ′-(5′-chloro) tricyclo [3.3.1.1 ^3,7 ] can] -4-yl} phenyl phosphate disodium Salt (CDP-Star ^™ ), 3- {4-methoxyspiro [1,2-dioxetane-3,2 ′-(5′-chloro) tricyclo [3.3.1.1 ^3,7 ] decane] -4 - yl} phenyl phosphate disodium salt ^(CSPD TM), [10- methyl -9 (10H) - acridinyl Louis den] phenoxymethyl phosphate disodium salt (Lumig n ^TM APS-5), and the like.

  When the enzyme is β-D-galactosidase, the amount of β-D-galactosidase can be measured by, for example, an absorbance method (colorimetric method), a luminescence method, or a fluorescence method. Examples of a method for measuring the amount of β-D-galactosidase by an absorbance method (colorimetric method) include o-nitrofel-β-D-galactopyranoside. As a method of measuring the amount of β-D-galactosidase by a luminescence method, for example, a method of reacting β-D-galactosidase and its substrate and measuring the luminescence intensity of the reaction solution with a luminescence intensity meter, a luminescence multiwell plate reader or the like Etc. Examples of the substrate of β-D-galactosidase include galacton-plus (Galacton-Plus, manufactured by Applied Biosystems) or a similar compound thereof. As a method of measuring the amount of β-D-galactosidase by a fluorescence method, for example, a method of reacting β-D-galactosidase and its substrate and measuring the fluorescence of the reaction solution with a fluorometer, a fluorescent multiwell plate reader or the like Etc. Examples of the substrate for β-D-galactosidase include 4-methylumbelliferyl-β-D-galactopyranoside.

  When the enzyme is luciferase, the amount of luciferase can be measured by, for example, a luminescence method. Examples of the method for measuring the amount of luciferase by the luminescence method include a method of reacting luciferase with its substrate and measuring the luminescence intensity of the reaction solution with a luminescence intensity meter, a luminescence multiwell plate reader, or the like. Examples of luciferase substrates include luciferin and coelenterazine.

  When the labeling substance is other than a fluorescent substance, luminescent substance, radioisotope and enzyme, a labeling substance labeled with a fluorescent substance, a luminescent substance, a radioisotope, an enzyme, etc., which is specifically bound to the labeling substance, A fluorescent substance, a luminescent substance, a radioisotope or a labeling substance that specifically binds to the labeling substance by binding to the labeling antibody or the labeling competing substance in the complex Detection can be performed using an enzyme in the same manner as described above. Examples of the substance that specifically binds to the labeling substance include an antibody that specifically binds the labeling substance, and when the labeling substance is biotin, avidin, streptavidin, and the like can be given. In addition, a substance that specifically binds to the labeling substance (an antibody that specifically binds to the labeling substance, avidin or streptavidin, etc.) is used after being bound to the labeling substance in the immune complex using an unlabeled substance, An antibody that binds to a substance that specifically binds to a labeling substance, such as an antibody that specifically binds to a constant region of an antibody or an antibody that specifically binds to avidin or streptavidin, a fluorescent substance, a luminescent substance, a radioisotope or Detection can also be carried out in the same manner as described above using a fluorescent substance, a luminescent substance, a radioisotope or an enzyme labeled with these antibodies after binding those labeled with an enzyme.

Antibodies used for these detections, antibodies that specifically bind to avidin or streptavidin and labeling substances, antibodies that specifically bind to the constant region of antibodies, antibodies that specifically bind to avidin or streptavidin, and polyclonal antibodies It may be a monoclonal antibody, or an antibody fragment from which Fc portions such as Fab, F (ab ′) ₂ obtained by pepsin treatment, and Fab ′ obtained by pepsin treatment-reduction treatment are removed.

(11) Quantification of measurement object In order to quantify the measurement object, a standard substance, that is, a solution of the measurement object having a known concentration is used. Preferably, a plurality of measurement object solutions having known concentrations are used. A calibration curve showing the relationship between the concentration of the measurement object and the measured value (the amount of information derived from the label), using the measurement object solution of this known concentration as the measurement sample, by the same method as the actual sample measurement. Create Next, the concentration of the measurement object in the sample can be determined by applying the measurement value obtained by the measurement of the sample to the calibration curve created previously.

(12) Aqueous medium and other coexisting materials In the immunoassay method of the present invention, examples of the aqueous medium for performing the antigen-antibody reaction of the measurement object include deionized water, distilled water, and buffer solution. Liquid is preferred. The buffer used for preparing the buffer solution is not particularly limited as long as it has a buffering capacity. For example, a lactic acid buffer, a citrate buffer, an acetate buffer, a succinate buffer, and phthalic acid having a pH of 1 to 11 Buffer, phosphate buffer, triethanolamine buffer, diethanolamine buffer, lysine buffer, barbitur buffer, imidazole buffer, malate buffer, oxalate buffer, glycine buffer, borate buffer, Examples thereof include carbonate buffer, glycine buffer, and Good buffer.

  Examples of the good buffer include 2-morpholinoethanesulfonic acid (MES) buffer, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (Bis-Tris) buffer, and tris (hydroxymethyl) aminomethane (Tris). Buffer, N- (2-acetamido) iminodiacetic acid (ADA) buffer, piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES) buffer, 2- [N- (2-acetamido) Amino] ethanesulfonic acid (ACES) buffer, 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) buffer, 2- [N, N-bis (2-hydroxyethyl) amino] ethanesulfonic acid (BES) buffer Agent, 3-morpholinopropanesulfonic acid (MOPS) buffer, 2- {N- [tris (hydroxymes L) Methyl] amino} ethanesulfonic acid (TES) buffer, N- (2-hydroxyethyl) -N ′-(2-sulfoethyl) piperazine (HEPES) buffer, 3- [N, N-bis (2- Hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO) buffer, 2-hydroxy-3-{[N-tris (hydroxymethyl) methyl] amino} propanesulfonic acid (TAPSO) buffer, piperazine-N, N′-bis (2-hydroxypropane-3-sulfonic acid) (POPSO) buffer, N- (2-hydroxyethyl) -N ′-(2-hydroxy-3-sulfopropyl) piperazine (HEPPSO) buffer, N- (2-hydroxyethyl) -N ′-(3-sulfopropyl) piperazine (EPPS) buffer, tricine [N-tris Hydroxymethyl) methylglycine] buffer, bicine [N, N-bis (2-hydroxyethyl) glycine] buffer, 3- [N-tris (hydroxymethyl) methyl] aminopropanesulfonic acid (TAPS) buffer, 2 -(N-cyclohexylamino) ethanesulfonic acid (CHES) buffer, 3- (N-cyclohexylamino) -2-hydroxypropanesulfonic acid (CAPSO) buffer, 3- (N-cyclohexylamino) propanesulfonic acid (CAPS) ) Buffering agent.

The concentration of the buffer solution is not particularly limited as long as it is suitable for measurement, but is preferably 0.001 to 2.0 mol / L, more preferably 0.005 to 1.0 mol / L, and 0.01 to 0.00. 1 mol / L is particularly preferable.
In the immunological quantification method of the present invention, metal ions, salts, saccharides, surfactants, preservatives, proteins, protein stabilizers and the like can coexist in the antigen-antibody reaction of the measurement object.

Examples of the metal ion include magnesium ion, manganese ion, zinc ion and the like.
Examples of the salts include sodium chloride and potassium chloride.
Examples of the saccharide include mannitol and sorbitol.
Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants, and nonionic surfactants are preferred. Examples of the nonionic surfactant include Tween 20 and Nonidet P-40. Examples of preservatives include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), Bioace, Procrine 300, Proxel GXL, and the like.

Examples of the protein include bovine serum albumin (BSA), fetal bovine serum (FBS), casein, block ace (Dainippon Pharmaceutical Co., Ltd.) and the like.
Examples of the protein stabilizer include peroxidase stabilizing buffer (Peroxidase Stabilizing Buffer, manufactured by DakoCytomation).

(13) Immunoassay reagent The immunoassay reagent of the present invention is an immunoassay reagent for immunoassay of a measurement object in a sample while suppressing the influence of hemoglobin. The cell lysate described in (3) above is used. It is characterized by containing. Examples of the immunoassay reagent of the present invention include an immunoassay reagent containing any of (i) to (iii) in addition to the cell lysate described in (3).
(I) a labeled antibody in which a label is bound to a first antibody that binds to a measurement object, and a second antibody that binds to the measurement object;
(Ii) a labeled competitor having a label bound to the competitor, and an antibody that binds to the analyte and the competitor,
(Iii) A labeled antibody in which a label is bound to a competitive substance, and an antibody that binds to an analyte and a competitive substance.

  The immunoassay reagent of the present invention can be used in the immunoassay method of the present invention. Examples of the cell lysate, the antibody that binds to the measurement object, the competitive substance, the labeled antibody that binds the label to the antibody that binds to the measurement object, and the labeled competitive substance used in the immunoassay reagent of the present invention include: Examples thereof include the above-mentioned cell lysate, antibody bound to the measurement target, competitive substance, labeled antibody bound to the antibody bound to the measurement target, and labeled competitive substance. Moreover, the immunoassay reagent of this invention contains the above-mentioned metal ion, salt, saccharides, surfactant, preservative, protein, protein stabilizer, etc. as needed.

  Further, a reagent such as a surfactant for dissolving blood cells in the sample described in (1), an aqueous medium and a coexisting substance of the reaction described in (12), a cleaning solution described in (6), The reagent used for the measurement of the amount of the described label and the standard substance described in (11) may be included. The cell lysate and the aqueous medium and coexisting substance of the reaction described in (12) may be used as separate reagents, but an aqueous medium containing the cell lysate and coexisting substance may be used as the specimen diluent.

Moreover, the reagent containing the cell lysate described in (3) above can be used as a reagent for suppressing the influence of hemoglobin on the immune reaction of the measurement object.
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, these do not limit the scope of the present invention at all.

Measurement of MxA protein by sandwich ELISA and suppression of hemoglobin effect by cell lysate [1] Preparation of anti-MxA protein monoclonal antibody As described below, two types of anti-human MxA protein monoclonal antibodies KM1124 with different epitopes (WO96 / 05230) ) And KM1135 (WO 96/05230). KM1124 is a mouse monoclonal antibody that binds to an epitope present in residues 220 to 297 from the amino terminus of human MxA protein, and KM1135 is a mouse monoclonal antibody that binds to an epitope present in residues 10 to 220 from the amino terminus of human MxA protein. is there.

Pristane-treated 8-week-old nude female mice (Balb / c) were each given 5 hybridoma strains KM1124 (FERM BP-4729) producing monoclonal antibody KM1124 and 5 hybridoma strains KM1135 (FERM BP-4731) producing monoclonal antibody KM1135. -20 × 10 ⁶ cells / mouse were injected intraperitoneally. After 10 to 21 days, the hybridoma strain became ascites tumor, and ascites was collected from the mice with ascites. The collected ascites was centrifuged at 3000 rpm for 5 minutes to remove solids, and the supernatant was collected. A monoclonal antibody purified from this supernatant by the caprylic acid precipitation method (Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) was used for the immunoassay of MxA protein.

[2] Preparation of recombinant MxA protein NdeI-BamHI fragment containing cDNA encoding human MxA protein was inserted between NdeI and BamHI of vector pET-14b [Novagen, EMD Biosciences] The Escherichia coli BL21 (DE3) pLysS strain was transformed with the human MxA protein expression vector pET14b-MxA (Nucleic Acids Res, 32 , 643-652, 2004) prepared as described above. This transformant expresses MxA protein with a His tag added to the N-terminus.

  The obtained transformant was inoculated into 5 mL of LB medium containing ampicillin and cultured with shaking at 37 ° C. until the absorbance at 600 nm (OD600) reached 0.5. This culture was inoculated into 250 mL of LB medium containing ampicillin and cultured with shaking at 37 ° C. until the absorbance at 600 nm reached 0.3 to 0.5. To this, isopropylthiogalactoside (IPTG) was added to a final concentration of 0.4 mmol / L, and further cultured with shaking at 37 ° C. for 2 hours to complete the culture. The culture was centrifuged at 3000 rpm for 10 minutes at 4 ° C. to recover the cells. The cells were stored at −80 ° C. until preparation of MxA protein.

  Since the MxA protein was present in an insoluble body (inclusion body) in the microbial cells, the microbial cells were thawed on ice and ice-cooled binding buffer (5 mmol / L imidazole, 0.5 mol / L sodium chloride, 20 mmol). / L Tris-HCl, pH 7.9) 20 mL was added and suspended. The bacterial cell suspension was sonicated 5 times for 30 seconds to disrupt the bacterial cells, and then centrifuged at 4 ° C. and 4000 rpm for 10 minutes. The supernatant was removed, and 20 mL of ice-cold binding buffer was added to the precipitate to suspend it, and sonication and centrifugation were again performed in the same manner. The supernatant was removed, and 20 mL of binding buffer containing 6 mol / L urea was added to the precipitate and suspended. Similarly, after ultrasonication, the mixture was allowed to stand on ice for 30 minutes to dissolve insoluble matter, and centrifuged at 4 ° C. and 10,000 rpm for 30 minutes. The supernatant was collected and filtered through a 0.45 nm Millipore filter.

  Add 0.5 mL of Ni-NTA His / Bind resin (Novagen, manufactured by EMD Biosciences) to the resulting solution, mix while rotating for 2 hours at 4 ° C., and bind the MxA protein to the resin via the His tag. I let you. The resin was collected by centrifugation at 3000 rpm for 2 minutes at 4 ° C. After adding 10 mL of a binding buffer containing 6 mol / L urea cooled on ice to the resin, the resin was centrifuged at 3000 rpm for 2 minutes at 4 ° C. to recover the resin. After repeating this washing operation again, 10 ml of an ice-cooled washing buffer (6 mol / L urea, 60 mmol / L imidazole, 0.5 mol / L sodium chloride, 20 mmol / L Tris-HCl, pH 7.9) was added to the resin. The resin was added and centrifuged at 3000 rpm for 2 minutes at 4 ° C. to recover the resin.

  Add 10 mL of ice-cold elution buffer (6 mol / L urea, 1 mol / L imidazole, 0.5 mol / L sodium chloride, 20 mmol / L Tris-HCl, pH 7.9) to the resin, and rotate at 4 ° C. for 2 hours. The MxA protein was eluted from the resin. The mixture was centrifuged at 3000 rpm for 2 minutes at 4 ° C., and the supernatant was collected and used as a standard MxA protein solution in the measurement of MxA protein.

[3] Preparation of anti-MxA protein antibody-immobilized plate The anti-MxA protein monoclonal antibody KM1135 prepared in [1] was diluted with PBS (pH 7.5) to 5 μg / mL, and a 96-well microtiter plate [Null The product was dispensed in a volume of 50 μL / well to Nalge Nunc International. After leaving overnight, the supernatant was removed by aspiration, 200 μL of pH 7.2 phosphate buffer containing 25% Block Ace (Dainippon Pharmaceutical Co., Ltd.) and 50 mmol / L sodium chloride was dispensed and allowed to stand at room temperature for 2 hours. Blocked. After removing the blocking solution, the plate was washed with PBS. What dried in the vacuum dryer overnight was used as an anti- MxA protein monoclonal antibody solid-phased plate.

[4] Preparation of peroxidase-labeled anti-MxA protein antibody The anti-MxA protein monoclonal antibody KM1124 prepared in [1] is bound to peroxidase (hereinafter abbreviated as POD) by the maleimide method as follows, and POD-labeled anti-MxA protein antibody Was made.

  First, the solvent of a solution containing 2 mg of KM1124 was replaced with 0.1 mol / L borate buffer (pH 8.0), and 0.086 mg of 2-iminothiolane hydrochloride (manufactured by Pierce) was added and stirred. Then, it was made to react at 30 degreeC for 30 minutes. Using a Sephadex G25 (Amersham Biosciences) column (diameter 1.5 cm × 30 cm) equilibrated with 0.1 mol / L phosphate buffer (pH 6.0), Unreacted 2-iminothiolane was removed, and sulfhydrylated KM1124 was recovered.

  On the other hand, 2.5 mg of POD (manufactured by Toyobo Co., Ltd., peroxidase IC) in a molar ratio of KM1124 was dissolved in 250 μL of 0.1 mol / L phosphate buffer (pH 7.0). This solution was heated at 30 ° C. for 5 minutes, and then 0.72 mg of N- (6-maleimidocaproyloxy) succinimide (EMCS, Dojin Chemical Research) dissolved in N, N-dimethylformamide (manufactured by Nacalai Tesque). (Made by Tosho Co., Ltd.) was added and stirred and reacted at 30 ° C. for 30 minutes. Using a Sephadex G25 column (diameter 1.5 cm x 30 cm) equilibrated with 0.1 mol / L phosphate buffer (pH 6.0), gel filtration of the solution after reaction was performed to remove unreacted EMCS. Then, maleimidated POD was recovered.

  The sulfhydrylated KM1124 solution obtained above and the maleimidated POD solution were mixed and reacted at 30 ° C. for 1 hour. After the reaction, gel filtration of the solution after the reaction is performed using a Sephacryl S-300 (Pharmacia) column (diameter 3 cm × 60 cm) equilibrated with 0.1 mol / L phosphate buffer (pH 7.0). Unreacted POD was removed and the labeled antibody was purified. The absorbance at 280 nm (A280) and the absorbance at 403 nm (A403) of each fraction of gel filtration were measured, and the number of moles of AKM from KM1124 and the number of moles of POD from A403 were calculated. The number of moles of POD / mol of KM1124 Fractions having a number of 1.5 or more were collected as fractions containing labeled antibody. The obtained labeled antibody was diluted 200 times with PBS (pH 7.5) and used.

[5] Construction of MxA protein measurement system by sandwich ELISA Buffer MxA protein solution prepared in [2] above [150 mmol / L sodium chloride, 0.1% BSA (manufactured by InterGen), 50 mmol / L Dilute with Tris-HCl, pH 7.2] to prepare MxA protein solutions of 0 (buffer only), 3.2, 6.3, 12.5, 25, 50, 100, 200 ng / mL. And used as a measurement sample.

  100 μL of a measurement sample was added to the anti-MxA protein antibody (KM1135) -immobilized plate prepared in [3] and incubated at room temperature for 1 hour to bind the MxA protein in the measurement sample to the antibody. After removing the measurement sample, a washing operation [400 μL of PBS containing 0.05% Tween 20 (manufactured by Kanto Chemical Co., Ltd.)] was added and removed five times. Next, 100 μL of the POD-labeled anti-MxA protein antibody (KM1124) prepared in [4] was added and reacted at room temperature for 30 minutes. The labeled antibody was removed, and a washing operation for removing 400 μL of the washing solution was performed 5 times. In a dark place, 100 μL of POD chromogenic substrate TMBlue (manufactured by Serological) containing 0.05% tetramethylbenzidine and hydrogen peroxide was added and allowed to react at room temperature for 10 minutes. The reaction was stopped by adding 100 μL of 0.5 mol / L sulfuric acid and incubating at room temperature for 10 minutes. Absorbance at a wavelength of 450 nm was measured with a plate reader. As a result, as the concentration of MxA protein in the measurement sample increased, the absorbance increased, indicating that MxA protein can be measured with high sensitivity.

[6] Preparation of cell lysate Suspension human Burkitt lymphoma cell line Raji cells (Lancet, 39 , 238-240, 1964, manufactured by Dainippon Pharmaceutical Co., Ltd.) were treated with 10% inactivated FBS, 1% Antibiotic-Antimycotic. [Invitrogen], RPMI 1640 medium containing 0.1% gentamicin (Invitrogen) [Sigma-Aldrich] 10 mL suspended to 1 × 10 ⁶ cells / mL I let you. The whole amount of the cell suspension was transferred to a 25 cm ² T flask, and suspended in a carbon dioxide culture apparatus (5% CO ₂ ) at 37 ° C. for 2 to 3 days. Thereafter, expansion culture was sequentially performed into a 150 cm ² T flask and a 225 cm ² T flask. When the cells became confluent in the 225 cm ² T flask, the culture solution containing the cells was collected in a sterilized container, and centrifuged at 1400 rpm for 5 minutes at 25 ° C. The supernatant was removed, suspended in a hypotonic buffer (10 mmol / L HEPES, 1.5 mmol / L MgCl ₂ , 10 mmol / L KCl) and collected to a cell number of 10 ⁷ cells / mL, and stored frozen. .

The solution containing 10 ⁷ cells / mL cultured cells obtained above was added to a cell lysis buffer (1% nonidet P40, 50 mmol / L Tris-HCl, pH 7.2) at a concentration of 5%. After incubating for 1 minute to lyse the cells, the mixture was centrifuged at 1400 rpm for 5 minutes at room temperature to precipitate insoluble components and collect the supernatant to obtain a cell lysate.
The protein content in the obtained cell lysate was measured using a protein assay kit by the Bradford method (manufactured by Bio-Rad). The protein content was serially diluted with 1 mg / mL BSA as a standard, the resulting absorbance and protein concentration were plotted, a calibration curve was prepared, and this was used to convert the concentration. The solution was stored at -80 ° C.

[7] Suppression of influence of hemoglobin in measurement of MxA protein by cell lysate The cell lysates of Raji cells prepared in [6] above were each at concentrations of 0, 0.63, 1.25, 2.5 and 5% ( Buffer concentrations (150 mmol / L sodium chloride, 0.1% BSA, 50 mmol / L Tris-) corresponding to protein concentrations of 0, 4.3, 9.3, 17.9, and 35.8 μg / mL, respectively. HCl, pH 7.2) was prepared and used as a specimen diluent.

  Four times the volume of PBS was added to the blood of 10 healthy MxA protein-negative individuals, centrifuged at 3000 rpm for 10 minutes, and the supernatant was removed. Four times the volume of PBS was added to the precipitated blood cells, suspended, centrifuged at 3000 rpm for 10 minutes, and the washing operation except the supernatant was repeated three times. The obtained washed blood cells were frozen at −30 ° C., thawed, and distilled water was added to burst red blood cells. The hemoglobin concentration of the obtained solution was measured with a hemoglobin measurement kit Hb Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.), and the hemoglobin concentration adjusted to 60 mg / mL with distilled water was used as a hemoglobin solution.

  Each solution in which a hemoglobin solution is added to a sample dilution solution containing cell lysate at each concentration so that the hemoglobin concentration is 30 mg / mL, 15 mg / mL, and 7.5 mg / mL, and each sample dilution solution to which no hemoglobin solution is added Then, the MxA protein solution prepared in [2] was diluted to a concentration of 50 ng / mL as a measurement sample, and a sandwich ELISA similar to [5] was performed to measure the absorbance.

  The influence of hemoglobin was examined by comparing the absorbance value of each measurement sample to which the hemoglobin solution was added with the absorbance value of the measurement sample to which no hemoglobin solution was added. As a result, as shown in FIG. 1, in the measurement sample to which the hemoglobin solution was added, the measurement value decreased as the concentration of hemoglobin increased compared to the measurement sample to which the hemoglobin solution was not added. It was shown to have an effect of lowering. Compared to the case where no cell lysate was added to the measurement sample, in the measurement sample to which the cell lysate was added, the decrease in the measurement value due to hemoglobin was suppressed as the concentration of the added cell lysate increased, and the cell It was shown that the influence of hemoglobin can be suppressed by adding a lysate.

Suppression of the effect of hemoglobin by cell lysate of T98G cells [1] Preparation of cell lysate of T98G cells Cell line T98G derived from an adherent human glioblastoma cell line (purchased from Dainippon Pharmaceutical Co., Ltd., J. Cell. Physiol. , 99 , 43-54, 1979) with 10% FBS, 1% non-essential amino acid (manufactured by Invitrogen), and 1 mmol / mL sodium pyruvate (manufactured by Invitrogen) Eagle MEM medium “Digo” (Nippon Pharmaceutical Co., Ltd.) The cells were cultured in a 6 cm petri dish for cell culture to which 10 mL was added using a carbon dioxide culture apparatus (5% CO ₂ , 37 ° C.) for 2 to 3 days until confluent. When the cells became confluent, the cells were transferred to a 15 cm petri dish and cultured in the same manner. When the cells became confluent in a 15 cm petri dish, the medium was removed by suction, washed with PBS, and washed with 0.02% EDTA. The cells were peeled from the petri dish by adding 0.25% trypsin solution, and then centrifuged (1400 rpm) at 25 ° C. for 5 minutes. The supernatant was removed, and a hypotonic buffer (10 mmol / L HEPES, 1.5 mmol / L MgCl ₂ , 10 mmol / L KCl) was added to suspend the cells and collect the solution.

  The solution containing the cultured cells obtained above was added to a cell lysis buffer (1% nonidet P40, 50 mmol / L Tris-HCl, pH 7.2) at a concentration of 5%, and incubated for 30 minutes. After the cells were lysed, the mixture was centrifuged at room temperature at 1400 rpm for 5 minutes to precipitate insoluble components and the supernatant was collected and used as a cell lysate. In the same manner as in Example 1 [6], the protein concentration of the cell lysate was measured and stored at −80 ° C.

[2] Suppression of influence of hemoglobin in MxA protein measurement by cell lysate of T98G cells The cell lysate of T98G cells prepared in [1] was adjusted to a protein concentration of 0.2, 0.4, 0.7, 1.4 mg / Samples diluted with buffer (150 mmol / L sodium chloride, 0.1% BSA, 50 mmol / L Tris-HCl, pH 7.2) and buffer without addition of cell lysate were prepared so that the volume was mL. A liquid was used. Each solution obtained by adding the 60 mg / mL hemoglobin solution prepared in Example 1 [7] to these specimen dilutions so that the hemoglobin concentration becomes 3.75, 7.5, 15, 30 mg / mL, and the hemoglobin solution Each sample diluted solution without addition of MxA protein solution prepared in Example 1 [2] was diluted to a concentration to obtain a measurement sample, and a sandwich ELISA similar to Example 1 [5] was performed to obtain the absorbance. Was measured.

  The influence of hemoglobin was examined by comparing the absorbance value of each measurement sample to which the hemoglobin solution was added with the absorbance value of the measurement sample to which no hemoglobin solution was added. As shown in FIG. 2, as in Example 1 [7], it was shown that hemoglobin has an effect of reducing the measured value. As in the case of the cell lysate of Raji cells in Example 1 [7], compared to the case where the cell lysate of T98G was not added to the measurement sample, the cell lysis added was added in the measurement sample to which the cell lysate of T98G was added. As the concentration of the substance increased, the decrease in the measured value due to hemoglobin was suppressed, and it was shown that the influence of hemoglobin can be suppressed by adding a cell lysate regardless of the cell type.

Measurement of MxA protein in whole blood The blood of 4 healthy subjects was collected, and each blood was divided into two, each of which was equivalent to 10% inactivated FBS, 1% Antibiotic-Antilytic, 0.1% RPMI1640 medium containing gentamicin is added, one induces the expression of MxA protein in leukocytes by adding interferon α at a concentration of 1000 U / mL and culturing for 24 hours, and the other for 24 hours without adding interferon α. Cultured. A 4-fold volume of a lysis solution [50 mmol / L Tris buffer (pH 7.2) containing 1% nonidet P40]] is added to the cultured whole blood sample to dissolve the hemocytes in the sample. The MxA protein in the solution was released into the solution, and a buffer solution (150 mmol / L sodium chloride, 0. 1%) was added to the specimen diluent [Raji cell lysate prepared in Example 1 [6] at a concentration of 5%. A sample diluted two-fold with 1% BSA, 50 mmol / L Tris-HCl, pH 7.2)] was used as a measurement sample. MxA protein in the measurement sample was measured in the same manner as in Example 1 [5].

  The MxA protein solution prepared in Example 1 [2] was diluted with the sample diluent, and 0 (sample diluent only), 3.2, 6.3, 12.5, 25, 50, 100, 200 ng / mL. Were prepared and measured in the same manner, and a calibration curve was prepared by plotting MxA protein concentration and absorbance. Based on the calibration curve, the MxA protein concentration in the measurement sample is determined from the absorbance of the measurement sample, and the dilution rate [dilution with the sample diluent (2 times) × dilution with the blood cell lysis buffer (5 times) = 10 times] Was multiplied to determine the MxA protein concentration in the whole blood sample. The quantitative results are shown in FIG. As shown in FIG. 3, MxA protein contained in healthy human blood stimulated with interferon α is 117.1 to 171.6 ng / mL, and MxA protein contained in healthy human blood not stimulated with interferon α is 0.8 to 9. It was determined to be 4 ng / mL.

Dilution Test of Whole Blood Specimen According to the method of Example 3, a dilution test of a clinical specimen positive for MxA protein was performed.
To the MxA protein-positive whole blood sample, 4 times the volume of hemocyte lysis buffer was added to lyse the hemocytes in the sample, and the MxA protein in the hemocytes was released into the solution. This was divided into two, one with a sample diluent (150 mmol / L sodium chloride, 0.1% BSA, 50 mmol / L Tris-HCl, pH 7.2) containing a cell lysate of Raji cells at a concentration of 5%. On the other hand, a sample dilution solution (PBS containing 0.1% BSA) containing no cell lysate, each diluted 2-fold, was used as a measurement sample. The sample diluted with the sample diluent containing the cell lysate was further diluted stepwise with the sample diluent containing the same cell lysate as 4/5, 3/5, 2/5, and 1/5. The measurement sample diluted with the other sample diluent without cell lysate is further diluted stepwise with 4/5, 3/5, 2/5, and 1/5 with the same sample diluent without cell lysate. did. The MxA protein was measured in the same manner as in Example 1 [5] for each of the measurement sample before dilution in stages and the measurement sample diluted in stages, and based on the calibration curve prepared in the same manner as in Example 3. The MxA protein was quantified. FIG. 4 shows the measurement results when diluted with a sample diluent containing cell lysate, and FIG. 5 shows the measurement results when diluted with a sample diluent without cell lysate. The value obtained by multiplying the measured value of the measurement sample before dilution (5/5) by the dilution rate). Table 1 shows the ratio (%) of the actual measured value to the theoretical value of the MxA protein concentration of each sample.

  As can be seen from FIGS. 4 and 5 and Table 1, in the dilution with the sample diluent not containing the cell lysate, the deviation rate between the theoretical value and the measured value increases as the dilution proceeds, and at the dilution rate of 1/5, the deviation occurs. The rate was + 20% or more. This was thought to be because the measurement was inhibited by hemoglobin in the sample at a dilution ratio of 5/5, and the inhibition was weakened by dilution of the sample. On the other hand, the specimen dilution solution containing cell lysate has good dilution linearity and the deviation rate is within ± 5%, and it is shown that the influence of hemoglobin can be suppressed by measuring in the presence of cell lysate. It was done.

MxA protein measurement kit A reagent kit for enzyme immunoassay of MxA protein comprising the following reagents was prepared.
(A) One anti-MxA protein antibody-immobilized plate In accordance with the method described in Example 1 [3], 5 μg / mL anti-MxA protein monoclonal antibody KM1135 in PBS (pH 7.5) was added in an amount of 50 μL / well. A 96-well microtiter plate, which was dispensed with and fixed, blocked with 25% Block Ace and dried.
(B) Hemocytolysis buffer Composition: 50 mmol / L Tris buffer (pH 7.2) containing 1% nonidet P40
Volume: 10 mL / bottle (c) Specimen diluent Composition: 5% Raji cell lysate (prepared according to the method described in Example 1 [2]), 150 mmol / L sodium chloride, 0.1% BSA, 50 mmol / L Tris-HCl (pH 7.2)
Volume: 30 mL / bottle (d) Labeled anti-MxA protein antibody POD-labeled anti-MxA protein monoclonal antibody KM1124 prepared according to the method described in Example 1 [4]. Dilute with labeled antibody diluent at the time of measurement.
(E) Labeled antibody diluent Composition: PBS (pH 7.5)
Capacity: 11 mL / bottle (f) Coloring substrate solution Composition: TMBlue (manufactured by Serological)
Capacity: 11 mL / bottle (g) Reaction stop solution Composition: 0.5 mol / L sulfuric acid Volume: 11 mL / bottle (h) Washing solution stock composition: 0.5% Tween 20, 10 mmol / 1.5 mol / L containing sodium chloride L phosphate buffer (pH 7.2)
Capacity: 100 mL / bottle Use 10 times diluted with distilled water when using.
(I) Standard substance MxA protein solution prepared in Example 1 [2] was adjusted to 1 μg / mL with a buffer [150 mmol / L sodium chloride, 0.1% BSA, 50 mmol / L Tris-HCl, pH 7.2]. Prepared solution

Suppression of the influence of hemoglobin by cell lysate in PAP measurement Using a PAP ELISA kit [manufactured by Dynabio], a PAP ELISA kit is used to determine whether the influence of hemoglobin on PAP measurement can be suppressed by cell lysate. And examined.
The cell lysate of Raji cells prepared in Example 1 [6] was added to the sample dilution solution contained in the PAP measurement kit so that the protein concentration was 0 (sample dilution solution only), 17.9, 35.8 μg / mL. did. Subsequently, the hemoglobin solution prepared in Example 1 [7] was added to these specimen dilutions so that the hemoglobin concentrations were 0.469, 0.938, 1.88, 3.75, 7.5, and 15 mg / mL. . A sample obtained by adding the recombinant pancreatic PAP antigen contained in the PAP measurement kit so as to be 1 ng / mL to each sample dilution solution to which these hemoglobin solutions were added and each sample dilution solution to which no hemoglobin solution was added was used as a measurement sample. Using the measurement kit, PAP in the measurement sample was measured. The measurement method was performed as follows according to the manual attached to the kit.

  100 μL of each measurement sample and standard solution was added to the wells of the anti-PAP antibody-immobilized 96-well plate of the kit, and incubated at room temperature for 3 hours to bind the PAP in the sample to the antibody. After removing the well solution, a washing operation for removing 400 μL of the washing solution was performed five times. Next, biotin-labeled anti-PAP antibody was added to the wells and incubated at room temperature for 30 minutes. The well solution was removed and washed 5 times as above. 100 μL of avidin-POD solution was added to the wells and incubated at room temperature for 15 minutes. The well solution was removed and washed 5 times as above. A coloring reagent was prepared by mixing the TMB (tetramethylbenzidine) solution of the kit and a hydrogen peroxide solution, and 100 μL was added to the well. After incubation in the dark for 10 to 15 minutes, 100 μL of the kit's orthophosphoric acid solution was added to stop the color reaction. Absorbance at a wavelength of 450 nm was measured with a plate reader. The results are shown in FIG.

  Similar to the MxA protein ELISA of Example 1, in the PAP measurement, the measurement value decreased as the hemoglobin concentration increased, indicating that hemoglobin has an effect of decreasing the measurement value. Compared to the case where the cell lysate is not added to the measurement sample, the measurement sample added with the cell lysate suppresses the decrease in the measured value due to hemoglobin, and the cell lysate is also added in the PAP immunoassay. It was shown that the influence of hemoglobin can be suppressed.

  The present invention provides a method for measuring a measurement object in a sample, which is useful in clinical diagnosis and in which the influence of hemoglobin is suppressed. By this measurement method, the measurement object in the sample containing hemoglobin can be accurately measured.

The suppression of the influence of hemoglobin in MxA protein measurement when adding cell lysates of Raji cells at various protein concentrations is shown. The horizontal axis represents the concentration of hemoglobin added, and the vertical axis represents the influence rate of absorbance with respect to the case where no hemoglobin is added. The suppression of the influence of hemoglobin in MxA protein measurement in the case of adding a cell lysate of T98G cells at each protein concentration is shown. The horizontal axis represents the concentration of hemoglobin added, and the vertical axis represents the influence rate of absorbance with respect to the case where no hemoglobin is added. The result of having measured the density | concentration of each MxA protein with respect to the sample of four types of whole blood when not stimulating with interferon alpha is shown. The measurement result of MxA protein when the sample of whole blood positive for MxA protein is diluted with a sample diluent containing cell lysate is shown. The measurement result of MxA protein when the sample of whole blood positive for MxA protein is diluted with a sample diluent not containing cell lysate is shown. The suppression of the influence of hemoglobin in PAP measurement when a cell lysate of Raji cells at each protein concentration is added is shown. The horizontal axis represents the concentration of hemoglobin added, and the vertical axis represents the influence rate of absorbance with respect to the case where no hemoglobin is added.

Claims (23)

A method for immunoassay of a measurement object, comprising reacting a measurement object in a sample and an antibody that binds to the measurement object in the presence of a cell lysate not derived from the sample. The method according to claim 1, wherein the immunoassay method is a sandwich method or a competitive method. A method for immunoassay of a measurement object, comprising reacting the measurement object in a sample with any one of (i) to (iii) in the presence of a cell lysate not derived from the sample.
(I) a labeled antibody in which a label is bound to a first antibody that binds to the measurement object, and a second antibody that binds to the measurement object;
(Ii) a labeled competitor with a label bound to the competitor, and an antibody that binds to the analyte and competitor,
(Iii) A competitive substance, and a labeled antibody that binds to the measurement target and the competitive substance and has a label bound thereto. Suppressing the influence of hemoglobin on the immune response of a measurement object, characterized by reacting the measurement object in a sample with an antibody that binds to the measurement object in the presence of a cell lysate not derived from the sample Method. The method according to any one of claims 1 to 4, wherein the sample is whole blood. 6. The method according to claim 5, wherein the whole blood is whole blood in which blood cells are lysed. The method according to claim 6, wherein the whole blood in which blood cells are dissolved is whole blood in which blood cells are dissolved with a surfactant. The method according to any one of claims 1 to 7, wherein the cell lysate is a cell lysate of animal cells. The method according to claim 8, wherein the animal cell is an animal cell line. The method according to claim 9, wherein the animal cell line is a Raji cell or a T98G cell. The method according to any one of claims 1 to 10, wherein the object to be measured is MxA protein or Pancreatitis associated protein. A reagent for suppressing the influence of hemoglobin on the reaction between a measurement object and an antibody that binds to the measurement object, comprising a cell lysate. An immunoassay reagent for a measurement object, comprising a cell lysate. The immunoassay reagent according to claim 13, further comprising any one of (i) to (iii).
(I) a labeled antibody in which a label is bound to a first antibody that binds to a measurement object, and a second antibody that binds to the measurement object;
(Ii) a labeled competitor having a label bound to the competitor, and an antibody that binds to the analyte and the competitor,
(Iii) A labeled antibody in which a label is bound to a competitive substance, and an antibody that binds to an analyte and a competitive substance. The immunoassay reagent according to claim 13 or 14, further comprising a reagent for dissolving blood cells in the sample. 16. The immunoassay reagent according to claim 15, wherein the reagent for dissolving blood cells is a surfactant. The immunoassay reagent according to claim 16, wherein the surfactant is a nonionic surfactant. The immunoassay reagent according to claim 17, wherein the nonionic surfactant is a polyoxyethylene surfactant. The immunoassay reagent according to claim 18, wherein the polyoxyethylene surfactant is polyoxyethylene alkylphenyl ether. The reagent according to any one of claims 12 to 19, wherein the cell lysate is a cell lysate of animal cells. The reagent according to claim 20, wherein the animal cell is an animal cell line. The reagent according to claim 21, wherein the animal cell line is a Raji cell or a T98G cell. The reagent according to any one of claims 12 to 22, wherein the object to be measured is MxA protein or Pancreatitis associated protein.

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