False high value inhibitor, immunoassay method using the same, and reagent kit containing the same

Abstract

To provide an inhibitor capable of reducing a false high value resulting from fibrin deposition in a specimen, an immunoassay method using the inhibitor, and a regent kit for measurement containing the inhibitor.SOLUTION: An immunoassay method is characterized by contacting a specimen containing a substance to be detected with a substance having affinity for fibrinogen (such as an anti-fibrinogen antibody) and, simultaneously or thereafter, with an antibody or antigen specific to the substance to be detected, thereby causing an immune reaction between the substance to be detected and the antibody or antigen specific thereto. A false high value inhibitor contains a substance having affinity for fibrinogen. A reagent kit contains the false high value inhibitor.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to an inhibitor that reduces false high values ​​caused by fibrin deposition in a specimen, an immunoassay using the same, and a reagent kit including the same. [Background technology]

[0002] One of the problems that frequently occurs in clinical testing is falsely high values ​​caused by fibrin precipitated in serum samples (Non-Patent Document 1).

[0003] Fibrin precipitation in serum occurs when serum is separated when blood coagulation is incomplete. Specifically, fibrin precipitation can occur when the blood is left for a short time before serum separation, when rapid clotting blood collection tubes are used and the mixture with the coagulation accelerator is insufficient, or when the serum is from a patient receiving anticoagulants (heparin, warfarin, etc.).

[0004] The mechanism by which falsely high values ​​occur due to fibrin precipitation in immunoassays is thought to be that minute fibrin (microfibrin) binds to the solid phase, causing poor B / F separation (a washing procedure to separate free components from the antigen-antibody complex) after the antigen-antibody reaction, resulting in nonspecific binding of labeled modified antibodies to the solid phase.

[0005] To prevent falsely high values ​​due to fibrin deposition, it is recommended to handle the blood collection tubes properly (enough inversions and clotting time) and re-centrifuge the sample according to the package insert. However, in recent years, there has been a demand for a faster turnaround time from blood collection to reporting of measurement results, and there is a demand for immunoassay methods that can provide accurate measurement values ​​even for serum samples that are incompletely coagulated and have fibrin deposition.

[0006] As a method for suppressing falsely high values ​​due to fibrin precipitation, a sample treatment solution containing a carrier carrying an active ester group and a method for removing contaminating proteins such as fibrin using the sample treatment solution have been disclosed (Patent Document 1). However, this method requires pretreatment by contacting the sample with the sample treatment solution prior to detection of the target substance, which poses a major problem in terms of ease of operation. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2022-35609 [Non-patent literature]

[0008] [Non-Patent Document 1] Midori Saito, "Non-specific reactions that are likely to be encountered in daily work and how to identify them," Biological Sample Analysis, NPO Society of Biological Sample Analysis Science, June 30, 2017, Vol. 40, No. 3, pp. 156-161 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to reduce the occurrence of falsely high values ​​due to fibrin deposition in a simpler manner. [Means for solving the problem]

[0010] As a result of intensive research conducted by the present inventors to solve the above problems, it was discovered that false high values ​​due to fibrin precipitation can be suppressed by contacting a specimen with a substance having affinity for fibrinogen and simultaneously or subsequently carrying out an immune reaction.The present inventors also discovered that false high values ​​due to fibrin precipitation can be reduced without pretreatment by using this substance as an inhibitor, and thus completed the present invention.

[0011] That is, the present invention includes the following aspects. [1] An immunoassay method characterized by contacting a sample containing a substance to be detected with a substance having affinity for fibrinogen, and simultaneously or thereafter contacting the sample with an antibody or antigen specific to the substance to be detected, thereby causing an immune reaction between the substance to be detected and the antibody or antigen specific to the substance. [2] The immunoassay method according to [1] above, wherein the substance having affinity for fibrinogen is an anti-fibrinogen antibody and / or a fibrinogen affinity peptide. [3] The immunoassay method according to [2] above, wherein the anti-fibrinogen antibody is a polyclonal antibody. [4] The immunoassay method according to [3] above, wherein the polyclonal antibody is a chicken polyclonal antibody and / or a rabbit polyclonal antibody. [5] The fibrinogen affinity peptide Contains any one of the following amino acid sequences (1) to (4): (1) the amino acid sequence set forth in SEQ ID NO: 1 (GPRVVERHQS); (2) an amino acid sequence having 70% or more identity to the amino acid sequence set forth in SEQ ID NO: 1; (3) an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 1; (4) An amino acid sequence in which another amino acid sequence is linked to the N-terminus and / or C-terminus of any of the amino acid sequences (1) to (3) above. and has fibrinogen binding activity, The immunoassay method according to [2] above. [6] A false high value suppressor containing a substance having affinity for fibrinogen, for use in the immunoassay method described in [1] above. [7] A reagent kit containing the false high value suppressor described in [6] above, for use in the immunoassay method described in [1] above.

[0012] The present invention will now be described in further detail.

[0013] [1] The immunoassay method of the present invention A first aspect of the present invention is an immunoassay method, which is characterized by contacting a specimen with a substance having affinity for fibrinogen and simultaneously or subsequently carrying out an immune reaction.

[0014] The specimen used in the present invention is not particularly limited, and examples thereof include serum, plasma, whole blood, urine, saliva, tears, ascites, peritoneal lavage fluid, cerebrospinal fluid, and cell or tissue extracts. Serum and plasma are preferred. The specimen may be appropriately diluted before use, and the dilution ratio is not particularly limited and can be appropriately selected, for example, within the range of undiluted to 1000-fold dilution, depending on the type and condition of the specimen used.

[0015] The animal species from which the specimen is derived is not particularly limited, and examples include humans, dogs, cats, rabbits, horses, and cows.

[0016] The substance to be detected in the present invention is not particularly limited as long as it is a substance that can be detected by immunoassay, and examples include proteins (including antibodies), peptides, nucleic acids, sugar chains, lipids, viruses, steroids, toxins, vitamins, drugs, and metabolites thereof.

[0017] In the present invention, a false high value refers to a measurement value obtained by a test that is higher than the true value, and in particular, it is preferable that the deviation from the true value is so large that it may cause a diagnostic error.

[0018] The criteria for determining false high values ​​can be set appropriately based on various conditions, such as the type of sample used, the substance to be detected, the sample volume, and the measurement method, and can be clinically significant. For example, if the ratio of the measured value to the true value is higher than a percentage set between 110% and 300%, the measured value can be determined to be false high. As another example, a cutoff value can be set to distinguish between negative and positive results, and if the measured value is higher than the cutoff value even though the true value is lower than the cutoff value, the measured value can be determined to be false high.

[0019] The substance having affinity for fibrinogen used in the present invention is not particularly limited, and examples thereof include an anti-fibrinogen antibody, a fibrinogen affinity peptide, and a fibrinogen affinity aptamer. These substances may be used alone or in combination of two or more types, and are not particularly limited. Preferably, they are anti-fibrinogen antibodies, or a mixture of an anti-fibrinogen antibody and a fibrinogen affinity peptide.

[0020] The above-mentioned anti-fibrinogen antibody will now be described in more detail.

[0021] The method for producing anti-fibrinogen antibodies is not particularly limited and may be appropriately selected from established methods. For example, they can be obtained by immunizing an animal with the full-length or partial region of fibrinogen as an immunogen. Another example is DNA immunization, in which an animal is immunized with an expression vector containing a cDNA encoding the amino acid sequence of the full-length or partial region of fibrinogen as an immunogen. Another example is by isolating antibodies that specifically bind to fibrinogen from a phage-display antibody library.

[0022] The animal species from which the fibrinogen used as the immunogen is derived is not particularly limited, and examples include humans, dogs, cats, rabbits, horses, cows, etc. It is preferable to use fibrinogen derived from the same animal species as the specimen to be measured, and for example, when measuring a human specimen, it is preferable to use human-derived fibrinogen as the immunogen.

[0023] The method for preparing fibrinogen to be used as the immunogen described above is not particularly limited, and examples include purification from plasma by the Cohn fractionation method, and production of full-length or partial regions of recombinant fibrinogen using yeast or animal cells into which an expression vector containing cDNA encoding the amino acid sequence of full-length or partial regions of fibrinogen has been introduced.

[0024] The immunized animal used to obtain the anti-fibrinogen antibody is not particularly limited as long as it has the ability to produce an antibody, and examples thereof include mice, rats, rabbits, chickens, goats, sheep, alpacas, dogs, cows, horses, cats, pigs, guinea pigs, hamsters, etc. Rabbits and chickens are preferred.

[0025] The anti-fibrinogen antibody may be a monoclonal antibody or a polyclonal antibody, preferably a polyclonal antibody.

[0026] The method for purifying the anti-fibrinogen antibody is not particularly limited and may be appropriately selected from established methods. For example, polyclonal antibodies can be purified by collecting serum from an immunized animal, concentrating it by ammonium sulfate precipitation, if necessary, and subjecting it to ion exchange chromatography, hydrophobic interaction chromatography, or affinity chromatography using a carrier immobilized with protein A, protein G, or protein L. As another example, chicken polyclonal antibodies can be purified by collecting egg yolk from eggs laid by immunized chickens, precipitating and removing lipids and lipoproteins by adding dextran sodium sulfate and calcium chloride, and then fractionating the yolk by ammonium sulfate precipitation.

[0027] The fibrinogen affinity peptides mentioned above will now be described in more detail.

[0028] The fibrinogen affinity peptide is not particularly limited, and examples thereof include a peptide containing the amino acid sequence GPRV (SEQ ID NO: 2) (Non-patent document: Proc Natl Acad Sci US A. 1978; 75(7): 3085-3089), a peptide consisting of the amino acid sequence EHIPA (SEQ ID NO: 3) (Non-patent document: Proc Soc Exp Biol Med. 1991; 198(1): 649-655), a peptide consisting of the amino acid sequence AHRPYAAC (SEQ ID NO: 4) (Non-patent document: Biomaterials 2015; 49: 27-36), a peptide consisting of the amino acid sequence CREKA (SEQ ID NO: 5) (Non-patent document: Proc Natl Acad Sci US A. 2007; 104(3): 932-936), and a cyclic peptide consisting of the amino acid sequence WECPYGLCWIQ (SEQ ID NO: 6) (Non-patent document: Bioconjugate Chem. 2012;23(3):548-556), and a peptide consisting of the amino acid sequence FLLVPL (SEQ ID NO: 7) (Non-Patent Document: Biotechnol Bioeng. 2002;77(3):278-289). A peptide containing the amino acid sequence GPRV (SEQ ID NO: 2) is preferred, and a peptide containing the amino acid sequence GPRVVERHQS (SEQ ID NO: 1) at positions 17-26 of the human fibrinogen Aα chain (UniProt Accession No. P02671) is more preferred.

[0029] The fibrinogen affinity peptides may be peptides that contain, in addition to the amino acid sequences of SEQ ID NOs: 1 to 7, an amino acid sequence that is, for example, 70% or more, preferably 80% or more, and more preferably 90% or more identical to these amino acid sequences, and that have fibrinogen-binding activity. Furthermore, any of the peptides may be peptides that contain an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added to the above sequence, and have fibrinogen-binding activity. Here, "several" preferably means 2 to 4, more preferably 2 to 3, and even more preferably 2. Furthermore, any of the peptides may be peptides that have fibrinogen-binding activity in which another amino acid sequence has been linked to either or both of the N-terminus and C-terminus of the amino acid sequence.

[0030] Examples of immunoassays in the present invention include the following: (A) a sandwich method, which uses two types of antibodies (one of which is labeled) with different epitopes that recognize the substance to be detected and allows a complex to form between the two antibodies and the substance to be detected; (B) a competitive method, which uses an antibody that recognizes the substance to be detected and a labeled antigen and utilizes the competitive binding of the labeled antigen and the substance to be detected contained in the sample to the antibody; and (C) a surface plasmon resonance method, which brings the sample into contact with a chip on which an antibody that recognizes the substance to be detected is immobilized and detects a change in refractive index as a signal that depends on the amount of the substance to be detected that binds to the antibody-immobilized chip. In particular, (A) or (B) are preferred in terms of simplicity and versatility.

[0031] The reagent for carrying out the above (A) sandwich method can be prepared, for example, by the following method.

[0032] (I) Of two types of antibodies (hereinafter referred to as "antibody 1" and "antibody 2") with different epitopes that recognize the substance to be detected, antibody 1 is bound to a carrier capable of B / F separation, such as an immunoplate or magnetic particles. The binding method may be physical binding using hydrophobic bonds, or chemical binding using a linker reagent that can crosslink the two substances.

[0033] (II) After binding the antibody 1 to the carrier, the surface of the carrier is blocked with bovine serum albumin, skim milk, casein, gelatin, a commercially available blocking agent for immunoassays, or the like to prevent non-specific binding, and the resulting product is used as a primary reagent.

[0034] (III) The antibody 2 is modified with a labeling substance, and a solution containing the resulting labeled and modified antibody is prepared as a secondary reagent. The labeling substance used to modify antibody 2 is preferably an enzyme such as peroxidase or alkaline phosphatase, a fluorescent substance, a chemiluminescent substance, a radioisotope, or another substance that can be detected by a detection device, or a substance that specifically binds to biotin, such as avidin. Furthermore, the solution used as the secondary reagent is preferably a buffer solution that allows for good antigen-antibody reactions, such as phosphate buffer or Tris buffer.

[0035] Furthermore, when preparing a reagent for carrying out the (B) competitive method, in step (III) of preparing a reagent for carrying out the sandwich method, instead of labeling and modifying antibody 2, the antigen can be labeled and modified to form a secondary reagent.

[0036] Measurement using the reagent prepared by the above method can be carried out by a two-step method or a one-step method.

[0037] The two-step method can be carried out in the following manner.

[0038] (IV) The primary reagent prepared in (II) is contacted with the sample for a certain period of time at a certain temperature to form an antigen-antibody complex. The reaction conditions are a temperature range of 4°C to 40°C and a time range of 1 minute to 180 minutes.

[0039] (V) Unreacted substances are removed by B / F separation, and then the mixture is contacted with the secondary reagent prepared in (III) above for a certain time at a certain temperature to form an antigen-antibody complex. The reaction conditions are a temperature range of 4°C to 40°C and a reaction time of 1 minute to 180 minutes.

[0040] (VI) Unreacted substances are removed by B / F separation, the labeled substance is quantified, and the concentration of the substance to be detected in the sample is quantified using a calibration curve prepared using a solution containing a known concentration of the substance to be detected as a standard.

[0041] The one-step method can also be carried out in the following manner.

[0042] (VII) The primary reagent prepared in (II), the secondary reagent prepared in (III), and the specimen are contacted for a certain time at a certain temperature to form an antigen-antibody complex. The reaction conditions are a temperature range of 4°C to 40°C and a reaction time of 1 minute to 180 minutes.

[0043] (VIII) Unreacted substances are removed by B / F separation, the labeled substance is quantified, and the concentration of the substance to be detected in the sample is quantified using a calibration curve prepared using a solution containing a known concentration of the substance to be detected as a standard.

[0044] The reagent can be used for manual measurement and for measurement using an automatic analyzer, and is particularly preferred for use in measurement using an automatic analyzer from the viewpoints of simplicity, speed, and accuracy.

[0045] In the present invention, the method of contacting a sample with a substance having affinity for fibrinogen and carrying out an immune reaction simultaneously with or thereafter is not particularly limited. For example, an inhibitor can be added when performing step (IV) of the measurement using the two-step method. As another example, an inhibitor can be added when performing step (VII) of the measurement using the one-step method. As yet another example, an inhibitor can be added in advance to the primary reagent and / or secondary reagent in steps (II) and / or (III) of preparing the reagent. Adding an inhibitor in advance to the primary reagent and / or secondary reagent is particularly preferred from the viewpoint of ease of measurement operations.

[0046] In the present invention, the concentration of the substance having affinity for fibrinogen that is brought into contact with the sample is not particularly limited, and may be added so that the final concentration is, for example, 0.01 mg / mL to 5 mg / mL, preferably 0.1 mg / mL to 0.5 mg / mL.

[0047] [2] The false high value suppressant of the present invention A second aspect of the present invention is a false high value suppressor containing a substance having affinity for fibrinogen. This false high value suppressor can be used in the immunoassay method of the present invention described above.

[0048] [3] Reagent kit of the present invention A third aspect of the present invention is a reagent kit, which contains the above-mentioned false high value suppressor and can be used in the above-mentioned immunoassay method of the present invention.

[0049] The reagent kit of the present invention may contain other reagents necessary for carrying out the immunoassay method of the present invention, as long as it contains the aforementioned false high value suppressor. It may also contain common reagents commonly used in the art. Examples include a washing solution for B / F separation, a substrate for detecting the labeled enzyme, a sample diluent, a standard for creating a calibration curve, a control sample for quality control, and a reagent for sample pretreatment. In addition, the kit may contain equipment such as a container for carrying out the antigen-antibody reaction, a sample cup for containing the sample, and an optical measurement cell. Furthermore, the kit may also contain instructions describing handling instructions, a barcode or two-dimensional code for viewing the electronic package insert, and the like.

[0050] The false high value suppressor of the present invention may be included in the kit as a single reagent, or may be included in the kit in a form in which it has been added to at least one of the above-mentioned reagents in advance. In particular, it is preferable that the false high value suppressor be included in the kit in a form in which it has been added to the reagents in advance from the viewpoint of ease of measurement operation.

[0051] The properties of the reagents contained in the kit of the present invention are not particularly limited, and examples include liquid reagents and freeze-dried reagents. In particular, freeze-dried reagents are preferred in terms of long-term storage stability. When the reagents are freeze-dried reagents, a dissolving solution for dissolving them before use may be included in the kit. [Effects of the Invention]

[0052] The present invention makes it possible to reduce false high values ​​caused by fibrin deposition in samples. This also provides an agent for suppressing false high values, which can suppress false high values ​​without pretreatment, and a reagent kit. [Brief explanation of the drawings]

[0053] [Figure 1] 1 is a graph showing the results of evaluating the affinity of antisera collected from chickens for fibrinogen in Example 2. [Figure 2] 1 is a chromatogram showing the results of an HPLC purity test of a chicken polyclonal antibody in Example 5. [Figure 3] 1 is a graph showing the results of evaluating the affinity of chicken polyclonal antibodies for fibrinogen in Example 6. [Figure 4] 10 is a graph showing the results of evaluating the affinity of rabbit polyclonal antibodies to fibrinogen in Example 9. [Example]

[0054] EXAMPLES In the following, examples are shown to specifically explain the present invention, but these examples are merely examples of the present invention and the present invention is not limited to these examples.

[0055] [Example 1] Immunization of chickens Chickens (Boris Brown, female, approximately 100 days old) were used for immunization. Human plasma-derived fibrinogen (Enzyme Research Laboratories, Inc., product number FIB3, concentration 13.75 mg / mL) was used as the immunogen. For the initial immunization, 2.0 mg of the immunogen emulsified with an equal volume of Freund's complete adjuvant (FCA) was injected subcutaneously per chicken. For the booster immunization, 2.0 mg of the immunogen emulsified with an equal volume of Freund's incomplete adjuvant (FIA) was injected subcutaneously per chicken. This was repeated every two weeks starting two weeks after the initial immunization. Test blood samples were taken from each chicken at 1 mL each time before the initial immunization and 21, 35, 49, and 63 days after the initial immunization. Antisera were prepared from the test blood samples using standard methods.

[0056] [Example 2] Antiserum titer test by ELISA The titer of the antiserum was evaluated by ELISA. The specific procedure is as follows. [1] Human plasma-derived fibrinogen (Enzyme Research Laboratories, Inc., product number FIB3) diluted to 1 μg / mL with phosphate-buffered saline (PBS) at pH 7.4 was dispensed into a black 96-well microplate (Greiner Bio-One GmbH, product number 655077) at 100 μL per well and left to stand at room temperature for 1 hour to allow immobilization. [2] Each well was washed three times with Tris-buffered saline (TBST) containing 0.05% Tween 20, pH 7.4, and 350 μL of blocking solution (Nacalai Tesque, product number 03953-66, product name: Blocking One) was added per well and allowed to stand at room temperature for 1 hour. [3] Each well was washed three times with TBST, and 100 μL of the sample (chicken antiserum) diluted with diluent (blocking solution diluted 10-fold with PBS) was dispensed into each well and left to stand at room temperature for 1 hour. [4] Each well was washed three times with TBST, and alkaline phosphatase-labeled anti-chicken IgY antibody (rabbit) (Jackson Immuno Research Laboratories, Inc., product number 303-055-003) diluted 10,000 times with diluent (blocking solution diluted 10 times with PBS) was dispensed at 100 μL per well, and the wells were left to stand at room temperature for 1 hour. [5] Each well was washed three times with TBST, and 100 μL of substrate solution (4-methylumbelliferyl phosphate) was dispensed into each well. The microplate was then shielded from light and allowed to stand at room temperature for 30 minutes. [6] The fluorescence intensity detected at a combination of an excitation wavelength of 360 nm and an emission wavelength of 465 nm was measured using a fluorescence microplate reader Spark (manufactured by Tecan Group Ltd.).

[0057] The evaluation results are shown in Figure 1. Antisera collected from chickens before sensitization showed no affinity for fibrinogen immobilized on a microplate. On the other hand, antisera collected after sensitization with the immunogen showed affinity for fibrinogen. Furthermore, the affinity of the antisera for fibrinogen improved with repeated sensitization.

[0058] [Example 3] Preparation of chicken polyclonal antibody Eggs laid by chickens 75 days or more after the initial immunization were collected. The eggshells were cracked, and the egg white was separated and removed to collect the egg yolk. The egg yolk was crushed with a spatula to disrupt the vitelline membrane, and then filtered through a 1 mm polyester mesh to separate and remove the vitelline membrane. The egg yolk liquid was collected. 1.5 mL of Tris-buffered saline (TBS) adjusted to pH 7.4 was added per gram of egg yolk liquid and stirred for 1 hour at room temperature. Next, 0.3 mL of 10% dextran sulfate sodium TBS solution was added per gram of egg yolk liquid and stirred for 10 minutes at room temperature. Next, 0.2 mL of 1 mol / L calcium chloride TBS solution was added per gram of egg yolk liquid and stirred for 10 minutes at room temperature, followed by standing for 1 hour. The reaction mixture was centrifuged (25°C, 8000 rpm, 15 minutes) to precipitate and remove lipids and lipoproteins, and the supernatant was collected. The supernatant was filtered through a paper filter (Advantec Toyo, product number 00131400), a 1.0 μm glass fiber filter (Merck Millipore, product number AP1504700), and a 0.45 μm membrane filter (Advantec Toyo, product number A045A047A) in that order to remove solids. To the filtrate, 0.245 g of ammonium sulfate per 1 mL of filtrate was added, stirred at room temperature for 10 minutes, and then allowed to stand for 1 hour for salting out. The reaction solution was centrifuged (25°C, 5000 rpm, 15 minutes) to separate and remove the supernatant, and the salting out precipitate was collected. To the salting out precipitate, 0.18 mL of TBS was added per 1 g of egg yolk liquid to suspend the precipitate. The suspension was transferred to a dialysis cellulose tube (Viskase Companies Inc., part number 521713, molecular weight cutoff 12,000-14,000) and dialyzed against 10 mL or more of TBS per mL (conditions: 4°C, 3 hours or more, with four external solution changes). The solution in the dialysis tube was collected and filtered through a 1.0 μm glass fiber filter (Merck Millipore, part number AP1504700) and a 0.45 μm membrane filter (Advantech Toyo, part number A045A047A) to remove solids, yielding a chicken polyclonal antibody solution. As a preservative, 0.9 mg of sodium azide was added per mL of chicken polyclonal antibody solution.

[0059] [Example 4] Evaluation of chicken polyclonal antibodies (1) Protein concentration Protein concentration was measured by absorbance. Specifically, the chicken polyclonal antibody solution was diluted approximately 100-fold with TBS adjusted to pH 7.4, transferred to a quartz cell with a 10 mm path length, and the absorbance at a wavelength of 280 nm was measured using a UV-visible spectrophotometer (Shimadzu Corporation, Model UV-1800). Protein concentration was calculated from the absorbance, assuming the extinction coefficient of chicken IgY at a wavelength of 280 nm to be 1.4.

[0060] The protein concentration of the chicken polyclonal antibody prepared in Example 3 was 39.36 mg / mL.

[0061] [Example 5] Evaluation of chicken polyclonal antibodies (2) HPLC purity test The high-performance liquid chromatograph used was an Agilent 1220 Infinity LC (Agilent Technologies, Inc.). The analytical column was a TSKgel SuperSW3000 (Tosoh, product number 0018675), the mobile phase was 50 mmol / L sodium phosphate + 150 mmol / L sodium sulfate aqueous solution (pH 6.5) containing 0.02% sodium azide, the flow rate was 0.35 mL / min, the column temperature was 25°C, and detection was by ultraviolet absorption (wavelength 280 nm). The sample used was a chicken polyclonal antibody solution diluted to approximately 0.5 mg / mL with Tris-buffered saline (TBS) adjusted to pH 7.4, and the injection volume was 100 μL. Purity was calculated by the area percentage method, using the ratio of the peak area of ​​chicken IgY (molecular weight approximately 180,000, retention time approximately 8 min) to the sum of the peak areas on the chromatogram.

[0062] 2 shows the chromatogram of the chicken polyclonal antibody prepared in Example 3. Chicken IgY appeared as a peak with a retention time of 7.985 min, and the purity calculated by the area percentage method was 75.59%.

[0063] [Example 6] Evaluation of chicken polyclonal antibodies (3) Titer test by ELISA The titer of the chicken polyclonal antibody was evaluated by ELISA. The specific procedure was the same as in Example 2.

[0064] The evaluation results are shown in Figure 3. The chicken polyclonal antibody showed affinity for fibrinogen, similar to the antiserum.

[0065] [Example 7] Immunization of rabbits Rabbits (Japanese white, female, 12 weeks old) were used as immunized animals. Human plasma-derived fibrinogen (Enzyme Research Laboratories, Inc., product number FIB3, concentration 13.75 mg / mL) was used as the immunogen. For the initial sensitization, 2.0 mg of the immunogen emulsified with an equal volume of Freund's complete adjuvant (FCA) was subcutaneously injected per rabbit. For booster sensitization, 2.0 mg of the immunogen emulsified with an equal volume of Freund's incomplete adjuvant (FIA) was subcutaneously injected per rabbit. This was performed three times, 14, 28, and 42 days after the initial sensitization. Seven days after the final sensitization, the rabbits were euthanized under anesthesia, exsanguinated, and antisera were prepared according to standard procedures.

[0066] [Example 8] Preparation of rabbit polyclonal antibody The rabbit polyclonal antibody was prepared by affinity purifying the antiserum prepared in Example 7. The specific procedure for affinity purification is described below. [1] An affinity column for antibody purification, SkillPak 5 AF-rProtein A HC-650F (manufactured by Tosoh Corporation, product number 0045258), was prepared. [2] The column was equilibrated by passing 10 column volumes (CV) of phosphate buffered saline (PBS) adjusted to pH 7.4 through the column at a flow rate of 2 mL / min. [3] The antiserum prepared in Example 7 was filtered through a 0.45 μm membrane filter (Advantec Toyo, product number A045A047A) to remove solid matter, and then passed through a column at a flow rate of 2 mL / min to adsorb the polyclonal antibody onto the column. [4] 10 CV of PBS was passed through the column at a flow rate of 2 mL / min to wash away non-adsorbed components. [5] Five CV of 100 mmol / L glycine hydrochloride buffer (pH 2.8) was passed through the column at a flow rate of 2 mL / min to elute the antibody adsorbed to the column, which was then collected. [6] The collected eluate was immediately neutralized by adding 0.1 mL of 1 mol / L Tris-HCl buffer (pH 9.0) per 1 mL of eluate to obtain rabbit polyclonal antibodies.

[0067] The rabbit polyclonal antibody obtained by affinity purification was dialyzed against PBS according to standard methods and filtered through a 0.45 μm membrane filter (Advantec Toyo, product number A045A047A) to remove solid matter. 0.9 mg of sodium azide was then added per 1 mL of antibody solution as a preservative.

[0068] [Example 9] Titer test of rabbit polyclonal antibodies by ELISA The titer of the rabbit polyclonal antibody was evaluated by ELISA. The specific procedure is as follows. [1] Human plasma-derived fibrinogen (Enzyme Research Laboratories, Inc., product number FIB3) diluted to 1 μg / mL with phosphate-buffered saline (PBS) at pH 7.4 was dispensed into a black 96-well microplate (Greiner Bio-One GmbH, product number 655077) at 100 μL per well and left to stand at room temperature for 1 hour to allow immobilization. [2] Each well was washed three times with Tris-buffered saline (TBST) containing 0.05% Tween 20, pH 7.4, and 350 μL of blocking solution (Nacalai Tesque, product number 03953-66, product name: Blocking One) was added per well and allowed to stand at room temperature for 1 hour.

[0069] [3] Each well was washed three times with TBST, and 100 μL of the sample (rabbit polyclonal antibody) diluted with diluent (blocking solution diluted 10-fold with PBS) was dispensed into each well and left to stand at room temperature for 1 hour. [4] Each well was washed three times with TBST, and 100 μL of alkaline phosphatase-labeled anti-rabbit IgG antibody (goat origin) (Merck KGaA, product number AP132A) diluted 10,000 times with diluent (blocking solution diluted 10 times with PBS) was dispensed into each well. The wells were then left to stand at room temperature for 1 hour. [5] Each well was washed three times with TBST, and 100 μL of substrate solution (4-methylumbelliferyl phosphate) was dispensed into each well. The microplate was then shielded from light and allowed to stand at room temperature for 30 minutes. [6] The fluorescence intensity detected at a combination of an excitation wavelength of 360 nm and an emission wavelength of 465 nm was measured using a fluorescence microplate reader Spark (manufactured by Tecan Group Ltd.).

[0070] The evaluation results are shown in Figure 4. The rabbit polyclonal antibody showed affinity for fibrinogen.

[0071] [Example 10] Preparation of fibrinogen-affinity peptide (Fib17-26 peptide) The Fib17-26 peptide, which is composed of the amino acid sequence GPRVVERHQS (SEQ ID NO: 1) at positions 17-26 of the human fibrinogen Aα chain, is a peptide containing the amino acid sequence (GPRV) (SEQ ID NO: 2) known to have affinity for fibrinogen (Non-patent document: Proc Natl Acad Sci USA. 1978;75(7):3085-3089). Synthesis and purification of the Fib17-26 peptide were contracted to Genscript Biotech Corporation.

[0072] [Example 11] Preparation of specimen The prepared samples are shown in Table 1, and the sample preparation methods are shown in Table 2. The preparation methods for each sample are explained below.

[0073] [Table 1]

[0074] [Table 2]

[0075] As shown in Table 1, serum was collected from 15 donors (donor IDs 1 to 15) under different sample preparation conditions A to F to prepare samples IDs 1-1 to 15-3. Specifically, serum was separated from the whole blood of each donor using the blood collection tubes, number of inversions immediately after collection, clotting time, and centrifugation conditions listed in Table 2. Under sample preparation conditions A, C, and E, serum separation was performed when blood coagulation was incomplete, resulting in fibrin precipitation. Under sample preparation conditions B and F, serum separation was performed when blood coagulation was complete, resulting in no fibrin precipitation. Under sample preparation condition D, fibrin-precipitated serum was centrifuged again to remove the fibrin.

[0076] Additionally, specimens ID16-1 and 16-2 were prepared using serum collected under appropriate conditions from each donor and sodium citrate plasma (collected using Terumo blood collection tubes VP-CA050K70). Specifically, specimen ID16-1 was prepared by pooling only the serum in which fibrin precipitation was not observed (specimen preparation condition G). Furthermore, specimen ID16-1 was prepared by adding 0.02 mL of sodium citrate plasma per 1 mL of serum specimen, followed by heating at 30°C for 10 minutes (specimen preparation condition H). In specimen ID16-2, fibrinogen contained in the sodium citrate plasma was converted to fibrin monomer, which then polymerized, resulting in fibrin precipitation.

[0077] [Example 12] Preparation of immunoreaction reagent for measuring thyroid-stimulating hormone (TSH) (1) A reagent container with two wells was prepared. A solution containing an alkaline phosphatase-labeled anti-TSH antibody was dispensed into one well of the reagent container. A solution containing magnetic microparticles immobilized with an anti-TSH antibody that recognizes a different epitope from the aforementioned anti-TSH antibody, and chicken polyclonal antibodies at different concentrations (0.1 mg / mL, 0.3 mg / mL, or 0.5 mg / mL) was dispensed into the other well of the reagent container. The solutions dispensed into each of the two wells of the reagent container were freeze-dried and then sealed with aluminum foil to prepare an immune reaction reagent for TSH measurement.

[0078] [Comparative Example 1] An immunoreaction reagent for measuring TSH was prepared in the same manner as in Example 12, except that no chicken polyclonal antibody was added.

[0079] [Example 13] Evaluation of the effect of suppressing false high values ​​in immunoreaction reagents for TSH measurement (1) The effect of suppressing false high values ​​was evaluated by the following method.

[0080] Using the immunoreaction reagents prepared in Example 12 and Comparative Example 1, fibrin-precipitated specimens were repeatedly measured within the same day, and the frequency of falsely high values ​​was counted.

[0081] The measuring devices used were the fully automated chemiluminescent enzyme immunoassay devices AIA-CL2400 (manufactured by Tosoh Corporation, manufacturing and sales notification number 13B3X90002000018) and AIA-CL1200 (manufactured by Tosoh Corporation, manufacturing and sales notification number 13B3X90002000020).

[0082] The criterion for determining a falsely high value was a value greater than 120% of the mean value (luminescence intensity, unit: cps) of the control sample (sample in which fibrin deposition was not observed) collected from the same donor.

[0083] The evaluation results are shown in Tables 3 to 6. Compared with the case where chicken polyclonal antibody was not added in the preparation of the immunoreaction reagent (Comparative Example 1, Table 3), the frequency of false high values ​​was reduced when chicken polyclonal antibody was added (Example 12, Tables 4 to 6), and the occurrence of false high values ​​was completely suppressed particularly when the concentration of chicken polyclonal antibody was 0.3 mg / mL or higher (Tables 5 and 6).

[0084] [Table 3]

[0085] [Table 4]

[0086] [Table 5]

[0087] [Table 6]

[0088] [Example 14] Preparation of immunoreaction reagent for measuring thyroid-stimulating hormone (TSH) (2) An immunoreaction reagent for measuring TSH was prepared in the same manner as in Example 12, except that rabbit polyclonal antibody was added at a concentration of 0.5 mg / mL instead of chicken polyclonal antibody.

[0089] [Example 15] Evaluation of the effect of suppressing false high values ​​in immunoreaction reagents for TSH measurement (2) Using the immune reaction reagents prepared in Examples 12, 14, and Comparative Example 1, fibrin precipitate samples prepared on a different day from Example 13 were repeatedly measured within the same day, and the frequency of false high values ​​was counted.

[0090] The measuring devices used were the fully automated chemiluminescent enzyme immunoassay devices AIA-CL2400 (manufactured by Tosoh Corporation, manufacturing and sales notification number 13B3X90002000018) and AIA-CL1200 (manufactured by Tosoh Corporation, manufacturing and sales notification number 13B3X90002000020).

[0091] The criterion for determining a falsely high value was a value greater than 120% of the mean value (luminescence intensity, unit: cps) of the control sample (sample in which fibrin deposition was not observed) collected from the same donor.

[0092] The evaluation results are shown in Tables 7 to 9. Compared to the case where neither chicken polyclonal antibody nor rabbit polyclonal antibody was added in the preparation of the immunoreaction reagent (Comparative Example 1, Table 7), the frequency of false high values ​​was reduced when either 0.5 mg / mL of chicken polyclonal antibody (Example 12, Table 8) or 0.5 mg / mL of rabbit polyclonal antibody (Example 14, Table 9) was added, and in particular, the occurrence of false high values ​​was completely suppressed in the case of 0.5 mg / mL of rabbit polyclonal antibody.

[0093] [Table 7]

[0094] [Table 8]

[0095] [Table 9]

[0096] [Example 16] Preparation of immunoreaction reagent for measuring hepatitis B virus surface antigen (HBsAg) A reagent container with two holes was prepared. A solution containing alkaline phosphatase-labeled anti-HBsAg antibody was dispensed into one hole of the reagent container. A solution containing magnetic microparticles immobilized with anti-HBsAg antibodies that recognize a different epitope from the aforementioned anti-HBsAg antibody, and chicken polyclonal antibodies at different concentrations, was dispensed into the other hole of the reagent container. The solutions dispensed into each of the two holes of the reagent container were freeze-dried and then sealed with aluminum foil to prepare an immune reaction reagent for HBsAg measurement.

[0097] Comparative Example 2 An immunoreaction reagent for measuring HBsAg was prepared in the same manner as in Example 16, except that chicken polyclonal antibody was not added.

[0098] [Example 17] Evaluation of the effect of suppressing false high values ​​on immunoreaction reagents for HBsAg measurement The effect of suppressing false high values ​​was evaluated by the following method.

[0099] Using the immunoreaction reagents prepared in Example 16 and Comparative Example 2, fibrin-precipitated specimens were repeatedly measured within the same day, and the frequency of falsely high values ​​was counted.

[0100] The measurement device used was a fully automated chemiluminescent enzyme immunoassay device AIA-CL2400 (manufactured by Tosoh Corporation, manufacturing and sales notification number 13B3X90002000018).

[0101] The criterion for falsely elevated values ​​was a measurement value of 0.030 international units (IU) / mL or higher. The maximum measurement value in a control sample (with no fibrin deposits) collected from the same donor was 0.013 IU / mL.

[0102] The evaluation results are shown in Tables 10 to 12. Compared with the case where chicken polyclonal antibody was not added in the preparation of the immunoreaction reagent (Comparative Example 2, Table 10), the frequency of false high values ​​was reduced when chicken polyclonal antibody was added (Example 16, Tables 11 and 12).

[0103] [Table 10]

[0104] [Table 11]

[0105] [Table 12]

[0106] [Example 18] Preparation of immunoreaction reagent for measuring cardiac troponin I (cTnI) A reagent container with two holes was prepared. A solution containing an alkaline phosphatase-labeled anti-cTnI antibody was dispensed into one hole of the reagent container. A solution containing magnetic microparticles immobilized with an anti-cTnI antibody that recognizes a different epitope from the aforementioned anti-cTnI antibody and a chicken polyclonal antibody at a concentration of 0.35 mg / mL was dispensed into the other hole of the reagent container. The solutions dispensed into each of the two holes of the reagent container were freeze-dried and then sealed with aluminum foil to prepare an immune reaction reagent for measuring cTnI.

[0107] Comparative Example 3 An immunoreaction reagent for measuring cTnI was prepared in the same manner as in Example 18, except that chicken polyclonal antibody was not added.

[0108] [Example 19] Evaluation of the effect of suppressing false high values ​​on immunoreaction reagents for cTnI measurement The effect of suppressing false high values ​​was evaluated by the following method.

[0109] Using the immunoreaction reagents prepared in Example 18 and Comparative Example 3, fibrin-precipitated specimens were repeatedly measured within the same day, and the frequency of falsely high values ​​was counted.

[0110] The measuring devices used were the fully automated chemiluminescent enzyme immunoassay devices AIA-CL2400 (manufactured by Tosoh Corporation, manufacturing and sales notification number 13B3X90002000018) and AIA-CL1200 (manufactured by Tosoh Corporation, manufacturing and sales notification number 13B3X90002000020).

[0111] The criterion for determining a falsely high value was a value greater than 150% of the mean value (luminescence intensity, unit: cps) of the control sample (sample in which fibrin deposition was not observed) collected from the same donor.

[0112] The evaluation results are shown in Tables 13 and 14. Compared with the case where chicken polyclonal antibody was not added in the preparation of the immunoreaction reagent (Comparative Example 3, Table 13), the frequency of false high values ​​was reduced when chicken polyclonal antibody was added (Example 18, Table 14).

[0113] [Table 13]

[0114] [Table 14]

[0115] [Example 20] Preparation of sample diluent for cardiac troponin I (cTnI) measurement The chicken polyclonal antibody and Fib17-26 peptide were added at different concentrations to phosphate buffered saline (PBS) adjusted to pH 7.4, and the resulting solution was used as a sample diluent for cTnI measurement.

[0116] Comparative Example 4 Phosphate buffered saline (PBS) adjusted to pH 7.4, which did not contain either the chicken polyclonal antibody or the Fib17-26 peptide, was used as the sample diluent for cTnI measurement.

[0117] Comparative Example 5 The sample diluent for cTnI measurement was prepared by adding 0.2 mg / mL of non-immunized chicken IgY antibody (manufactured by R&D Systems, Inc., product number AB-101-C) to phosphate-buffered saline (PBS) adjusted to pH 7.4. The antibody had no specific affinity for fibrinogen.

[0118] [Example 21] Evaluation of the effect of sample dilution solution for cTnI measurement on suppressing false high values The effect of suppressing false high values ​​was evaluated by the following method.

[0119] Using the specimen dilutions prepared in Example 20, Comparative Example 4, and Comparative Example 5, a fibrin precipitate specimen (specimen ID16-2 prepared by adding sodium citrate plasma to serum specimen ID16-1) was diluted two-fold and measured repeatedly within the same day, and the frequency of false high values ​​was counted.

[0120] The measurement device used was the fully automated chemiluminescent enzyme immunoassay AIA-CL2400 (manufactured by Tosoh Corporation, manufacturing and sales notification number 13B3X90002000018), and the immunoreaction reagent used was AIA-Pack CL Troponin I (manufactured by Tosoh Corporation, manufacturing and sales certification number 228ABEZX00051000).

[0121] The criterion for determining a falsely high value was a value greater than 150% of the measured value (luminescence intensity, unit: cps) of a control sample (serum sample ID16-1 in which fibrin deposition was not observed) diluted 2-fold with phosphate-buffered saline (PBS) adjusted to pH 7.4.

[0122] The evaluation results are shown in Tables 15 to 20. Compared with the case where no chicken polyclonal antibody was added in the preparation of the specimen dilution solution (Comparative Example 4, Table 15) and the case where an IgY antibody derived from non-immunized chickens was added (Comparative Example 5, Table 16), the frequency of false high values ​​was reduced when chicken polyclonal antibody and Fib17-26 peptide were added at a certain concentration or higher (Example 20, Tables 18 to 20). In particular, the occurrence of false high values ​​was completely suppressed when the chicken polyclonal antibody concentration was 0.2 mg / mL (Table 18) and when 0.1 mg / mL of chicken polyclonal antibody and 0.5 mg / mL of Fib17-26 peptide were added simultaneously (Table 20).

[0123] [Table 15]

[0124] [Table 16]

[0125] [Table 17]

[0126] [Table 18]

[0127] [Table 19]

[0128] [Table 20] [Industrial Applicability]

[0129] The present invention provides a suppressor that reduces false high values ​​caused by fibrin deposition in a sample, an immunoassay using the suppressor, and a reagent kit containing the suppressor. These are suitable for use in simple, rapid, and accurate immunoassays in clinical tests, and are therefore extremely useful industrially.

Claims

1. An immunoassay method characterized by contacting a specimen containing a substance to be detected with a substance having affinity for fibrinogen, and simultaneously or thereafter contacting the specimen with an antibody or antigen specific to the substance to be detected, thereby causing an immune reaction between the substance to be detected and the antibody or antigen specific to the substance.

2. 2. The immunoassay method according to claim 1, wherein the substance having affinity for fibrinogen is an anti-fibrinogen antibody and / or a fibrinogen affinity peptide.

3. The immunoassay method according to claim 2, wherein the anti-fibrinogen antibody is a polyclonal antibody.

4. The immunoassay method according to claim 3, wherein the polyclonal antibody is a chicken polyclonal antibody and / or a rabbit polyclonal antibody.

5. The fibrinogen affinity peptide - Contains any of the following amino acid sequences (1) to (4): (1) the amino acid sequence set forth in SEQ ID NO: 1 (GPRVVERHQS); (2) an amino acid sequence having 70% or more identity to the amino acid sequence set forth in SEQ ID NO: 1; (3) an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted and / or added to the amino acid sequence set forth in SEQ ID NO: 1; (4) An amino acid sequence in which another amino acid sequence is linked to the N-terminus and / or C-terminus of any of the amino acid sequences (1) to (3) above; - and has fibrinogen binding activity, The immunoassay method according to claim 2.

6. A false high value suppressant for use in the immunoassay method according to claim 1, comprising a substance having affinity for fibrinogen.

7. A reagent kit for use in the immunoassay method of claim 1, comprising the false high value suppressant of claim 6.

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