WO2019138898A1 - Éprouvette d'immunochromatographie, kit de mesure et procédé de mesure - Google Patents

Éprouvette d'immunochromatographie, kit de mesure et procédé de mesure Download PDF

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Publication number
WO2019138898A1
WO2019138898A1 PCT/JP2018/048024 JP2018048024W WO2019138898A1 WO 2019138898 A1 WO2019138898 A1 WO 2019138898A1 JP 2018048024 W JP2018048024 W JP 2018048024W WO 2019138898 A1 WO2019138898 A1 WO 2019138898A1
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Prior art keywords
antibody
hba1c
measurement
detection reagent
line detection
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PCT/JP2018/048024
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English (en)
Japanese (ja)
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岡本　淳
裕 川南
真希子 平岡
圭三 米田
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東洋紡株式会社
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Priority to JP2019564637A priority Critical patent/JP7352831B2/ja
Publication of WO2019138898A1 publication Critical patent/WO2019138898A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a method of measuring the ratio of hemoglobin A1c amount to hemoglobin amount in a measurement sample by immunochromatography: hemoglobin A1c (%), an immunochromatographic test strip used in the measuring method, and a kit including the immunochromatographic test strip. More specifically, the ratio of the amount of hemoglobin A1c to the amount of hemoglobin in the measurement sample without measuring the amount of hemoglobin A1c and the amount of hemoglobin in the measurement sample by the immunochromatography method: hemoglobin A1c (%)
  • the present invention relates to a measurement method of direct quantification from absorbance, an immunochromatographic test strip used in the measurement method, and a measurement kit and a measurement method including the immunochromatographic test strip.
  • Hemoglobin A1c which is one of the diagnostic items for diabetes, refers to hemoglobin (which may be abbreviated as Hb hereinafter) which plays a role of transporting oxygen in blood.
  • HbA1c (%) which is the ratio of the amount of HbA1c to the total Hb amount, refers to a substance in which the valine residue located on the N-terminal side of the ⁇ chain of Hb is glycated among bound glycated Hbs for the past 1 to 2 months It reflects the average blood glucose level of and is used to observe the long-term course of diabetes.
  • POCT is an abbreviation of Point Of Care Testing, and refers to a clinical test performed by a medical worker beside a subject. POCT is different from the clinical examination performed in the central laboratory of a large-scale hospital, etc., and the examination result can be obtained instantly on the spot, so POCT is being spread also in diabetes diagnosis.
  • the immunochromatography method is an immunoassay using capillary action, and is widely used in pregnancy test and influenza test worldwide.
  • visual judgment quantitative evaluation
  • a technology has been developed to quantify the amount of the analyte contained in the measurement sample using an analyzer such as an immunochromator reader. It is getting worse.
  • One of the methods for quantifying the amount of a substance to be analyzed using the immunochromatography method is a sandwich method using an antigen-antibody reaction.
  • sandwich method two kinds of antibodies having different epitopes for the substance to be analyzed are used.
  • One of the antibodies is used as a detection antibody sensitized with detection particles such as gold colloids, colored latex particles, and fluorescent particles.
  • detection particles such as gold colloids, colored latex particles, and fluorescent particles.
  • the other antibody forms a test line as a capture antibody immobilized linearly on the surface of the porous support.
  • the antibody that specifically captures the detection antibody is linearly immobilized on the surface of the porous support at a position different from the test line to form a control line.
  • the analyte contained in the measurement sample is developed from one end (upstream side) of the porous support, moves while forming an immune complex with the detection antibody, and is captured on the test line in contact with the capture antibody. Color.
  • the free detection reagent that did not form an immune complex with the analyte passes over the test line, and is captured by the antibody in the control line to develop color.
  • the amount of the substance to be analyzed can be quantified by using an apparatus such as an immunochromator reader for these color development intensities.
  • Patent Document 1 discloses a technique for measuring HbA1c (%) by immunochromatography for the purpose of improving storage stability.
  • HbA1c which is an analyte
  • Patent Document 2 discloses a measurement technique of HbA1c (%) that can stabilize the color development intensity of a control line without being affected by the concentration of a detection target substance in a biological sample.
  • gold colloid or latex particles are used as detection particles, it was not enough from a viewpoint of correlation with a sensitivity, accuracy, and an HPLC method.
  • Patent Documents 3 and 4 disclose a technique for measuring HbA1c (%) by immunochromatography for the purpose of developing a pretreatment method for exposing the N-terminus of the ⁇ chain, which is an epitope of HbA1c, to a protein surface. .
  • the invention is expected to have a certain effect in that HbA1c (%) can be directly quantified from the reflection absorbance (mAbs) of the test line.
  • mAbs reflection absorbance
  • the above-described invention is not sufficient in terms of accuracy because it does not have a control line.
  • gold colloid or latex particles are used as detection particles, it was not sufficient from the viewpoint of sensitivity, accuracy, and correlation with the HPLC method.
  • the anti-Hb antibody is used as a detection antibody and the anti-HbA1c antibody is used as a capture antibody, the Hb amount dependency can not be completely avoided.
  • the ratio of HbA1c amount to Hb amount in the measurement sample by immunochromatography method HbA1c (%), which has higher correlation with sensitivity, accuracy, and HPLC method than the prior art
  • An object of the present invention is to provide a method and a kit including an immunochromatographic test strip and an immunochromatographic test strip used in the measurement method. More specifically, the ratio of the amount of HbA1c in the measurement sample to the amount of Hb in the measurement sample without measuring the amount of HbA1c and the amount of Hb in the measurement sample by the immunochromatography method, which is less affected by the amount of Hb in the measurement sample than before.
  • An object of the present invention is to provide a measurement method of directly quantifying HbA1c (%) from the reflection absorbance of a line on a membrane, and a kit including an immunochromatographic test strip and an immunochromatographic test strip used in the measurement method.
  • the inventors of the present invention conducted intensive studies to solve the above problems, and as a result, as a test line detection reagent carried on a conjugation pad, an anti-Hb antibody or an anti-HbA1c antibody (detection antibody), cellulose-based colored microparticles (detection particles), And blocking protein (blocking agent), as a control line detection reagent carried on the conjugation pad, cellulose-based colored microparticles (detection particle), and blocking protein chemically labeled with a labeling substance (blocking agent)
  • an anti-Hb antibody or an anti-HbA1c antibody detection antibody
  • cellulose-based colored microparticles detection particles
  • blocking protein blocking protein
  • Blocking Peptide Fragment BPF for protein for blocking.
  • an anti-HbA1c antibody as an antibody (detection antibody) carried on the conjugation pad and an anti-Hb antibody as an antibody (capture antibody) immobilized linearly on a membrane, Hb in the measurement sample is better than in the prior art. The inventors have found that they are less susceptible to the amount and completed the present invention.
  • the representative invention is as follows. 1. (1) Sample pad, (2) a conjugation pad carrying a test line detection reagent and a control line detection reagent; (3) Lines of antibody A for specifically capturing hemoglobin or hemoglobin A1c in the measurement sample, and antibody B for specifically capturing the labeling substance in the control line detection reagent, at different positions Membrane fixed in the shape of (4) and an absorbent pad,
  • the test line detection reagent is a complex of an antibody C for capturing hemoglobin or hemoglobin A1c in the measurement sample, a cellulose-based colored fine particle, and a blocking protein
  • the immunochromatographic test strip, wherein the control line detection reagent is a complex of cellulose-based colored fine particles and a blocking protein chemically labeled with a labeling substance.
  • a measurement kit for quantifying the ratio of the amount of hemoglobin A1c to the amount of hemoglobin which comprises the immunochromatographic test strip according to any one of 1 to 7, the measurement sample dilution liquid, and the immunochromato reader. 9.
  • the immunochromatographic test strip of the present invention carries a specific antibody, detection particles, and a blocking protein in a specific configuration, it has high correlation with the high sensitivity, high accuracy, HPLC method, and Hb in the measurement sample.
  • the ratio of HbA1c amount to Hb amount in the measurement sample: HbA1c (%) can be measured without being affected by the amount.
  • the measurement sample used in the present invention is not particularly limited, and examples thereof include biological samples such as blood, lymph, spinal fluid, sweat, urine, tears, saliva, skin, mucous membrane, hair and the like.
  • biological samples such as blood, lymph, spinal fluid, sweat, urine, tears, saliva, skin, mucous membrane, hair and the like.
  • serum, blood cells or plasma obtained by centrifuging blood can be used as a sample.
  • the measurement sample is not limited to human origin, and biological samples derived from mammals such as dogs, cats and cattle are also targets.
  • the measurement item in the present invention is the ratio of the amount of HbA1c in the measurement sample to the amount of Hb: HbA1c (%).
  • the composition of the immunochromatographic test strip of the present invention is arranged in order of the sample pad having the addition portion, the conjugation pad, the membrane, and the absorption pad with the addition portion (dropping portion) of the measurement sample solution of the immunochromatographic test piece as the upstream side. ing.
  • 1 is a sample pad
  • 2 is a conjugation pad
  • 3 is a membrane
  • 4 is an absorbent pad
  • 5 is a backing sheet
  • 6 is a test line
  • 7 is a control line
  • 8 is an adhesive sheet.
  • the immunochromatographic test strip is in the form of an elongated strip having a width of 3 to 5 mm (preferably about 4 mm) and a length of 40 to 100 mm (preferably about 60 mm).
  • a test line 6 is formed at a position of about 15 mm from the upstream end of the immunochromatographic test strip at a position of about 15 mm, in which the antibody A for specifically capturing Hb or HbA1c in the measurement sample is linearly fixed.
  • a control line 7 is formed in which an antibody B for specifically capturing the labeling substance of the control line detection reagent is linearly fixed at a position of about 20 mm from the end.
  • a test line detection reagent which is a complex of the antibody C for capturing Hb or HbA1c in the measurement sample, a cellulose based colored fine particle and a blocking protein, and a cellulose based colored
  • a control line detection reagent which is a complex of a microparticle and a blocking protein chemically labeled with a labeling substance, is carried.
  • the material of the sample pad 1 used in the present invention is not particularly limited as long as the material can be developed to the downstream conjugation pad, membrane, and absorbent pad after absorbing the measurement sample quickly, for example, cellulose filter paper Or non-woven fabric, glass filter paper or non-woven fabric, polyester filter paper or non-woven fabric, polyethylene filter paper or non-woven fabric. Among these, cellulose filter paper is preferable.
  • the thickness of the sample pad 1 is preferably 0.1 to 2.0 mm, and more preferably 0.2 to 1.0 mm. If the thickness is small, the flow of the measurement sample downstream may be uneven and the measurement accuracy may be reduced. On the other hand, if the thickness is large, the downstream development may be delayed and the measurement time may be increased. In addition, the required amount of measurement sample required for downstream deployment is increased.
  • the material of the conjugation pad 2 used in the present invention can hold the test line detection reagent and the control line detection reagent in a dry state, and can rapidly release both the detection reagents with the downstream development of the measurement sample.
  • No particular limitation is imposed on the material, and examples thereof include filter paper or nonwoven fabric made of cellulose, filter paper or nonwoven fabric made of glass, filter paper or nonwoven fabric made of polyester, filter paper or nonwoven fabric made of polyethylene. Among these, glass filter paper is preferable.
  • the thickness of the conjugation pad 2 is preferably 0.1 to 2.0 mm, and more preferably 0.2 to 1.0 mm. If the thickness is thin, it may not be possible to keep the target amount of test line detection reagent and control line detection reagent dry. On the other hand, if the thickness is large, the downstream development may be delayed and the measurement time may be increased. In addition, the required amount of measurement sample required for downstream deployment is increased.
  • the material of the membrane 3 used in the present invention is not particularly limited as long as it can expand the measurement sample accurately and uniformly, but, for example, cellulose, cellulose derivative, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, polyvinyl chloride And polyvinylidene fluoride or nylon membranes. Among these, nitrocellulose membranes are preferred.
  • the material of the absorbent pad 4 used in the present invention is not particularly limited as long as the material can be retained so as not to cause backflow after rapidly absorbing the measurement sample developed from the upstream, for example, cellulose filter paper or nonwoven fabric Glass filter paper or nonwoven fabric, polyester filter paper or nonwoven fabric, polyethylene filter paper or nonwoven fabric can be mentioned. Among these, cellulose filter paper is preferable.
  • the thickness of the absorbent pad 4 is preferably 0.2 to 5.0 mm, and more preferably 0.5 to 2.0 mm. If the thickness is small, the measurement sample absorbed by the absorption pad may flow back to the membrane side depending on the amount of dropped measurement sample. On the other hand, when the thickness is large, the size of the immunochromatographic test piece and the housing case covering the immunochromatographic test piece also becomes large, which is not preferable from the viewpoint of POCT.
  • the test line detection reagent carried on the conjugation pad 2 used in the present invention is an antibody C for capturing Hb or HbA1c in the measurement sample as a detection antibody, cellulose-based colored fine particles as a detection particle, and a blocking agent Is a complex with a blocking protein of
  • the optimum blocking agent differs depending on the detection particles used and the target performance, so the effect of the amount of Hb in the measurement sample is highly correlated with the high sensitivity, high precision, and HPLC method, which is the object of the present invention.
  • HbA1c %
  • the antibody C is an anti-Hb antibody or an anti-HbA1c antibody, preferably an anti-HbA1c antibody.
  • the antibody C needs to be an anti-HbA1c antibody
  • the antibody A is an anti-HbA1c antibody
  • the antibody C needs to be an anti-Hb antibody.
  • HbA1c in the measurement sample can not be captured and detected, and measurement of HbA1c (%) is impossible.
  • the measurement result (correction value) largely varies depending on the amount of Hb, so it is necessary to measure the amount of Hb by another means . That is, HbA1c (%) can not be quantified directly from the reflection absorbance of the line on the membrane.
  • the anti-Hb antibody or anti-HbA1c antibody may be a commercially available product, or may be produced separately by a known method. In addition, it may be a monoclonal antibody or a polyclonal antibody, and the molecular size is not particularly limited.
  • the cellulose-based colored fine particles have a large amount of hydroxyl groups, they can not only retain many reactive dyes by covalent bonding, but also can maintain stable dispersibility in water and the like even after being concentrated.
  • As the cellulose-based colored fine particles regenerated cellulose, purified cellulose, natural cellulose or the like can be used, or partially derivatized cellulose may be used. It is preferable that 20 to 90 wt% of the mass of the cellulose-based colored fine particles is derived from cellulose, more preferably 20 to 80 wt%, and still more preferably 20 to 70 wt%.
  • the average particle size of the cellulose-based colored fine particles is not particularly limited, but is preferably 100 to 1000 nm, and more preferably 200 to 800 nm.
  • the average particle size is larger than 1000 nm, downstream development is delayed and measurement time is lengthened. In addition, it becomes easy to be captured on the membrane, and the background itself becomes colored, whereby the coloration in the test line and the control line becomes unclear.
  • the average particle size is small, the amount of antibody capable of physical adsorption or chemical binding may be reduced, and the measurement sensitivity may be reduced.
  • the color of the cellulose-based colored fine particles is not particularly limited, and examples thereof include red, blue, yellow, green, black, white and fluorescent colors. Among these, blue and black that are not easily affected by the background Hb-derived red are preferable, and blue is more preferable.
  • Examples of such cellulose-based colored fine particles include colored cellulose nanobeads (NanoAct (registered trademark)) manufactured by Asahi Kasei Co., Ltd., among which Navy (BL1), Dark Navy (BL2) and Black (KR1) are preferable, and Navy (BL1), Dark Navy (BL2) are more preferable.
  • the binding amount of the antibody C to the cellulose-based colored fine particles can be controlled by adjusting the preparation mass ratio of the cellulose-based colored microparticles and the antibody C and is not particularly limited, but the preparation mass of the cellulose-based colored microparticles and the antibody C
  • the ratio is preferably 1: 0.01 to 1: 1, more preferably 1: 0.02 to 1: 0.5, and still more preferably 1: 0.02 to 1: 0.2.
  • the mass ratio is out of the above range, the binding amount of the antibody C to the cellulose-based colored fine particles is insufficient, or the binding amount of the antibody C to the cellulose-based colored fine particles is excessively increased, and the antibody C does not contribute to the antigen-antibody reaction.
  • the measurement sensitivity may decrease due to the increase of
  • the blocking protein is not particularly limited, but preferably is a blocking peptide fragment of a microorganism-derived protein (hereinafter may be abbreviated as BPF), bovine serum albumin of an animal-derived protein (hereinafter sometimes abbreviated as BSA), casein And BPF, which is a microorganism-derived protein, is more preferable.
  • BPF about 22 kDa
  • BSA bovine serum albumin of an animal-derived protein
  • casein And BPF which is a microorganism-derived protein
  • BPF about 22 kDa
  • BSA bovine serum albumin of an animal-derived protein
  • casein And BPF which is a microorganism-derived protein
  • the antibody since it is necessary to use a borate buffer under alkaline conditions (pH 8.5 to 10) for dissolving casein, the antibody may be inactivated to lower the sensitivity. Borate buffers also have high environmental risks. On the other hand, it is preferable to dissolve BPF because tris in neutral conditions, phosphoric acid, PIPES, etc. can be used.
  • the blocking protein may be a commercially available product, or may be produced by a known method separately.
  • the molecular size is also not particularly limited, but preferably 100 kDa or less in average molecular weight. In general, the smaller the molecular size of the blocking protein, the more the binding amount of the blocking protein to the detection particle 1 particle increases, which contributes to the improvement of the measurement accuracy.
  • the method for binding the antibody C to the cellulose-based colored fine particles is not particularly limited, but it is preferable to sensitize by physical adsorption by hydrophobic bond or chemical bond by covalent bond, physical adsorption for easy operation and inexpensive cost. Is more preferred.
  • the reactive active group is not particularly limited, and examples thereof include a carboxyl group, an amino group, an aldehyde group, a thiol group, an epoxy group and a hydroxyl group. Among these, a carboxyl group and an amino group are preferable. In the case of a carboxyl group, carbodiimide can be used to form a covalent bond with the amino group of the ligand.
  • the control line detection reagent carried on the conjugation pad 2 used in the present invention is a complex of cellulose-based colored fine particles as detection particles and a blocking protein chemically labeled with a labeling substance as a label.
  • the optimum blocking agent differs depending on the detection particles used and the target performance, so the effect of the amount of Hb in the measurement sample is highly correlated with the high sensitivity, high precision, and HPLC method, which is the object of the present invention.
  • HbA1c %
  • the average particle size of the cellulose-based colored fine particles of the test line detection reagent and the average particle size of the cellulose-based colored fine particles of the control line detection reagent are preferably substantially the same.
  • substantially identical means that the average particle diameter is within ⁇ 50 nm. If the average particle sizes of the two are different, the developing speed to the downstream and the coloring intensity may differ, the correction of the test line by the control line may not function well, and the measurement accuracy may be lowered.
  • the color of the cellulose-based colored fine particles of the test line detection reagent and the color of the cellulose-based colored fine particles of the control line detection reagent are preferably substantially the same.
  • substantially identical means that the maximum absorption wavelength is within ⁇ 20 nm.
  • a test line and a control line A combination of a plurality of LED-PDs corresponding to the respective colors is required, and the size of the device is increased.
  • the label in the blocking protein chemically labeled with the labeling substance is not particularly limited, but is preferably biotin or digoxigenin because it is relatively inexpensive, easily available, and proven in the field of protein labeling and the like. More preferable.
  • the labeling method for the blocking protein chemically labeled with the labeling substance is not particularly limited as long as it forms a covalent bond by a chemical reaction, but an N-hydroxysuccinimide method can be exemplified.
  • N-hydroxysuccinimide method for example, the carboxyl group of biotin is condensed with an N-hydroxyamine compound in the presence of a dehydration condensation agent to be selectively activated, and via the amino group and the amide bond of the blocking protein. Can be labeled.
  • the N-hydroxyamine compound used for the condensation reaction is not particularly limited, and examples thereof include N-hydroxysuccinimide, N-hydroxynorbornene-2,3-dicarboximide, and 2-hydroxyimino-2-cyanoacetic acid ethyl ester.
  • N-hydroxysuccinimide hereinafter sometimes abbreviated as NHS
  • NHS N-hydroxysuccinimide
  • the dehydration condensation agent used for the condensation reaction is not particularly limited, and examples thereof include 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride, 1-cyclohexyl- (2-morpholinyl-4-ethyl) -carbodiimide, and meso p-Toluene sulfonate is mentioned.
  • 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride hereinafter sometimes abbreviated as EDC
  • EDC 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride
  • the reaction temperature is not particularly limited, but is preferably 10 to 50 ° C., and more preferably 20 to 40 ° C.
  • the reaction time varies depending on the reaction temperature, but is usually 5 minutes to 24 hours.
  • the unreacted N-hydroxyamine compound and the dehydrating agent contained in the reaction solution can be easily separated from the aqueous solvent by filtration, centrifugation or the like.
  • the blocking protein in the blocking protein chemically labeled with the labeling substance is not particularly limited, but may be a microorganism-derived protein Blocking Peptide Fragment (hereinafter sometimes abbreviated as BPF), an animal-derived protein bovine serum albumin (described below) , BSA (which may be abbreviated), casein is preferable, and BPF of a microorganism-derived protein is more preferable. Since BPF (about 22 kDa) has a smaller molecular weight than BSA (about 66 kDa), more sensitive measurement is possible. In addition, BSA has a risk of contamination due to foreign substances since it is bovine origin, but there is no such risk because BPF is microbial.
  • BPF microorganism-derived protein Blocking Peptide Fragment
  • BSA animal-derived protein bovine serum albumin
  • casein is preferable
  • BPF of a microorganism-derived protein is more preferable. Since BPF (about 22 kDa) has a smaller mo
  • the blocking protein may be a commercially available product, or may be produced by a known method separately.
  • the molecular size is also not particularly limited, but preferably 100 kDa or less in average molecular weight. In general, the smaller the molecular size of the blocking protein, the more the binding amount of the blocking protein to the detection particle 1 particle increases, which contributes to the improvement of the measurement sensitivity.
  • the blocking protein for the test line detection reagent and the blocking protein for the control line detection reagent are preferably substantially identical from the viewpoint of simplification of the production process.
  • the introduction amount of the label in the blocking protein chemically labeled with the labeling substance can be controlled by adjusting the molar ratio of the blocking protein to the preparation of the label (hereinafter sometimes referred to as introduction ratio), and is particularly limited.
  • the introduction ratio is preferably 1: 1 to 1: 100, more preferably 1: 1 to 1:50.
  • the introduction ratio is less than 1: 1, the introduction amount of the label may be insufficient, and the measurement sensitivity may be reduced.
  • the introduction ratio is greater than 1: 100, the amount of introduction of the label becomes excessive, and the correction of the test line by the control line does not function well due to nonspecific coloration in the control line, etc., and the measurement accuracy decreases. is there.
  • the binding amount of the blocking protein chemically labeled with the labeling substance to the cellulose-based colored fine particles is adjusted by adjusting the mass ratio of the cellulose-based colored microparticles and the blocking protein chemically labeled with the labeling substance.
  • the blocking protein chemically labeling with a labeling substance to the cellulose-based colored fine particles Is preferably present in large excess.
  • the mass ratio of the cellulose-based colored fine particles and the blocking protein chemically labeled with the labeling substance is preferably 1: 100 or more. That is, the control line detection reagent preferably does not contain a blocking protein which has not been chemically labeled with a labeling substance.
  • the test line detection reagent and the control line detection reagent are preferably loaded on the conjugation pad at a mixing ratio (mass ratio) of 2: 1 to 50: 1, more preferably 3: 1 to 30: 1, and 6 More preferably, it is from 1 to 15: 1. If the mixing ratio is out of the above range, the amount of test line detection reagent becomes relatively low, and the amount of control line detection reagent becomes relatively low, so that the correction of the test line by the control line does not work well. Measurement accuracy may decrease.
  • the method of loading the test line detection reagent and the control line detection reagent on the conjugation pad 2 is not particularly limited.
  • the mixed solution can be uniformly applied, sprayed or impregnated onto a conjugation pad and then dried at a suitable temperature for a given period of time in a thermostatic bath.
  • the application amount of the mixed solution is not particularly limited, but preferably 5 to 50 ⁇ L per 1 cm of line length.
  • the concentration of the cellulose-based colored fine particles (sensitized with blocking protein chemically labeled with antibody C or a labeling substance) in the mixed solution is not particularly limited, but is preferably 0.01 to 0.5 wt%, 0.02 to 0.2 wt% is more preferable, and 0.02 to 0.1 wt% is more preferable.
  • concentration is lower than 0.01 wt%, Hb and HbA1c can not be sufficiently captured and detected, and the measurement sensitivity may be lowered.
  • the concentration is higher than 0.5 wt%, the measurement sensitivity is not improved, and only the cost is increased.
  • the drying temperature is not particularly limited, but 20 ° C. to 80 ° C. is preferable, and 20 ° C. to 60 ° C. is more preferable.
  • the drying time varies depending on the drying temperature, but is usually 5 to 120 minutes.
  • the linearly immobilized capture antibody that forms the test line 6 on the membrane 3 used in the present invention is the antibody A for capturing Hb or HbA1c in the measurement sample.
  • the antibody A is an anti-Hb antibody or an anti-HbA1c antibody, preferably an anti-Hb antibody.
  • the antibody C is an anti-HbA1c antibody
  • the antibody A needs to be an antibody Hb antibody
  • the antibody C is an anti-Hb antibody
  • the antibody A needs to be an anti-HbA1c antibody.
  • HbA1c in the measurement sample can not be captured and detected, and measurement of HbA1c (%) is impossible.
  • the measurement result (correction value) largely varies depending on the amount of Hb, so it is necessary to measure the amount of Hb by another means . That is, HbA1c (%) can not be quantified directly from the reflection absorbance of the line on the membrane.
  • the anti-Hb antibody or anti-HbA1c antibody may be a commercially available product, or may be produced separately by a known method. In addition, it may be a monoclonal antibody or a polyclonal antibody, and the molecular size is not particularly limited.
  • the linearly immobilized capture antibody that forms the control line 7 on the membrane 3 used in the present invention is the antibody B for specifically capturing the labeling substance of the control line detection reagent.
  • the antibody B is an anti-biotin antibody when the labeling substance is biotin, and is an anti-digoxigenin antibody when the labeling substance is digoxigenin.
  • the anti-biotin antibody or anti-digoxigenin antibody may be a commercially available product, or may be produced separately by a known method. In addition, it may be a monoclonal antibody or a polyclonal antibody, and the molecular size is not particularly limited.
  • the method for immobilizing the capture antibody forming the test line and the capture antibody forming the control line on the membrane 3 is not particularly limited.
  • the capture antibody forming the test line and the control line are formed.
  • the capture antibody can be prepared by applying a fixed amount at different positions on each line and then drying in a thermostat at a suitable temperature for a fixed time.
  • the application amount of the two capture antibodies is not particularly limited, but 0.1 to 2 ⁇ L per 1 cm of line length is preferable.
  • the application concentration of the two capture antibodies is not particularly limited, but is preferably 0.1 to 10 mg / mL, more preferably 0.2 to 5 mg / mL, and still more preferably 0.5 to 2 mg / mL.
  • the drying temperature is not particularly limited, but 20 ° C. to 80 ° C. is preferable, and 20 ° C. to 60 ° C. is more preferable.
  • the drying time varies depending on the drying temperature, but is usually 5 to 120 minutes.
  • the prepared membrane 3 is attached near the center of the adhesive sheet 8 and then the conjugation pad 2 is partially overlapped and attached on one end of the membrane 3 and then the sample pad 1 Is applied partially on top of the opposite end of the conjugation pad 2 to the membrane 3 and then on the other end of the membrane 3 and then fixed. It can be produced by cutting into strips of width.
  • the test line 6 and the control line 7 may be prepared after producing the test piece, or may be prepared before producing the test piece.
  • the immunochromatographic assay kit preferably includes, in addition to the immunochromatographic test strip, an assay sample dilution solution for pre-processing and / or diluting the assay sample, and an immunochromator.
  • the measurement sample dilution solution preferably contains a nonionic surfactant that improves the spreadability of the measurement sample and does not affect the immune reaction.
  • the nonionic surfactant is not particularly limited, but polyoxyethylene alkyl phenyl ether (Triton (registered trademark) surfactant etc.), polyoxyethylene alkyl ether (Brij (registered trademark) surfactant etc.) And polyoxyethylene sorbitan fatty acid ester (Tween (registered trademark) surfactant and the like), polyoxyethylene fatty acid ester, sorbitan fatty acid ester, alkyl glucoside, sucrose fatty acid ester and the like.
  • the surfactants may be used alone or in combination of two or more.
  • the concentration of the nonionic surfactant is preferably 0.01 wt% to 5.0 wt%, more preferably 0.05 wt% to 4.0 wt%, and still more preferably 0.1 wt% to 3.0 wt%. If the concentration is low, downstream deployment may be difficult. In addition, the development may be uneven and the measurement accuracy may be reduced. On the other hand, if the concentration is high, the physically adsorbed detection particles and the antibody, and / or the membrane and the antibody may be separated, and measurement values may not be obtained.
  • the measurement sample dilution solution may be added with inorganic salts or a buffer used for pH adjustment.
  • a buffer used for pH adjustment.
  • any kind of buffer may be used as long as it has sufficient buffer capacity in the target pH range, for example, tris, phosphoric acid, phthalic acid, citric acid, maleic acid, Succinic acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, Good buffer (MES, ADA, PIPES, ACES, colamine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycinamide, bicine) can be mentioned.
  • Tris, phosphate, MES, PIPES, TES, HEPES are preferable, Tris, phosphate, and the like, because they have sufficient buffering ability around 7.0 which is the optimum pH range of the antibody used in the present invention.
  • the acid, PIPES is more preferred.
  • the dilution factor of the measurement sample by the measurement sample dilution liquid is not particularly limited, but 50 to 1000-fold dilution is preferable, and 100 to 500-fold dilution is more preferable. If the dilution factor of the measurement sample is low and the concentration is high, downstream development may be difficult. In addition, it may be easily influenced by contaminants in the measurement sample, and the measurement accuracy may be lowered. On the other hand, when the dilution factor of the measurement sample is high and the concentration is low, the concentrations of Hb and HbA1c in the measurement sample may decrease, and the measurement sensitivity may decrease.
  • the immunochromatographic test strip has at least a first opening for dropping a measurement sample on the sample pad 1, and a suitable second opening for measuring the test line 6 and the control line 7 on the membrane 3. It may be housed in a plastic housing case.
  • the method to measure HbA1c (%) on a membrane using the immunochromatographic test piece of this invention is not specifically limited, The following method can be illustrated. First, the measurement sample and the measurement sample dilution liquid are mixed at a predetermined dilution ratio to obtain a developable diluted measurement sample. Then, the diluted measurement sample is dropped onto the sample pad 1 so that the diluted measurement sample passes through the sample pad 1 and develops on the conjugation pad 2 by capillary action.
  • the diluted measurement sample was sensitized with the HbA1c in the diluted measurement sample and the anti-HbA1c antibody in the test line detection reagent while dissolving the test line detection reagent and the control line detection reagent carried on the conjugation pad 2 Cellulose-based colored fine particles form an immune complex.
  • Hb for example, HbA0 etc.
  • HbA0 etc. other than HbA1c in the diluted measurement sample does not form an immune complex with the test line detection reagent.
  • the diluted measurement sample is spread on the membrane 3.
  • the immunocomplex is captured and accumulated by a capture antibody (anti-Hb antibody) forming the test line, and the test line 6 is colored.
  • Hb other than HbA1c (for example, HbA0 etc.) in the dilution measurement sample is also captured and accumulated by the capture antibody (anti-Hb antibody) that forms the test line, so on the test line Hb other than the immune complex and HbA1c
  • the competitive capture with for example, HbA0 etc.
  • HbA1c (%) the probability of capture depends on the ratio of the amount of HbA1c to the amount of Hb in the measurement sample: HbA1c (%). That is, HbA1c (%) can be directly measured from the coloring intensity of the test line.
  • the cellulose colored fine particles sensitized with the blocking protein chemically labeled with the labeling substance (biotin) in the control line detection reagent form a control line. It is captured by (anti-biotin antibody) and accumulated, and the control line 7 develops color. Finally, the diluted measurement sample is absorbed by the absorption pad 4.
  • the HbA1c (%) may be measured directly from the color intensity of the test line, or in consideration of the flow spots of the diluted measurement sample, the color intensity of the test line is measured from the correction value divided by the color intensity of the control line. It is also good.
  • the measurement principle of HbA1c (%) using the immunochromatographic test strip of the present invention is based on competitive capture between the immunocomplex on the test line and Hb other than HbA1c (for example, HbA0 etc.) For this reason, the total amount of Hb in the diluted measurement sample needs to be sufficiently larger than the amount of capture antibody (anti-Hb antibody) forming the test line.
  • the molar ratio of total Hb in the measurement sample to the capture antibody (anti-Hb antibody) forming the test line is preferably 5: 1 to 2000: 1, and more preferably 10: 1 to 1500: 1. And 20: 1 to 1000: 1 are more preferable.
  • the measurement result (correction value) largely fluctuates depending on the amount of Hb, so it may be necessary to measure the amount of Hb by another means. That is, HbA1c (%) may not be quantified directly from the reflection absorbance of the line on the membrane.
  • the test line may be thinned and the measurement sensitivity may be reduced.
  • the measuring method of the test line and the control line of the immunochromatographic test strip of the present invention is not particularly limited, and a commercially available immunochromator may be used, or the immunochromator may be manufactured by a separately known method.
  • the detection system is not particularly limited, and, for example, LED-FD, LED-CMOS, and LED-CCD can be used.
  • HbA1c (%) described in the examples is all NGSP values.
  • Example 1 (1) Preparation of test line detection reagent for HbA1c measurement Anti-HbA1c monoclonal antibody (HbA1c Antibody, OAMA02329, manufactured by AVIVA SYSTEM BIOLOGY) with 8.57 mg / mL was diluted with distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical). It adjusted to 0 mg / mL.
  • cellulose-based colored fine particles NaIn (registered trademark), BL2: Dark Navy, average particle diameter 365 nm, manufactured by Asahi Kasei Corp.) 100 ⁇ L, 10 mM Tris buffer (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.) ) (PH 7.0) 900 ⁇ L and 100 ⁇ L of the above 1.0 mg / mL (0.1 wt%) anti-HbA1c monoclonal antibody were added to a 15 mL centrifuge tube and vortexed. Next, it was placed in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 37 ° C. and allowed to stand for 120 minutes.
  • a low temperature incubator IIR 604, manufactured by Yamato Scientific Co., Ltd.
  • a blocking solution consisting of 1.0 wt% of Blocking Peptide Fragment: BPF (BPF-301, manufactured by Toyobo Co., Ltd.) and 10 mM Tris buffer (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.)
  • BPF Blocking Peptide Fragment
  • Tris buffer 204-07885, manufactured by Wako Pure Chemical Industries, Ltd.
  • a washing solution consisting of 10 mM Tris buffer (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the resultant was treated with an ultrasonic disperser (UH-50, manufactured by SMT) for 10 seconds.
  • UH-50 manufactured by SMT
  • a centrifuge MX-307, made by Tomy Seiko Co., Ltd.
  • a rack-in rotor TMA-300, made by Tomy Seiko Co., Ltd.
  • AR510-04 made by Tomy Seiko Co., Ltd.
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride: EDC (15022-86, manufactured by Nacalai Tesque) 20 mg, N-hydroxysuccinimide: NHS (18948-02, manufactured by Nacalai Tesque) 20 mg, And 100 ⁇ L of distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical Co., Ltd.) were added to a 1.5 mL microtube and vortexed to obtain an EDC / NHS solution.
  • cellulose-based colored fine particles NaAct (registered trademark), BL2: Dark Navy, average particle diameter 365 nm, manufactured by Asahi Kasei Corp.) 100 ⁇ L, 10 mM Tris buffer (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.) ) (PH 7.0) and 100 ⁇ L of the D biotin-BPF solution were added to a 15 mL centrifuge tube and vortexed. Next, it was placed in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 37 ° C. and allowed to stand for 120 minutes.
  • a low temperature incubator I 604, manufactured by Yamato Scientific Co., Ltd.
  • a blocking solution consisting of 1.0 wt% of Blocking Peptide Fragment: BPF (BPF-301, manufactured by Toyobo Co., Ltd.) and 10 mM Tris buffer (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.)
  • BPF Blocking Peptide Fragment
  • Tris buffer 204-07885, manufactured by Wako Pure Chemical Industries, Ltd.
  • a washing solution consisting of 10 mM Tris buffer (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the resultant was treated with an ultrasonic disperser (UH-50, manufactured by SMT) for 10 seconds.
  • UH-50 manufactured by SMT
  • a centrifuge MX-307, made by Tomy Seiko Co., Ltd.
  • a rack-in rotor TMA-300, made by Tomy Seiko Co., Ltd.
  • AR510-04 made by Tomy Seiko Co., Ltd.
  • a 60 mm ⁇ 300 mm membrane card (Hi-Flow Plus 120 Membrane Cards) consisting of an adhesive tape portion of 20 mm ⁇ 300 mm on the upstream side, a membrane portion of 25 mm ⁇ 300 mm at the center, and an adhesive tape portion of 15 mm ⁇ 300 mm on the downstream side.
  • test line detection reagent for HbA1c measurement and the control line detection reagent for HbA1c measurement were mixed at a ratio of 6: 1 (mass ratio).
  • mixing may be performed at a volume ratio of 6: 1.
  • a 20 mm ⁇ 300 mm absorbent pad (CELLULOSE FIBER SAMPLE PADS, CFSP 002000, manufactured by Millipore) was attached to the downstream 15 mm ⁇ 300 mm adhesive tape portion of the membrane card for measuring HbA1c so as to overlap the membrane portion by 2 mm.
  • a guillotine-type cutting module (CM5000, manufactured by BIODOT) was used to cut into a strip having a width of 4 mm and a length of 63 mm to obtain an immunochromatographic test piece for HbA1c measurement.
  • HbA1c measurement performance evaluation sample QRM HbA1c 2007-1, manufactured by Jikken Medical Standard Materials Organization, L1 to L5 levels.
  • the total hemoglobin common reference standard substance JCCRM 9112, 3 levels of L, M, and H manufactured by Tokushu Medical Standard Substances Organization, which is a commercially available Hb standard substance
  • M 0.274 g / L
  • H 0.359 g / L
  • Diluted Hb samples (L, M, H) were obtained.
  • N 10 (in total, 20 of the immunochromatographic test strips for HbA1c measurement were used).
  • the sensitivity of the immunochromatographic test piece for HbA1c measurement of Example 1 was able to
  • the CV (L1, L4) (%) at N 10 of the corrected value (L1, L4) is calculated, and the average value of CV (L1) (%) and CV (L4) (%): CV (%) Is calculated, CV ⁇ 3% is 3 points (excellent), 3% ⁇ CV ⁇ 5% is 2 points (good), 5% ⁇ CV ⁇ 10% is 1 point (average), 10% ⁇ CV Was rated 0 (bad).
  • the reflective absorbance (mAbs) of the test line was evaluated as 2 points (less than 10 mAbs), 1 point (average) of 10 to 20 mAbs, and 0 point (bad) more than 20 mAbs.
  • the nonspecific adsorption of the immunochromatographic test strip for HbA1c measurement of Example 1 was confirmed at 2 points at reflection absorbance ⁇ 10 mAbs (below the measurement lower limit) of the test line, that is, blocking of the detection particles in the test line detection reagent was not a problem. .
  • Example 2 As an antibody used for preparation of a test line detection reagent for measuring HbA1c, an anti-Hb monoclonal antibody (HBA1 Antibody, OAMA02326, manufactured by AVIVA SYSTEM BIOLOGY) instead of an anti-HbA1c monoclonal antibody (HbA1c Antibody, OAMA02329, manufactured by AVIVA SYSTEM BIOLODY) is used.
  • HBA1 Antibody OAMA02326, manufactured by AVIVA SYSTEM BIOLOGY
  • HbA1c Antibody OAMA02329, manufactured by AVIVA SYSTEM BIOLODY
  • Anti-HbA1c monoclonal antibody HbA1c Antibody, OAMA02329, AVIVA SYSTEM BIOLODY
  • anti-Hb monoclonal antibody HBA1 Antibody, OAMA02326, AVIVA SYSTEM BIOLOGY
  • Example 3 As blocking protein (including labeled blocking protein) used for preparation of test line detection reagent for HbA1c measurement and control line detection reagent for HbA1c measurement, instead of Blocking Peptide Fragment: BPF (BPF-301, Toyobo Co., Ltd.) Bovine serum albumin: In the same manner as Example 1, except that BSA (A7906, manufactured by Sigma-Aldrich) (Example 3) and casein (030-01505, manufactured by Wako Pure Chemical Industries, Ltd.) (Example 4) were used. The immunochromatographic test piece for HbA1c measurement was produced and evaluated.
  • BPF labeled blocking protein
  • Bovine serum albumin Bovine serum albumin
  • test line detection reagent for HbA1c measurement and the control line detection reagent for HbA1c measurement are cellulose-based colored fine particles (NanoAct (registered trademark), BL2: Dark Navy, average particle diameter 365 nm , Instead of mixing in a ratio of 6: 1 in terms of mass conversion by Asahi Kasei Corporation 1: 1 (Example 5), 3: 1 (Example 6), 15: 1 (Example 7), 30: 1 (implementation) Example 8) An immunochromatographic test piece for measuring HbA1c was prepared and evaluated in the same manner as in Example 1 except that the mixture was mixed at a ratio of 60: 1 (Example 9). The obtained evaluation results are shown in Table 1.
  • Example 10 As a detection particle used for preparation of a test line detection reagent for HbA1c measurement and a control line detection reagent for HbA1c measurement, instead of cellulose-based colored fine particles (NanoAct®, BL2: Dark Navy, average particle diameter 365 nm, manufactured by Asahi Kasei Corp.) Cellulose-based colored fine particles (NanoAct (registered trademark), RE1: Red, average particle size 330 nm, manufactured by Asahi Kasei Corporation) (Example 10), cellulose-based colored fine particles (NanoAct (registered trademark), RE2, Dark Red, average particle size 340 nm, manufactured by Asahi Kasei Corp.
  • Example 11 and using an immunochromator reader (C10060-10, measurement mode: Gold C) instead of the immunochromator reader (C10060-10, measurement mode: Latex, Line, Hamamatsu Photonics) lloid, Line, except that was measured using a Hamamatsu Photonics Co., Ltd.) was evaluated to produce a HbA1c measurement immunochromatographic specimen in the same manner as in Example 1. The obtained evaluation results are shown in Table 1.
  • Example 12 As a detection particle used for preparation of a test line detection reagent for HbA1c measurement and a control line detection reagent for HbA1c measurement, instead of cellulose-based colored fine particles (NanoAct®, BL2: Dark Navy, average particle diameter 365 nm, manufactured by Asahi Kasei Corp.) An immunochromatographic test piece for measuring HbA1c was prepared and evaluated in the same manner as in Example 1 except that a cellulose-based colored fine particle (NanoAct (registered trademark), BL1: Navy, average particle size 325 nm, manufactured by Asahi Kasei Corp.) was used. The obtained evaluation results are shown in Table 1.
  • Example 13 As a detection particle to be used for preparation of a test line detection reagent for HbA1c measurement, a cellulose-based colored fine particle (NanoAct (NanoAct (registered trademark), BL2: Dark Navy, average particle diameter 365 nm, manufactured by Asahi Kasei Corp.) instead RE1: Red, average particle size 330 nm, manufactured by Asahi Kasei Corp., and used as an immunochromator reader (C10060-10, C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics Co., Ltd.).
  • Measurement mode An immunochromatographic test piece for HbA1c measurement was prepared and evaluated in the same manner as in Example 1 except that measurement was performed using Gold Colloid (Line, manufactured by Hamamatsu Photonics K.K.). The obtained evaluation results are shown in Table 1.
  • Example 14 In the preparation process of the dilution measurement sample, it is a commercially available HbA1c standard substance, HbA1c measurement performance evaluation sample (QRM HbA1c 2007-1, manufactured by Medicated Laboratory Medical Standards substance mechanism, five levels of L1 to L5) and a commercially available Hb standard substance Instead of diluting one total hemoglobin common reference standard substance (JCCRM 9112, 3 levels of L, M, H manufactured by Tokushu Medical Standards Corp., L, M, H) with the measurement sample dilution solution 500 times each, Example 14 An immunochromatographic test piece for HbA1c measurement was prepared and evaluated in the same manner as in Example 1 except that the dilution was made 100 times (Example 15), 1000 times (Example 16), 2000 times (Example 17). . The obtained evaluation results are shown in Table 1.
  • Anti-Hb monoclonal antibody for use in preparation of membrane card for HbA1c measurement is 1.0 mg / mL in distilled water (Otsuka distilled water, Otsuka Pharmaceutical)
  • 0.2 mg / mL Example 18
  • 0.5 mg / mL Example 19
  • 2.0 mg / mL Example 20
  • Anti-HbA1c monoclonal antibody for use in preparation of test line detection reagent for HbA1c measurement is 1.0 mg in distilled water (Otsuka distilled water, Otsuka Pharmaceutical) Instead of 0.2 mg / mL (the antibody concentration in the test line detection reagent for measuring HbA1c is 0.01 mg / mL instead of 0.2 mg / mL (the antibody concentration in the test line detection reagent for measuring HbA1c is adjusted to / mL) ) (Example 21), 0.4 mg / mL (antibody concentration in the test line detection reagent for measuring HbA1c is 0.02 mg / mL) (Example 22), 2.0 mg / mL (test line detection reagent for measuring HbA1 c) The concentration of antibody in the solution was adjusted to 0.1 mg / / mL
  • Test line detection reagent for HbA1c measurement Anti-HbA1c monoclonal antibody (HbA1c Antibody, OAMA02329, manufactured by AVIVA SYSTEM BIOLOGY) of 8.57 mg / mL was diluted with distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical). It adjusted to 05 mg / mL.
  • a blocking solution consisting of 10 wt% of bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) was added and lightly stirred.
  • BSA bovine serum albumin
  • a centrifuge MX-307, made by Tomy Seiko Co., Ltd.
  • TMA-300 made by Tomy Seiko Co., Ltd.
  • AR510-04 made by Tomy Seiko Co., Ltd.
  • bovine serum albumin BSA (A7906, manufactured by Sigma-Aldrich), 150 mM sodium chloride (192-13925, manufactured by Wako Pure Chemical Industries, Ltd.), 20 mM Tris buffer (204-07885, Japanese sum) 30 mL of a washing solution (pH 8.2) made of Kojun Chemical Industries, Ltd. was added, and the mixture was treated with an ultrasonic disperser (UH-50, manufactured by SMT Co.) for 10 seconds.
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride: EDC (15022-86, manufactured by Nacalai Tesque) 20 mg, N-hydroxysuccinimide: NHS (18948-02, manufactured by Nacalai Tesque) 20 mg, And 100 ⁇ L of distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical Co., Ltd.) were added to a 1.5 mL microtube and vortexed to obtain an EDC / NHS solution.
  • bovine serum albumin BSA (A7906, manufactured by Sigma-Aldrich), 150 mM sodium chloride (192-13925, manufactured by Wako Pure Chemical Industries, Ltd.), 20 mM Tris buffer (204-07885, Japanese sum) 30 mL of a washing solution (pH 8.2) made of Kojun Chemical Industries, Ltd. was added, and the mixture was treated with an ultrasonic disperser (UH-50, manufactured by SMT Co.) for 10 seconds.
  • Example 2 Thereafter, in the same manner as in Example 1, immunochromatographic test pieces for measuring HbA1c were produced. Then, the procedure was carried out except that an immunochromator reader (C10060-10, measurement mode: Gold Colloid, Line, Hamamatsu Photonics) was used instead of the immunochromator (C10060-10, measurement mode: Latex, Line, Hamamatsu Photonics). Evaluation was made in the same manner as in Example 1. The evaluation results obtained are shown in Table 2. When gold colloid particles were used for detection particles, sensitivity, accuracy and correlation were insufficient. In addition, blocking was insufficient and nonspecific adsorption was confirmed.
  • an immunochromator reader C10060-10, measurement mode: Gold Colloid, Line, Hamamatsu Photonics
  • C10060-10 measurement mode: Latex, Line, Hamamatsu Photonics
  • Blocking Peptide Fragment An immunochromatographic test piece for measuring HbA1c was prepared and evaluated in the same manner as in Comparative Example 1 except that BPF (BPF-301, manufactured by Toyobo Co., Ltd.) was used. The evaluation results obtained are shown in Table 2. When gold colloid particles were used for detection particles, sensitivity, accuracy and correlation were insufficient.
  • test line detection reagent for HbA1c measurement Anti-HbA1c monoclonal antibody (HbA1c Antibody, OAMA02329, manufactured by AVIVA SYSTEM BIOLOGY) of 8.57 mg / mL with 50 mM potassium dihydrogen phosphate (166-04255, Wako Pure Chemical Industries, Ltd.
  • a washing solution consisting of 1.0 wt% of bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) and 50 mM of potassium dihydrogen phosphate (166-04255, manufactured by Wako Pure Chemical Industries, Ltd.) And treated with an ultrasonic disperser (UH-50, manufactured by SMT Co., Ltd.) for 10 seconds.
  • BSA bovine serum albumin
  • UH-50 ultrasonic disperser
  • a coating solution consisting of 1.0 wt% of bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) and 50 mM potassium dihydrogen phosphate (166-04255, manufactured by Wako Pure Chemical Industries, Ltd.) 2 mL was added, and the mixture was treated with an ultrasonic disperser (UH-50, manufactured by SMT Co., Ltd.) for 10 seconds to obtain a test line detection reagent for HbA1c measurement.
  • the detection particle concentration in the test line detection reagent for HbA1c measurement was 0.5 mg / mL (0.05 wt%), and the antibody concentration was 0.05 mg / mL (0.005 wt%).
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride: EDC (15022-86, manufactured by Nacalai Tesque) 20 mg, N-hydroxysuccinimide: NHS (18948-02, manufactured by Nacalai Tesque) 20 mg, And 100 ⁇ L of distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical Co., Ltd.) were added to a 1.5 mL microtube and vortexed to obtain an EDC / NHS solution.
  • a washing solution consisting of 1.0 wt% of bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) and 50 mM of potassium dihydrogen phosphate (166-04255, manufactured by Wako Pure Chemical Industries, Ltd.) And treated with an ultrasonic disperser (UH-50, manufactured by SMT Co., Ltd.) for 10 seconds.
  • BSA bovine serum albumin
  • UH-50 ultrasonic disperser
  • a coating solution consisting of 1.0 wt% of bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) and 50 mM potassium dihydrogen phosphate (166-04255, manufactured by Wako Pure Chemical Industries, Ltd.) 2 mL was added, and the mixture was treated with an ultrasonic disperser (UH-50, manufactured by SMT Co., Ltd.) for 10 seconds to obtain a control line detection reagent for HbA1c measurement.
  • the detection particle concentration in the control line detection reagent for measuring HbA1c was 0.5 mg / mL (0.05 wt%), and the labeling ratio of blocking protein was 1: 7.
  • Blocking Peptide Fragment An immunochromatographic test piece for HbA1c measurement was prepared and evaluated in the same manner as in Comparative Example 3 except that BPF (BPF-301, manufactured by Toyobo Co., Ltd.) was used. The evaluation results obtained are shown in Table 2. When latex particles were used for the detection particles, the sensitivity, accuracy and correlation were insufficient.
  • Comparative example 5 An immunochromatographic test piece for HbA1c measurement was prepared and evaluated in the same manner as in Comparative Example 4 except that the test line detection reagent for HbA1c measurement of Example 1 was used instead of the test line detection reagent for HbA1c measurement of Comparative Example 3. The evaluation results obtained are shown in Table 2. The use of latex particles only for the detection particles of the test line detection reagent resulted in insufficient sensitivity, accuracy and correlation.
  • the measurement result (correction value) largely varies depending on the amount of Hb, so it was found that the amount of Hb needs to be measured by another means. That is, HbA1c (%) could not be quantified directly from the reflection absorbance of the line on the membrane.
  • the ratio of the amount of HbA1c in the measurement sample to the amount of Hb: HbA1c (%) with high sensitivity, high accuracy, high correlation with the HPLC method and without the influence of the amount of Hb in the measurement sample It is possible to provide an immunochromatographic test strip that can be used.

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Abstract

La présente invention aborde le problème de la réalisation d'une éprouvette d'immunochromatographie qui permet de mesurer le rapport (% HbA1c) de la quantité d'HbA1c à la quantité d'Hb dans un échantillon de mesure avec une sensibilité élevée, une précision élevée et une corrélation élevée à la CLHP. La solution selon l'invention porte sur une éprouvette d'immunochromatographie qui est conçue à partir : d'un tampon d'échantillon ; d'un tampon de conjugaison qui est chargé avec un réactif de détection de ligne de test et un réactif de détection de ligne de contrôle ; une membrane à laquelle un anticorps A, qui est destiné à capturer spécifiquement l'Hb ou l'HbA1c dans un échantillon de mesure, et un anticorps B, qui est destiné à capturer spécifiquement un marqueur dans le réactif de détection de ligne de contrôle, ont été fixés dans des lignes à des emplacements différents ; et un tampon d'absorption. Le réactif de détection de ligne de test est un composite d'un anticorps C qui est destiné à capturer l'Hb ou l'HbA1c dans l'échantillon de mesure, de fines particules de cellulose colorées et d'une protéine de blocage. Le réactif de détection de ligne de contrôle est un composite de particules de cellulose colorées et fines, et d'une protéine de blocage qui a été marquée avec un marqueur.
PCT/JP2018/048024 2018-01-09 2018-12-27 Éprouvette d'immunochromatographie, kit de mesure et procédé de mesure WO2019138898A1 (fr)

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CN111647080A (zh) * 2020-02-14 2020-09-11 北京纳百生物科技有限公司 一种免疫胶体金混合标记用c线抗体及其应用
CN111732656A (zh) * 2020-02-14 2020-10-02 北京纳百生物科技有限公司 一种适用于特定pH的胶体金混合标记用C线抗体及其应用
JP2021071316A (ja) * 2019-10-29 2021-05-06 東洋紡株式会社 イムノクロマト試験片およびそれを用いた測定方法
WO2022265106A1 (fr) * 2021-06-17 2022-12-22 東洋紡株式会社 Éprouvette d'immunochromatographie
WO2023163155A1 (fr) * 2022-02-28 2023-08-31 株式会社カネカ Dispositif de détection
WO2024026855A1 (fr) * 2022-08-05 2024-02-08 柯正浩 Procédé de lecture et de mesure de période d'anticorps neutralisant basé sur une bandelette de test de criblage rapide

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