WO2019138898A1

WO2019138898A1 - Immunochromatography test piece, measurement kit, and measurement method

Info

Publication number: WO2019138898A1
Application number: PCT/JP2018/048024
Authority: WO
Inventors: 岡本　淳; 裕川南; 真希子平岡; 圭三米田
Original assignee: 東洋紡株式会社
Priority date: 2018-01-09
Filing date: 2018-12-27
Publication date: 2019-07-18
Also published as: JPWO2019138898A1; JP7352831B2

Abstract

[Problem] Provided is an immunochromatography test piece that makes it possible to measure the ratio (% HbA1c) of the amount of HbA1c to the amount of Hb in a measurement sample with high sensitivity, high accuracy, and high correlation to HPLC. [Solution] The present invention is an immunochromatography test piece that is configured from: a sample pad; a conjugation pad that is loaded with a test line detection reagent and a control line detection reagent; a membrane to which an antibody A that is for specifically capturing the Hb or HbA1c in a measurement sample and an antibody B that is for specifically capturing a marker in the control line detection reagent have been fixed in lines at different locations; and an absorption pad. The test line detection reagent is a composite of an antibody C that is for capturing the Hb or HbA1c in the measurement sample, fine colored cellulose particles, and a blocking protein. The control line detection reagent is a composite of fine colored cellulose particles and a blocking protein that has been marked with a marker.

Description

Immunochromatographic test strip, assay kit and assay method

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.

According to the International Diabetes Federation, the number of diabetes patients in the world is rapidly increasing, including in developed countries such as America and Europe, as well as in emerging countries such as China, India and Brazil. There are 200 million people, and it is predicted to reach about 640 million by 2040, and the importance of diabetes diagnosis is increasing.

Hemoglobin A1c (which may hereinafter be abbreviated as HbA1c), 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 β 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.

Conventionally, measurement techniques such as HPLC, capillary electrophoresis, enzyme colorimetric assay, and immunoturbidimetric assay have been used to measure HbA1c (%), but these measurement methods have specialized knowledge and analysis It is mainly used in large-scale hospitals and inspection centers that carry out a large number of examinations because the equipment is large and expensive, and there is a problem that HbA1c (%) can not be easily measured in small-scale hospitals. The

In recent years, the word POCT has attracted attention in medical practice. 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.

Among POCT for the purpose of measuring HbA1c (%), a technique using an immunochromatographic method has been proposed. The immunochromatography method is an immunoassay using capillary action, and is widely used in pregnancy test and influenza test worldwide. In the conventional immunochromatography method, visual judgment (qualitative evaluation) was common, but in recent years, 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. In the 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. The other antibody forms a test line as a capture antibody immobilized linearly on the surface of the porous support. In addition, 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. However, since the above-mentioned invention sandwiches HbA1c, which is an analyte, with two kinds of anti-HbA1c antibodies, it is possible to measure the amount of HbA1c in the measurement sample, but it is possible to measure HbA1c (%). It was necessary to measure the Hb amount by another means. Further, 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. However, in

patent documents

1 and 2, since 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 β 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. However, the above-described invention is not sufficient in terms of accuracy because it does not have a control line. In addition, since 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. Furthermore, since 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.

JP, 2017-129533, A International Publication WO 2017/065213 JP 2012-251789 A JP, 2015-158515, A

In the present invention, in view of the above problems, 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) By using it, it discovered that correlation with a sensitivity, precision, and an HPLC method improves significantly rather than a prior art. Moreover, it discovered that correlation with a sensitivity, precision, and an HPLC method improves further by using Blocking Peptide Fragment: BPF for protein for blocking. Furthermore, by using 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.

That is, 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.
2. The immunochromatographic test strip according to 1, wherein the antibody A is an antibody for specifically capturing hemoglobin in a measurement sample, and the antibody C is an anti-hemoglobin A1c antibody.
3. The immunochromatographic test strip according to 1 or 2, wherein the labeling substance is biotin and the antibody B is an anti-biotin antibody.
4. The immunochromatographic test strip according to any one of 1 to 3, wherein the blocking protein is a microorganism-derived blocking peptide fragment.
5. The immunochromatography according to any one of 1 to 4, wherein the test line detection reagent and the control line detection reagent are carried on the conjugation pad at a mixing ratio (mass ratio) of 2: 1 to 50: 1. Test pieces.
6. The immunochromatographic test strip according to any one of 1 to 5, wherein the color of the cellulose-based colored fine particles is blue or black.
7. The immunochromatographic test strip according to any one of 1 to 6, wherein the cellulose-based colored fine particles used for the test line detection reagent and the cellulose-based colored fine particles used for the control line detection reagent are substantially the same.
8. 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. A method of quantifying the ratio of the amount of hemoglobin A1c to the amount of hemoglobin in the measurement sample using at least the following steps (i) to (iii) in this order using the immunochromatographic test strip or the measurement kit according to any one of 1 to 8.
Step (i): Step of mixing and diluting the measurement sample and the measurement sample dilution liquid in a volume ratio of 1:49 to 1: 999 Step (ii): step of spotting the mixed solution of step (i) onto the sample pad Step (iii): The step of colorimetrically quantifying the ratio of the amount of hemoglobin A1c to the amount of hemoglobin from the reflection absorbance of the test line of the immunochromatographic test strip and the reflection absorbance of the control line. The measuring method according to 9, wherein the measurement sample and the measurement sample dilution liquid are mixed and diluted in a volume ratio of 1:99 to 1: 499.

Since 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.

It is a figure (upper surface) which shows an example of the immuno chromatography test piece of this invention. It is a (side) figure showing an example of the immuno chromatography test piece of the present invention.

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. In blood, in addition to whole blood, serum, blood cells or plasma obtained by centrifuging blood can be used as a sample. Moreover, 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. Next, an example of the immunochromatographic test strip of the present invention will be described with reference to the drawings. In FIGS. 1 and 2, 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, and 8 is an adhesive sheet.

In the examples of FIGS. 1 and 2, 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. . In addition, 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. Furthermore, in the conjugation pad 2 of the immunochromatographic test strip, 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. In order to measure the ratio of the amount of HbA1c to the amount of Hb in the measurement sample: HbA1c (%), it is necessary to optimize the combination of the detection particles and the blocking agent.

The antibody C is an anti-Hb antibody or an anti-HbA1c antibody, preferably an anti-HbA1c antibody. In addition, when the antibody A described later is an anti-Hb antibody, the antibody C needs to be an anti-HbA1c antibody, and when the antibody A is an anti-HbA1c antibody, the antibody C needs to be an anti-Hb antibody. In a configuration in which both the antibody A and the antibody C sandwich an anti-Hb antibody, that is, an anti-Hb antibody, HbA1c in the measurement sample can not be captured and detected, and measurement of HbA1c (%) is impossible. On the other hand, in the configuration in which both the antibody A and the antibody C sandwich the anti-HbA1c antibody, that is, the anti-HbA1c antibody, 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.

Since 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. When 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. On the other hand, when 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. When 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. Because BPF (about 22 kDa) has a smaller molecular weight than BSA (about 66 kDa), more accurate 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. On the other hand, 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. In addition, in order to improve sensitization efficiency, you may introduce | transduce a reactive active group into cellulose type coloring microparticles. 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. In order to measure the ratio of the amount of HbA1c to the amount of Hb in the measurement sample: HbA1c (%), it is necessary to optimize the combination of the detection particles and the blocking agent.

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. Here, 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. Here, substantially identical means that the maximum absorption wavelength is within ± 20 nm. When the two colors are different, for example, when measuring with a general light emitting diode (hereinafter sometimes abbreviated as LED) -photodiode (hereinafter sometimes abbreviated as PD) measurement device, 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. By using the 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. 2-hydroxyimino-2-cyanoacetic acid amide, N-hydroxypiperidine, N-hydroxyphthalimide, N-hydroxyimidazole, N-hydroxymaleimide and the like. Two or more of these compounds may be used. Among these, N-hydroxysuccinimide (hereinafter sometimes abbreviated as NHS) is preferable because it is relatively inexpensive, easily available, and proven in the field of peptide synthesis and the like. 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. Among these, 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride (hereinafter sometimes abbreviated as EDC) is preferable because it has been widely used as a water-soluble condensing agent in the field of peptide synthesis and the like. 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. On the other hand, it is necessary to use borate buffer under alkaline conditions (pH 8.5 to 10) for dissolving casein, but borate buffer has high environmental risk. 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 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. Although not preferred, the introduction ratio is preferably 1: 1 to 1: 100, more preferably 1: 1 to 1:50. When the introduction ratio is less than 1: 1, the introduction amount of the label may be insufficient, and the measurement sensitivity may be reduced. On the other hand, if 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. In order to make the blocking protein chemically labeled with a labeling substance function as an ordinary blocking agent, although not particularly limited, the blocking protein chemically labeling with a labeling substance to the cellulose-based colored fine particles Is preferably present in large excess. Specifically, 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. For example, after mixing the test line detection reagent and the control line detection reagent in a fixed ratio, the mixed solution The composition 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 μL per 1 cm of line length. Further, 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. When the concentration is lower than 0.01 wt%, Hb and HbA1c can not be sufficiently captured and detected, and the measurement sensitivity may be lowered. On the other hand, even if the concentration is higher than 0.5 wt%, the measurement sensitivity is not improved, and only the cost is increased. Then, it is preferable to dry after the application. 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. When the above-mentioned antibody C is an anti-HbA1c antibody, the antibody A needs to be an antibody Hb antibody, and when the antibody C is an anti-Hb antibody, the antibody A needs to be an anti-HbA1c antibody. In the configuration in which both the antibody A and the antibody C sandwich an anti-Hb antibody, that is, an anti-Hb antibody, HbA1c in the measurement sample can not be captured and detected, and measurement of HbA1c (%) is impossible. On the other hand, in the configuration in which both the antibody A and the antibody C sandwich the anti-HbA1c antibody, that is, the anti-HbA1c antibody, 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. For example, 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 μL 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. If the concentration is low, Hb and HbA1c can not be sufficiently captured and detected, which may lower the measurement sensitivity. On the other hand, even if the concentration is increased, the measurement sensitivity is not improved, and only the cost is increased. Then, it is preferable to dry after the application. 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.

In the immunochromatographic test strip of the present invention, 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.

In the present invention, 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. As the buffer, 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. Among these, 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.

Although 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. At that time, 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. In addition, Hb (for example, HbA0 etc.) other than HbA1c in the diluted measurement sample does not form an immune complex with the test line detection reagent. Then, the diluted measurement sample is spread on the membrane 3. When the diluted measurement sample reaches the test line 6, the immunocomplex is captured and accumulated by a capture antibody (anti-Hb antibody) forming the test line, and the test line 6 is colored. In addition, 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.) occurs, and 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. Thereafter, when the diluted measurement sample reaches the control line 7, 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.

As described above, 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. Specifically, 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. When the molar ratio is small, 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. On the other hand, when the molar ratio is large, 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.

Hereinafter, the present invention will be described in detail based on examples. The present invention is not limited to these examples. In addition, 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. Next, 1.0 wt% of cellulose-based colored fine particles (NanoAct (registered trademark), BL2: Dark Navy, average particle diameter 365 nm, manufactured by Asahi Kasei Corp.) 100 μL, 10 mM Tris buffer (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.) ) (PH 7.0) 900 μL and 100 μL 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. Next, 12 mL of a blocking solution (pH 7.0) 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.) In addition, it was left still for 60 minutes in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 37 ° C. Then, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.), 10,000 × g Centrifugation was performed at 25 ° C. for 15 minutes to remove antibody-sensitized cellulose-based colored fine particles, and then the supernatant was removed. Then, 12 mL of a washing solution (pH 7.0) 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. Then, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.), 10,000 × g Centrifugation was performed at 25 ° C. for 15 minutes to remove antibody-sensitized cellulose-based colored fine particles, and then the supernatant was removed. Then, 15 wt% sucrose (196-00015, manufactured by Wako Pure Chemical Industries, Ltd.), 0.2% Blocking Peptide Fragment: BPF (BPF-301, manufactured by Toyobo Co., Ltd.), 10 mM Tris buffer (204-07885, Japanese sum) A coating solution (pH 7.0) 2.0 mL consisting of Kojun Pharmaceutical Co., Ltd. was added, and treated with an ultrasonic disperser (UH-50, 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%).

(2) Preparation of control line detection reagent for HbA1c measurement D Biotin (04822-91, manufactured by Nacalai Tesque) 16 mg and dimethyl sulfoxide: DMSO (08904-14, manufactured by Nacalai Tesque) 1000 μL are added to a 1.5 mL microtube The mixture was vortexed to obtain D-biotin solution. Next, 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 μL 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. Next, 100 μL of the D biotin solution and 100 μL of the EDC / NHS solution are mixed, placed in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 25 ° C., and allowed to stand for 15 minutes, and D biotin / EDC / NHS solution Obtained. Next, 10 mg of Blocking Peptide Fragment: BPF (BPF-301, manufactured by Toyobo Co., Ltd.) and 100 μL of distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical Co., Ltd.) are added to a 1.5 mL microtube and vortexed to obtain a BPF solution. The Next, 100 μL of the D-biotin / EDC / NHS solution and 100 μL of the BPF solution are mixed and then placed in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 25 ° C. and allowed to stand for 30 minutes. Obtained. Next, 1.0 wt% of cellulose-based colored fine particles (NanoAct (registered trademark), BL2: Dark Navy, average particle diameter 365 nm, manufactured by Asahi Kasei Corp.) 100 μL, 10 mM Tris buffer (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.) ) (PH 7.0) and 100 μL 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. Next, 12 mL of a blocking solution (pH 7.0) 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.) In addition, it was left still for 60 minutes in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 37 ° C. Then, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.), 10,000 × g Centrifugation was carried out at 25 ° C. for 15 minutes to remove the supernatant after sedimentation of the D-biotin-sensitized cellulose-based fine particles. Then, 12 mL of a washing solution (pH 7.0) 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. Then, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.), 10,000 × g Centrifugation was carried out at 25 ° C. for 15 minutes to remove the supernatant after sedimentation of the D-biotin-sensitized cellulose-based fine particles. Then, 15 wt% sucrose (196-00015, manufactured by Wako Pure Chemical Industries, Ltd.), 0.2% Blocking Peptide Fragment: BPF (BPF-301, manufactured by Toyobo Co., Ltd.), 10 mM Tris buffer (204-07885, Japanese sum) A coating solution (pH 7.0) 2.0 mL consisting of Kojun Pharmaceutical Co., Ltd.) was added, and treated for 10 seconds with an ultrasonic dispersion machine (UH-50, manufactured by SMT Co., Ltd.) 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.

(3) Preparation of membrane card for HbA1c measurement Anti-Hb monoclonal antibody (HBA1 Antibody, OAMA02326, AVIVA SYSTEM BIOLOGY) of 3.6 mg / mL in distilled water (Otsuka distilled water, Otsuka Pharmaceutical) 1.0 mg / Prepared in mL. Then, 1.0 mg / mL of an anti-biotin polyclonal antibody (Biotin Antibody, A150-111A, manufactured by BETHYL) was prepared to 0.5 mg / mL with distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical). Next, 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. Using a dispensing platform (XYZ3060, manufactured by BIODOT) and a Bio Jet nozzle (BHQHR-XYZ, manufactured by BIODOT) at a position of 15 mm from the upstream side of the membrane part of HF120 manufactured by Millipore After applying / mL anti-Hb monoclonal antibody at a coating amount of 1.0 μL / cm, it was dried for 30 minutes with a dryer (WFO-510, manufactured by Tokyo Rika Kikai Co., Ltd.) adjusted to 45 ° C, and the line width was about 1 mm A test line was formed. Furthermore, using a dispensing platform (XYZ3060, manufactured by BIODOT) and a Bio Jet nozzle (BHQHR-XYZ, manufactured by BIODOT) at a position 20 mm from the upstream side of the membrane part of the membrane card in which the test line is formed, the above After applying 0.5 mg / mL of anti-biotin polyclonal antibody at a coating amount of 1.0 μL / cm, it was dried for 30 minutes with a dryer (WFO-510, manufactured by Tokyo Rika Kikai Co., Ltd.) adjusted to 45 ° C. By forming a control line of about 1 mm, a membrane card for HbA1c measurement was obtained.

(4) Preparation of Conjugation Pad for HbA1c Measurement The 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). When the particle concentrations in the HbA1c measurement test line detection reagent and the HbA1c measurement control line detection reagent are the same, mixing may be performed at a volume ratio of 6: 1. Then, using a dispensing platform (XYZ3060, manufactured by BIODOT) and an Air Jet nozzle (AJQHR-XYZ, manufactured by BIODOT) on the entire surface of a 10 mm × 300 mm conjugation pad (GLASSFIBER DIAGNOSTIC PAD, GFDX001050, manufactured by Millipore) After uniformly applying the mixture at a coating amount of 15 μL / cm, the mixture was dried for 30 minutes with a dryer (WFO-510, manufactured by Tokyo Rika Kikai Co., Ltd.) adjusted to 45 ° C. to obtain a conjugation pad for HbA1c measurement. The

(5) Preparation of immunochromatographic test piece for HbA1c measurement The HbA1c measurement conjugation pad was attached to a 20 mm × 300 mm adhesive tape portion on the upstream side of the HbA1c measurement membrane card so as to overlap the membrane portion by 5 mm. Then, a 20 mm × 300 mm sample pad (CELLULOSE FIBER SAMPLE PADS, CFSP 002000, manufactured by Millipore) was laminated further upstream so as to overlap the conjugation pad by 5 mm. Next, 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. Then, 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.

(6) Preparation of measurement sample dilution solution Phosphate buffered saline powder (162-19321, manufactured by Wako Pure Chemical Industries, Ltd.) 1 bag, polyoxyethylene (20) sorbitan monolaurate: Tween (registered trademark) 20 (166- 21213, manufactured by Wako Pure Chemical Industries, Ltd. 2 g, polyoxyethylene (10) octyl phenyl ether: Triton X (registered trademark) -100 (160-24751, manufactured by Wako Pure Chemical Industries, Ltd.) 2 g, distilled water (Otsuka distilled water, 200 mL of Otsuka Pharmaceutical Co., Ltd.) was added to a 500 mL glass bottle to prepare a measurement sample diluent (50 mM PBS, 1.0 wt% Tween (registered trademark) 20, 1.0 wt% Triton X (registered trademark) -100).

(7) Preparation of diluted sample The above-mentioned sample dilution solution was used as a commercially available HbA1c standard substance, for HbA1c measurement performance evaluation sample (QRM HbA1c 2007-1, manufactured by Jikken Medical Standard Materials Organization, L1 to L5 levels). HbA1c (%) at L1 = 5.01%, L2 = 5.65%, L3 = 7.35%, L4 = 9.51%, L5 = 11.26% by diluting 500 times with And a diluted HbA1c sample (L1 to L5) having an Hb concentration of L1 to L5 = 0.280 g / L. Then, 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) is respectively multiplied by 500 with the measurement sample dilution solution. By dilution, HbA1c (%) is L, M, H = 5.20%, and the Hb concentration is L = 0.156 g / L, M = 0.274 g / L, H = 0.359 g / L Diluted Hb samples (L, M, H) were obtained.

(8) Evaluation of immunochromatographic test piece for HbA1c measurement: Evaluation of sensitivity The above-mentioned immunochromatographic test piece for HbA1c measurement was placed on a horizontal table. Next, 100 μL of the diluted HbA1c sample (L1, L4) was taken with a micropipette, slowly dropped on a sample pad, and allowed to stand at 25 ° C. for 10 minutes. Then, the reflection absorbances (mAbs) of the control line and the test line on the membrane were measured using an immunochromator (C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics K. K.). The experiment was performed on each of the diluted HbA1c samples (L1 and L4) at N = 10 (in total, 20 of the immunochromatographic test strips for HbA1c measurement were used). As an index of sensitivity, N = 10 mean value of reflection absorbance (mAbs) of the test line of the diluted HbA1c sample (L4) and N = 10 average of reflection absorbance (mAbs) of the test line of the diluted HbA1c sample (L1) Difference with value: Calculate ΔL4-L1 (mAbs), 3 points of ΔL4-L1 ≧ 200 mAbs (excellent), 2 points of 150 mAbs ≦ ΔL4-L1 <200 mAbs (good), 100 mAbs ≦ ΔL4-L1 <150 mAbs Points (average), ΔL4-L1 <100 mAbs were evaluated as 0 points (bad). The sensitivity of the immunochromatographic test piece for HbA1c measurement of Example 1 was able to be measured with high sensitivity, with three points at ΔL4-L1 = 260 mAbs.

(9) Evaluation of immunochromatographic test piece for HbA1c measurement: evaluation of accuracy The immunochromatographic test piece for HbA1c measurement was placed on a horizontal table. Next, 100 μL of the diluted HbA1c sample (L1, L4) was taken with a micropipette, slowly dropped on a sample pad, and allowed to stand at 25 ° C. for 10 minutes. Then, the reflection absorbances (mAbs) of the control line and the test line on the membrane were measured using an immunochromator (C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics K. K.). The experiment was performed on each of the diluted HbA1c samples (L1 and L4) at N = 10 (in total, 20 of the immunochromatographic test strips for HbA1c measurement were used). Obtained after calculating correction values (L1, L4) obtained by dividing the reflection absorbances (mAbs) of the test line of the diluted HbA1c sample (L1, L4) by the reflection absorbance (mAbs) of the control line as an index of accuracy. 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 precision of the immunochromatographic test piece for HbA1c measurement of Example 1 was as high as 3 points at CV = 2.0%, and high precision measurement was possible.

(10) Evaluation of immunochromatographic test piece for HbA1c measurement: Evaluation of correlation with HPLC method The immunochromatographic test piece for HbA1c measurement was placed on a horizontal table. Next, 100 μL of the diluted HbA1c sample (L1 to L5) was taken with a micropipette, slowly dropped on a sample pad, and allowed to stand at 25 ° C. for 10 minutes. Then, the reflection absorbances (mAbs) of the control line and the test line on the membrane were measured using an immunochromator (C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics K. K.). The experiment was performed on each of the diluted HbA1c samples (L1 to L5) at N = 10 (in total, 50 of the immunochromatographic test strips for measuring the HbA1c were used). After calculating the correction values (L1 to L5) obtained by dividing the reflection absorbances (mAbs) of the test line of the diluted HbA1c sample (L1 to L5) by the reflection absorbance (mAbs) of the control line as an index of correlation, The obtained correction values (L1 to L5) and HbA1c (%) of the diluted HbA1c sample (L1 to L5), L1 = 5.01%, L2 = 5.65%, L3 = 7.35%, L4 = Pearson correlation coefficient: 9.51%, L5 = 11.26%: r is calculated, 3 points (excellent) of r ≧ 0.95, 2 points (good) of 0.90 ≦ r <095, 0 .85 ≦ r <0.90 was evaluated as 1 point (average), and r <0.85 as 0 point (bad). The correlation of the immunochromatographic test piece for measuring HbA1c of Example 1 had a high correlation between HbA1c (%) and 3 points at r = 0.98.

(11) Evaluation of immunochromatographic test piece for HbA1c measurement: Evaluation of Hb concentration dependency The immunochromatographic test piece for HbA1c measurement was placed on a horizontal table. Next, 100 μL of the diluted Hb sample (L, M, H) was taken with a micropipette, slowly dropped onto a sample pad, and allowed to stand at 25 ° C. for 10 minutes. Then, the reflection absorbances (mAbs) of the control line and the test line on the membrane were measured using an immunochromator (C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics K. K.). The experiment was performed on each of the diluted Hb samples (L, M, H) at N = 10 (in total, 30 of the immunochromatographic test strips for HbA1c measurement were used). Correction values (L, M, H) obtained by dividing the reflection absorbances (mAbs) of the test line of the diluted Hb sample (L, M, H) by the reflection absorbances (mAbs) of the control line as an indicator of Hb concentration dependency. Is calculated, CV (Hb) (%) of total N = 30 is calculated with each N = 10 of the obtained correction values (L, M, H), CV (Hb) <3% at 3 points (Excellent), 3% ≦ CV (Hb) <5% at 2 points (good), 5% ≦ CV (Hb) <10% at 1 point (average), 10% ≦ CV (Hb) at 0 points (bad) It evaluated as). The Hb concentration dependency of the immunochromatographic test piece for HbA1c measurement of Example 1 is constant regardless of the amount of Hb if the HbA1c (%) in the measurement sample is the same at three points at CV (Hb) = 2.8%. It confirmed that the measurement result (correction value) of was obtained.

(12) Evaluation of immunochromatographic test piece for HbA1c measurement: Evaluation of nonspecific adsorption The above-mentioned immunochromatographic test piece for HbA1c measurement was placed on a horizontal table. Next, 100 μL of distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical Co., Ltd.) was taken with a micropipette, slowly dropped onto a sample pad, and allowed to stand at 25 ° C. for 10 minutes. Next, the reflection absorbance (mAbs) of the test line on the membrane was measured using an immunochromator reader (C10060-10, measurement mode: Latex, Line, manufactured by Hamamatsu Photonics K.K.). 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. Anti-HbA1c monoclonal antibody (HbA1c Antibody, OAMA02329, AVIVA SYSTEM BIOLODY) instead of anti-Hb monoclonal antibody (HBA1 Antibody, OAMA02326, AVIVA SYSTEM BIOLOGY) as an antibody used for preparation of a membrane card for HbA1c measurement In the same manner as in Example 1 except that To prepare a bA1c for measuring immunochromatographic test piece was evaluated. The obtained evaluation results are shown in Table 1.

(Examples 3 and 4)
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. When casein is used, 100 mM of boro- dine instead of 10 mM Tris buffer (204-07885, manufactured by Wako Pure Chemical Industries, Ltd.) (pH 7.0) as a buffer for blocking liquid, washing liquid and coating liquid. An acid buffer solution (021-02195, manufactured by Wako Pure Chemical Industries, Ltd.) (pH 9.0) was used. The obtained evaluation results are shown in Table 1.

(Examples 5 to 9)
In the preparation step of the conjugation pad for HbA1c measurement, the 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.

(Examples 10 and 11)
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.

(Examples 14 to 17)
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.

(Examples 18 to 20)
Anti-Hb monoclonal antibody (HBA1 Antibody, OAMA02326, AVIVA SYSTEM BIOLOGY) for use in preparation of membrane card for HbA1c measurement is 1.0 mg / mL in distilled water (Otsuka distilled water, Otsuka Pharmaceutical) In the same manner as in Example 1 except that 0.2 mg / mL (Example 18), 0.5 mg / mL (Example 19) and 2.0 mg / mL (Example 20) were prepared instead of The immunochromatographic test piece for HbA1c measurement was produced and evaluated. The obtained evaluation results are shown in Table 1.

(Examples 21 to 23)
Anti-HbA1c monoclonal antibody (HbA1c Antibody, OAMA02329, AVIVA SYSTEM BIOLOGY) 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 (Example 23). Examples were evaluated to produce a HbA1c measurement immunochromatographic specimen in the same manner as 1. The obtained evaluation results are shown in Table 1.

(Comparative example 1)
(1) Preparation of 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. Then, 13.5 mL of gold colloid particles having an OD 520 of 1.0 (EMGC 40: Red, average particle size 40 nm, manufactured by BBI Solutions), 50 mM potassium dihydrogen phosphate (166-04255, manufactured by Wako Pure Chemical Industries, Ltd.) 1.5 mL of pH 7.0) and 1.5 mL of the 0.05 mg / mL anti-HbA1c monoclonal antibody were added to a 50 mL centrifuge tube and gently stirred. Next, it was placed in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 25 ° C. and allowed to stand for 10 minutes. Next, 1 mL of a blocking solution consisting of 10 wt% of bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) was added and lightly stirred. Next, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.) The centrifugation was carried out at 25 ° C. for 15 minutes to remove antibody-sensitized gold colloid particles and then the supernatant was removed. Then, 1.0 wt% of 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. Next, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.) The centrifugation was carried out at 25 ° C. for 15 minutes to remove antibody-sensitized gold colloid particles and then the supernatant was removed. Then, 2.5 wt% sucrose (196-00015, manufactured by Wako Pure Chemical Industries, Ltd.), 0.25 wt% Bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich), 37.5 mM sodium chloride (192- A coating solution (pH 8.2) consisting of 13925, Wako Pure Chemical Industries, Ltd. and 20 mM Tris buffer (204-07885, Wako Pure Chemical Industries, Ltd.) is added, and an ultrasonic dispersion machine (UH-50, SMT Co., Ltd.) Treatment for 10 seconds, and the solution amount of the coating solution was adjusted to OD 520 = 4.0 to obtain a test line detection reagent for HbA1c measurement. The detection particle concentration in the test line detection reagent for measuring HbA1c was OD520 = 4.0, and the antibody concentration was 0.02 mg / mL (0.002 wt%).

(2) Preparation of control line detection reagent for HbA1c measurement D Biotin (04822-91, manufactured by Nacalai Tesque) 16 mg and dimethyl sulfoxide: DMSO (08904-14, manufactured by Nacalai Tesque) 1000 μL are added to a 1.5 mL microtube The mixture was vortexed to obtain D-biotin solution. Next, 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 μL 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. Next, 100 μL of the D biotin solution and 100 μL of the EDC / NHS solution are mixed, placed in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 25 ° C., and allowed to stand for 15 minutes, and D biotin / EDC / NHS solution Obtained. Next, 10 mg of Bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) and 100 μL of distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical) are added to a 1.5 mL microtube and vortexed to obtain a BSA solution. The Next, 100 μL of the D-biotin / EDC / NHS solution and 100 μL of the BSA solution are mixed and then placed in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 25 ° C. and allowed to stand for 30 minutes. Obtained. Then, 13.5 mL of gold colloid particles having an OD 520 of 1.0 (EMGC 40: Red, average particle size 40 nm, manufactured by BBI Solutions), 50 mM potassium dihydrogen phosphate (166-04255, manufactured by Wako Pure Chemical Industries, Ltd.) 1.5 mL of pH 7.0) and 100 μL of the D biotin-BSA solution were added to a 50 mL centrifuge tube and gently stirred. Next, it was placed in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 25 ° C. and allowed to stand for 10 minutes. Next, 1 mL of a blocking solution consisting of 10 wt% of bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) was added and lightly stirred. Next, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.) Centrifugation was performed at 25 ° C. for 15 minutes to remove the supernatant after sedimentation of D-biotin-sensitized gold colloid particles. Then, 1.0 wt% of 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. Next, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.) Centrifugation was performed at 25 ° C. for 15 minutes to remove the supernatant after sedimentation of D-biotin-sensitized gold colloid particles. Then, 2.5 wt% sucrose (196-00015, manufactured by Wako Pure Chemical Industries, Ltd.), 0.25 wt% Bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich), 37.5 mM sodium chloride (192- A coating solution (pH 8.2) consisting of 13925, Wako Pure Chemical Industries, Ltd. and 20 mM Tris buffer (204-07885, Wako Pure Chemical Industries, Ltd.) is added, and an ultrasonic dispersion machine (UH-50, SMT Co., Ltd.) C. for 10 seconds, and the solution volume of the coating solution was adjusted to OD 520 = 4.0 to obtain a control line detection reagent for HbA1c measurement. The detection particle concentration in the control line detection reagent for measuring HbA1c was OD520 = 4.0, and the labeling ratio of blocking protein was 1: 7.

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.

(Comparative example 2)
As a blocking protein (including a labeled blocking protein) used to prepare a test line detection reagent for HbA1c measurement and a control line detection reagent for HbA1c measurement, instead of Bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) 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.

(Comparative example 3)
(1) Preparation of 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. 10 wt% latex particles (Estapor (registered trademark), K 030: Blue, average particle size 300 nm, Merk Millipore, Inc.) at a concentration of 1.0 mg / mL (manufactured by Kogyo Co., Ltd.) (pH 7.0) to 50 mM potassium dihydrogen phosphate The solution was adjusted to 1.0 wt% with (166-04255, manufactured by Wako Pure Chemical Industries, Ltd.) (pH 7.0). Then, 100 μL of the 1.0 wt% latex particles and 100 μL of the 1.0 mg / mL 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 25 ° C. and allowed to stand for 60 minutes. Then, 12 mL of a blocking solution consisting of 1.0 wt% of bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) was added, and the mixture was allowed to stand for 60 minutes in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 25 ° C. did. Next, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.) Centrifugation was performed at 25 ° C. for 15 minutes to remove antibody-sensitized latex particles and then supernatant. Then, 12 mL of a washing solution (pH 7.0) 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. Next, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.) Centrifugation was performed at 25 ° C. for 15 minutes to remove antibody-sensitized latex particles and then supernatant. Next, a coating solution (pH 7.0) 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%).

(2) Preparation of control line detection reagent for HbA1c measurement D Biotin (04822-91, manufactured by Nacalai Tesque) 16 mg and dimethyl sulfoxide: DMSO (08904-14, manufactured by Nacalai Tesque) 1000 μL are added to a 1.5 mL microtube The mixture was vortexed to obtain D-biotin solution. Next, 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 μL 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. Next, 100 μL of the D biotin solution and 100 μL of the EDC / NHS solution are mixed, placed in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 25 ° C., and allowed to stand for 15 minutes, and D biotin / EDC / NHS solution Obtained. Next, 10 mg of Bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) and 100 μL of distilled water (Otsuka distilled water, manufactured by Otsuka Pharmaceutical) are added to a 1.5 mL microtube and vortexed to obtain a BSA solution. The Next, 100 μL of the D-biotin / EDC / NHS solution and 100 μL of the BSA solution are mixed and then placed in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 25 ° C. and allowed to stand for 30 minutes. Obtained. Next, 10 wt% latex particles (Estapor (registered trademark), K 030: Blue, average particle size 300 nm, manufactured by Merk Millipore), 50 mM potassium dihydrogen phosphate (166-04255, manufactured by Wako Pure Chemical Industries, Ltd.) (pH 7). It adjusted to 1.0 wt% with 0). Then, 100 μL of the 1.0 wt% latex particles and 100 μL of the D biotin-BSA 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 25 ° C. and allowed to stand for 60 minutes. Then, 12 mL of a blocking solution consisting of 1.0 wt% of bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) was added, and the mixture was allowed to stand for 60 minutes in a low temperature incubator (IN 604, manufactured by Yamato Scientific Co., Ltd.) adjusted to 25 ° C. did. Next, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.) Centrifugation was carried out at 25 ° C. for 15 minutes to remove the supernatant after sedimentation of the D-biotin-sensitized latex particles. Then, 12 mL of a washing solution (pH 7.0) 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. Next, using a centrifuge (MX-307, made by Tomy Seiko Co., Ltd.), a rack-in rotor (TMA-300, made by Tomy Seiko Co., Ltd.) and a rack (AR510-04, made by Tomy Seiko Co., Ltd.) Centrifugation was carried out at 25 ° C. for 15 minutes to remove the supernatant after sedimentation of the D-biotin-sensitized latex particles. Next, a coating solution (pH 7.0) 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.

Thereafter, in the same manner as in Example 1, immunochromatographic test pieces for HbA1c measurement were prepared and evaluated. 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. In addition, blocking was insufficient and nonspecific adsorption was confirmed.

(Comparative example 4)
As a blocking protein (including a labeled blocking protein) used to prepare a test line detection reagent for HbA1c measurement and a control line detection reagent for HbA1c measurement, instead of Bovine serum albumin: BSA (A7906, manufactured by Sigma-Aldrich) 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.

(Comparative example 6)
An anti-HbA1c monoclonal antibody (Human HbA1c monoclonal antibody, MAB0030-MO6A, manufactured by Abnova) instead of an anti-Hb monoclonal antibody (HBA1 Antibody, OAMA02326, AVIVA SYSTEM BIOLOGY) is used as an antibody for preparation of a membrane card for HbA1c measurement. An immunochromatographic test piece for measuring HbA1c was prepared and evaluated in the same manner as in Example 1 except that it was used. The evaluation results obtained are shown in Table 2. In the configuration in which the anti-HbA1c antibody is sandwiched, 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.

According to the present invention, 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.

1: Sample pad 2: Conjugation pad 3: Membrane 4: Absorbent pad 5: Backing sheet 6: Test line 7: Control line 8: Adhesive sheet

Claims

(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.
The immunochromatographic test strip according to claim 1, wherein the antibody A is an antibody for specifically capturing hemoglobin in a measurement sample, and the antibody C is an anti-hemoglobin A1c antibody.
The immunochromatographic test strip according to claim 1 or 2, wherein the labeling substance is biotin and the antibody B is an anti-biotin antibody.
The immunochromatographic test strip according to any one of claims 1 to 3, wherein the blocking protein is a microorganism-derived blocking peptide fragment.
The test line detection reagent according to any one of claims 1 to 4, wherein the test line detection reagent and the control line detection reagent are carried on the conjugation pad at a mixing ratio (mass ratio) of 2: 1 to 50: 1. Immunochromatographic test strip.
The immunochromatographic test strip according to any one of claims 1 to 5, wherein the color of the cellulose-based colored fine particles is blue or black.
The immunochromatographic test strip according to any one of claims 1 to 6, wherein the cellulose-based colored fine particles used for the test line detection reagent and the cellulose-based colored fine particles used for the control line detection reagent are substantially the same.
A measurement kit for quantifying the ratio of the amount of hemoglobin A1c to the amount of hemoglobin, comprising the immunochromatographic test strip according to any one of claims 1 to 7, the measurement sample dilution liquid and the immunochromato reader.
A ratio of the amount of hemoglobin A1c to the amount of hemoglobin in the measurement sample is quantified using at least the following steps (i) to (iii) in this order using the immunochromatographic test strip or the measurement kit according to any one of claims 1 to 8 Method.
Step (i): Step of mixing and diluting the measurement sample and the measurement sample dilution liquid in a volume ratio of 1:49 to 1: 999 Step (ii): step of spotting the mixed solution of step (i) onto the sample pad Step (iii): The step of colorimetrically quantifying the ratio of the amount of hemoglobin A1c to the amount of hemoglobin from the reflection absorbance of the test line of the immunochromatographic test strip and the reflection absorbance of the control line
The method according to claim 9, wherein the measurement sample and the measurement sample dilution liquid are mixed and diluted at a volume ratio of 1:99 to 1: 499.