JP2018151185A - Immunochromatographic test piece - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunochromatographic test piece of a biological sample which can remove contaminant such as bilirubin resulting in measurement error selectively and efficiently when measuring hemoglobin concentration in a biological sample quantitatively.SOLUTION: In the immunochromatographic test piece which is configured so that a sample pad, a conjugate pad, a membrane and an absorption pad are connected to each other in turn, the immunochromatographic test piece contains a removal material of the contaminant on the upstream side of the membrane.SELECTED DRAWING: None

Description

  The present invention relates to an immunochromatographic test piece having a function of removing contaminants such as bilirubin and milky milk that cause measurement errors (overlapping absorption wavelengths) when quantitatively measuring hemoglobin in a biological sample.

  In recent years, the term POCT has attracted attention in the medical field. POCT is an abbreviation for Point Of Care Testing, and refers to a clinical test performed by a medical worker alongside a subject. Unlike clinical tests performed in a central laboratory of a large-scale hospital or the like, POCT can obtain test results instantly, so POCT is also spreading in diabetes diagnosis.

  In the POCT, a technique using an immunochromatography method has been proposed. The immunochromatography method is an immunoassay method using a capillary phenomenon, and pregnancy test drugs and influenza test drugs using the immunochromatography method are widely used worldwide. Conventional immunochromatography is generally performed by visual determination (qualitative evaluation), but in recent years, a technique for quantifying the concentration of a substance contained in a sample using an analyzer such as a chromatographic reader is being developed.

  However, in biological samples, there are reducing substances such as bilirubin and ascorbic acid, and especially the absorption wavelength of bilirubin and milky milk overlaps with the maximum absorption wavelength of hemoglobin, so the presence of these substances causes the hemoglobin and glycated hemoglobin. The measured value was greatly affected, and an error was sometimes caused in the measured value.

  In order to determine the hemoglobin concentration in a biological sample by absorptiometry, it is preferable to measure the absorbance in the maximum absorption wavelength region of hemoglobin. However, absorption of contaminants such as bilirubin and milk powder mixed in the biological sample is preferable. Since it overlaps with the wavelength, it is difficult to quantify using single wavelength photometry in the maximum absorption wavelength region. Therefore, conventionally, the absorbance of the main wavelength region where the influence of the contaminant substance remains and the sub-wavelength region where the influence of the contaminant substance is small are measured, and the concentration of the measurement target substance is calculated by subtracting the absorbance derived from the contaminant substance. It was unavoidable (Non-Patent Document 1).

  As a method of avoiding the influence of bilirubin, a method of adding an amphoteric surfactant to a measurement reagent is known. For example, Patent Document 1 describes that an amphoteric surfactant is added to the first reagent for the purpose of avoiding the influence of bilirubin. Furthermore, in Patent Document 2, in a method for measuring a biological component in which hydrogen peroxide generated by an enzymatic reaction is detected with peroxidase and an oxidizable color former, both the first reagent and both the first reagent and the second reagent are amphoteric. A method for measuring biological components in the presence of a surfactant and a ferrocyan compound is described.

Biological sample analysis Vol. 36, No3 (2013)

Japanese Patent Laid-Open No. 7-039394 JP-A-7-155196

  The present invention provides an immunochromatographic test piece capable of selectively and efficiently removing contaminants such as bilirubin that cause measurement errors when quantitatively measuring the hemoglobin concentration in a biological sample. Let it be an issue.

  As a result of intensive studies to solve the above problems, the present inventor can selectively and instantaneously remove contaminants such as bilirubin by using an immunochromatographic test piece including a contaminant removal material having a specific configuration. The present invention has been completed.

That is, the present invention has the following configuration.
(1) An immunochromatographic test piece in which a sample pad, a conjugate pad, a membrane, and an absorption pad are sequentially connected, and contains an impurity removing material upstream of the membrane.
(2) The immunochromatographic test piece according to (1), wherein the removal material is at least one selected from the group consisting of fibers, woven fabrics, nonwoven fabrics, particles and powders.
(3) The removal material is one or more selected from the group consisting of activated carbon, activated carbon fiber, titanium oxide, divinylbenzene / glycidyl methacrylate copolymer and hydrophobic silica (1) or ( The immunochromatographic test piece according to 2).
(4) The immunochromatographic test piece according to any one of (1) to (3), wherein the contaminant is bilirubin or milk.

  According to the present invention, by bringing a biological sample into contact with an immunochromatographic test piece having a specific configuration, contaminants such as bilirubin in the biological sample can be selectively, efficiently, and rapidly removed. The hemoglobin concentration can be measured quantitatively without receiving. In addition, since the influence of contaminants is reduced, measurement in the maximum absorption wavelength region of hemoglobin is possible, and measurement can be performed with high accuracy even when the hemoglobin concentration in the biological sample is low.

It is a schematic diagram which shows an example of the immunochromatography test piece of this invention. It is a schematic diagram which shows another example of the immunochromatographic test piece of this invention. It is a schematic diagram which shows another example of the immunochromatographic test piece of this invention. It is a schematic diagram which shows another example of the immunochromatographic test piece of this invention. It is a schematic diagram which shows another example of the immunochromatographic test piece of this invention. It is a schematic diagram which shows the modification of the immunochromatography test piece of this invention. It is a schematic diagram which shows another modification of the immunochromatographic test piece of this invention. It is a schematic diagram which shows an example of the conventional immunochromatographic test piece.

  In the present invention, the form of the immunochromatographic test piece is not particularly limited. For example, an immunochromatographic test piece connected in the order of a sample pad, a conjugate pad, a membrane, and an absorption pad with the biological sample introduction part of the immunochromatographic test piece as an upstream side can be mentioned. FIG. 1 shows an example of the immunochromatographic test piece of the present invention. 1 is a contaminant removal material, 2 is a sample pad, 3 is a conjugate pad, 4 is a membrane, 5 is an absorbent pad, and 6 is an adhesive sheet (backing sheet) for connecting the above 1-5. ). As shown in FIG. 1, it is preferable that a part of the end portions of the members 1 to 5 are connected so as to overlap each other.

  In FIG. 1, the contaminant removal material 1 is shown as being disposed on the upstream side of the sample pad 2. However, as shown in FIG. 2, the contaminant removal material 1 is a part of the upper surface of the sample pad 2. Or the aspect arrange | positioned so that all (not shown) may be covered is also within the scope of the present invention. Also, as shown in FIG. 3, the contaminant removal material 1 is disposed so as to contact the lower surface of the sample pad 2, and the contaminant removal material 1 and the sample pad 2 are integrated as shown in FIG. Such an embodiment is also within the scope of the present invention. Further, as shown in FIG. 5, a mode in which the contaminant removing material 1 replaces the sample pad 2 is also included in the present invention. Further, the contaminant removal material 1 is located downstream of the sample pad 2 and upstream of the conjugate pad 3, ie, between the sample pad 2 and the conjugate pad 3 (FIG. 6), An embodiment (FIG. 7) in which the removal material 1 is disposed downstream of the conjugate pad 3 and upstream of the membrane 4, that is, between the conjugate pad 3 and the membrane 4 is also within the scope of the present invention. Furthermore, the aspect (not shown) arrange | positioned in the state by which the contaminant removal material 1 was disperse | distributed everywhere in the upstream of the membrane 4 is not excluded.

  In the present invention, the contaminant removal material is preferably at least one selected from the group consisting of fibers, woven fabrics, nonwoven fabrics, particles and powders. In the case of a woven fabric or a non-woven fabric, it can be used in the mode shown in FIGS. 1 to 3 and FIG. It can be used in an embodiment.

  In the present invention, the contaminant removal material is at least one selected from the group consisting of activated carbon, activated carbon fiber (fibrous activated carbon), titanium oxide, divinylbenzene / glycidyl methacrylate copolymer and hydrophobic silica. preferable. Here, the titanium oxide and the hydrophobic silica can be obtained in the form of particles or powders, and in the other cases, any of fibers, nonwoven fabrics, particles or powders can be obtained and prepared. Among these, activated carbon or activated carbon fiber (hereinafter sometimes simply referred to as activated carbon) is preferable from the viewpoint of availability and cost. Of these, those for water treatment are more preferred.

  The raw material (precursor) of the activated carbon is not particularly limited. For example, phenol, cellulose, rayon, polyacrylonitrile, pitch, aramid, polyimide, polyamide, polyamideimide, polyphenylenebenzobisoxazole, polyvinyl alcohol, Examples thereof include polyethersulfone, polysulfone, and polyphenylene oxide. These materials can be used to produce fibers, woven fabrics, nonwoven fabrics, particles, or powders, and then carbonized, activated, and purified as necessary.

  In the present invention, when a porous material is used as the contaminant removal material, the total pore volume is preferably in the range of 0.25 to 1.00 cc / g. More preferably, it is 0.3-0.95cc / g, More preferably, it is 0.40-0.90cc / g. If the total pore volume is too small, contaminants in the biological sample may not be sufficiently removed, and if it is too large, the strength of the single fiber may be significantly reduced and the form may not be maintained. The total pore volume can be measured using, for example, a specific surface area / pore distribution measuring device Gemini 2375 (manufactured by Micromeritics).

In the present invention, the contaminant removal material preferably has a specific surface area in the range of 100 to 2500 m ² / g. More preferably 500~2000m ² / g, more preferably from 900~2000m ² / g. If the specific surface area is too small, impurities in the biological sample may not be sufficiently removed, or the immunochromatographic test piece needs to be made larger than necessary. If the specific surface area is too large, the strength of the single fiber will be increased. It may be significantly reduced and the form may not be maintained. The specific surface area means a specific surface area determined by a BET method using a nitrogen gas adsorption isotherm at a liquid nitrogen temperature, and can be measured using, for example, a specific surface area / pore distribution measuring apparatus Gemini 2375 (manufactured by Micromeritics). .

According to the study of the present inventors, in a bilirubin removal test using activated carbon fibers having a total pore volume of 0.42 cc / g and a specific surface area of 973 m ² / g, approximately 2 mg of bilirubin per 50 mg of activated carbon fibers. It was found that can be removed.

  In the present invention, when a porous body is used as a contaminant removal material, the average pore diameter is preferably 10 to 35 mm. More preferably, it is 10-30cm, More preferably, it is 10-25cm. If the average pore diameter is too small, contaminants do not sufficiently enter the pores (mesopores, micropores), and the desired removability cannot be exhibited. On the other hand, the average pore diameter is too large to cause a problem, but the upper limit is about 35 mm.

  In the present invention, when the contaminant removal material is in a fiber shape, the single fiber diameter is preferably 5 to 30 μm. If the single fiber diameter is too small, sufficient effective pores (mesopores, micropores) for removing contaminants may not be ensured. On the other hand, if the single fiber diameter is too large, compactness may be impaired. When the contaminant removal material is in the form of particles or powder, the average particle diameter is preferably 1 nm to 1 mm. If the average particle size is too small, sufficient effective pores (mesopores, micropores) for removing contaminants may not be ensured. On the other hand, if the average particle size is too large, the compactness may be impaired.

  In the present invention, contaminants mainly target bilirubin and milk. Bilirubin is a substance having a yellow color tone present in blood and urine, and has an absorption at a wavelength of about 350 to 600 nm. On the other hand, in milk, fat that is ingested as a meal is not decomposed so much and remains in the blood, which causes the biological sample to become cloudy.

  In the present invention, the sample pad is a part into which a biological sample is introduced, and is not limited as long as it is a material and a form that absorbs a liquid biological sample in a state of being molded into the pad and can be developed by capillary action. Examples thereof include porous materials such as glass fiber, acrylic fiber, cellulose filter paper and nonwoven fabric, polyethylene, polypropylene, and ethylene / vinyl acetate copolymer.

  In the present invention, the conjugate pad is impregnated with a detection reagent that specifically reacts with an analyte in a biological sample and dried. The conjugate pad only needs to have a function of forming a complex of the detection reagent and the analyte when the biological sample passes through the conjugate pad. For example, the conjugate pad may be glass fiber, cellulose filter paper, polyester, etc. And non-woven fabrics such as acrylonitrile copolymer or rayon.

  In the present invention, the membrane has a test line in which an antibody to the analyte is immobilized in a spot shape or a line, and a control line in which an antibody to the analyte is immobilized in a spot shape or a line. . Examples of such a membrane include cellulose, cellulose derivatives, nitrocellulose, cellulose acetate, polyurethane, polyester, polyethylene, polyvinyl chloride, polyvinylidene fluoride, and nylon. Membranes, fabrics, fibrous or non-woven matrices made of these materials are suitable.

  In the present invention, the absorbent pad only needs to be able to rapidly absorb and hold a biological sample, and is porous such as glass fiber, acrylic fiber, cellulose filter paper or nonwoven fabric, polyethylene, polypropylene, or ethylene / vinyl acetate copolymer. There is a mass.

  In the present invention, the reason why the immunochromatographic test piece constituted using the above-mentioned material does not remove hemoglobin but exhibits the selectivity of removing bilirubin is not clear. However, as will be described later, the absorbance does not substantially decrease before and after the treatment when the hemoglobin-dissolved solution is brought into contact with the contaminant removal material. However, when the solution in which the bilirubin is dissolved is brought into contact with the contaminant removal material, the absorbance increases. It is clear that contaminants such as bilirubin are selectively removed from the decrease.

  The immunochromatographic test piece of the present invention is a kit housed in a plastic housing case having an opening for introducing a biological sample into the most upstream part, a membrane test line, a control line, and an opening for measuring hemoglobin. Offered as. In addition, the immunochromatographic test piece has an opening for introducing a biological sample into the most upstream part, an opening for measuring hemoglobin at a part of the sample pad, an opening for measuring a membrane test line, and a control line. Provided as a kit housed in a plastic housing case. In addition, these kits may have an air vent opening in the absorbent pad portion.

  With the kit configuration as described above, it is possible to measure hemoglobin at the membrane portion, and it is also possible to measure hemoglobin at the sample pad portion. Any housing case can be employed depending on the application and purpose. Needless to say, when measuring hemoglobin at the sample pad portion, an opening for measuring hemoglobin is provided downstream of the contaminant removal material.

  The present invention envisages measuring the hemoglobin concentration in a biological sample using absorptiometry. Since hemoglobin A1c (%) is calculated as hemoglobin A1c concentration / hemoglobin concentration × 100, it is difficult to obtain an accurate numerical value if the denominator includes an error. By adopting the present invention, a more accurate value can be obtained.

(Measurement of total pore volume)
About 30 mg of an activated carbon sample was collected, vacuum-dried at 120 ° C. for 12 hours, weighed, and measured using a specific surface area / pore distribution measuring device Gemini 2375 (manufactured by Micromeritics). The adsorption amount of nitrogen gas at the boiling point of liquid nitrogen (-195.8 ° C.) was measured in the range of relative pressure of 0.02 to 0.95, and the adsorption isotherm of the sample was created. The total pore volume (unit: cc / g) was calculated from the result at a relative pressure of 0.95.

(Measurement of specific surface area)
About 30 mg of an activated carbon sample was collected, vacuum-dried at 120 ° C. for 12 hours, weighed, and measured using a specific surface area / pore distribution measuring device Gemini 2375 (manufactured by Micromeritics). The adsorption amount of nitrogen gas at the boiling point of liquid nitrogen (-195.8 ° C.) was measured in the range of relative pressure of 0.02 to 0.95, and the adsorption isotherm of the sample was created. Based on the results in the relative pressure range of 0.02 to 0.15, the specific surface area per unit weight (unit: m ² / g) was determined by the BET method.

(Calculation of average pore diameter)
The average pore diameter (unit: Å) was determined by dividing the total pore volume (unit: cc / g) by the specific surface area (unit: m ² / g) and multiplying it by 40000.

[Example 1]
As a contaminant removal material, activated carbon fiber having a specific surface area of 973 m ² / g and total pore volume of 0.42 cc / g obtained by carbonizing and activating phenol-based fibers, sample pad (trade name: PIORAS sheet EVA (Aion) Co., Ltd.), conjugate pad (trade name: Glass Fiber Diagnostic Pad GFDX (manufactured by Merck Millipore)), membrane (trade name: Hi-Flow Plus type HF075 (manufactured by Merck Millipore)), absorption pad (trade name) : Cellulose Fiber Sample Pads CFSP (manufactured by Merck Millipore)), each end is overlapped about 1 mm in order and is successively connected on the backing sheet, and the width is about 3 mm and the length is about 60 m. The immunochromatography test piece 1 was prepared. The weight of the activated carbon fiber used was 8 mg.

  The following experiment was conducted using the prepared immunochromatographic test piece 1. Specifically, interference check A plus hemolyzed hemoglobin and bilirubin F were dissolved in distilled water to 135 mg / mL and 2 mg / mL, respectively, and then diluted (1% Triton X-100, 1% Tween-20, 50 mM PBS , PH 7.1) was diluted 500 times to obtain a sample solution. 150 μL of the obtained sample solution was dropped in the vicinity of the most upstream part of the test piece 1. After confirming that the tip of the sample solution reached the absorption pad by capillary action, the reflection absorbance at a wavelength of 420 nm irradiated on the membrane portion was measured. For the measurement of reflection absorbance, an immunochromatographic reader (C10060-10, manufactured by Hamamatsu Photonics) was used. The results are shown in Table 1.

[Example 2]
As in Example 1, except that activated carbon fiber having a specific surface area of 1962 m ² / g and a total pore volume of 0.85 cc / g obtained by carbonizing and activating phenol-based fibers was used as a contaminant removal material. Thus, an immunochromatographic test piece 2 was prepared. The weight of the activated carbon fiber used was 8 mg.

  In the same manner as in Example 1, the sample solution was dropped in the vicinity of the most upstream part of the test piece 2, and the reflection absorbance was measured at the membrane part. The results are shown in Table 1.

[Example 3]
Using the same material as in Example 2, an immunochromatographic test piece 3 as shown in FIG. 3 was produced. The weight of the activated carbon fiber used was 10 mg.

  In the same manner as in Example 1, the sample solution was dropped near the uppermost stream portion of the test piece 3, and the reflection absorbance was measured at the membrane portion. The results are shown in Table 1.

[Example 4]
Using the same material as in Example 2, an immunochromatographic test piece 4 as shown in FIG. 5 was produced. The weight of the activated carbon fiber used was 18 mg. In this example, since the sample pad was substituted with activated carbon fiber, the substantial sample pad was not used.

  In the same manner as in Example 1, the sample solution was dropped near the most upstream part of the test piece 4, and the reflection absorbance was measured at the membrane part. The results are shown in Table 1.

[Example 5]
As in Example 1, except that activated carbon fibers having a specific surface area of 1840 m ² / g and a total pore volume of 0.71 cc / g obtained by carbonizing and activating phenol-based fibers were used as the contaminant removal material. Thus, an immunochromatographic test piece 5 was produced. The weight of the activated carbon fiber used was 8 mg.

  In the same manner as in Example 1, the sample solution was dropped in the vicinity of the most upstream part of the test piece 5, and the reflection absorbance was measured at the membrane part. The results are shown in Table 1.

[Example 6]
As in Example 1, except that activated carbon fiber having a specific surface area of 1677 m ² / g and a total pore volume of 0.80 cc / g obtained by carbonizing and activating the pitch fiber was used as a contaminant removal material. Thus, an immunochromatographic test piece 6 was produced. The weight of the activated carbon fiber used was 8 mg.

  In the same manner as in Example 1, the sample solution was dropped in the vicinity of the most upstream part of the test piece 6, and the reflection absorbance was measured at the membrane part. The results are shown in Table 1.

[Comparative Example 1]
An immunochromatographic test piece 7 (FIG. 8) was prepared in the same manner as in Example 1 except that no contaminant removal material was used.

  In the same manner as in Example 1, the sample solution was dropped near the most upstream part of the test piece 7, and the reflection absorbance was measured at the membrane part. The results are shown in Table 1.

[Reference Example 1]
Interference check A plus hemolyzed hemoglobin is dissolved in distilled water to 135 mg / mL, and then diluted 500 times with a diluent (1% Triton X-100, 1% Tween-20, 50 mM PBS, pH 7.1). A sample solution was obtained. 150 μL of the obtained sample solution was dropped in the vicinity of the most upstream part of the test piece 7, and the reflection absorbance was measured at the membrane part in the same manner as in Example 1. The results are shown in Table 1.

[Reference Example 2]
A sample solution similar to that in Reference Example 1 was prepared, dropped near the uppermost stream portion of the test piece 1, and the reflection absorbance was measured at the membrane portion in the same manner as in Example 1. The results are shown in Table 1.

[Reference Example 3]
Interference check A plus bilirubin F is dissolved in distilled water to 2 mg / mL, and then diluted 500 times with a diluent (1% Triton X-100, 1% Tween-20, 50 mM PBS, pH 7.1). A sample solution was obtained. 150 μL of the obtained sample solution was dropped in the vicinity of the most upstream part of the test piece 7, and the reflection absorbance was measured at the membrane part in the same manner as in Example 1. The results are shown in Table 1.

[Reference Example 4]
A sample solution similar to that in Reference Example 3 was prepared and dropped in the vicinity of the most upstream portion of the test piece 1, and the reflection absorbance was measured at the membrane portion in the same manner as in Example 1. The results are shown in Table 1.

  From the results of Reference Examples 1 and 2, even if a sample solution in which hemoglobin is dissolved is dropped onto an immunochromatographic test piece provided with a contaminant removal material, no decrease in absorbance is observed. On the other hand, from the results of Reference Examples 3 and 4, when a sample solution in which bilirubin was dissolved was dropped onto an immunochromatographic test piece provided with a contaminant removal material, the absorbance was greatly reduced. This clearly shows that the immunochromatographic test strip of the present invention does not remove hemoglobin but removes bilirubin.

  As for the result of Example 1-6, the light absorbency of wavelength 420nm is falling compared with the comparative example 1. FIG. This is because bilirubin was removed from the sample solution in contact with the contaminant removal material, as is apparent from the results of Reference Example 1-4. Hemoglobin A1c (%) is an index for diagnosing diabetes obtained by hemoglobin A1c concentration / hemoglobin concentration × 100, and accurate measurement cannot be performed if the measured value of hemoglobin concentration includes an error. The present invention shows that a more accurate hemoglobin concentration can be measured.

  According to the present invention, the hemoglobin concentration can be quantitatively measured without being affected by contaminants. In addition, since the influence of contaminants is reduced, measurement in the maximum absorption wavelength region of hemoglobin is possible, and even when the concentration of hemoglobin in a biological sample is low, it can be measured with high accuracy. Is preferred.

1. 1. Removal material for contaminants 2. Sample pad Conjugate pad 4. Membrane 5 Absorption pad 6. Adhesive sheet (backing sheet)

Claims (4)

  An immunochromatographic test piece in which a sample pad, a conjugate pad, a membrane, and an absorption pad are connected in order, and an immunochromatographic test piece comprising a contaminant removal material upstream of the membrane.   The immunochromatographic test piece according to claim 1, wherein the removal material is one or more selected from the group consisting of fibers, woven fabrics, nonwoven fabrics, particles, and powders.   The said removal material is 1 or more types chosen from the group which consists of activated carbon, activated carbon fiber, a titanium oxide, a divinylbenzene / glycidyl methacrylate copolymer, and hydrophobic silica, The Claim 1 or 2 characterized by the above-mentioned. Immunochromatographic test piece.   The immunochromatographic test piece according to any one of claims 1 to 3, wherein the contaminant is bilirubin or milk.