JP6103776B2 - Method for measuring hematocrit value - Google Patents

Description

  The present invention relates to a method for measuring an object to be measured in a sample obtained by diluting a blood specimen using immunochromatography. In particular, the present invention relates to the measurement method including a step of calculating a hematocrit value by measuring a reflected light intensity of a member that has absorbed a sample, and correcting the measurement value of the measurement object.

  In recent years, in clinics and small hospitals, the need to “perform various tests while examining patients” has increased, and from the conventional outsourced test, the test by Point of Care Testing (POCT) has been increased. It is getting done. A typical example of a reagent used for such POCT is a lateral flow type immunochromatographic test strip. Common immunochromatographic test strips include a test line for measuring an object to be measured and a control line indicating that a reaction has been established. The complex of the measurement target and the label on which the antibody against the measurement target is immobilized is captured by the antibody immobilized on the test line of the insoluble membrane to form the test line, and is not captured by the test line. The remaining label is captured by the control line anti-immunoglobulin antibody to form a control line.

  Measurement methods using immunochromatography include a qualitative method for visually determining the color development of the test line and a quantitative method for measuring the color intensity of the test line with a dedicated device. Corresponding immunochromatographic test strips and test devices are used.

  In the quantitative method of measuring the color intensity such as the test line of immunochromatographic test strips with a dedicated device, various pretreatments may be performed depending on the type of sample, amount of sample used, measurement principle, etc. is there. When supplying a sample to a test strip or test device, the sample may be dropped as it is, when the sample is diluted with a dedicated diluent and then dropped, or when a dedicated diluent is dropped after a small amount of sample is dropped. is there. When it is difficult to collect a large amount of samples, such as samples of infants and children, or when adjusting the concentration of the measurement target to a concentration suitable for measurement when the measurement target in the sample is high, The specimen is often diluted with a dedicated diluent.

  Normally, the concentration of blood components is measured using serum or plasma separated from whole blood as a sample, but in order to obtain serum or plasma from whole blood, a device such as a centrifuge is required and takes time. . Therefore, measurement using whole blood is desired in the POCT region. When measuring a sample obtained by diluting whole blood to hemolyze, the measurement result is obtained by diluting the blood cell volume, unlike the case of measuring serum or plasma. Therefore, it is necessary to correct the measurement result with a hematocrit value (ratio of the volume of blood cells in the blood). Hereinafter, correcting the measurement value of the measurement object in the blood sample using the hematocrit value may be simply referred to as “hematocrit correction”.

  Conventionally, correction for uniformly multiplying the measurement result by a correction coefficient obtained from an average hematocrit value in a healthy person has been performed for the above problem. However, the hematocrit value varies depending on the individual, and the standard value ranges from 39 to 52% for men and 35 to 48% for women. Therefore, an accurate concentration of the measurement object cannot be obtained by the conventional correction that is multiplied by a uniform coefficient. In order to measure the exact concentration of the measurement object, it is necessary to correct the concentration of the measurement object with the hematocrit value of the same sample as the sample from which the measurement object concentration was measured.

The hematocrit value has heretofore been obtained by calculation from the red blood cell count and the average red blood cell volume using a microhematocrit method by centrifugation or an automatic blood cell counter.
As another method, in the measurement that converts the measurement result obtained from whole blood into a measurement value when serum or plasma is measured, the hemoglobin concentration (g / dl) in the whole blood is measured and obtained. A method has been reported in which a value obtained by multiplying the obtained hemoglobin concentration by about 3 times is used as the hematocrit value (%), and that the hematocrit value is used to convert the measurement result of whole blood into the measurement value when serum or plasma is measured. (Patent Document 1).
However, even in these methods, the hemoglobin concentration and the hematocrit value must be obtained by a method different from the measurement of the concentration of the measurement object by the immunochromatography method, which is complicated, time-consuming, and costly. It cannot satisfy your needs.

  Patent Document 2 discloses a method of measuring a hematocrit value in a control line of a test strip in a measurement method using an immunochromatographic test strip. However, since this method uses an immune reaction, there is a problem that an antibody for obtaining a hematocrit value is further required in addition to an antibody for measuring a measurement object.

JP 2001-272403 A International publication 2011/125877 pamphlet

An object of the present invention is to provide a measurement method comprising a step of calculating a hematocrit value with good reproducibility in a method for measuring a measurement object in a sample obtained by diluting a blood sample using immunochromatography. There is.
It is another object of the present invention to provide a test device that embodies the measurement method.

In the method of measuring an object to be measured in a sample obtained by diluting a blood specimen using immunochromatography, the present inventors reflect the reflection of a member holding (filtering or adsorbing) a hemoglobin component in the sample. Hemoglobin (Hb concentration in a blood sample can be measured using light intensity, and hematocrit value is calculated from the Hb concentration, and it is confirmed that hematocrit correction of the measurement value of the measurement object can be performed. The present invention has been completed, that is, the present invention has the following configuration.
[1] A measurement method for measuring an object to be measured in a sample obtained by hemolyzing a blood sample, using a test strip for immunochromatography,
The immunochromatographic test strip has the following 1) to 3):
1) A sample pad that absorbs a sample and retains a hemoglobin component in the sample. 2) It is located downstream of the sample pad and contains a conjugate in which a first antibody against the measurement target is immobilized on a label. Conjugate pad 3) The measurement method comprising an insoluble membrane having a test line on the downstream side of the conjugate pad and having a second antibody against the measurement target immobilized thereon, and the following steps A) and B).
A) A step of supplying a sample to the sample pad, optically detecting hemoglobin held in the sample pad to obtain a hematocrit value of a blood sample, and B) a step of measuring an object to be measured in a test line on an insoluble membrane [ 2] The measuring method according to [1], further comprising the following step C).
C) Step of correcting the measurement value of the measurement object in step B) by hematocrit using the hematocrit value obtained in step A) [3] Immunochromatography for measuring the measurement object in the sample in which the blood sample is hemolyzed A test device in which a test strip is housed in a housing,
The immunochromatographic test strip has the following 1) to 3):
1) A sample pad that absorbs a sample and retains a hemoglobin component in the sample. 2) It is located downstream of the sample pad and contains a conjugate in which a first antibody against the measurement target is immobilized on a label. Conjugate pad 3) An insoluble membrane located on the downstream side of the conjugate pad and having a test line on which a second antibody against the measurement target is immobilized, and a housing having the following 4) and 5).
4) Sample detection window for optically detecting the hemoglobin component held on the sample pad 5) Test line detection window for optically detecting the test line

By using the measurement method of the present invention, a measurement system in a sample obtained by diluting a blood sample is measured using immunochromatography, and a new measurement system is constructed or a complicated measurement operation is performed. Without doing so, the hematocrit value is determined.
In the measurement method of the present invention, since the measurement object and hemoglobin in the same sample are measured by the same immunochromatography, the concentration of the measurement object in the sample can be corrected based on the hematocrit value of the same sample, The concentration of the measurement object can be measured more accurately.
Furthermore, since the measurement method of the present invention measures the hemoglobin concentration in the sample by the reflected light intensity, it is not necessary to prepare an antibody for measuring the hemoglobin concentration, and the cost is reduced. In addition, it is possible to avoid the risk of divergence of hemoglobin measurement values in the presence of non-specific substances, and more accurate hematocrit correction of the measurement object is possible.
Moreover, the test device of the present invention is suitably used for the above-described measurement method.

It is a schematic diagram which shows the structure of the test strip for immunochromatography used by this invention.

The measurement method of the present invention will be described in detail.
The measurement method of the present invention is a method for measuring an object to be measured in a sample obtained by hemolyzing a blood sample using an immunochromatographic test strip, and the immunochromatographic test strip includes the following 1) to 3). And including the following steps A) and B).
1) Sample pad that absorbs the sample and retains the hemoglobin component in the sample 2) Contains a conjugate that is located downstream of the sample pad and in which the first antibody to the measurement target is immobilized on the label Conjugate pad 3) An insoluble membrane located downstream of the conjugate pad and having a test line on which the second antibody against the analyte is immobilized A) Sample was supplied to the sample pad and held in the sample pad A step of optically detecting hemoglobin to determine a hematocrit value of a blood sample B) A step of measuring an object to be measured in a test line on an insoluble membrane A dropping of a sample onto the sample pad of the immunochromatographic test strip comprises: A certain amount of support, as is usually done in the laboratory area. Or by any method as long as it is a method capable dropwise pull. For example, a method using a pipette or a dropper capable of dropping a certain amount of droplets can be used. Moreover, it may be dropped manually or a device capable of automatic operation may be used.
As a method for optically measuring the hemoglobin concentration, a known method such as the SLS-Hb method may be used. For example, absorbance or reflected light intensity may be measured.
By extrapolating the amount of change in reflected light intensity to a calibration curve (calibration curve) of a sample having a known concentration, the concentration (Hb value) of hemoglobin is calculated.
The calibration curve of the hematocrit value can be created by converting the Hb value of the calibration curve into a hematocrit value using the general correlation (1).

Hematocrit value (%) = Hb value (g / dL) × 2.9 (1)

Next, the measurement method of the present invention will be described by taking as an example the case where the correction using the hematocrit value is applied to the CRP measurement method.
The CRP measurement method of the present invention is a method for measuring the CRP concentration in a sample obtained by hemolyzing a blood sample using an immunochromatography test strip. The immunochromatography test strip comprises the following 1) to 3): And a measurement method having the following steps A) to C).
1) A sample pad that absorbs a sample and retains a hemoglobin component in the sample. 2) A conjugate that is located downstream of the sample pad and contains a conjugate in which a first antibody against CRP is immobilized on a label. Pad 3) An insoluble membrane located downstream of the conjugate pad and having a test line on which the second antibody against CRP is immobilized (where the second antibody is monovalent when the epitope of the first antibody against CRP is monovalent) The epitope of the first antibody is different from that of the first antibody, but when the first antibody etope is multivalent, the epitope of the second antibody is the same as the first antibody, or the first antibody and the second antibody Including the same case.)
A) A step of supplying a sample to the sample pad, optically detecting hemoglobin held in the sample pad, and determining a hematocrit value of a blood sample B) A step of measuring CRP in a test line on an insoluble membrane C) step The step of correcting the CRP measurement value in step B) by hematocrit using the hematocrit value obtained in A) The measurement of CRP concentration may be performed by directly obtaining the signal change amount in the test line as the measurement value. It may be calculated from the amount of change.
The signal derived from the labeled body may be measured according to a known method. For example, when the labeled body is a colloidal gold particle, the absorbance or reflected light intensity may be measured. The amount of change in absorbance or reflected light intensity is extrapolated to a calibration curve of a sample having a known concentration to calculate the CRP concentration.
If the method of measuring the signal of the labeled body is the same as the method of measuring the signal of hemoglobin, it is desirable that the measurement system can be simplified because measurement can be performed with one measuring instrument.

In the step C), specifically, the CRP concentration measured on the test strip test line is corrected with the hematocrit value calculated from the hemoglobin value measured on the test strip sample pad. The method of correcting the CRP concentration by hematocrit is calculated as follows, as in the case of conventional hematocrit correction.

(Measurement object)
The measurement object in the present invention is a substance present in blood and requires hematocrit correction. For example, inflammation-related markers such as C-reactive protein (CRP), IgA, IgG, IgM, fibrin degradation products (eg, D dimer), soluble fibrin, thrombin-antithrombin complex (TAT), plasmin-plasmin inhibitor complex ( Coagulation / fibrinolysis markers such as PIC), oxidative LDL, circulation-related markers such as brain natriuretic peptide (BNP), metabolism-related markers such as adiponectin, carcinoembryonic antigen (CEA), α-fetoprotein (AFP), CA19- 9, tumor markers such as CA125, prostate specific antigen (PSA), hepatitis B virus (HBV), hepatitis C virus (HCV), allergen-specific immunoglobulin E (IgE), hormones, drugs and the like.

(Specimen and sample)
The specimen in the present invention is blood, and those blood specimens diluted with a specimen diluent and hemolyzed are referred to as samples. Regarding the dilution of a blood sample, a case where the measurement object is C-reactive protein (CRP) will be described in detail. The dilution of the blood sample is performed in consideration of the concentration suitable for the measurement of the measurement object. In general, the dilution factor is 2 to 10,000 times. In addition, it is desirable to use a sample that has been diluted 50 to 200 times and hemolyzed. For example, it is preferable to set the dilution rate to 100 times and the CRP measurement range to 0.2 to 20 mg / mL.
In the case of the CRP measurement method, the immunochromatographic test strip is designed so that an infant or a child is a subject to be measured, and the sample amount is 1 to 10 μL. Therefore, in order to secure a sample amount necessary for measurement by immunochromatography, the blood specimen is diluted 10 to 200 times with a specimen diluent. Moreover, when measuring Hb density | concentration by reflected light intensity, it is diluted 10 to 100 times also from the point of catching the change of coloring.

  Next, the immunochromatographic test strip and test device used in the measurement method of the present invention will be described in detail.

(Antibodies to measurement object)
An antibody against the measurement target is immobilized on a test line of a label and an immunochromatography test strip described later.
The antibody to the measurement object may be any antibody that specifically reacts to the measurement object, and is not limited in any way by the method for producing them, whether it is a polyclonal antibody or a monoclonal antibody. Good. For example, when the measurement target is human CRP, the anti-human CRP antibody may be an antibody that specifically reacts with human CRP, and is not limited by the method for producing them. It may be an antibody or a monoclonal antibody. In general, hybridomas producing such antibodies are myeloma cells (myeloma) of the same type as the spleen cells of animals immunized with human CRP according to the method of Kohler and Milstein (see Nature, Vol. 256, page 495 (1975)). Cell) and cell fusion.
Here, when the antibody used in the present invention is a monoclonal antibody, the relationship between the antibody immobilized on the label (first antibody) and the antibody immobilized on the insoluble membrane (second antibody) is as follows. When the epitope of one antibody is monovalent, the epitope of the second antibody is different from that of the first antibody. When the epitope of the first antibody is multivalent, the epitope of the second antibody is the first epitope. The same antibody may be used, and the first antibody and the second antibody may be the same antibody.
For example, in the case of a CRP measurement method using a sample obtained by diluting a blood sample to about 10 to 200 times, a combination of antibodies capable of measuring 0.2 to 20 mg / dL at a CRP concentration of about 100 times dilution is desirable.

(Sample pad)
In the present invention, a sample pad is a part that receives a sample. The sample pad may be of any material and form as long as it absorbs the sample while it is molded into the pad and allows the liquid component of the sample and the measurement target to pass through.
The sample pad holds at least a part of hemoglobin in the sample without passing it through filtration or adsorption.
In the present invention, the sample pad serves as a hemoglobin measurement site for measuring the hemoglobin concentration in the sample held without passing through the sample pad by the reflected light intensity at the same time as receiving the sample.
Suitable materials for the sample pad include, but are not limited to, glass fiber (glass fiber), acrylic fiber, hydrophilic polyethylene material, dry paper, paper pulp, and fabric. More preferably, a glass fiber pad is used. The sample pad may have the function of a conjugate pad described later. The sample pad may contain a commonly used blocking reagent for the purpose of preventing / suppressing nonspecific reaction (adsorption) on an insoluble membrane or the like.
Examples of the blocking reagent include trade name “NEO PROTEIN SAVER” (manufactured by TOYOBO), sericin, trade name “ImmunoBlock” (produced by Dainippon Pharmaceutical), trade name “ApplieBlock” (produced by Seikagaku Biobusiness), trade name “ SEA BLOCKTM / EIA / WB "(PIERC), brand name" Blocking One "(Nacalai Tesque), BSA, brand name" Blocking Peptide Fragment "(TOYOBO), brand name" StartingBlock "(PBS), brand name" From among “Blocking Buffer” (manufactured by PIERCE), trade name “SmartBlock” (manufactured by CANDOR bioscience GmbH), trade name “HeteroBlock” (manufactured by Omega Biologicals), etc., those that do not adversely affect the specific reaction itself can be selected as appropriate. .

(Conjugate)
The conjugate used in the present invention is obtained by immobilizing an antibody against the measurement target described above on a label.
Examples of the label include those conventionally used as an antibody immobilization carrier in an immunochromatography method. For example, colloidal gold particles, metallic colloidal particles such as platinum colloidal particles, color latex particles, magnetic particles, and the like are preferable, and colloidal gold particles are particularly preferable.
Although it is known that the particle size of the gold colloid particles greatly affects the sensitivity of the measurement, in the present invention, the particle size of the gold colloid particles is preferably 20 to 60 nm, particularly preferably 30 to 40 nm. The particle diameter is a value measured by a dynamic light scattering method (manufactured by Particles sizing systems, NICOMP).
The colloidal gold particles are produced by a generally known method, for example, by dropping and stirring a trisodium citrate aqueous solution into a heated tetrachlorogold (III) acid aqueous solution.
Hereinafter, the case where colloidal gold particles are used will be described in detail.

(Sensitization of antibody to label)
The conjugate is obtained by sensitizing the antibody against the measurement target to the label.
Immobilization of the antibody to the measurement object on the label, for example, immobilization of the antibody against CRP on the gold colloid particles is usually performed by physical adsorption. At this time, the antibody concentration is preferably adjusted to 0.5 μg / mL to 5 μg / mL, and the buffer and its pH are preferably 0.1 to 10 mmol / L phosphate buffer (pH 6 to 7), more 2 mmol / L phosphate buffer (pH 6-7) is preferable, and 2 mmol / L phosphate buffer (pH 7.0) is more preferable. In addition, it is preferable that the region where the antibody on the gold colloid particle is not bound is subjected to a blocking treatment by binding BSA or the like.
The conjugate thus prepared is dispersed and stored in a storage reagent containing a component for preventing denaturation (denaturation inhibitor). As this denaturation inhibitor, proteins such as BSA, glycerin, sugars and the like are used.

(Detection reagent)
In the present invention, the detection reagent is specifically a solution containing at least a conjugate.
The detection reagent keeps the conjugate in a stable state, and promotes the specific reaction of the antibody immobilized on the conjugate with the analyte (eg, CRP) when mixed with the sample, or the conjugate. For example, one or more kinds of stabilizers, solubilizing aids, and the like may be contained for the purpose of quickly and effectively dissolving and fluidizing. Examples of stabilizers and solubilizers include bovine serum albumin (BSA), sucrose, casein, and amino acids.
In the present specification, the term “detection” or “measurement” includes any of proof, qualification, quantification, and the like of the existence of the measurement object, and is not limitedly interpreted.

(Conjugate pad)
In the present invention, the conjugate pad is obtained by impregnating a detection reagent containing a conjugate that specifically reacts with a measurement object into a material suitable for a conjugate pad described later and drying it. The conjugate pad has a function of forming a complex between the conjugate and the measurement target when the sample passes through the conjugate pad.
The conjugate pad may be disposed so as to contact the insoluble membrane alone. Alternatively, a sample that is placed in contact with the sample pad and that has passed through the sample pad by capillary flow is received, and the sample is subsequently contacted with the conjugate pad by a capillary flow on a surface that is different from the contact surface with the sample pad. May be arranged so as to be transferred to the pad (hereinafter referred to as “3rd pad”). Note that how the sample pad, the conjugate pad, and the like are arranged on the insoluble membrane can be appropriately changed.
Suitable materials for the conjugate pad include, but are not limited to, paper, cellulose blends, nitrocellulose, polyester, acrylonitrile copolymers, non-woven fibers such as glass fibers (glass fibers) or rayon. Preferably, a fiberglass pad is used.
The conjugate pad also keeps the conjugate stable and promotes a specific reaction with the analyte (eg CRP) in the sample when the conjugate comes into contact with the sample, or is quick and effective. For the purpose of dissolution and fluidization, for example, one or more kinds of stabilizers, solubilizing agents and the like may be contained. Examples of stabilizers and solubilizers include bovine serum albumin (BSA), sucrose, casein, amino acids and the like. In particular, the anti-CRP antibody, in the Ca ²⁺ ions presence and absence may reactivity differ greatly, in order to control the reactivity, is the chelating agent suitable Ca ²⁺ ions in the conjugate pad EDTA, EGTA, etc. may be included. On the contrary, for the purpose of adding Ca ²⁺ ions, calcium salts such as CaCl ₂ may be added.

(3rd pad)
In the present invention, it is preferable to place a 3rd pad between the sample pad and the insoluble membrane. The 3rd pad may be located either between the sample pad and the conjugate pad, or between the conjugate pad and the insoluble membrane, but is preferably between the conjugate pad and the insoluble membrane.
The 3rd pad is disposed for the purpose of removing components unnecessary for the measurement of the measurement target that can be included in the sample and allowing the components necessary for the measurement to smoothly develop the insoluble membrane. For example, blood cells or insoluble blood cell fragments in a sample are components unnecessary for measurement of a measurement object such as CRP, and therefore it is desirable to remove them with a 3rd pad.
The 3rd pad can also have an additional function of previously removing aggregates that have become so large that the insoluble membrane cannot be developed smoothly among aggregates generated by the antigen-antibody reaction. .
The 3rd pad may be of any substance and form as long as the liquid component of the sample and the measurement target can pass through. Specific examples of the substance constituting the 3rd pad include, but are not limited to, glass fiber (glass fiber), acrylic fiber, hydrophilic polyethylene material, dry paper, paper pulp, and woven fabric. Preferably, a blood cell separation membrane or a similar membrane is used.

(Insoluble membrane)
In the present invention, an insoluble membrane (hereinafter sometimes simply referred to as “membrane”) may be made of any material. Examples thereof include, but are not limited to, polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, and ceramics. Specific examples include glass fiber filter paper and cellulose filter paper sold by Millipore, Toyo Filter, and Whatman. In addition, by appropriately selecting the pore size and structure of the insoluble membrane, it is possible to control the speed at which the immune complex of the conjugate and the measurement target flows through the membrane. By controlling the flow rate through the membrane, the amount of immune complex bound to the antibody immobilized on the membrane test line can be adjusted, so the pore size and structure of the membrane can be controlled by other immunochromatographic test strips. It is desirable to optimize in consideration of the combination with the constituent material of the part.

(Immobilization of antibody on insoluble membrane)
A test line for measuring the measurement object is formed on the insoluble membrane, and an antibody against the measurement object is immobilized on the test line.
Immobilization of the antibody to the measurement object on the insoluble membrane can be performed by a known method. For example, when the immunochromatographic test strip is of the lateral flow type, an apparatus having a mechanism capable of preparing the antibody to a predetermined concentration and moving it horizontally while discharging the liquid from the nozzle at a constant speed, etc. Is applied to an insoluble membrane in a line shape.
In this case, the concentration of the antibody is preferably 0.1 mg / mL to 5 mg / mL, and more preferably 0.5 mg / mL to 2 mg / mL. In addition, the amount of immobilized antibody insoluble membrane can be optimized by adjusting the ejection speed from the nozzle of the above-described apparatus in the case of the lateral flow type. In the case of the lateral flow type, 0.5 μL / cm to 2 μL / cm is preferable. In the present invention, the lateral flow method is a method in which a sample solution or the like is developed so as to move in a parallel direction with respect to the insoluble membrane, and the flow-through method is a method in which the sample solution or the like is perpendicular to the insoluble membrane. This is a method of expanding to pass through.
Further, in the present invention, the position of the test line, that is, the position where the antibody to the measurement object is applied to the insoluble membrane is not particularly limited, and in the case of a lateral flow type, from the conjugate pad, the measurement object It is only necessary that the immunocomplex of conjugated with the conjugate develops by capillary action and passes through the test line.
The insoluble membrane is preferably provided with a control line for confirming the completion of the measurement. An antibody that captures the developed conjugate is immobilized on the control line. Examples of such an antibody include an anti-mouse IgG antibody.
Preferably, the test line to which the antibody for the measurement target is applied is upstream, and the control line to which the anti-mouse IgG antibody is applied is preferably positioned downstream thereof. At this time, it is desirable that the distance between the lines is sufficiently long so that the signal of the label can be detected. Even in the case of the flow-through type, the position where the antibody is applied to the measurement target may be arranged so that the signal of the label can be detected.
The antibody solution to be applied to the insoluble membrane can usually be prepared using a predetermined buffer solution. Examples of the buffer solution include a buffer solution usually used such as a phosphate buffer solution, a Tris buffer solution, and a Good buffer solution. The pH of the buffer is preferably in the range of 6.0 to 9.5, more preferably 6.5 to 8.5, and even more preferably 7.0 to 8.0. The buffer may further contain salts such as sodium chloride, stabilizers such as sucrose, preservatives such as procrine, and the like. The salts include those added in the step of adjusting the pH of the buffer solution such as sodium hydroxide in addition to those included for adjusting the ionic strength such as sodium chloride.
After immobilizing the antibody on the insoluble membrane, blocking can also be carried out by coating a portion other than the antibody immobilization site with a commonly used blocking agent in the form of a solution or mist. In the present specification, the insoluble membrane on which the antibody is immobilized as described above may be referred to as “antibody-immobilized membrane”.
In the present invention, the sample pad, the conjugate pad, and the insoluble membrane may be arranged in this order from upstream to downstream where the sample is developed, and whether they are the same pad or individual pads. It doesn't matter. That is, the sample pad and the conjugate pad may be the same pad, the upstream portion of this pad may be the sample pad, and the downstream portion may be the conjugate pad. The conjugate pad and the insoluble membrane may be the same pad, and the upstream portion of the pad may be a conjugate pad and the downstream portion may be an insoluble membrane. Furthermore, the sample pad, the conjugate pad, and the insoluble membrane may be the same pad, and the upstream portion of the pad may be a sample pad, the midstream portion may be a conjugate pad, and the downstream portion may be an insoluble membrane. In general, each is often a separate pad.

(Absorption pad)
It is preferable that an absorption pad is formed on the immunochromatographic test strip.
In the present invention, the absorption pad is a part having liquid absorbency that controls the development of the sample by absorbing the sample that has moved and passed through the insoluble membrane. In the lateral flow type, it may be provided at the most downstream side of the test strip. In the flow through type, for example, it may be provided at the lower part of the antibody-immobilized membrane.
For example, filter paper can be used as the absorbent pad, but the absorbent pad is not limited to this. Preferably, Whatman 740-E or the like is used.

(Test strip for immunochromatography)
In the present invention, the immunochromatographic test strip is sufficient if it contains at least a sample pad, a conjugate pad, and an insoluble membrane on which an antibody against a measurement target is immobilized, and if necessary, reagent components and other membranes. Etc. Other membranes include 3rd pads, absorption pads, and the like.
The immunochromatographic test strip is usually arranged on a solid support such as a plastics adhesive sheet. It is preferable that the solid support is made of a substance that does not hinder the capillary flow of the sample, and the adhesive component is a substance that does not hinder the capillary flow of the sample. A polyester film or the like can be laminated for the purpose of increasing the mechanical strength of the antibody-immobilized membrane and preventing evaporation (drying) of moisture during the assay.

(Test device)
The test device of the present invention includes a sample detection window for optically detecting a hemoglobin component held at least on the sample pad by the test strip for immunochromatography, and a test line for optically detecting the test line. It is stored in a housing having a detection window.
As the housing, an appropriate container (housing) is used in consideration of the size of the test strip, the sample addition method / position, the antibody immobilization position on the antibody-immobilized membrane, the signal detection method, and the like.
The housing is provided with a sample addition window, a sample detection window, and a test line detection window, but the sample addition window and the sample detection window are the same as long as they can play their respective roles. It may be a different window or a different window.
The sample addition window is formed to supply a sample to the immunochromatographic test strip, and the shape, formation site, etc. are not limited as long as it plays a role.
The sample detection window is formed to optically detect the hemoglobin component held on the sample pad, and the shape, formation site, etc. are not limited as long as it plays a role.
The test line detection window part is formed to measure the measurement object on the test line, and the shape, formation site, and the like are not limited as long as it plays a role.

(Sample dilution liquid)
The specimen diluent used in the present invention significantly inhibits the antigen-antibody reaction between the measurement object and the antibody against it, or conversely promotes the reaction significantly, resulting in excessive aggregation of the label and poor development due to capillary action. As long as it is possible to detect the signal of the antigen-antibody reaction according to the concentration of the measurement object, a diluent having any composition may be used. Examples of such a diluent include purified water and a low concentration buffer having a pH of 6.0 to 10.0. Examples of the low-concentration buffer include 10 mmol / L to 20 mmol / L phosphate buffer, 10 mmol / L to 20 mmol / L Tris hydrochloride buffer, 10 mmol / L to 20 mmol / L glycine hydrochloride buffer, and the like.
In addition, a surfactant may be added to these diluted solutions for the purpose of controlling the developing speed of the sample solution on the test strip. In particular, the measurement range can be adjusted by including Tween 20 (manufactured by Kishida Chemical Co., Ltd.) in the diluent. Since a preferable concentration response curve is obtained, it is preferable to add 0.01 to 0.1% by weight of Tween20. When this diluted solution is used, a desirable dilution ratio is 10 to 50 times. In addition, EDTA or EGTA, which is a chelating agent for Ca ²⁺ ions, may be included in the diluent.

  Next, the present invention will be specifically described with reference to examples in which the measurement object is CRP, but these do not limit the scope of the present invention.

[Test Example 1] 1 Preparation Example of Test Strip for Immunochromatography of the Present Invention 1) Preparation of Colloidal Gold Labeled Anti-CRP Antibody (Conjugate) Anti-CRP Monoclonal Antibody (Clone: 08207) 2 mmol / L Phosphate Buffer (pH 7) 0.0) to an antibody solution having an antibody concentration of 20 μg / mL, 1 mL of the antibody solution was added to 20 mL of 1 OD / mL colloidal gold particle (particle size 30 nm) solution, and the mixture was stirred at room temperature for 10 minutes. Add 2 mL of 5% by weight bovine serum albumin (BSA) aqueous solution to the colloidal gold particle-antibody mixture, stir for 5 minutes, and then centrifuge at 10,000 rpm for 45 minutes at 10 ° C., and precipitate (conjugate) Got. To the resulting conjugate, 1.2 mL of Conjugate Dilution Buffer (Scripps) was added to suspend the conjugate.
The absorbance at the maximum absorption wavelength of each conjugate was measured.
For convenience, the antibodies are represented by clone names of the hybridomas that produce them (the same applies hereinafter).

2) Preparation of conjugate pad The conjugate prepared in 1) above was diluted with 20 mmol / L Tris-HCl buffer (pH 7.2) containing 15 OD / mL, 3 wt% BSA, and 4 wt% sucrose. A gate solution was prepared. This was impregnated into a glass fiber pad (Nippon Pole Co., No. 8964) having a constant volume by 1.2 times the pad volume. The conjugate pad was dried by heating at 70 ° C. for 30 minutes in a dry oven. Moreover, when adding additives, such as a sensitizer, as needed, after adding a required quantity to the said conjugate solution, what is necessary is just to perform the same operation.

3) Preparation of anti-CRP antibody-immobilized membrane An anti-CRP monoclonal antibody (Clone: 08210) was prepared as 1 mmol / L phosphate buffer (pH 7.2) containing 2.5 wt% sucrose so as to be 1 mg / mL. Then, an anti-CRP monoclonal antibody (test line) is placed inside one end of the short side of the nitrocellulose membrane (Millipore, HF240 or HF180), and an anti-immunoglobulin antibody (control line) at an interval of about 5 mm, Using an immunochromatographic dispenser “XYZ3050” (BIO DOT), coating was performed in a line shape so as to be 0.75 μL / cm. The membrane was dried in a dry oven at 70 ° C. for 45 minutes to obtain an antibody-immobilized membrane.

4) Preparation of sample pad Glass fiber pad obtained by cutting 20 mmol / L Tris-HCl buffer (pH 7.2) containing 24 mmol / L sodium chloride, 0.5 wt% sucrose and 30 mmol / L ethylenediaminetetraacetic acid into a constant volume. (Lydall) was impregnated with 1.15 times the volume of the pad. The sample pad was dried at 70 ° C. for 45 minutes in a dry oven.

5) Preparation of test strip The above antibody-immobilized membrane (b) is attached to a plastics adhesive sheet (a), the anti-CRP monoclonal antibody (test line) (c) on the upstream side of the development, and then the anti-immunoglobulin antibody (control) The application part was arrange | positioned in order of line) (d1), and also 3rd pad (i) which consists of a glass fiber pad was mounted | worn. Next, the conjugate pad (e) prepared in 2) above is placed and mounted, and the sample pad (f) prepared in 4) above is placed and mounted so as to overlap this conjugate pad, and the opposite end is placed on the opposite end. An absorbent pad (g) was placed and mounted. Finally, a polyester film (h) was placed and mounted on the upper surface so as to cover the antibody-immobilized membrane and the absorption pad, and laminated. In this way, a test strip was produced by cutting each structural element into a stacked structure. The test strip is housed and mounted in a dedicated plastic housing (having a sample addition window, a sample detection window, and a test line detection window, not shown in FIG. 1) during the assay, It was in the form of FIG. 1 shows a schematic configuration diagram of a test strip.

6) Preparation of specimen dilution liquid Preservative was added to 10 mmol / L phosphate buffer (pH 7.2) containing 0.01% by weight of Tween 20 as a final concentration, and the liquid filtered through a 0.45 μm filter was used as the specimen of this reagent. Diluted solution.

[Test Example 2] 1 Preparation Example of Conventional Immunochromatographic Test Strip 1) Antibody-sensitized conjugate (conjugate in which anti-CRP monoclonal antibody is immobilized on colloidal gold particles) and anti-hemoglobin antibody-sensitized conjugate ( Preparation of anti-hemoglobin monoclonal antibody immobilized on gold colloid particles) Anti-CRP monoclonal antibody (Clone: 08207) and anti-hemoglobin monoclonal antibody (Clone: 69202) The antibody concentration was adjusted, 1 mL each was added to 20 mL of 1 OD / mL colloidal gold particle (particle size 30 nm) solution, and the mixture was stirred at room temperature for 10 minutes. 2 mL of a 5 wt% bovine serum albumin (BSA) aqueous solution is added to the gold colloid particle-each antibody mixture, and the mixture is further stirred for 5 minutes, and then centrifuged at 10,000 rpm for 45 minutes at 10 ° C. CRP antibody-sensitized conjugate and anti-hemoglobin antibody-sensitized conjugate) were obtained. 1.2 mL of Conjugate Dilution Buffer (manufactured by Scripts) was added to each of the obtained conjugates to suspend the conjugate. The absorbance at the maximum absorption wavelength of each conjugate was measured.
i) Clone: 08202 (80 μg / mL), 2 mmol / L borate buffer, pH 9.0
ii) Clone: 69202 (140 μg / mL), 2 mmol / L borate buffer, pH 9.0

2) Preparation of conjugate pad 3 wt% BSA, 4 wt so that the anti-CRP antibody sensitized conjugate prepared in 1) above is 15 OD / mL, and the anti-hemoglobin antibody sensitized conjugate is 5 OD / mL. A conjugate solution was prepared by mixing with 20 mmol / L Tris-HCl buffer (pH 7.2) containing a% sucrose solution. This was impregnated into a glass fiber pad (Nippon Pole Co., No. 8964) having a constant volume by 1.2 times the pad volume. The conjugate pad was dried by heating at 70 ° C. for 30 minutes in a dry oven. Moreover, when adding additives, such as a sensitizer, as needed, after adding a required quantity to the said conjugate solution, what is necessary is just to perform the same operation.

3) Preparation of insoluble membrane carrier (antibody-immobilized membrane) on which anti-CRP antibody and anti-hemoglobin antibody are immobilized Anti-CRP monoclonal antibody (Clone: 08210) and anti-hemoglobin monoclonal antibody (Clone: 69213) at 1 mg / mL Thus, it was prepared as a 10 mmol / L phosphate buffer solution (pH 7.2) containing 2.5% sucrose and resisted the position (CRP measurement line) inside one end of the short side of the nitrocellulose membrane (Millipore SHF180). The CRP monoclonal antibody was set to 0.75 μL / cm using an immunochromatographic dispenser “XYZ3050” (BIO DOT) with an anti-hemoglobin monoclonal antibody on the outside (Hb measurement line) at an interval of about 5 mm. Painted in line Clothed. The membrane was dried in a dry oven at 70 ° C. for 45 minutes to obtain an antibody-immobilized membrane.

4) Preparation of sample pad Same as Test Example 1.

5) Preparation of test strip On the plastic adhesive sheet (a), the application part (CRP measurement line) of the anti-CRP antibody (c) on the upstream side of the development, and then the application part (Hb measurement line) of the anti-hemoglobin antibody (d2) The antibody-immobilized membrane (b) prepared in 3) above was attached, and 3rd Pad (i) consisting of a glass fiber pad was attached. Next, the conjugate pad (e) prepared in 2) above is placed and mounted, and the sample pad (f) prepared in 4) above is placed and mounted so as to overlap this conjugate pad, and the opposite end is placed on the opposite end. An absorbent pad (g) was placed and mounted. Finally, a polyester film (h) was placed and mounted on the upper surface so as to cover the antibody-immobilized membrane and the absorption pad and laminated. Thus, the test strip for immunochromatography was produced by cutting each structural element into a superposed structure. In the assay, the test strip has a dedicated housing made of plastics (a sample supply window formed on the sample pad and a test line detection window formed on the test line, not shown in FIG. 1). ) In the form of a test device. FIG. 1 shows a schematic configuration diagram of a test strip.

[Example 1] Method for measuring hematocrit value according to the present invention (1) Specimen From 3 healthy subjects (hematocrit low value, medium value, high value) with consent to blood sampling, 5 mL of EDTA-2K vacuum blood collection tube was used. Blood was collected.
(2) Preparation of Sample A part of the specimen diluted 30-fold with the specimen diluent of Test Example 1 was used as a sample.
(3) Test method 120 μL of each sample was added to the sample addition window part (also serving as the sample addition window part) of the test device of Test Example 1, and 5 minutes using an immunochromato reader ICA-1000 (Hamamatsu Photonics) The reflected light intensity of the later sample pad was measured from the sample detection window.
(4) Calibration A calibrator prepared using certified practical reference material JCCRM622 (General Medical Institution for Medical Reference Materials) for total hemoglobin measurement is added to the sample supply window of a 120 μL test strip, and 5 with an immunochromatographic reader ICA-1000. The reflected light intensity of the sample pad after the minute was measured, and a calibration curve of Hb was prepared. The calibration curve Hb value was converted into a hematocrit value using the general correlation (1), and a calibration curve of the hematocrit value was created.

Hematocrit value (%) = Hb value (g / dL) × 2.9 (1)

(5) Results Table 1 shows conversion results to hematocrit values when the hemoglobin concentrations of three specimens (hematocrit low value, medium value, and high value) were measured at N = 5, respectively. According to the hematocrit value measurement method of the present invention, all three samples have better reproducibility than the hematocrit value (Table 2) obtained by the Hb measurement method based on antigen-antibody reaction (conventional method) that has been adopted by the inventors so far. Measurement results (CV: 2.4 to 9.4%) were obtained.

[Comparative example] Method for measuring Hb by antigen-antibody reaction (conventional example)
1. Test Method Using the test device prepared in Test Example 2, 120 μL of the same sample as in Example 1 was dropped on the sample addition window, and the test was performed 5 minutes later using an immunochromatography reader ICA-1000 (Hamamatsu Photonics). The reflected light intensity of the Hb measurement line was measured from the line detection window. The hematocrit value was obtained from the calibration curve obtained in advance.
2. Test results The results are shown in Table 2 below.

３．結果
本発明のヘマトクリット補正により各検体のＣＲＰ濃度を正確に求めることができた。[Example 2] Measurement of CRP using the test device of the present invention The CRP concentration was measured, and hematocrit correction was performed by the reflected light intensity of hemoglobin on the sample pad.
1. Test Method Using the test device prepared in Test Example 1, the CRP concentration and hemoglobin concentration of the blood sample were measured. That is, 10 μL of each blood sample was diluted 150-fold with 10 mmol / L phosphate buffer (pH 7.2) containing 0.01 wt% Tween 20 and hemolyzed, and 120 μL of the sample was added to the sample supply window (sample) of the test device. And the reflected light intensity of the test line was measured from the test line detection window part of the test device 5 minutes later using an immunochromato reader ICA-1000 (Hamamatsu Photonics). The obtained reflected light intensity was extrapolated to a calibration curve obtained in advance to obtain the CRP concentration.
In addition, the hematocrit value of each blood sample was determined by the method of Example 1.
2. Hematocrit correction according to the present invention The CRP concentration of each blood sample was corrected for hematocrit according to the following equation.

3. Results The CRP concentration of each specimen could be accurately determined by the hematocrit correction of the present invention.

(A) Plastics adhesive sheet (b) Antibody-immobilized membrane (c) Anti-CRP antibody (d1) Anti-immunoglobulin antibody (d2) Anti-hemoglobin antibody (e) Conjugate pad (f) Sample pad (g) Absorption pad (H) Polyester film (i) 3rd pad

According to the present invention, in a method of measuring an object to be measured in a sample obtained by diluting a blood sample using immunochromatography, the object to be measured is measured in order to measure the object to be measured and hemoglobin in the same sample. The object concentration can be corrected based on the hematocrit value of the same sample, and the measurement object concentration can be measured more accurately.
Furthermore, the measurement method of the present invention can perform hematocrit correction without measuring a hemoglobin concentration in a sample by reflected light intensity, and without performing a new measurement system or a complicated measurement operation. .

Claims (2)

A measurement method for measuring an object to be measured in a sample obtained by hemolyzing a blood sample using an immunochromatographic test strip,
The immunochromatographic test strip has the following 1) to 3):
1) A sample pad that absorbs a sample and retains a hemoglobin component in the sample , and in which an antibody to hemoglobin is not immobilized 2) A sample pad that is located downstream of the sample pad and is the first to the measurement object 3) an insoluble membrane having a test line on the downstream side of the conjugate pad and having a second antibody against the analyte immobilized thereon, The said measuring method which has the process of following A) and B).
A) A step of supplying a sample to the sample pad, optically detecting hemoglobin held in the sample pad, and determining a hematocrit value of a blood sample B) A step of measuring a measurement object in a test line on an insoluble membrane The measurement method according to claim 1, further comprising the following step C).
C) Step of correcting the measurement value of the measurement object in step B) by hematocrit using the hematocrit value obtained in step A)

Images