JP5936797B1 - Immunochromatographic test strips - Google Patents

Abstract

In a detection method using immunochromatography that detects a substance to be detected in a sample by developing a sample derived from stool in an insoluble membrane, and a method for suppressing a non-specific reaction peculiar to stool, and the immunochromatography used for this method It is an object to provide a test strip. By using a test strip in which a porous third pad made of polysulfone or cellulose acetate is placed upstream of the insoluble membrane, a method for suppressing non-specific reactions in stool-derived samples and the immunochromatographic test used therefor Provide a strip.

Description

  The present invention relates to an immunochromatographic test strip and a detection method using the same. In particular, the present invention relates to a test strip suitable for detecting a substance to be detected in a sample derived from stool and a detection method using the test strip. Furthermore, it is related with the suppression method of a nonspecific reaction in the detection using the test strip for immunochromatography.

Detection methods using immunochromatography are widely used because detection with high specificity and sensitivity is possible. Examples of samples applied for detection using immunochromatography include urine, feces, saliva, blood, serum, and the like, which are suitable as clinical laboratory samples and are widely used as diagnostically useful samples.
In order to detect a substance to be detected contained in these samples using immunochromatography, it is necessary to exclude various detection interfering substances contained in the sample. As an interfering substance, a color reaction is carried out in spite of the fact that there is no substance that causes clogging of the insoluble membrane for development, which hinders the development of the sample itself by immunochromatography, or there is no substance to be detected. There are substances that cause so-called non-specific reactions as shown. Various factors can be considered as the cause of this non-specific reaction, but it is not yet clear.
In general, in order to eliminate substances that may cause clogging in the insoluble membrane, the separation function of the sample pad is ensured, or when whole blood is used as a sample, blood cell separation is performed to eliminate blood cells. A method is known in which the membrane is disposed downstream of the sample addition section.
Patent Document 1 discloses a method in which a buffer containing a specific polymer is used as a developing solvent for immunochromatography in order to suppress nonspecific reactions.
Patent Document 2 discloses an immunochromatographic detection device in which serum albumin and a linear water-soluble polymer are coexisting with a conjugate in order to suppress nonspecific reactions.
Furthermore, gelatin, casein, bovine serum albumin and the like are generally known as reagents for blocking non-specific reactions in antigen-antibody reactions, and various methods for coexisting them in the immunochromatographic reaction system are also known.
As described above, various methods for eliminating the influence of the detection interfering substance are known. In the detection method using immunochromatography, a method for suppressing these non-specific reactions when a sample derived from stool is applied. So far there has been no investigation.

JP 2003-344406 A JP 2002-148266 A

  The present invention relates to a method for suppressing nonspecific reactions peculiar to stool in a detection method using immunochromatography for detecting a substance to be detected in a sample by developing a sample derived from stool in an insoluble membrane, and An object is to provide an immunochromatographic test strip to be used.

As a result of diligent research to solve the above-mentioned problems, the present inventors have used a test strip in which a porous third pad made of polysulfone or cellulose acetate is arranged upstream of an insoluble membrane, so that a fecal sample can be used. However, the present inventors have found that the non-specific reaction can be suppressed and detected accurately, and the present invention has been completed. These materials have been used as blood cell separation membranes in immunochromatography for developing whole blood as a sample, but have been completely used in immunochromatography for developing fecal samples. And the possibility has not been studied. The present inventors have found that a nonspecific reaction can be unexpectedly suppressed by allowing a complex of a stool-derived sample and a conjugate to pass through using such a material suitable for blood cell separation, and the present invention. It came to complete.
That is, the present invention has the following configuration.
<1>
An immunochromatographic test strip for detecting a complex of a detected substance and a conjugate in a sample by spreading a sample derived from stool in an insoluble membrane, wherein the conjugate is immunized against the detected substance. The test strip, wherein an antibody or antigen that reacts chemically is immobilized on a label and has the following constitutions (1) to (3).
(1) Sample pad having a sample supply part (2) Porous third pad made of polysulfone or cellulose acetate disposed downstream of the sample pad and upstream of the insoluble membrane (3) Immunity against a substance to be detected Insoluble membrane <2> having a detection part for detecting a complex of a substance to be detected and a conjugate in a sample, to which a chemically reactive antibody or antigen is immobilized
The test strip according to <1>, wherein the conjugate is impregnated in the conjugate pad, and the conjugate pad is disposed downstream of the sample pad and upstream of the third pad.
<3>
The test strip according to <1> or <2>, wherein the porous third pad has an average pore diameter of 1 to 100 μm.
<4>
A detection method using immunochromatography that detects a complex of a substance to be detected and a conjugate in a sample by developing a sample derived from stool in an insoluble membrane, and includes the following steps (a) to (c) Comprising the steps of:
(A) A sample is brought into contact with a conjugate in which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized on a label to form a complex of the substance to be detected and the conjugate in the sample. (B) passing the composite through a porous third pad made of polysulfone or cellulose acetate (c) developing the composite that has passed through the third pad in an insoluble membrane to Step of detecting the complex <5> in a detection part on which an immunologically reactive antibody or antigen is immobilized
The detection method according to <4>, wherein the conjugate is impregnated in the conjugate pad, and the conjugate pad is disposed downstream of the sample pad and upstream of the third pad.
<6>
The detection method according to <4> or <5>, wherein an average pore size of the porous third pad is 1 to 100 μm.
<7>
A non-specific reaction suppression method in detection using immunochromatography for detecting a complex of a substance to be detected and a conjugate in a sample by developing a sample derived from stool in an insoluble membrane, comprising the following steps (a ) To (c).
(A) A sample is brought into contact with a conjugate in which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized on a label to form a complex of the substance to be detected and the conjugate in the sample. (B) passing the complex through a porous third pad made of polysulfone or cellulose acetate to reduce non-specific reactive substances (c) developing the complex that has passed through the third pad in an insoluble membrane A step of detecting the complex in a detection part on which an antibody or antigen that immunologically reacts with the substance to be detected is immobilized

  According to the immunochromatographic test strip including a third pad made of a specific material according to the present invention, a substance to be detected in a sample derived from stool can be detected without causing a nonspecific reaction peculiar to stool. Therefore, the substance to be detected in stool can be accurately detected using immunochromatography.

It is a schematic block diagram of the test strip which has arrange | positioned the 3rd pad of this invention. It is a schematic block diagram of the test strip without a third pad.

(sample)
In the present invention, stool is used as a sample. The stool may be used as a sample as it is, or may be diluted with a diluent as appropriate. Moreover, you may use as a sample what was diluted and filtered suitably.

(Substance to be detected)
The substance to be detected of the present invention may be any substance as long as it is contained in feces as a sample and can be detected using an antigen-antibody reaction, and examples thereof include viruses, parasites, and proteins.
For example, infectious gastroenteritis is mainly caused by viruses and parasites. Examples of viruses that can be detected include norovirus, adenovirus, rotavirus, sapovirus, enterovirus, etc. Examples of insects include cryptospiridium, amoeba dysentery, giardia and the like. In addition, influenza virus as a virus to be detected, and proteins to be detected include human hemoglobin, hepatitis B virus antibody, hepatitis C virus antibody, human immunodeficiency virus antibody, and the like.

(Test strip for immunochromatography)
The immunochromatographic test strip of the present invention is a test strip for detecting a complex of a substance to be detected and a conjugate in a sample by developing a sample derived from stool in an insoluble membrane, and has the following configuration ( 1) to (3).
(1) Sample pad having a sample supply part (2) Porous third pad made of polysulfone or cellulose acetate disposed downstream of the sample pad and upstream of the insoluble membrane (3) Immunity against a substance to be detected An insoluble membrane having a detection part for detecting a complex of a substance to be detected and a conjugate in a sample, to which a chemically reactive antibody or antigen is immobilized

  The conjugate is an antibody or antigen that immunologically reacts with a substance to be detected and is immobilized on a label. The conjugate is present as a conjugate pad, a sample pad, a third pad, It may exist in a state where it is impregnated in another pad other than the insoluble membrane (type A), may exist as a conjugate part in a part of the sample pad (type B), and further, a test strip Alternatively, it may exist as a separate conjugate reagent to be mixed with the specimen (type C).

Hereinafter, the test strip of the type A in which the conjugate is present will be described.
The sample pad, the conjugate pad, the third pad, and the insoluble membrane are arranged in this order from upstream to downstream in the sample flow direction, and are arranged so that at least a part of the upper and lower layers overlap each other. A test strip of such an arrangement is shown in FIG.
When a sample containing a substance to be detected is supplied to the sample pad of such a test strip, the substance to be detected flows through the sample pad to the conjugate pad on the downstream side. In the conjugate pad, the substance to be detected and the conjugate contact with each other and pass through the pad while forming a complex (aggregate). The composite is then passed through a porous third pad placed in contact with the bottom surface of the conjugate pad and developed into an insoluble membrane.
Since an insoluble membrane is immobilized with an antibody or antigen that immunologically reacts with a substance to be detected, a part of the insoluble membrane is bound and immobilized by an immune reaction. Become. The immobilized complex is detected by a means for detecting absorbance or reflected light derived from the conjugate.

Next, a test strip having a conjugate type of type B will be described.
The difference from the type A test strip is that the sample pad and the conjugate pad are integrated, that is, the sample supply part and the conjugate part are formed in a part of the sample pad.
The sample supply part is a part for supplying a sample containing a substance to be detected, the conjugate part is a part containing a conjugate, and the sample supply part is on the upstream side of the conjugate part.

Next, a test strip having a conjugate type of type C will be described.
The difference from the above type A test strip is that there is no conjugate pad and the conjugate is present as a separate conjugate reagent. For example, a filter chip in which a conjugate is incorporated in a filter can be mentioned. Using such a filter chip, the conjugate and the substance to be detected are bonded to each other by passing the specimen diluent, thereby forming a complex (aggregate). By supplying this to the same test strip as type A except that it does not have a conjugate pad, the substance to be detected can be detected.

(Sample pad)
The sample pad used in the present invention is a portion that receives a sample, and includes any substance and form that can absorb a liquid sample while being molded into the pad and allow the liquid and the detection target to pass through. Specific examples of materials suitable 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. Preferably, a fiberglass pad is used. The sample pad can also have the function of a conjugate pad described later. The sample pad can contain a blocking reagent that is usually used for the purpose of preventing / suppressing nonspecific reaction (adsorption) in the antibody-immobilized membrane.

(Third pad)
The third pad may be any one that can permeate the complex of the substance to be detected and the conjugate in the sample. The purpose of the present invention is to capture a nonspecific reaction substance derived from stool. Therefore, the third pad needs to be a porous member made of polysulfone or cellulose acetate. Although the reason is not clear, the third pad made of these materials allows the non-specific reaction substance due to the stool sample to be captured and the non-specific reaction can be reduced, although the complex of the conjugate and the substance to be detected is passed. . On the other hand, a third pad made of a material other than polysulfone and cellulose acetate did not show the effect of reducing non-specific reactions. That is, polyamide, polycarbonate, polyethersulfone, and glass fiber did not have such a capturing function.
Further, the average pore diameter of the porous third pad of the present invention is 1 μm or more as a lower limit, preferably 5 μm or more, more preferably 10 μm or more, further preferably 15 μm or more, particularly preferably 20 μm or more, and preferably 25 μm or more. Most preferred. As an upper limit, 100 micrometers or less are illustrated, 80 micrometers or less are preferable, 60 micrometers or less are more preferable, 55 micrometers or less are further more preferable, 50 micrometers or less are especially preferable, and 45 micrometers or less are the most preferable. Moreover, as a range of an average pore diameter, 1-100 micrometers which is a combination of the said minimum and upper limit is illustrated, 5-80 micrometers is preferable, 10-60 micrometers is more preferable, 15-55 micrometers is more preferable, 20-50 micrometers is especially preferable. Most preferred is 25 to 45 μm. This is because if it is less than 1 μm, clogging is caused and the flow of the specimen itself becomes slow, and if it is more than 100 μm, it is considered that the function of capturing the non-specific reaction substance is lowered.
The said average hole diameter is calculated | required from the electron micrograph of the surface or back surface of a 3rd pad.

(Insoluble membrane)
The insoluble membrane used in the present invention has at least one detection unit on which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized. Immobilization of an antibody or antigen that reacts immunologically with a substance to be detected on an insoluble membrane carrier can be carried out by a conventionally known method. In the case of a lateral flow type immunochromatographic reagent, it has a mechanism capable of preparing a liquid containing the above-mentioned antibody or antigen at a predetermined concentration and moving it horizontally while discharging the liquid from the nozzle at a constant speed. The solution can be immobilized by applying the solution to the insoluble membrane carrier in a line using a device or the like and drying. The concentration of the antibody or antigen in the above solution is preferably 0.1 to 5 mg / mL, and more preferably 0.5 to 2 mg / mL. In addition, the amount of antibody or antigen immobilized on the insoluble membrane carrier can be optimized by adjusting the ejection speed from the nozzle of the above apparatus in the case of the lateral flow type, and is preferably 0.5 to 2 μL / cm. It is.
The measurement method using the lateral flow type immunochromatography reagent is a measurement method in which the sample is developed so as to move in a parallel direction with respect to the insoluble membrane carrier by capillary action.
In addition, a solution containing the antibody or antigen described above at a predetermined concentration can be prepared by adding the antibody or antigen to a buffer solution. Examples of the buffer include normal buffers such as phosphate buffer, Tris buffer, and Good's buffer. 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, preservatives such as procrine, and the like. The salts include those added for the purpose 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 or antigen on the insoluble membrane, blocking can also be carried out by coating a region other than the region where the antibody or antigen is immobilized with a commonly used blocking agent in the form of a solution or vapor.
It should be noted that a control capture reagent conventionally used in immunochromatographic reagents may be immobilized on the insoluble membrane. The control capture reagent is a reagent for ensuring the reliability of the assay, and captures the control reagent contained in the conjugate pad. For example, when KLH labeled on the conjugate pad is included as a control reagent, an anti-KLH antibody or the like corresponds to the control capture reagent. The position at which the control capture reagent is immobilized can be appropriately selected to suit the design of the assay system.

  As the membrane constituting the insoluble membrane used in the present invention, a known membrane conventionally used as an insoluble membrane carrier for an immunochromatographic reagent can be used. For example, there are membranes composed of fibers made of polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, ceramics, and the like. Specific examples include glass fiber filter paper and cellulose filter paper commercially available from Sartorius, Millipore, Toyo Roshi, Whatman and the like. Of these, Sartorius and UniSart CN140 are preferable. In addition, by appropriately selecting the pore size and structure of the insoluble membrane carrier, it is possible to control the speed at which the complex of the conjugate and the substance to be detected in the sample flows through the insoluble membrane carrier.

  The immunochromatographic test strip is preferably disposed on a solid support such as a plastic adhesive sheet. The solid support is composed of a material that does not interfere with the capillary flow of the sample and conjugate. Further, the immunochromatographic test strip may be fixed on a solid support with an adhesive or the like. In this case, the adhesive component and the like are also composed of a substance that does not interfere with the capillary flow of the sample and the conjugate. The immunochromatographic test strip is stored in an appropriate container (housing) taking into account the size of the immunochromatographic test strip, the method and position of sample addition, the position where the insoluble membrane detector is formed, and the signal detection method. -It can be mounted and used, and the state stored and mounted in this way is called "device".

(Antibodies or antigens that react immunologically with the substance to be detected)
The antibody or antigen that immunologically reacts with the substance to be detected used in the present invention is an antibody or antigen that can bind to the substance to be detected. When the substance to be detected is a virus or an antigen, the antibody or the substance to be detected When is an antibody, an antigen is preferred. An antibody or antigen that reacts immunologically with a substance to be detected is immobilized on a label and a detection part described later. The antibody or antigen immobilized on the label and the detection unit may be the same, but it is preferable that the label and the detection unit are different.

(Marker)
As the label used in the present invention, a known label conventionally used for a test strip for immunochromatography can be used. For example, colloidal metal particles such as gold colloid particles and platinum colloid particles, color latex particles, magnetic particles, and fluorescent particles are preferable, and gold colloid particles and color latex particles are particularly preferable.

(Conjugate)
The conjugate used in the present invention is obtained by immobilizing an antibody or antigen that immunologically reacts with a substance to be detected on the label as described above. When the detected substance detects rotavirus and adenovirus, the conjugate is preferably one in which an anti-rotavirus monoclonal antibody and an anti-adenovirus monoclonal antibody are immobilized on colloidal gold particles.
Examples of a method for immobilizing an antibody or antigen that immunologically reacts with a substance to be detected to a label include physical adsorption and chemical bonding, and are generally immobilized by physical adsorption.

(Other)
The immunochromatographic test strip of the present invention may further contain other reagents and components depending on measurement conditions and samples.
Examples of other reagents include blocking agents that prevent non-specific reactions.
Other configurations include, for example, an absorption pad that controls the development of the sample by absorbing the sample that has moved and passed through the insoluble membrane.
The production of the immunochromatographic test strip of the present invention can be carried out by appropriately modifying or altering the method described in the examples.

(kit)
The detection kit using the immunochromatography of the present invention only needs to include the immunochromatographic test strip. The detection kit may further include a reagent necessary for detection, a sample diluent, a test tube, a swab for collecting stool, an instruction manual, a housing for storing a test strip, and the like.

(Other)
In this specification, the meaning of upstream or downstream is used to mean the upstream side or the downstream side in the direction of sample flow. In other words, when the test pad of the present invention is laminated so that the sample pad, conjugate pad, third pad, and insoluble membrane partially overlap from the top, the sample pad is the most upstream and the insoluble membrane is the downstream. Become. In addition, the end pad may be stacked on the downstream side of the insoluble membrane so as to overlap with each other. In this case, the end pad is the most downstream.

1. Test Method (i) Preparation of Immunochromatographic Test Strip FIG. 1 is a schematic configuration diagram of the immunochromatographic test strip of the present invention.
Place the insoluble membrane (b) on the plastic adhesive sheet (a), then place and install the third pad (g), conjugate pad (d), sample pad (e) in this order, and the absorbent pad on the opposite end. (f) was placed and mounted. The conjugate pad is impregnated with a conjugate of gold colloid sensitized with an anti-adenovirus monoclonal antibody, and the insoluble membrane contains anti-adenovirus monoclonal antibody (c1) and control reagent (c2) in the flow direction. It is fixed on the line vertically. Each pad is laminated so that the upper and lower pads are in contact with a part thereof. A line made of the anti-adenovirus monoclonal antibody (c1) is called a test line, and a line made of the control reagent (c2) is called a control line.
Thus, the structure which piled up each component was cut | disconnected by the fixed width | variety, and the test strip for immunochromatography was produced.
A comparative “no third pad” test strip is shown in FIG. Since this test strip does not have a third pad, the sample pad is placed in place of the longer one so that the downstream end of the conjugate pad in the flow direction is in contact with the insoluble membrane and the upstream end is in contact with the sample pad. did.

(Ii) How to determine the average pore size of the third pad
A third pad cut out to an appropriate size was fixed to a sample stage, and Pt sputter coating was applied to prepare a sample for speculum. Using the Hitachi scanning electron microscope S-4800, the front and back surfaces of the third pad were photographed at an acceleration voltage of 1.5 kV and an observation magnification of 200 to 2000 times.
The holes observed on the front and back surfaces were selected 6 points at a time, and the diameters of these holes were measured, and the average value of the diameters of the 4 points of holes excluding the maximum value and the minimum value was obtained. Here, since the membrane made of polysulfone is an asymmetric membrane on both sides, the average pore diameter of the surface used as the upstream side was determined.
The measurement results are shown in Table 1.

(Iii) Test procedure Feces from four normal adults were used as specimens (all specimens are adenovirus-free negative specimens). 0.1 mg of each specimen was suspended in 1000 ml of specimen diluent, and the supernatant was used as a sample.
120 μL of a sample was dropped on a test strip on which a third pad made of the material shown in Table 1 was placed, and after 10 minutes, the color intensity of the test line was evaluated using a color sample called “color chart”. The color intensity of the color chart is quantified in increments of 0.25 from 0 to 4, and extremely weak non-specific reactions less than 0.25 were further evaluated on the basis of SS (very weak) and S (weak). . From the evaluation result of the color intensity, the strength of the non-specific reaction was evaluated, and further, the comprehensive evaluation was performed from the results of the four samples. Each evaluation standard is shown below Table 1.

2. Test results The test results are shown in Table 1.
From this result, when a third pad made of polysulfone and cellulose acetate was used as the third pad, the nonspecific reaction could be suppressed, and in particular, the case of polysulfone could suppress the nonspecific reaction better. In this case, the average pore diameter of the porous holes of the third pad was 20 to 50 μm.
On the other hand, when a third pad made of polyamide, polycarbonate, polyethersulfone, or glass fiber was used, the nonspecific reaction could not be suppressed. In addition, even when the third pad itself was not used, coloring was observed on the test line, and the nonspecific reaction could not be suppressed (not shown in the table).

  According to the immunochromatographic test strip including a third pad made of a specific material according to the present invention, a substance to be detected in a sample derived from stool can be detected without causing a nonspecific reaction peculiar to stool. Therefore, the substance to be detected in stool can be accurately detected using immunochromatography.

(A) Plastic adhesive sheet (b) Insoluble membrane (c1) Antibody against detection target (c2) Control reagent (d) Conjugate pad (e) Sample pad (f) Absorption pad (g) Third pad

Claims (4)

A non-specific reaction suppression method in detection using immunochromatography for detecting a complex of a substance to be detected and a conjugate in a sample by developing a sample derived from stool in an insoluble membrane, comprising the following steps (a ) To (c).
(A) A sample is brought into contact with a conjugate in which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized on a label to form a complex of the substance to be detected and the conjugate in the sample. Process
(B) The step of passing the composite through a porous third pad made of polysulfone and having an average pore diameter of 20 to 50 μm to reduce non-specific reactants
(C) The complex that has passed through the third pad is developed in an insoluble membrane, and the complex is detected by a detection unit in which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized. Process A detection method using immunochromatography that detects a complex of a substance to be detected and a conjugate in a sample by developing a sample derived from stool in an insoluble membrane, and includes the following steps (a) to (c) Comprising the steps of:
(A) A sample is brought into contact with a conjugate in which an antibody or antigen that immunologically reacts with a substance to be detected is immobilized on a label to form a complex of the substance to be detected and the conjugate in the sample. said composite step (b) the polysulfone down or Ranaru average pore diameter complex passed through the step (c) third pad to reduce non-specific reaction substance a porous third pad passed in 20~50μm of A step of developing the insoluble membrane and detecting the complex in a detection unit in which an antibody or antigen that immunologically reacts with a target substance is immobilized 3. An immunochromatographic test strip used in the nonspecific reaction suppression method according to claim 1 or the detection method according to claim 2, wherein the conjugate is an antibody that immunologically reacts with a substance to be detected. Alternatively, the test strip in which an antigen is immobilized on a label and has the following configurations (1) to (3).
(1) A downstream of the sample pad (2) Sample pad having a sample supply unit, polysulfone down or Ranaru average pore diameter of 20~50μm porous third pad (3) located upstream of the insoluble membrane to be An insoluble membrane having a detection part for detecting a complex of a substance to be detected and a conjugate in a sample, to which an antibody or antigen that immunologically reacts with the detection substance is immobilized The test strip according to claim 3 , wherein the conjugate is impregnated in a conjugate pad, the conjugate pad being disposed downstream of the sample pad and upstream of the third pad.

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