JP2003083975A - Test piece for immunoassay and immunoassay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a test piece for immunoassay capable of specifically detecting only Listeria viable cells simply and rapidly with high accuracy and sensitivity, and immunoassay using the test piece. SOLUTION: The test piece having a fixed phase (a) wherein a specific bonding substance capable of being specifically bonded to Listeria bacteria is fixed on a water absorbable substrate. A specimen liquid receiving part (b) for dropping a specimen liquid and a color forming substrate solution receiving part (c) for dropping a color forming substrate solution containing a color forming substance of β-glucosidase are arranged to the region between the fixed phase and one end on the upstream side of the water absorbable substrate.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a test strip for immunoassay and an immunoassay.

[0002]

2. Description of the Related Art In recent years, mass infection by pathogenic bacteria such as O157 and Salmonella has become a problem. Among them, food poisoning due to Listeria monocytogenes is expected to become a serious problem in the future.

Although food poisoning due to Listeria monocytogenes mainly occurs in Europe and America, there is a concern that outbreaks may occur in Japan as the food becomes Westernized. One of the reasons why Listeria monocytogenes is regarded as a problem is its ability to grow even at low temperatures. Since many causative bacteria that cause food poisoning hardly grow at low temperature, the occurrence of food poisoning can be prevented by paying attention to cold storage of food. However, since Listeria monocytogenes grows only during low temperature storage, it is difficult to suppress the growth during storage.

A culture method for detecting Listeria monocytogenes has also been defined, but the culture takes a very long time and labor, and it takes about one week before the test results can be obtained. These are also one of the factors that make it difficult to control Listeria monocytogenes.

There is a demand for a highly sensitive, rapid and simple detection method for predicting and preventing Listeria monocytogenes, and various simple test kits and test instruments have been developed. Among them, the immunochromatography method is a technique that enables detection of a target substance by a relatively simple operation. The immunochromatographic method is, for example, a test piece having a stationary phase on which a first specific binding substance capable of specifically binding to the test substance is immobilized, so that the presence of the test substance is expected. A method of developing a sample solution is mentioned, and it is possible to simultaneously perform selective capture and concentration of a test substance.

[0006] The detection of the test substance captured in the stationary phase is carried out by detecting the labeled complex in which the second specific binding substance capable of specifically binding to the test substance and the labeling substance are immobilized on the carrier. This can be achieved by binding to a substance and detecting the labeled substance of the complex composed of [test substance-labeled complex]. Colored particles, colloidal gold particles and enzymes are generally used as the labeling substance.

[0007] The immunochromatographic method is a rapid and highly sensitive method for detecting a test substance. On the other hand, when a target of detection by the immunochromatographic method is a microorganism, a problem may occur. Even if the presence of a microorganism is confirmed, it does not matter in most cases if it is dead. However,
The first specific binding substance used in the stationary phase often traps the microorganism regardless of whether the microorganism is alive or dead. Furthermore, since the test substance is concentrated in the stationary phase, the probability that dead microorganisms will be detected is higher than in other immunological tests that do not involve concentration in the stationary phase. .

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a test strip for an immunoassay method, which enables specific detection of only live Listeria monocytogenes, simply and quickly, with high precision and high sensitivity, and It is to provide an immunoassay method using a test piece.

[0009]

Means for Solving the Problems That is, the present invention provides [1]
A test piece comprising: (a) a stationary phase having a specific binding substance capable of specifically binding to Listeria monocytogenes immobilized on a water-absorbing substrate, the stationary phase being upstream of the stationary phase and the water-absorbing substrate. The following (b) and (c): (b) a test sample solution receiving part for dropping the test sample solution, (c) a color containing a chromogenic substrate of β-glucosidase in a region between the one end A color-developing substrate solution receiving part for dropping the chromogenic substrate solution, [2] a test sample for immunoassay, [2] a test sample solution receiving part of the test piece according to 1 above Including the step of dropping and developing the sample solution and the step of dropping and developing the chromogenic substrate solution of β-glucosidase in the chromogenic substrate solution receiving part, the presence or absence of live Listeria monocytogenes in the test sample solution is fixed. Β- contained in live Listeria monocytogenes
An immunoassay for detecting the presence or absence of color development by glucosidase and a chromogenic substrate, and [3] when live Listeria monocytogenes is not detected by the immunoassay described in 2, the stationary phase portion of the test piece used is placed on a medium. The immunization according to 2 above, further comprising a step of arranging, culturing under conditions suitable for culturing live Listeria monocytogenes, and testing for the presence or absence of growth of live Listeria monocytogenes using a chromogenic substrate for β-glucosidase. Measurement method.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The test piece of the present invention is a test piece having (a) a stationary phase on which a specific binding substance capable of specifically binding to Listeria monocytogenes is immobilized on a water-absorbent substrate. hand,
In the region between the stationary phase and one end on the upstream side of the water absorbent substrate,
(B) and (c) below: (b) a test sample solution receiving part for dropping the test sample solution, (c) for dropping a chromogenic substrate solution containing a chromogenic substrate of β-glucosidase A chromogenic substrate solution receiving part is arranged.
By using the test piece of the present invention, the Listeria bacterium in the test sample solution is selectively and densely captured on the stationary phase, and the color development by the β-glucosidase and the chromogenic substrate of the live Listeria bacterium is used as an index. As a result, it is possible to detect live Listeria monocytogenes trapped on the stationary phase when live Listeria monocytogenes exists regardless of the presence or absence of Listeria dead cells.

The water-absorbent substrate used as the test piece is not particularly limited as long as it is a substrate capable of absorbing an aqueous liquid. In the present invention,
It is preferable to use a water-absorbing substrate having an appropriate water-absorbing property that can secure a sufficient time for sufficient reaction between live Listeria monocytogenes, a specific binding substance and a chromogenic substrate during development. Specific examples of such water-absorbent substrate include non-woven fabric, filter paper, glass fiber cloth, glass filter, nitrocellulose membrane, nylon membrane and the like.

The degree of water absorption of the water-absorbent substrate is, for example,
When one end of a water-absorbent substrate having a thickness of 140 μm is immersed in water, the water absorption distance of 40 mm from the part immersed in water is 4 mm.
It is preferable that it develops in 0 to 300 seconds. The degree of water absorption is appropriately adjusted by, for example, coating the surface of the water-absorbent substrate with a hydrophilic polymer such as hydroxypropylmethyl cellulose, polyvinyl alcohol, or hydroxyethyl cellulose, or impregnating the water-absorbent substrate. You can also

The shape of the water-absorbent substrate is not particularly limited as long as it can spread the above liquids, and for example, a rectangular sheet shape (piece shape) or rod shape is preferable.

The stationary phase can be formed by immobilizing a specific binding substance capable of specifically binding to Listeria monocytogenes described below on a water-absorbent substrate used as a test piece. Examples of the immobilization method include known physical adsorption methods and covalent bonding methods. Further, after applying a solution containing a specific binding substance used for the stationary phase and a hydrophilic polymer in a desired range on a water-absorbent substrate, the water-absorbing group is added to a coagulating solvent for coagulating the hydrophilic polymer. The stationary phase can also be formed by dipping the material. The hydrophilic polymers include those exemplified above, and the coagulation solvent includes acetone, ethanol, methanol, ether and the like.

The stationary phase is preferably formed at a desired position on the water-absorbent substrate with a width similar to the width of the water-absorbent substrate in the direction perpendicular to the developing direction of each component.

Further, the stationary phase is preferably one in which the specific binding substance is immobilized in an amount equal to or more than the amount capable of capturing the presence of live Listeria monocytogenes in a detectable range. The immobilized amount of the specific binding substance in the stationary phase can be appropriately determined depending on the type of the specific binding substance and the like.

The water-absorptive substrate on which the stationary phase is formed is used to prevent non-specific adsorption of a protein not to be detected on the substrate, ease of development, and immobilization of immobilized specific binding substance. From the viewpoint of storage stability, it is preferably used by immersing it in a solution containing a blocking agent, a surfactant and sugar (hereinafter referred to as a treatment solution) and drying it according to a known method. Here, examples of the blocking agent used include proteins having adsorptivity to a water-absorbent substrate such as bovine serum albumin, casein, gelatin, and skim milk. As the surfactant, polyoxyethylene (1
0) Octyl phenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene alkyl allyl ether phosphate, polyoxyethylene alkyl ether phosphate, polyoxyethylene alkyl phenyl ether and the like can be mentioned. As sugar,
Examples include sucrose and trehalose. The content of the blocking agent in the treatment liquid is preferably 0.1 to 10% by weight. The content of the surfactant in the treatment liquid is preferably 0.01 to 1% by weight. The sugar content in the treatment liquid is preferably 0.1 to 10% by weight.

The specific binding substance used in the stationary phase may be any substance that can specifically bind to Listeria monocytogenes, and examples thereof include anti-Listeria antibody.

The test sample solution receiving part (the part for dropping the test sample solution) and the chromogenic substrate solution receiving part (the chromogenic substrate solution containing the chromogenic substrate) of the test piece used in the present invention are dropped. Part) is, in order from the one end on the upstream side of the water-absorbent substrate, the chromogenic substrate solution receiving part, the test sample solution receiving part,
It is preferably arranged so as to be a stationary phase. The positions of the chromogenic substrate solution receiving section and the test sample solution receiving section may be reversed. Further, the test sample solution receiving section and the chromogenic substrate solution receiving section do not necessarily have to be provided separately, for example, only the test sample solution receiving section is created,
You may use it as a chromogenic substrate solution receiving part.

Further, a water-absorbing pad portion, which is a means for accelerating the development of each of the above-mentioned liquids in the test piece by the absorbing power of the aqueous liquid, is provided on the opposite side of the sample liquid receiving portion with the stationary phase interposed therebetween. Good. As the water-absorbing pad used for the water-absorbing pad portion, a material having excellent absorbency of an aqueous liquid is suitable, and for example, a glass filter, filter paper, non-woven fabric or the like can be used.

In the present specification, the term "upstream side of the stationary phase" means the terminal side of the side containing the sample liquid receiving part as viewed from the stationary phase.

The test sample solution receiving portion and the color-developing substrate solution receiving portion can be produced by, for example, adhering a dropping pad onto the water-absorbent substrate by adhesion. As the dropping pad, for example, a glass filter,
Filter paper and non-woven fabric can be used. Further, the test sample solution receiving section and the chromogenic substrate solution receiving section may be directly provided on the water-absorbent substrate.

The test sample solution receiving part and the chromogenic substrate solution receiving part may be provided in a shape suitable for dropping and developing each solution, and the shapes are not particularly limited, but at least The width is preferably the same as the width of the water-absorbent substrate in the direction perpendicular to the developing direction of each component.

The development movement distance (distance from the sample liquid receiving part to the stationary phase) in the test piece used in the present invention is
From the viewpoint of color development uniformity and color sensitivity in the stationary phase, preferably 0.5 cm or more, more preferably 1 cm or more, from the viewpoint of reachability of live Listeria monocytogenes to the stationary phase, color sensitivity and measurement time. , Preferably 8 cm or less, more preferably 6 cm or less. That is, the expansion movement distance is preferably 0.5 to 8 cm, more preferably 1 to 6 cm.

It should be noted that it is desirable that the sample liquid receiving portion, the stationary phase and the like are provided at positions on the test piece where the expansion and movement distance can be secured.

Further, as another aspect of the present invention, an immunoassay method using the test piece of the present invention is provided. The method includes a step of dropping a test sample solution into a test sample solution receiving section to develop it and a step of dropping a chromogenic substrate solution of β-glucosidase into a chromogenic substrate solution receiving section to develop it. The presence or absence of live Listeria monocytogenes in the sample solution is detected by the presence or absence of color development by the β-glucosidase and the chromogenic substrate possessed by the live Listeria monocytogenes captured in the stationary phase.

Listeria monocytogenes measured by the immunoassay method of the present invention include, for example, L. monocytogenes and L. inn.
ocua, L. seeligeri, L. welshimeri, L. ivanovii,
L. grayi is mentioned.

The test sample solution means a liquid containing a test sample for testing for the presence of live Listeria monocytogenes,
It is not particularly limited. For example, food extract,
Examples include culture fluid, blood, serum, urine, stool, sweat, saliva and the like. Further, it may be a diluting solution obtained by diluting them with an appropriate diluting solvent (for example, an arbitrary buffer solution or the like). The test sample may be a liquid sample or a solid sample.
In the case of a solid sample, for example, the solid sample is appropriately crushed,
The test sample solution can be prepared by dissolving or diluting it in a solvent such as a phosphate buffer solution or physiological saline.

Examples of the chromogenic substrate for β-glucosidase include, for example, esculin and ammonium iron citrate, 5
-Bromo-4-chloroindol-3-yl-β-D-
Examples include glucoside.

The chromogenic substrate solution is a chromogenic substrate,
Those dissolved in any aqueous solvent are suitable.

The test sample solution and the chromogenic substrate solution can be dropped into the test sample solution receiving section and the chromogenic solution receiving section using, for example, a micropipette or a dropper. The chromogenic substrate solution is preferably dropped after the test sample solution is dropped. To the chromogenic substrate solution receiving part or the test sample solution receiving part, from the viewpoint of preventing background coloration after dropping the test sample solution and before dropping the chromogenic substrate solution, for example, an aqueous solution of NaCl or the like is used. You may drop. The expansion of each liquid is performed in the test piece in a form of natural expansion by the capillary phenomenon, and the components contained in each liquid are expanded with the expansion.

The amount of the chromogenic substrate to be added is not particularly limited as long as it is the amount at which color development can be confirmed, and may be appropriately determined, for example, according to the test sample to be tested.

Then, the presence or absence of color development by the β-glucosidase and the chromogenic substrate possessed by the live Listeria monocytogenes captured in the stationary phase is detected. The color of the chromogenic substrate may be detected by any method as long as the color can be detected, but it is preferable to detect it visually because it is quick and simple. At that time, even if the killed Listeria monocytogenes was trapped on the stationary phase, β-glucosidase activity was not observed in the killed bacterial cells, so when the color development was detected on the stationary phase, the test was performed. It serves as an index for the presence of live Listeria monocytogenes in the sample solution. The conditions for color development with β-glucosidase and the chromogenic substrate may be known conditions depending on the combination of β-glucosidase and the chromogenic substrate used.

Further, as another embodiment of the immunoassay method of the present invention, when live Listeria monocytogenes is not detected by the immunoassay method, the stationary phase portion of the test piece used is placed on a medium,
The immunoassay method further comprises a step of culturing live Listeria monocytogenes under conditions suitable for culturing, and examining the presence or absence of growth of Listeria monocytogenes using a chromogenic substrate for β-glucosidase.

Since a small amount of live Listeria monocytogenes is present in the test sample solution, after the step of dropping and developing the test sample solution in the test sample solution receiving portion of the test piece There are few live Listeria monocytogenes captured by
Even when color development due to glucosidase and chromogenic substrate cannot be detected, the stationary phase portion of the obtained test piece is placed on a medium and cultured live Listeria monocytogenes cells are grown to increase the degree of color development. Can be increased. That is, by culturing and growing Listeria monocytogenes on the stationary phase, β-glucosidase increases accordingly, and thus the color development of the chromogenic substrate can be detected. Therefore, even if a small amount of live Listeria monocytogenes is contained in the test sample solution and the presence thereof cannot be detected by the immunoassay alone, in the immunoassay of the present embodiment, By examining the presence or absence of proliferation using a chromogenic substrate for β-glucosidase, it is possible to detect live Listeria monocytogenes.

The medium for arranging the stationary phase portion (that is, the medium for culturing live Listeria monocytogenes) is Oxfor.
Examples include d agar medium and PALCAM agar medium.

Conditions suitable for culturing live Listeria include an optimum temperature of 37 ° C. and an optimum pH of 5.5 to 9.0, which may be appropriately changed depending on other conditions during the culture, such as salt concentration. You may let me.

In the step of inspecting the presence or absence of growth of Listeria monocytogenes using a chromogenic substrate for β-glucosidase, the color development by β-glucosidase and the chromogenic substrate occurs at least in the stationary phase portion arranged on the medium. It is preferred to add a chromogenic substrate so that it can be detected. For example, the chromogenic substrate solution may be dropped directly onto the stationary phase portion on the medium, or the chromogenic substrate may be added to the medium in advance. From the viewpoint of simplifying the operation, it is preferable to add it to the medium in advance. The chromogenic substrate and the chromogenic substrate solution are the same as above. The amount of the chromogenic substrate added is not particularly limited as long as it is an amount at which color development can be detected, and may be appropriately determined, for example, according to the test sample to be tested. For example, when the chromogenic substrate is added to the medium in advance, the amount to be added may be appropriately determined according to the chromogenic substrate used and is not particularly limited. By adding a chromogenic substrate to the medium, during or after culturing the viable Listeria monocytogenes, the presence or absence of color development by the β-glucosidase and the chromogenic substrate at the stationary phase part and its surroundings causes the growth of viable Listeria monocytogenes. The presence or absence of can be easily detected.

As another aspect of the present invention, an immunoassay kit is provided. Examples of such a kit include a kit containing the test piece of the present invention. These kits may further contain a chromogenic substrate, a diluent solvent for the test sample and the like.

[0040]

EXAMPLES The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples.

Preparation Example 1: Preparation of Test Pieces As shown in FIG. 1, a nitrocellulose membrane (pore size 8 μm, 6 × 40 m) shown as the water-absorbent substrate 1 was prepared.
m) on top of Rabbit anti Listeria,
0.5 μl of an aqueous solution containing 0.2% by weight of Fitzgerald
Was applied in a line (6 × 1 mm) using a dispenser and dried at 37 ° C. for 1 hour to obtain a membrane in which stationary phase 2 was arranged. The obtained membrane was immersed in a 1% by weight bovine serum albumin aqueous solution for 20 minutes. The immersed membrane was dried overnight at room temperature.

A polyester non-woven fabric (6
× 8 mm, thickness 1 mm) and polyester non-woven fabric (6 ×
6 mm, thickness 1 mm) were adhered to each other to prepare a test sample solution receiving section 3 and a chromogenic substrate solution receiving section 4, respectively.
Further, as the water-absorbing pad 5 for absorbing the developed liquid component, a glass fiber non-woven fabric (manufactured by Whatman, GF
/ B, 15 x 30 mm, thickness 1 mm)
A test piece was prepared.

Example 1: Detection of live Listeria monocytogenes in a test sample solution 1) Preparation of a test sample solution Listeria monocytogenes (ATCC76)
44) Brain Heart Infusion Broth (Br
ain Heart Infusion Agar (manufactured by Nissui Pharmaceutical Co., Ltd.) and cultured at 35 ° C. for 24 hours,
The obtained culture solution was used as a test sample solution 1 (live listeria). In addition, the obtained culture solution was autoclaved (1
The test sample solution 2 (dead Listeria monocytogenes) was obtained by killing the Listeria monocytogenes at 21 ° C. for 15 minutes. Using the two types of test sample solutions obtained as described above,
The test was conducted as follows.

2) Test method 100 μl of each test sample solution was dropped on the test sample solution receiving section 3. After 5 minutes, 50 μl of 0.9 wt% NaCl aqueous solution was added dropwise to the chromogenic substrate solution receiving part 4. After a further 5 minutes, the chromogenic substrate solution (0.1 wt% esculin, 0.05 wt% ammonium iron citrate-containing aqueous solution) was added dropwise to the chromogenic substrate solution receiving part 4 and developed. After the development, it was visually confirmed after 5 to 10 minutes whether or not the stationary phase 2 of the test piece exhibited a blackish purple color.

The criteria for determining the coloration are as follows. +: Black purple is displayed −: Black purple is not displayed

The results are shown in Table 1.

[0047]

[Table 1]

As shown in Table 1, according to the method of Example 1, it is possible to detect only live Listeria monocytogenes without detecting dead Listeria monocytogenes.

Example 2: Detection of low-concentration live Listeria monocytogenes in test sample solution 1) Preparation of test sample solution In the same manner as in Example 1 1), Listeria monocytogenes
Culture of monocytogenes, ATCC 7644) was performed. The obtained culture solution was diluted 10 ⁷ times with a 0.9 wt% NaCl aqueous solution. The concentration of the bacterial solution in the obtained diluted culture solution was 10 ² CFU / μl. This diluted culture solution was used as a test sample solution.

2) Test method 100 μl of the test sample solution was dropped on the test sample solution receiving section 3. After a further 5 minutes, the chromogenic substrate solution (0.1 wt% esculin, 0.05 wt% ammonium iron citrate-containing aqueous solution) was added dropwise to the chromogenic substrate solution receiving part 4 and developed. After the development, it was visually confirmed after 5 to 10 minutes whether the stationary phase 2 of the test piece exhibited a black purple color.
No color development was observed and it was negative (Table 2).

From the above-mentioned test piece which was confirmed to be negative, the water-absorbing pad 5, the test sample solution receiving section 3 and the chromogenic substrate solution receiving section 4 which were laminated were removed. The stationary phase portion of the test piece thus obtained was treated with 0.1 wt% esculin and 0.0
The cells were placed on a solid medium (Listeria selection agar medium, Oxford composition, manufactured by Merck) containing 5 wt% ammonium iron citrate, and cultured at 35 ° C. for 24 hours. After culturing the stationary phase Listeria monocytogenes on the water-absorbent substrate, it was visually confirmed whether the stationary phase 2 and the agar medium around the stationary phase exhibited a blackish purple color. Table 2 shows the results of coloration before and after the culture treatment.

[0052]

[Table 2]

As shown in Table 2, according to the method of Example 2, even if the number of viable Listeria monocytogenes in the test sample solution was very small and it was difficult to immediately detect it, the test sample was not detected. It can be seen that by culturing live Listeria monocytogenes captured in the stationary phase of No. 1, it is possible to selectively detect live Listeria monocytogenes.

[0054]

INDUSTRIAL APPLICABILITY The present invention provides a test strip for an immunoassay method, which enables specific detection of only live Listeria monocytogenes in a simple and rapid manner with high accuracy and high sensitivity. According to the immunoassay method of the present invention using the test piece, when detecting Listeria monocytogenes, without detecting dead Listeria monocytogenes, only live Listeria monocytogenes can be simply and quickly, and highly accurate and highly sensitive. Can be detected. Further, even if the number of live Listeria monocytogenes in the test sample solution is extremely small, it is possible to simply culture the Listeria monocytogenes and selectively detect the live Listeria monocytogenes.

[Brief description of drawings]

FIG. 1 is a schematic view (cross-sectional view) showing one embodiment of the immunoassay test piece of the present invention. The arrow indicates the developing direction, and the dimensions indicate those of the test piece used in the examples.

[Explanation of symbols]

1 Water-absorbent substrate 2 stationary phase 3 Test sample receiving part 4 Coloring substrate solution receiving part 5 Water absorption pad

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Claims (3)

[Claims] 1. A test piece comprising a (a) stationary phase having a specific binding substance capable of specifically binding to Listeria monocytogenes immobilized on a water absorbent substrate, the stationary phase and a water absorbing group. The following (b) and (c): (b) the test sample solution receiving part for dropping the test sample solution in the area between the upstream side end of the material, (c) the coloring property of β-glucosidase An immunoassay test piece, comprising a chromogenic substrate solution receiving part for dropping a chromogenic substrate solution containing a substrate. 2. The step of dropping and developing the test sample solution in the test sample solution receiving section of the test piece according to claim 1, and dropping the chromogenic substrate solution of β-glucosidase in the chromogenic substrate solution receiving section. Immunoassay to detect the presence or absence of live Listeria monocytogenes in the test sample liquid by detecting the presence or absence of color development by β-glucosidase and the chromogenic substrate of the live Listeria monocytogenes captured in the stationary phase. Law. 3. When no live Listeria monocytogenes is detected by the immunoassay according to claim 2, the stationary phase portion of the test piece used is placed on a medium and the conditions are suitable for culturing the live Listeria monocytogenes. The immunoassay method according to claim 2, further comprising the step of culturing and inspecting the presence or absence of growth of live Listeria monocytogenes with a chromogenic substrate for β-glucosidase.