JPH09184840A - Testing jig for immunochromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a testing jig for immunochromatography and a measuring method using the jig whereby a substance to be measured in a liquid sample can be measured accurately with high sensitivity. SOLUTION: The testing jig is provided with a first part 3 including a first antibody and a second antibody, and a second part 4. The first antibody is held to be movable through capillarity, labeled by a marker from which visually detectable signals can be obtained and specific to a substance to be measured. The second arntibody is specific to the substance to be measured, recognizes a different part from the first antibody and is coupled with biotin. The second part 4 has a part, 5 where avidin is immobilized. The first part 3 and the second part, 4 are coupled to bring about capillarity therebetween.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an immunochromatographic test device for measuring the amount of a substance to be measured in a liquid sample and a measuring method used therefor.

[0002]

2. Description of the Related Art A measuring method combining a chromatography technique utilizing a capillary phenomenon and an immunological technique, a so-called immunochromatography method (immunoflow method),
This method is widely used as a particularly excellent semi-quantitative analysis method, which is a simple method capable of easily and quickly determining without using a special device.

[0003] The basic principle of the immunochromatography method is that a liquid sample containing a substance to be measured is supplied to a site containing an antibody labeled with a marker and allowed to react, and the resulting compound of the substance to be measured and a marker-labeled antibody is reacted. Is moved by capillary action to a second portion on which an antibody that specifically binds to the substance to be measured is immobilized, and so-called sandwich-type immunization of the immobilized antibody, the substance to be measured, and the marker-labeled antibody is performed on the second part. A complex is formed, and the amount of the substance to be measured is measured based on the color development derived from the immobilized marker.

[0004] For example, Japanese Patent Publication No. Hei 7-13640 discloses that a) vesicle markers which are coplanar and linked so as to cause capillary action with each other, and which are movably held therein. A first portion containing an antibody (tracer) specific to the substance to be measured and serving as a sample addition site;
b) An immunochromatographic test tool having a second portion in which an antibody that specifically binds to a measurement target substance is immobilized, and a method for measuring a measurement target substance using the same are disclosed.

[0005] International Publication No. WO84 / 04171 discloses that
A method utilizing an avidin-biotin reaction for immobilizing an antibody against a substance to be measured on a part of a test device for immunochromatography is disclosed.

[0006]

However, in these methods, since one of the antibodies involved in the so-called antigen-antibody reaction for detecting the substance to be measured is immobilized on the solid phase, immobilization is performed. The reaction rate of the sandwich-type immune complex formation reaction consisting of antibody-substance to be measured-marker-labeled antibody is naturally slower than when such complex formation is carried out in the liquid phase. That is, in a reaction for forming a sandwich-type immune complex on a solid phase like a conventional immunochromatographic test device, it takes time to complete the reaction. In some cases, the measurement had to be performed in a state where the complex formation reaction was not sufficiently completed. The slow reaction rate of the sandwich-type immune complex-forming reaction on the solid phase is due to, for example, immunology using the sandwich-type immune complex-forming reaction on the solid phase such as enzyme-linked immunosorbent assay (ELISA). It can be expected from the fact that the reaction time course in the static measurement method does not reach the plateau in several hours.

Therefore, in a measuring method such as an immunochromatographic method which requires completion of the measurement in a short time (often within 10 minutes), the sandwich-type immune complex forming reaction is not sufficiently completed. However, there is a problem in that the measurement accuracy is lowered and the detection sensitivity is lowered.

[0008]

Means for Solving the Problems The present invention has been made in view of the above-mentioned situation, and (a) is movably held by a capillary phenomenon, and 1) a visually detectable signal is A first antibody, which is labeled with the obtained marker and is specific to the substance to be measured, and 2) a second antibody specific to the substance to be measured, which has a recognition site different from that of the first antibody and to which biotin is bound. And a second part having a part to which avidin is immobilized, and (c) the first part and the second part are connected so that capillarity occurs between them. , A test tool for immunochromatography.

The present invention also provides: 1) a first antibody specific for a substance to be measured, which is labeled with a marker that gives a visually detectable signal; and 2) a first antibody specific for the substance to be measured, The second, which has a different recognition site from the antibody and is bound by biotin
The first that holds the antibody and capillaries so that they can move by capillarity
A liquid sample is supplied to the portion to start the reaction in the liquid phase, and the reaction product is transported by capillarity to the second portion having the portion to which avidin is immobilized, and the avidin-immobilized portion of the second portion. The present invention is a method for measuring the amount of a substance to be measured in the liquid sample, based on the degree of color development derived from the marker in the reaction product captured by.

That is, the present inventor has a problem in the conventional immunochromatographic test device, that is, a short time (for example, 10 minutes) in order to carry out the sandwich type immune complex forming reaction for the measurement on the solid phase. As a result of intensive research to develop a test tool for the immunochromatography method that solves the problem that the measurement accuracy cannot be completed and the detection sensitivity is lowered, the measurement target substance, 1) A first antibody specific to a substance to be measured, which is labeled with a marker that gives a visually detectable signal (hereinafter, referred to as marker labeled first antibody).
Abbreviated as antibody. ), And 2) specific to the substance to be measured,
The first antibody is reacted with a second antibody having a different recognition site and bound to biotin (hereinafter abbreviated as biotin-conjugated second antibody) in the liquid phase of the first portion, and first, a marker-labeled first antibody- A substance to be measured-a biotin-conjugated second antibody sandwich-type immune complex (that is, a reaction product) is formed in a liquid phase, and then this is developed film (second part) having an avidin-immobilized part (judgment part). Of the sandwich type immune complex (reaction product) in the sandwiched immune complex (reaction product) captured in the determination unit as a result of the avidin-biotin reaction. When the immunochromatographic test device configured to measure the amount of the substance to be measured based on the degree of color development derived from the marker labeled on the first antibody is used, the conventional immunochromatographic test device can be used. Kick to solve the problems such as described above,
The inventors have found that the substance to be measured can be measured with high sensitivity and high accuracy, and have completed the present invention.

The first portion and the second portion of the present invention, which can be connected to each other by capillarity, are usually formed by using a water-absorbing film or sheet which can cause capillarity. However, such a film or sheet is not particularly limited as long as it is usually used in this field as a film or sheet having water absorbability, and examples thereof include nitrocellulose. Membranes, cellulose nitrate membranes, glass filter membranes, nylon membranes, modified nylon membranes, membrane materials such as polyvinylidene difluoride (PVDF) membranes, such as filter papers, non-woven fabrics (eg, cellulosic fibers such as rayon and cotton threads, glass fibers, For example, nylon, polyester, polypropylene,
Preferred are sheet-shaped products such as those prepared from chemical fibers prepared from polyurethane and the like).

The antibody used to prepare the marker-labeled first antibody or biotin-conjugated second antibody to be retained in the first part of the present invention may be any antibody to the substance to be measured, and is not particularly limited. Not done. That is, according to a conventional method, for example, a method described in “Introduction to Immunological Experiments, Second Edition, Nao Matsuhashi et al., Academic Publishing Center (1981)”, etc.
Polyclonal antibodies prepared by immunizing animals such as cows, sheep, rabbits, goats, rats, mice, and the like, with antibodies to be measured, or by a conventional method, ie, Keller and Milstein [Nature, vol.
95 (1975)], a monoclonal antibody produced by a hybridoma or the like obtained by fusing cells from a tumor line of a mouse with splenocytes of a mouse previously immunized with a measurement target, according to the cell fusion method. You can
It is optional to use these singly or in an appropriate combination. Considering the ease of obtaining an antibody having uniform properties, a monoclonal antibody is preferred over a polyclonal antibody. These antibodies may be digested with enzymes such as pepsin and papain if necessary to obtain F (ab ') ₂ ,
Needless to say, Fab 'or Fab may be used.

The marker in the marker-labeled first antibody according to the present invention is one that gives a visually detectable signal, and the one labeled with the first antibody can be moved by capillarity. It is not particularly limited as long as it is one, but specifically, for example, gold colloid, iron colloid,
Metal colloids such as silver colloids, non-metal colloids such as selenium colloids, colored latexes prepared from high molecular polymers such as polystyrene and polyacrylamide, fluorescent dyes such as rhodamine B and carboxylfluorescein, and methyl orange, for example. , Colored liposomes containing dyes such as Coomassie Brilliant Blue R250, fluorescent dyes such as rhodamine isothiocyanate, fluorescein isothiocyanate (FITC), dyes such as Coomassie Brilliant Blue R250, etc. Gold colloid is particularly preferable in consideration of the sheath and sensitivity.

The gold colloid used in the present invention includes:
Usually, a commercially available product may be used, but a conventional method, for example, a method of reducing chloroauric acid with sodium citrate [Nature Phys.
Sci., Vol. 241, 20 (1973)]. The particle size of the gold colloid is not particularly limited, but is usually 5 nm to 90 nm, preferably 10 nm to 70 nm.

The method for preparing the marker-labeled first antibody of the present invention by labeling the antibody with these markers is as follows:
Physical adsorption methods used in this field [eg Tech
niques in Immunocytochemistry, volume 1, p108-133,
Acadenic Press, J.Histochem.Cytochem., Vol.25,
1187 to 1200 (1977), Experientia, vol.31, 1147 (1975), etc.], chemical bonding method [for example, Japanese Patent Publication No. 7-107535
Publication, J. Immunol. Methods, vol. 75, 351 (1984), BB
A., vol.640,66 (1981), BBRC, VOL.89,1114 (1979),
J.Immunol.Methods, vol.22,165 (1984) etc.]
Etc. are all listed, and the desired marker-labeled first antibody may be prepared by appropriately selecting according to the type of marker.

The preparation of the marker-labeled first antibody using colloidal gold as a marker is a method known per se [eg
Techniques in Immunocytochemistry, volume 1, p108-
133, Academic Press, J. Histochem. Cytochem., Vol.2
5,1187 to 1200 (1977), Experientia, vol.31, 1147 (1975)
Etc.] and the like.

Specifically, it can be easily obtained as follows, for example. That is, a commercially available gold colloid or a gold colloid prepared by a conventional method, for example, a method of reducing chloroauric acid with sodium citrate [Nature Phys. Sci., Vol. 241, 20 (1973)], The first antibody of
Histochem.Cytochem., Vol.25, 1187 ~ 1200 (1977), Exper
ientia, vol.31, 1147 (1975)]]. More specifically, colloidal gold and 0.5 to 100 μg is usually added to 1 mg of colloidal gold.
Preferably, 1 to 50 μg of the first antibody against the substance to be measured is reacted in an appropriate buffer solution at room temperature for 5 to 30 minutes, and then a dispersant such as polyethylene glycol is added, followed by centrifugation or the like. It can be easily obtained by collecting the first antibody labeled with colloidal gold. The obtained gold colloid-labeled first antibody may be uniformly dispersed and stored in a solution containing a dispersant such as polyethylene glycol.

The buffer used in the above reaction is of any type, concentration, pH if it does not inhibit the binding reaction between the gold colloid and the first antibody for the substance to be measured.
Etc. are not particularly limited.

The method for preparing the biotin-conjugated second antibody used in the present invention is, for example, a commercially available biotinylation reagent, more specifically, biotin (for example, NHS-biotin) into which a succinimide group has been introduced, or N. -A method of reacting hydroxysuccinimide (NHS) and biotin bound via a spacer with an amino group of an antibody [eg, J. Biol. Chem., Vol. 264, 272-279 (1989),
Shinsei Chemistry Laboratory 12, Molecular Immunology III, p103-104, Tokyo Kagaku Dojinsha, etc.], eg commercially available N- [6- (Biotinamide) hex
yl] -3 '-(2'-pyridyldithio) propionamide (biotin-H
PDP) and N-Iodoacetyl-N-biotinylhexylenediamine
To react with the thiol group of the antibody [eg Annals
of the New York Academy of Science, vol.254, 203 (1
975) etc.], a method of reacting biotin introduced with a hydrazino group with an aldehyde group of an aldehyde-modified antibody [eg J. Biol. Chem., Vol. 172, 71 (1948); Biotech.App.
l.Biochem., vol.9, 488-496 (1987)], prepared from an antibody using a derivative in which a maleimide group is introduced into biotin.
Method of binding to SH group of Fab ', method of binding biotin introduced with hydrazino group to sugar chain of antibody, method of binding biotin introduced with hydrazino group to carboxyl group of antibody in the presence of water-soluble carbodiimide, etc. Any method may be used as long as it does not lose the activity of the antibody usually used in this field.

As a method for holding the marker-labeled first antibody and biotin-conjugated second antibody thus prepared in the first part of the present invention so that they can be moved by capillarity, a method generally used in this field is used. There is no particular limitation as long as it is present. Specifically, for example, the membrane-like material or sheet-like material as described above is immersed in a solution containing a marker-labeled first antibody and a biotin-conjugated second antibody, and then blow-dried or freeze-dried. And a method in which a solution containing a marker-labeled first antibody and a biotin-conjugated second antibody is applied to, sprayed on, or dripped onto a film-like or sheet-like material as described above, and then blown or freeze-dried. To be The solution containing the marker-labeled first antibody and biotin-conjugated second antibody is usually used in this field, for example, Tris buffer, phosphate buffer, veronal buffer, borate buffer,
All buffers such as Good's buffer which are usually used in the assay method utilizing the antigen-antibody reaction are mentioned, and the pH thereof is not particularly limited as long as it does not suppress the antigen-antibody reaction, but usually 5 to 9 It is preferably selected from the range.
In such a solution, a stabilizer such as albumin, globulin, water-soluble gelatin, polyethylene glycol, a surfactant, a saccharide, and the like are appropriately contained as long as the solution does not inhibit the target antigen-antibody reaction. It may be. The film-like or sheet-like material used for holding the marker-labeled first antibody and the biotin-conjugated second antibody is
In order to prevent these antibodies from being non-specifically adsorbed and affecting the measurement, it is desirable to perform a so-called blocking treatment in advance. Such a blocking treatment is a method usually used in this field, for example, a blocking agent such as albumin, globulin, casein, polyvinyl alcohol, and a surfactant (however, the influence on the measurement of the film or sheet is not affected. A suitable buffer solution (for example, Tris buffer solution, phosphate buffer solution, veronal buffer solution, borate buffer solution, Good buffer solution or the like having a pH of about 5 to 9) is used. It may be carried out by a method in which it is dipped in a suitable time and then dried.

The amounts of the marker-labeled first antibody and biotin-conjugated second antibody to be retained in the first part of the present invention vary depending on the kind of the substance to be measured and the detection limit or the calibration limit. Although not particularly limited, for example, the retention amount per unit area (cm ² ) of the first portion of the present invention (as immunoglobulin amount) is usually 0.01 μg to 10 m.
g, preferably 0.05 μg to 1 mg, more preferably 0.1 to 100
It is appropriately selected from the range of μg.

The second part of the present invention, so-called avidin immobilized on a so-called spreading membrane, is not particularly limited as long as it has a binding ability to biotin, and specifically, for example, avidin derived from egg white and a microorganism. Derived streptavidin,
Examples thereof include derivatives thereof [eg, neutroavidin (NeutrAvidin, manufactured by Pierce) obtained by cleaving the sugar chain of avidin]. The commercially available avidin used in the present invention can be used as it is, and its quality, purification degree, and the like are not particularly limited as long as it does not contain a component that inhibits the intended measurement.

The method for immobilizing avidin on the second part (developing membrane) of the present invention is not particularly limited as long as it is a method usually used in this field, but specifically, it is used as the second part, for example. A method of applying an avidin solution to a part of a film or sheet made of the above-mentioned material, dropping or spraying, then drying and immobilizing by physical adsorption, for example, an amino group in the film or sheet. And a method of chemically bonding avidin using a functional group such as a carboxyl group. The film-like material or sheet-like material having the avidin-immobilized portion thus obtained is preferably subjected to so-called blocking treatment in order to prevent nonspecific adsorption from affecting the measurement. Such a blocking treatment is carried out by a method usually used in this field, for example, by using these film-like or sheet-like materials as a blocking agent such as albumin, globulin, casein, polyvinyl alcohol, a surfactant, etc. Select a suitable buffer (eg, pH).
Of about 5 to 9 for example, a Tris buffer, a phosphate buffer, a veronal buffer, a borate buffer, a good buffer, etc.) for an appropriate time and then drying.

The amount of avidin to be immobilized in the second part of the present invention varies depending on the kind of the substance to be measured and the detection limit or the calibration limit, and is not particularly limited. The amount of avidin retained per unit area (cm ² ) of the avidin-immobilized portion in the second part of the invention is usually 0.01 μg to 1 mg, preferably 0.1 μg to 100 μg, more preferably 0.5 μg to 50 μg. It

In order to sufficiently complete the sandwich-type immune complex forming reaction, or to influence the measurement by the coloring substance in the liquid sample or the measurement by the marker-labeled first antibody that is not bound to the substance to be measured. In order to avoid the influence, the avidin-immobilized portion in the second portion should be located at a position apart from the connecting end with the first portion and the other end of the second portion to some extent (for example, in the middle of the second portion). It is desirable to have it.

The substance to be measured by the immunochromatographic test device of the present invention is not particularly limited, but examples thereof include biological samples such as blood, plasma, serum, cerebrospinal fluid, urine, feces and the like, and appropriate buffer solutions thereof. Proteins such as albumin, hemoglobin, myoglobin, transferrin, protein A, C-reactive protein (CRP), etc. contained in a liquid sample obtained by diluting or suspending with, for example, high specific gravity lipoprotein (HDL), low Specific gravity lipoprotein (L
DL), lipid proteins such as very low density lipoproteins, for example, nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), for example, enzymes such as alkaline phosphatase, lactate dehydrogenase, lipase and amylase, for example, IgG, Ig
Immunoglobulins such as M, IgA, IgD and IgE (or fragments thereof such as Fc, Fab and F (ab) ₂ );
For example, fibrinogen, fibrin degradation product (FD
P), blood coagulation-related factors such as prothrombin and thrombin, for example, antibodies to viruses such as hepatitis B virus, hepatitis C virus, and AIDS virus, for example, microorganisms such as hepatitis B virus, syphilis tryponema (TP), chlamydia, and Candida. Related antigens, for example antibodies against microorganisms such as chlamydia, such as human chorionic gonadotropin (h
Hormones such as CG).

The immunochromatographic test device of the present invention may be prepared, for example, as follows. That is,
First, for example, a polyvinyl chloride sheet, polyethylene sheet, polypropylene sheet, polystyrene sheet or the like having an appropriate size is used as a carrier, and a double-sided tape or the like is used on the carrier. A film-like material or a sheet-like material having a (judgment part) is adhered to form a second portion (that is, a developed film), and then a marker-labeled first antibody (for example, a gold colloid-labeled first antibody) and a biotin-bound second The membrane-shaped or sheet-shaped material holding the antibody can be prepared by, for example, using a double-sided tape or the like to bond the second portion and the second portion such that capillarity occurs between them to form the first portion. In order to facilitate capillarity between the first portion and the second portion, the first portion is
You may make it adhere | attach so that the lower end of a part may overlap about 1-2 mm. Further, in the immunochromatographic test device of the present invention, in order to smoothly perform the absorption of the liquid sample and the sandwich-type immune complex forming reaction in the first part, the liquid sample is further absorbed at the lower end of the first part. Part is provided, the liquid sample is first dropped on this part, and the first
You may make it carry out this to a part and start a reaction. The liquid sample absorbing unit may be a film or sheet (which may be the same as the film or sheet used to form the first portion or the second portion) capable of absorbing the liquid sample. It can be prepared by using a double-sided tape or the like to adhere the first portion and the first portion so as to cause capillary action.
Note that, in order to easily cause a capillary phenomenon between the first portion and the liquid sample absorbing portion, the liquid absorbing portion may be adhered so as to overlap the lower end of the first portion by about 1 to 2 mm. In addition, as the whole size, the time from the dropping of the liquid sample to the liquid sample absorbing portion to the development of the liquid sample to the end of the second portion due to the capillary phenomenon is usually 15
It is set to be within minutes, preferably within 10 minutes.
One preferred embodiment of the immunochromatographic test device of the present invention thus obtained is shown in FIG.

Incidentally, in FIG. 1, each numeral indicates the following respectively. 1: Carrier 2: Liquid sample absorption part 3: First part 4: Second part 5: Judgment part (avidin immobilization part)

The measurement using the measuring method of the present invention is
For example, the following may be performed. That is, first, for example, a liquid sample is dropped onto the first portion of the immunochromatographic test device of the present invention prepared as described above, or the first portion is immersed in the liquid sample (the test is performed). When the tool is provided with the liquid sample absorption section, the liquid sample is dropped onto the liquid sample absorption section, the liquid sample absorption section is dipped in the liquid sample, and the like. The liquid sample is supplied to the portion), the substance to be measured in the liquid sample in the first portion, 1) the marker-labeled first antibody, and
2) By reacting with a biotin-conjugated second antibody, first, a sandwich type immune complex (that is, a reaction product) of a marker-labeled first antibody-a substance to be measured-biotin-conjugated second antibody is formed in a liquid phase. Then, this is transferred to a development membrane (second part) having an avidin-immobilized part (judgment part) by capillarity, and the sandwich-type immune complex is captured by the judgment part using the avidin-biotin reaction. The marker-labeled first antibody that has not bound to the substance to be measured is carried further downstream due to the capillary phenomenon. Then, as a result, the degree of color development derived from the marker labeled with the first antibody in the sandwich-type immune complex captured in the determination part was visually observed, and the result was compared with the marker prepared in advance. The amount of the substance to be measured can be quantitatively measured by applying it to a color tone table showing the relationship between the degree of color development derived and the amount of the substance to be measured.

If the degree of color development is less than a specific color tone, it is negative, and if the degree of color development is darker, it is possible to semi-quantify the substance to be measured by the method of the present invention. The degree of color development is determined using a urine test paper tester such as Pretester RM-405 and Pretester RM-505 (both manufactured by Wako Pure Chemical Industries, Ltd.), for example, a densitometer. It goes without saying that you may go there.

Further, since the immunochromatographic test device of the present invention utilizes the avidin-biotin reaction, the developing membrane (the second part) has a reagent which is directly involved in the intended measurement other than the part where avidin is immobilized. Is not included. Therefore, the immunochromatographic test device of the present invention can be used for various measurement targets only by appropriately changing the first part to a film-like or sheet-like product holding an appropriate marker-labeled first antibody and biotin-conjugated second antibody. It also has a manufacturing advantage that a test tool for immunochromatography that can be used for measuring a substance can be manufactured. Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.

[0032]

Embodiment 1 (Production of immunochromatographic test device) (1) Production of development film having determination part (streptavidin-immobilized part) Biodyne C film (modified nylon film: manufactured by Nippon Pall Ltd.).
Streptavidin solution (2 mg / ml: 50 mM phosphate buffer, pH 7.4. Streptavidin; manufactured by Wako Pure Chemical Industries, Ltd.) at a position 20 mm from the lower end of 5 mm × 40 mm) TLC applicator (trade name: Renomate). After being linearly applied using Kamag Co., Ltd., it was dried to prepare a determination part (streptavidin-immobilized part). The obtained membrane with the determination part was blocked with a blocking solution [0.5% Tween20 (Kao Corporation trade name), 0.5% casein and 0.15M NaCl containing 50m].
M phosphate buffer, pH 7.4. And subjected to a blocking treatment for 2 hours and then dried again to obtain a developed film.

(2) Preparation of biotin-binding antibody solution Anti-BJP-λ chain (Bence Jones protein) monoclonal antibody solution (IgG content: 0.05 mg / ml in 0.1M NaHCO ₃ s)
ol .. 4 ml of Wako Pure Chemical Industries, Ltd. and N-Hydroxysuccinimido-bio
tin. Made by Sigma. ) A solution (13 mg / ml in DMF) (100 μl) was mixed and reacted at 4 ° C. overnight. After that, add 50
Dialyze against mM phosphate buffer (pH 7.4) (1 liter x
2. ), The resulting solution is a biotin-conjugated antibody solution (5 ml)
And

(3) Preparation of Gold Colloid-Labeled Antibody Solution An anti-BJP-λ chain monochromat having a recognition site different from that of the monoclonal antibody used for preparing the biotin-conjugated antibody.
10 μl of null antibody solution (IgG content: 20 mg / ml) and the method of Frens [Nature Phys. Sci., Vol. 241, 20-22
(1973)] and adjusted the pH to 6.5 with 0.1 MK ₂ CO ₃ (average particle size of gold colloid: 15 _nm. OD _{520 nm}
= 1.2) 6 ml was mixed and reacted for 1 hour, and then polyethylene glycol (manufactured by Sigma Co., trade name: Carbowax 20M) was added to the reaction solution to a final concentration of 0.05%. Then, the reaction solution was centrifuged at 15000 rpm × 20 minutes, the supernatant was discarded, and the resulting residue was treated with 50 mM phosphate buffer (containing 0.05% polyethylene glycol, pH 7.4).
After suspending in 3 ml, it was centrifuged again at 15000 rpm × 20 minutes. The obtained residue was suspended in 3 ml of 50 mM phosphate buffer (containing 0.05% polyethylene glycol, pH 7.4) to give a gold colloid-labeled antibody solution.

(4) Retaining the biotin-conjugated antibody and the colloidal gold-labeled antibody on the glass fiber membrane A solution obtained by mixing equal amounts of the biotin-conjugated antibody solution prepared in (2) and the gold colloid-labeled antibody solution prepared in (3). To
Blocking solution [0.5% Tween 20 (Kao Corporation trade name), 0.5% casein and 0.15 M NaCl in 50 mM phosphate buffer, pH 7.4. ] The glass fiber membrane (5 mm × 6 mm; manufactured by Millipore) that had been subjected to blocking treatment with] was immersed in the glass fiber membrane and dried to obtain a glass fiber membrane having a biotin-conjugated antibody and a gold colloid-labeled antibody as a tracer.

(5) Preparation of immunochromatographic test tool Vinyl chloride sheet (5 mm x 60 mm; Shinei Sangyo Co., Ltd.)
Made. ) Is adhered to the developing film having the determination part (streptavidin-immobilized part) prepared in (1) above using a double-sided tape to form a second part, and then the biotin-bonded prepared in (4) The glass fiber film holding the antibody and the colloidal gold-labeled antibody is adhered to the lower end of the spreading film by using a double-sided tape so as to be overlapped by about 1 to 2 mm.
Formed part. Further, a liquid sample absorption film (Roprosorb: manufactured by Nippon Pall Co., Ltd.) (5 mm × 16 mm) is adhered to the lower end of the glass fiber film by using a double-sided tape to form a liquid sample absorption part. A test tool for immunochromatography as shown in schematic FIG. 1 was prepared.

(Measurement procedure) The liquid sample (BJP-λ) was placed in the liquid sample absorption part of the immunochromatographic test device prepared above.
10 mM phosphate buffered saline containing 1 μg / ml of chains. pH 7.
4) 80 μl was added dropwise. On the other hand, 80 μl of a 10 mM phosphate buffered saline (pH 7.4) was added dropwise as a negative control to the liquid sample absorption part of another immunochromatographic test device. When the liquid sample was developed to the tip of the immunochromatographic test device (the end of the development membrane) by capillary action (about 8 minutes), the color of the determination part (streptavidin-immobilized part) was observed, and the BJP-λ chain was observed. A clear red-purple line was observed when a liquid sample containing was added, while a negative purple liquid line was observed when a negative control liquid sample was dropped onto the determination part (streptavidin-immobilized part). No line was observed. From these results, it is understood that the immunoassay test tool of the present invention can be used to quantify or semiquantify BJP-λ chain in a liquid sample.

[0038]

As described above, the present invention provides a test tool for immunochromatography for measuring the amount of a substance to be measured in a liquid sample, and a measuring method used therefor. According to the present invention, there is a problem in the conventional immunochromatographic test device, that is, a sandwich type immune complex forming reaction for measurement is carried out in a short time (for example, within 10 minutes) in order to perform the reaction on a solid phase. It is possible to accurately measure the substance to be measured in the liquid sample with high sensitivity without causing the problem that the measurement accuracy and the detection sensitivity are deteriorated due to the inability to complete the measurement.

Further, since the immunochromatographic test utensil of the present invention utilizes the avidin-biotin reaction, the developing membrane (second portion) is a reagent which is directly involved in the intended measurement in addition to the portion where avidin is immobilized. Is not included. Therefore, the immunochromatographic test device of the present invention can be used for various measurement targets only by appropriately changing the first part to a film-like or sheet-like product holding an appropriate marker-labeled first antibody and biotin-conjugated second antibody. It also has a manufacturing advantage that a test tool for immunochromatography that can be used for measuring a substance can be manufactured. Therefore, the present invention is a major invention that contributes to the industry.

[Brief description of the drawings]

FIG. 1 shows one of preferred embodiments of the immunochromatographic test device of the present invention.

[Explanation of symbols]

 In FIG. 1, each numeral indicates the following. 1: Carrier 2: Liquid sample absorption part 3: First part 4: Second part 5: Judgment part (avidin immobilization part)

Claims (2)

[Claims] 1. A first antibody specific to a substance to be measured, which is (a) movably held by capillarity, 1) labeled with a marker that gives a visually detectable signal,
And 2) a first part which is specific to the substance to be measured and which has a second antibody having a recognition site different from that of the first antibody and having biotin bound thereto, and (b) a second part having a site to which avidin is immobilized. (C) The immunochromatographic test device, wherein (c) the first part and the second part are connected so that capillarity occurs between them. 2. A first antibody specific to a substance to be measured, which is labeled with a marker capable of obtaining a visually detectable signal, and 2) a first antibody specific to the substance to be measured and a recognition site. Of the second antibody bound to the biotin-bound second antibody, which is different from that of the biotin, and is allowed to move by capillarity to provide a liquid sample to start the reaction in the liquid phase. Second having a fixed part
A method for measuring the amount of a substance to be measured in the liquid sample based on the degree of color development derived from the marker in the reaction product captured by the avidin-immobilized site of the second part.