JPH10267929A - Simple measurement method and reaction plate and reagent kit used therein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To eliminate an ampul for dilution or the like, simplify diluting operation, and reduce the cost of a kit, by forming a specimen dilution part and a reaction part in a reaction plate. SOLUTION: Three circles 2, 3, 4 for reaction (reaction parts) and a circle 5 for dilution (specimen dilution part) are formed on a plane 15 made of black synthetic resin by printing. A circle 17 for cleaning is formed in the vicinity of the hole 5 by printing, thereby forming a reaction plate 16. Each specified amount of diluent is dripped on both the circle 5 for dilution and the circle 17 for cleaning. Specimen is collected with a trace pipette, and added to the circle 5 for dilution. The diluent in the circle 17 for cleaning is suck and discharged several times with the trace pipette, thereby cleaning the inside of the trace pipette. The specimen is diluted in this manner, and then a specified amount of the diluted specimen is transferred to the reaction circles 2, 3, 4 with the trace pipette. The specimen is reacted with immunity agglutination reagent, so that an ampul containing diluent for dilution, a cuvette containing diluent, etc., are made unnecessary and diluting operation is simplified.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a simple measuring method using an immunoagglutination reagent, a reaction plate and a reagent kit used therefor.

[0002]

2. Description of the Related Art Latex reagents obtained by sensitizing latex particles with antibodies, heat-denatured gamma globulin, antigens, etc., are reacted with a test sample on a slide plate, and the corresponding antigens, rheumatoid factors, antibodies, etc. An antibody reaction occurs and the latex particles aggregate, making it possible to visually determine the presence or absence of aggregation.

[0003] This latex slide reagent is widely used because a result can be obtained in a few minutes.

[0004] However, when a corresponding antigen or the like is present in a test sample in a large excess, it is necessary to dilute the sample in advance because it causes a false negative due to a zone phenomenon.

[0005] Further, since the reaction is a qualitative reaction of + or-, it is necessary to dilute the sample to near the detection sensitivity of the latex reagent in order to know the concentration of the target substance in the sample.

For example, in the measurement of urinary estrogen, urine is diluted 50-fold, 100-fold, and 200-fold using a diluting ampule, and a slide is prepared by adding an anti-estriol antibody-sensitized latex to one drop of each diluted urine. Stir well on the plate, then add 1 drop of estriol sensitized latex and add 2 more drops.
The mixture was allowed to react with gentle stirring for a minute, and the presence or absence of aggregation was visually determined to obtain a semi-quantitative value.

[0007]

Therefore, conventionally, it is necessary to use a test tube for dilution or an ampoule containing a diluent to dilute a sample, and a measuring kit for 50 times requires 150 tubes for dilution. Since the ampoule and the like are incorporated as a set, the dilution operation is not only complicated, but also the kit itself becomes large and the cost is high.

[0008]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned conventional problems, and the first invention is to dilute a sample, and then to dilute the diluted sample on a reaction plate. In a measuring method in which a reaction with a reagent is performed and the presence or absence of particle aggregation is determined as an index, a sample is diluted in a sample diluting section of a reaction plate, and then a test dilution sample is added to the reaction section of the reaction plate. It depends on the measuring method.

A second invention relates to a reaction plate having a sample dilution section and a reaction section.

The third invention relates to a simple measuring reagent kit comprising a reaction plate having a sample diluting section, a pipette with a scale, a diluent, and an immunoagglutination reagent.

Samples to be measured in the present invention include:
Urine, serum, plasma, blood, tears, saliva, nasal discharge, ascites, cervical mucus and the like are used.

As a diluent used for diluting a sample, each buffer such as physiological saline, phosphoric acid, Tris, glycine and the like can be used, and the pH is arbitrary from acidic to alkaline in consideration of the stability of the substance to be measured. Can be selected.

As an immunoagglutination reagent, any reaction principle of an agglutination reaction or an agglutination inhibition reaction can be used, and as the carrier particles, latex, gelatin, bacterial cells, liposomes and the like can be used.

As a reaction plate used for diluting a sample, a reaction plate having at least one sample diluting portion near a circular or elliptical circle or a concave reaction portion is used.

The sample diluting section is formed on the reaction plate as a circle, a concave portion, or a cuvette for dilution and a holding section for setting the cuvette, and an appropriate number thereof are provided as necessary.

As a material for the reaction plate, glass, plastic, coated paper, metal or the like is used.

The circle for dilution forms a circular or elliptical ring on the reaction plate with a paint or the like in the same manner as the reaction section.

The concave portion for dilution may be formed in a circular or elliptical shape when the reaction plate is molded, and it may be formed by press for coated paper, metal and the like.

In addition to the case where the sample dilution section is provided integrally with the reaction plate, a holding section for setting the dilution cuvette may be formed on the reaction plate so that a dilution cuvette can be used. .

The holding portion for setting the dilution cuvette may be any one of a hole, a notch, and a notched bent portion.

The shape of the hole may be circular, but the shape is not limited as long as it can hold the dilution cuvette.

The shape of the notch is preferably a U-shape or an ellipse, but may be any shape as long as it can be fitted with the dilution cuvette.

The shape of the cut-and-folded portion may be any of a + shape, a V shape, and a U shape.
The cuvette is inserted into the hole formed and used.

The diluting cuvette is convenient when a large amount of diluent is used to perform high dilution.

In the above description, the size of the circle, the concave portion, and the cuvette can be appropriately determined according to the dilution factor of the sample.

The washing section can be formed in the same manner as the above-mentioned dilution section.

The pipette used for collecting the test sample is several μm.
A micropipette capable of collecting several tens μl from 1 is preferable, and a capillary tube, an automatic pipette and the like can also be used.

It is convenient to use the graduated pipette shown in FIG.

The graduated pipette is provided with a scale at one end of a heat-resistant thin tube having an inner diameter of about 1 mm and having both ends opened, and has a slightly larger diameter at the other end than at least the sample collection / addition capacity. A heat-shrinkable flexible tube having an internal capacity and easily deformable by the pressing force of a finger is fitted and fixed by heat shrinkage, and the other end of the flexible tube is flattened from both sides by heat and pressure. The length of the sealing end of the flexible tube and the heat-shrinkable fixing portion is such that the finger does not touch the sealed end when sandwiched between the fingers.

[0029]

[Effect] For example, urine of the pregnant woman in the third trimester

FIG. 2 shows a micropipette 7 shown in FIG.

In FIG. 1, 5 μl is collected in a dilution circle 5 of a reaction plate 6 shown in FIG. 1, 45 μl of a diluent is added to the dilution circle 5, and the reaction plate 6 is shaken to mix to prepare a 10-fold diluted sample. I do.

5 μl, 2.5 μl, 1.25 of the diluted sample
Then, 1 μl of the mixture was added to each of reaction circles 2, 3, and 4, and then a drop of anti-estriol antibody-sensitized latex was added thereto.
One drop of the OA conjugate-sensitized latex is added, and the reaction plate 6 is shaken to react for 2 minutes.

The anti-estriol antibody-sensitized latex is
Sensitivity is set so that anti-estriol antibody is saturated with 1.25 μl of a 10-fold diluted specimen of estriol 20 μg / ml in raw urine, and no agglutination reaction occurs.

That is, the estriol concentration of a 10-fold diluted sample of 20 μg / ml is 2 μg / ml, and the amount of estriol in 1.25 μl is 2.5 ng.

Therefore, if 2.5 ng of estriol is present in the reaction circle, the anti-estriol antibody of the antibody-sensitized latex is saturated. Does not occur and is determined to be positive. .

When estriol in the reaction circle is 2.5
In the case of less than ng, the anti-estriol antibody on the surface of the antibody-sensitized latex is not completely saturated, so that it undergoes an agglutination reaction with the next antigen-sensitized latex to be added to give an agglutination image,
The result of the determination is negative.

[0035]

[Table 1] As shown in FIG.
In the case where the estriol concentration was observed in the circle to which was added (determination: negative), the estriol concentration in the raw urine was determined to be less than 5 μg / ml.

When 5 μl of a 10-fold diluted urine sample did not show an agglutination image (judgment: positive), when 2.5 μl showed an agglutination image (judgment: negative), the estriol concentration in the raw urine was determined. Is semi-quantified to be greater than or equal to 5 μg / ml and less than 10 μg / ml.

Furthermore, 5 μl of a 10-fold diluted urine sample and 2.5
Although no aggregation image was observed with μl (judgment: positive),
When an aggregation image is observed in 25 μl (judgment: negative),
The estriol concentration in the raw urine is 2 when the concentration is 10 μg / ml or more.
It is semi-quantified to be less than 0 μg / ml.

Then, urine specimens 10-fold diluted 5, 2.5,
When no aggregation image is observed in all the circles of 1.25 μl (judgment: positive), the estriol concentration in the raw urine is semi-quantified to be 20 μg / ml or more.

In this way, a 10-fold dilution of the raw urine is collected in each of the reaction circles at 5, 2.5, and 1.25 μl, and the antibody-sensitized latex and the antigen-sensitized latex are added and reacted. Semi-quantitative measurement can be easily performed.

[0040]

【Example】

 Example 1

FIG. 1 shows an example of a reaction plate of the present invention, in which three oval reaction circles 2, 3, and 4 are formed by printing on a mount 1 having a black upper surface and a non-water-absorbing coating treatment. A single oval dilution circle 5 is similarly formed by printing over the reaction circles 2, 3, and 4 to form a reaction plate 6.
It was created.

Embodiment 2

FIG. 2 shows an example of a micro pipette 7 used in the present invention. 11 are sequentially provided at three locations, and the other end side has a slightly larger diameter than the thin tube, has at least the content of the sample collection / addition capacity, and can be easily deformed by finger pressure. The flexible tube 12 is fitted and fixed by heat shrinkage, and the other end of the flexible tube 12 is flatly sealed from both sides by heat and pressure to form a sealed end portion 13, and the flexible tube 12 is sealed. When the length between the end portion 13 and the heat-shrinkable fixing portion 14 is sandwiched between fingers, the length is such that the finger does not touch the sealed end portion 13.

Example 3 A 0.1 M glycine buffer (pH 8.2) was diluted with 0.1% of bovine serum albumin (BSA) and 0.9% of NaCl.

Example 4 A rabbit anti-estriol antibody was physically adsorbed to a commercially available polystyrene latex particle having a particle diameter of 0.2 μm by a conventional method for sensitization. The suspension was suspended in a 0.2 M glycine-NaCl buffer (pH 8.2) containing 0.1% BSA to obtain an antibody-sensitized latex reagent.

Example 5 A conjugate of estriol-16-glucuronide and ovalbumin was prepared according to the method of BF ERLANGER et al. (JBC 234 (5), 1090, 1959). did.

The conjugate was physically adsorbed to commercially available 0.8 μm polystyrene latex particles by a conventional method, and 0.2M glycine-NaCl containing 0.1% BSA was used.
The antigen-sensitized latex reagent was obtained by floating in a buffer solution (pH 8.2).

Example 6 45 μl of the diluent of Example 3 was placed in the diluting circle 5 of the reaction plate 6, then urine was drawn up to a 5 μl scale 9 with a trace pipette 7 and added to the diluting circle 5. Shake to mix to prepare 10-fold diluted urine.

The inside of the tip of the micropipette 7 is washed or renewed with purified water, and 8.75 μl of the diluted sample in the diluting circle 5 is sucked up to the scale 11. 25μl, 2.5μl, 5μ
Extrusion is performed according to the scales 11, 10 and 9 sequentially. Next, the anti-estriol antibody-sensitized latex reagent of Example 4 was added dropwise to each of the circles 4, 3, and 2 one by one.
The reaction plate 6 was shaken to mix, and one drop of the estriol-sensitized latex of Example 5 was added and mixed.
After reacting for 2 minutes, the presence or absence of an aggregated image was visually determined.

Embodiment 7

FIG. 3 is another example of the reaction plate of the present invention, in which three reaction circles 2, 3, 4 and one dilution circle 5 are provided by printing on a black synthetic resin flat plate 15, and A cleaning circle 17 was provided by printing next to the dilution circle 5 to form a reaction plate 16.

Example 8 In the same manner as in Example 6, 45 μl of the diluent was dropped on both the diluting circle 5 and the washing circle 17, and 5 μl of urine was collected with a small amount of a pipette 7 and extruded into the diluting circle 5. Then, the diluent in the cleaning circle 17 is sucked and discharged several times with the micropipette to wash the inside of the thin tube 8.

By following the same procedure as in Example 6, the estriol concentration in urine can be semi-quantified. By setting the neutralization amount of estriol in one drop of the antibody-sensitized latex to 2.5 ng,

As shown in Table 1, when no aggregation was obtained with 1.25 μl of a 10-fold diluted sample (determination: positive), 20 μg / m
1 or more estrogen is contained, and
In the case of non-aggregation at 5 μl and aggregation at 1.25 μl, 10
It becomes less than 20 μg / ml at μg / ml or more, becomes non-aggregated at 5 μl, and is less than 10 μg / ml at 5 μg / ml or more when it is 2.5 μl and is less than 5 μg / ml when it is aggregated at 5 μl. You.

[0051]

As described above, according to the present invention, the following various excellent effects can be obtained.

1. According to the method of the present invention, the sample is diluted in the sample diluting section on the reaction plate, and then the diluted sample is reacted with the immunoagglutination reagent on the reaction plate. In addition, a cuvette or the like containing a diluting liquid is not required, and the diluting operation is significantly simplified.

2. The use of a graduated pipette facilitates the collection of the original sample and the collection of the diluted sample, and can be performed with a single pipette, so that there is no waste in the operation and the kit, and semi-quantitative measurement can be performed rationally. .

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing one embodiment of the present invention.

FIG. 2 is an explanatory view showing an example of a graduated pipette used in the present invention.

FIG. 3 is an explanatory view showing another embodiment of the present invention.

Claims (18)

[Claims] In a measurement method for diluting a sample and then reacting the diluted sample with an immunoagglutination reagent on a reaction plate and determining whether or not particles have aggregated as an index, the sample is diluted in a sample dilution section of the reaction plate. And then adding the diluted sample to the reaction section of the reaction plate. 2. The simple measurement method according to claim 1, wherein the sample is any one of urine, serum, plasma, blood, tear, saliva, nasal discharge, ascites, and cervical mucus. 3. The simple measurement method according to claim 1, wherein the particles are any of latex, gelatin, liposome, and metal colloid. 4. The simple measurement method according to claim 1, wherein the determination method is one of a latex agglutination reaction and a latex agglutination inhibition reaction. 5. A reaction plate having a sample dilution section and a reaction section. 6. The reaction plate according to claim 5, for determining the presence or absence of aggregation of particles. 7. The reaction plate according to claim 5, wherein the sample dilution section is one of a circle and a concave. 8. The reaction plate according to claim 5, wherein the sample diluting section comprises a diluting cuvette and a holding section for setting the diluting cuvette. 9. The reaction plate according to claim 8, wherein the holding portion is a hole. 10. The reaction plate according to claim 8, wherein the holding portion has a semicircular, U-shaped, or V-shaped notch. 11. The reaction plate according to claim 8, wherein the holding portion is an X-shaped, V-shaped, U-shaped or concave cut or bent portion. 12. A reaction plate comprising a sample diluting section, a washing section, and a reaction section. 13. A simple measurement reagent kit comprising a reaction plate having a sample dilution section, a graduated pipette, a diluent, and an immunoagglutination reagent. 14. A simple measurement reagent kit comprising a reaction plate having a sample diluting part and a washing part, a pipette with a scale, a diluent, and an immunoagglutination reagent. 15. The simple measurement reagent kit according to claim 13, wherein the immunoparticle agglutination reagent comprises an antibody reagent and an antigen-sensitized latex. 16. The simple measurement reagent kit according to claim 13, wherein the antibody reagent is an antibody-sensitized latex. 17. The simple measurement reagent kit according to claim 13, wherein the immunoparticle agglutinating reagent comprises an antibody-sensitized latex and a polymer antigen. 18. The simple measurement reagent kit according to claim 13, wherein the immunoagglutination reagent is an antibody-sensitized latex or an antigen-sensitized latex.