JPH10221334A - Hemolytic measuring method and microblood collecting tube and reagent kit used therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To easily measure the main components and trace components of a blood sample by adding a diluted hemolytic sample to an immunological measuring system and measuring antigens or antibodies after dissolving blood cell components by diluting collected blood with a hemolytic diluting solution. SOLUTION: Both ends of a capillary tube 1 having a required length and a uniform inside diameter are opened, with one opening 2 being cut in a circular shape perpendicularly to the axial center of the tube 1, and scale divisions 3 are marked on the surface of the tube 1 on the opening 2 side. Then a flexible tube 5 having an inside diameter which is slightly larger than the outside diameter of the tube 1 is put on the other opening 4 of the tube 1. The tube 1 can be made of glass or a heat-resistant synthetic resin and the flexible tube 5 can be made of a synthetic resin, such as the polyethylene, etc., so that the tube 5 may become flat when the tube 5 is pressed between fingers and can return to its original pipe-like shape when the tube 5 is released from the pressing force. A diluted hemolytic sample is collected by diluting collected blood with a hymolytic diluting solution so as to dissolve blood cell components and added to an immunological measuring system for measuring antigens or antibodies.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hemolytic measuring method, a micro blood collection tube and a reagent kit used for the method.

[0002]

2. Description of the Related Art Conventionally, in the pharmacological test or the side effect / toxicity test of pharmaceuticals, a drug is screened using small animals such as mice, rats, and guinea pigs, and then rabbits,
Normally, clinical trials are started after transfer to medium-sized animals such as cats and dogs and sufficient efficacy and safety have been confirmed.

[0003] As a method for confirming and judging the effectiveness of a drug, various test methods have been established according to the purpose. However, methods for judging the side effect or toxicity of the drug include reproduction, developmental toxicity tests, and carcinogenesis. Hematocrit measurement has become widespread for the measurement of the basic components of animal body fluids, as there are only irritation tests, irritation tests for skin, eye mucosa, blood vessels, etc., photosensitization tests, antigenicity tests, etc. In fact, the measurement of components such as albumin, IgG, IgA, IgM, and complement, which are main constituent proteins in blood, is hardly performed.

[0004] Generally, albumin, IgG, I
It is said that gA, IgM, complement protein, fibrinogen, etc. occupy about 70% of the serum protein, and play a very large role in their body, making them the most basic measurement items in clinical tests.

Therefore, it is important to know the fluctuation of these serum proteins at the stage of animal experiments for confirming the efficacy, side effects and toxicity of the drug. The measurement of IgG, A, and M is effective in knowing the effect on immune function.

[0006] Therefore, conventionally, these serum proteins have been measured using a one-way immunodiffusion method (S
RID).

[0007]

However, in order to collect a sufficient amount of serum necessary for the measurement of the serum component from a small animal such as a mouse or rat, the blood must be collected in an amount of 0.5 ml to 1.0 ml.
Since it is necessary to collect ml of blood, an extremely large burden or damage is given to an animal individual, and it has been difficult to observe temporal or daily fluctuations in blood components due to drug administration.

[0008]

Means for Solving the Problems The present invention has been made to solve the above-mentioned conventional problems, and the first invention is to dilute blood collected using a micro blood collection tube with a hemolytic diluent. The hemolysis method is characterized by hemolyzing blood cell components and then adding the diluted hemolyzed sample to an immunological measurement system to measure an antigen or an antibody.

The second invention has an inner diameter of 1 mm with both ends opened.
A scale is provided on one end side of the heat-resistant thin tube around φ, and the other end side has a slightly larger diameter than the thin tube, has at least the capacity for collecting and adding a sample or the like, and is easily pressed by a finger. A deformable heat-shrinkable flexible tube is fitted and fixed by heat shrink, and the other end of the flexible tube is partially or completely opened, and the open end of the flexible tube and the heat shrink A micro blood collection tube, characterized in that the length between the fixed portion and the fixed portion is such that the open end side can be sealed by compression and the contents can be released by sandwiching with fingers.

A third aspect of the present invention relates to a hemolytic reagent kit comprising a micro blood collection tube, a hemolytic diluent, an antibody-sensitized latex, and a diluent.

The animals to be measured according to the present invention include small animals such as mice, rats, hamsters, guinea pigs and chickens which are widely used as experimental animals, and medium-sized animals such as rabbits, dogs and cats, and goats. , Sheep,
It can also be carried out on large animals such as cows and horses.

As a blood collection method, in the case of a small animal, it is convenient to puncture the fundus venous plexus, but it is also possible to collect blood by cutting the tail vein or the ear vein, and it is also possible to use blood collected from the heart or jugular vein. It is possible.

When blood is collected, if a capillary having an inner diameter of about 1 mmφ is used, blood is naturally drawn up by capillary action. Therefore, it is possible to easily collect a required amount of blood by grading the capillary.

In order to discharge blood collected from the capillary tube, the blood may be blown directly into the mouth or by connecting a discharge tube to the mouth.

As shown in FIG. 1, a scale is provided at one end of a heat-resistant thin tube having an inner diameter of about 1 mmφ with both ends opened, and has a slightly larger diameter than the thin tube at the other end and has at least the capacity for collecting and adding a sample or the like. 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 partially or entirely opened. A micro blood collection tube having a length between the open end of the flexible tube and the heat-shrinkable fixing portion, the length being such that the open end side can be sealed by compression and the contents can be released by sandwiching with fingers. May be used.

With the use of the above-mentioned micro blood collection tube, the blood is easily sucked up by the capillary phenomenon at the time of blood collection because the inside of the capillary is open. At the time of discharge, the open end of the flexible tube is sealed by bending or the like. By pressing the flexible tube with the fingers while keeping it, the blood in the tubule is released.

The micro blood collection tube is convenient for collecting blood from the fundus venous plexus of a small animal, but it is also easy to collect blood that has been bled by tail vein cutting or ear vein incision.

Heart puncture, carotid blood sampling, decapitation blood sampling,
It is also possible to collect a certain amount of blood collected by an abdominal aorta cutting method, an axillary artery cutting method, a hip artery cutting method, or the like using an automatic pipette or the like. it can.

In addition, an anticoagulant such as heparin may be used to prevent coagulation of blood at the time of blood collection.

Examples of the hemolytic diluent for hemolyzing and diluting the blood collected by the above operation include water, chemicals such as saponin, animal hemolysin such as snake venom, plant hemolysin such as ritin, and staphylo. Physiological saline containing at least any one of bacterial hemolytic toxins such as lysine or a buffer such as a phosphate buffer isotonic with blood can be used.

The concentration of the hemolysin varies depending on the type of the hemolysin, but in the case of saponin, it is suitably about 0.1 to 1.0%.

The blood protein components to be measured include:
Albumin, IgG, IgA, IgM, IgD, IgE
Such as immunoglobulins, complement components such as C3 and C4, anemia markers such as ferritin and transferrin, hemoglobin and other hemoglobins, and blood clotting factors such as fibrinogen.
It is applicable to almost the same items as blood components that can be measured in humans at present.

The measurement sensitivity of the latex immunoturbidimetry is as follows:
The measurement sensitivity varies depending on the affinity of the antibody used and the size of the latex particles. However, since it is at a technical level that can measure up to 1 ng / ml at present, the concentration is 1 ng / ml in a 50-fold diluted hemolyzed sample, that is, 50 ng in blood. /
Those with a concentration of ml or more can be measured.

As an immunological assay system for measuring a diluted hemolyzed sample, a latex immunoturbidimetric assay is convenient, but immunoassays such as an enzyme immunoassay, an immunoluminescence assay, and a radioimmunoassay are also used. be able to.

In the latex immunoturbidimetry, a sample containing the antigen or antibody to be measured is added to a buffer, and a latex reagent sensitized with an antibody or antigen corresponding to the antigen or antibody to be measured is added to the buffer. And the change in turbidity after a certain time is measured as the absorbance in the visible or far-infrared region and the scattered light intensity.

The measurement wavelength is 500 nm to 950 nm.
Is suitable because latex particles having a particle diameter of 0.2 to 0.5 μm can be measured with high sensitivity and are not affected by hemoglobin.

As a method similar to the latex immunoturbidimetric method, a measuring method for measuring the number of aggregation of latex particles by an antigen-antibody reaction is also applicable to the present invention.

[0027]

[Action] For example, the mouse is gripped with the left hand to fix the head, the tip of the micro blood collection tube held in the right hand is inserted along the rear surface of the eyeball of the mouse, and protruded to break the fundus. Thereafter, when the micro blood collection tube is slightly pulled, the flexible tube fixed to the other end is in an open state, and blood rises due to the capillary phenomenon. When the blood reaches the scale, the micro blood collection tube is pulled out.

Next, with the blood collection side tip of the micro blood collection tube facing the opening of the sample cup or test tube containing the hemolytic diluent, a flexible tube fixed to the rear end of the micro blood collection tube is used. When the flexible tube is compressed by a finger with the open end bent and held, the collected blood is released into the hemolytic diluent of the sample cup, and the sample is quickly shaken by shaking the container and stirring. Is obtained.

The hemolyzed sample is placed in a sample rack of a general-purpose automatic analyzer together with the sample cup, and 20 μl of the hemolyzed sample is collected by a sampling probe of the automatic analyzer, and then 250 μl of a buffer is added by a reagent probe, and the sample is added for about 5 minutes. After incubation at 37 ° C., for example, anti-mouse I
When 50 μl of the latex sensitized with the gM antibody was added, the IgM in the hemolyzed sample and the anti-IgM antibody sensitized on the latex particle surface reacted with the antigen-antibody, and the latex particles aggregated in proportion to the IgM amount, Turbidity increases.

The increase in the turbidity can be measured as an increase in absorbance after a certain period of time (about 5 minutes), and a calibration curve is prepared in advance in the same manner using an IgM standard solution having a known concentration. Thus, the IgM concentration in the diluted sample can be quantitatively measured, and the IgM concentration in the mouse blood can be known by multiplying the concentration by a dilution factor.

The IgM concentration in serum can be obtained by dividing the IgM concentration in the blood by the hematocrit value and multiplying by 100.

The ratio of serum to blood cells in whole blood is approximately 4: 3.
Therefore, the IgM concentration in serum can be estimated by multiplying 4/7 by the IgM concentration in the blood.

[0033]

【Example】

 Example 1

FIG. 1 shows an example of a micro blood collection tube of the present invention.
Both ends of a thin tube 1 having a required length and a substantially uniform inner diameter are opened, and one opening 2 of the thin tube 1 is cut by a plane perpendicular to the axis of the thin tube 1 to form a circle. Scales 3 are provided at required intervals on the opening 2 side, and a flexible tube 5 having an inner diameter slightly larger than the outer diameter of the thin tube 1 is fitted on the other opening 4 side. It is fixed to the thin tube 1.

Here, the length and inner diameter of the thin tube 1 can be appropriately determined according to the amount of blood to be collected and added.
The length can be used as long as it is 2 cm or more. However, in consideration of operability and hygiene, 5 to 10 cm is preferable, and the inner diameter is 1 m.
It may be around mφ. Glass or heat-resistant synthetic resin can be used as the material of the thin tube 1.

The flexible tube 5 can be made of a synthetic resin such as polyethylene, polypropylene, or vinyl. However, the flexible tube 5 is easily crushed when pinched with fingers, and the original cylinder is released when the pressing force is reduced. Any shape can be used as long as the shape can be restored.

The length of the flexible tube 5 is the same as that of the thin tube 1.
However, even when the open-side end 6 of the flexible tube 5 is bent and sealed, at least the collection and addition of a sample or the like can be ensured.

The flexible tube 5 is fitted into the thin tube 1,
As means for fixing, there is fixing using heat shrinkage of the flexible tube 5 or the like. When utilizing the heat shrinkage of the flexible tube 5, after fitting the flexible tube 5 made of a heat-shrinkable synthetic resin such as Sumitube into the thin tube 1,
The flexible tube 5 of the fitting portion may be partially heated and heat-shrinked to be fixed to the thin tube 1, or the end of the thin tube 1 may be heated and inserted into the flexible tube 5, It may be contracted.

Example 2 0.5 g of saponin was dissolved in 100 ml of physiological saline to prepare a hemolytic diluent.

Example 3 A commercially available polystyrene latex particle having a particle size of 0.2 μm is sensitized by physically adsorbing a goat anti-rat IgG antibody by a conventional method. 0.2M glycine containing 0.1% BSA
The sensitized latex reagent was obtained by floating with a NaCl buffer (pH 7.2).

0.1 g of BSA was added to 0.1 M glycine-Na
A reaction buffer was prepared by dissolving in a Cl buffer.

Example 4 The limbs of Wistar rats were fixed to a fixed base with a cord, and the tail vein was cut with a scalpel at a distance of about 2 cm from the base of the tail. The oozing blood was collected in 7 μl using the micro blood collection tube obtained in Example 1. Collected.

Next, the blood collection side tip of the micro blood collection tube was directed toward a sample cup containing 200 μl of the hemolytic diluent prepared in Example 2, and the blood was released into the sample cup. The release was performed by pressing the flexible tube with fingers while bending and sealing the open end of the flexible tube of the micro blood collection tube.

The sample cup is shaken to stir and hemolyze. The buffer 300 of Example 3 was added to 3 μl of the hemolyzed sample.
Then, 50 μl of anti-rat IgG antibody-sensitized latex reagent was added, and 0.57 min at 1 minute and 5 min after the start of the reaction.
The absorbance at μm (OD × 10000) was measured and the difference in absorbance was determined to be 1849.

Using a rat IgG standard solution, the same operation as above was performed to determine the relationship between the IgG concentration and the absorbance difference.

The standard curve shown in FIG. 2 was created.

Using the standard curve, the IgG concentration was determined from the absorbance difference of the hemolyzed sample of the rat.
g / dl, multiplied by a dilution factor of 31 times, the IgG concentration in whole blood was 888.8 mg / dl, and since the hematocrit value was 43%, the IgG concentration in serum was 1555.4 mg / dl. It has been found.

Example 5 A rabbit anti-guinea pig albumin antibody was sensitized to latex particles in the same manner as in Example 3 to obtain a sensitized latex reagent.

Five guinea pigs were fixed on a fixed base, respectively, and 14 μl of serum was sequentially collected from the fundus venous plexus using a micro blood collection tube, and added to 400 μl of the hemolytic diluent in the same manner as in Example 4 for measurement. . The standard curve is

FIG. 3 shows the results of calculating the albumin concentration in the serum corrected by the albumin concentration in the hemolyzed sample and the estimated hematocrit value of 45%.

[Table 1] It was as follows.

Example 6 A sensitized latex reagent was obtained by sensitizing a sheep anti-rat IgE antibody to commercially available 0.5 μm polystyrene latex particles in the same manner as in Example 3.

The rat was grasped with the left hand to fix the head, the tip of the micro blood collection tube held in the right hand was inserted along the rear surface of the rat's eyeball, and after protruding to break the fundus, When the blood collection tube is pulled slightly, the flexible tube fixed to the other end is in an open state, and blood rises in the capillary tube due to capillary action. When the blood rises to the 7 μl scale, the micro blood collection tube is withdrawn.

Thereafter, the same operation as in Example 4 was carried out, 250 μl of the buffer solution of Example 3 was added to 20 μl of the obtained hemolyzed sample, and then 5 μl of the anti-rat IgE antibody-sensitized latex reagent was added.
0 μl was added, and the absorbance at 0.7 μm at 1 minute and 5 minutes after the start of the reaction was measured to determine the difference in absorbance.

[Table 2] It was as follows.

Using a rat IgE standard solution, the same operation as above was performed to determine the relationship between the IgE concentration and the absorbance difference.

The standard curve shown in FIG. 4 was created.

Using the standard curve, the IgE concentration of the hemolyzed sample of the rat was determined, and then the IgE concentration in whole blood multiplied by a dilution factor of 31 and the IgE concentration in serum using the estimated hematocrit value were determined. Where

Table 2 shows the results.

[0053]

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, blood collected using a micro blood collection tube is diluted and hemolyzed with a hemolytic diluent, and measured by an immunoassay, so that the main constituent components and the micro components in the blood can be easily quantified. Can be measured.

2. By using a small blood collection tube, it is only necessary to collect a small amount of blood, so blood collection is easy and the load on individual animals is minimized, blood collection can be repeated, and the action or side effects / toxicity etc. of drugs etc. can be reduced. It can be measured over a long period of time.

3. Since the hemolysis measurement is performed, the centrifugal separation operation for separating the blood cell component from the plasma or serum, which has been conventionally required, becomes unnecessary, and the operation becomes extremely simple. Also, since no separation operation is required, the amount of blood collected can be reduced.

4. Because of the above effects, the drug efficacy or side effect range of drugs and the like can be evaluated and determined over a long period of time at the animal test stage at the same level as in the human body, and therefore, drugs with safe and excellent drug efficacy Can be developed economically and reliably.

[Brief description of the drawings]

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

FIG. 2 is an explanatory diagram showing an effect of the present invention.

FIG. 3 is an explanatory diagram showing an effect of the present invention.

FIG. 4 is an explanatory diagram showing an effect of the present invention.

Claims (12)

[Claims] 1. A blood sample collected using a micro blood collection tube is diluted with a hemolytic diluent to lyse blood cell components, and thereafter, the diluted hemolyzed sample is added to an immunological assay system, and an antigen or antibody is added. A hemolytic measuring method characterized by measuring. 2. The method according to claim 1, wherein blood is collected from a small animal using a micro blood collection tube. 3. The small animal is a mouse, rat, hamster,
The method according to claim 2, wherein the method is a guinea pig or a chicken. 4. The method according to claim 1, wherein blood collection is performed by puncture of the fundus venous plexus or blood collection of the tail vein. 5. The method according to claim 1, wherein the hemolytic diluent is water or a hemolytic agent containing a hemolytic toxin. 6. The method according to claim 5, wherein the hemolysin is saponin. 7. The method according to claim 1, wherein the immunological measurement system is any one of latex immunoagglutination turbidimetry, immunoturbidimetry, enzyme immunoassay, and immunoluminescence analysis. 8. The antigen to be measured is albumin, IgG, I
The method according to claim 1, wherein the method is any one of gA, IgM, C3, C4, fibrinogen, transferrin, ferritin, hemoglobin, β2-microglobulin, CRP, and IgE. 9. A scale is provided at one end of a heat-resistant thin tube having an inner diameter of about 1 mmφ with both ends opened, and the other end has a slightly larger diameter than the thin tube and has an inner capacity larger than at least the collection / addition capacity of a sample or the like. A heat-shrinkable flexible tube, which is capable of being deformed easily by the pressing force of a finger, is fitted and fixed by heat shrinkage, and the other end of the flexible tube is partially or entirely opened. The length between the open end of the flexible tube and the heat-shrinkable fixing portion is set to a length that allows the content to be released by sealing the open end side by compression and sandwiching it with fingers. Blood collection tube. 10. A hemolytic reagent kit comprising a micro blood collection tube, a hemolytic diluent, an antibody-sensitized latex, and a diluent. 11. The hemolytic reagent kit according to claim 10, comprising a micro blood collection tube for collecting 7 μl or 14 μl, a sample chip containing 200 μl or 400 μl of a hemolytic diluent, an antibody-sensitized latex reagent, and a reaction buffer. 12. The hemolytic reagent kit according to claim 10, wherein the hemolytic diluent is a saponin solution.