JPS60244861A - Hemolysis type measuring method and reagent kit used therein - Google Patents

Abstract

PURPOSE:To make easily an immunological measurement by diluting blood with a hemolyzing agent contg. hemolysis poison to hemolyze blood cell components then adding the dilute hemolysis sample into an immunological measuring reaction system and measuring an antigen or antibody. CONSTITUTION:The unclotted blood is diluted with the hemolyzing agent contg. water or hemolysis poison such as saponin. The volumetric ratio between the blood and the hemolyzing agent is preferably blood:hemolyzing agent >=(1:10). The hemolysis is nearly perfect in about 10 seconds but the mixture is preferably shaked. The measuring reaction system for the antigen or antibody in blood can be utilized for the immunological mearuing reaction system. The earlobe 1 is scored by a knife to bleed the same in the figure, and after 10mul blood is sampled by a microsampling tube 2, the sample is put into a vial 3 contg. 100mul distilled water and is diluted. The blood is hemolyzed by shaking and thereafter the entire volume is transferred into a vial 4 contg. 300mul diluent and the antigen or antibody is measured by an R-PHA method.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a hemolytic measurement method for measuring antigens or antibodies in blood by diluting and hemolyzing blood cell components in blood without separating them, and reagents used therein. It's about the kit.

[Prior art]

In recent years, cases of developing hepatitis B after blood transfusion have increased, and this has become a social problem.

Therefore, as a method for detecting HB virus-related antigens in blood for transfusion, various measurement systems such as the radioimmunoassay method have been developed, but the standard method that can be easily implemented is the reverse passive hemagglutination method ( Below R-PH
Method A) is widely used.

However, even in the R-PHA method, blood is fractionated or collected, transferred to a sedimentation test tube for centrifugation, centrifuged in a centrifuge, and the supernatant, plasma or serum, is gently sucked out with a pipette and tested. It must be transferred to a tube, and then a certain amount must be taken from the test tube with a pipette and diluted before being subjected to the measurement reaction. Therefore, there are problems in that the operation is time-consuming and requires a large number of instruments.

[Problem that the invention seeks to solve]

The present invention eliminates the need for time-consuming operations and numerous instruments required for separating measurement samples such as plasma or serum from blood, and simplifies immunoassay methods using red blood cells or latex as carriers. I am trying to make it possible.

It is also possible to dilute whole blood as it is and add it to the measurement reaction system without separating blood cell components from blood, but in this method, as shown in the comparative example in Example 1 below, blood cells derived from the test subject are It may settle and affect the judgment results. If this effect is to be avoided, it will be necessary to increase the dilution, resulting in a decrease in detection sensitivity.

In addition, blood cell components, which are microparticles of about 7 to 8 microns, can be easily r
It was also not possible to find a suitable p-material that could be used.

The present inventor conducted further studies and found that it is possible to eliminate this effect by destroying blood cell components, and has completed the present invention.

[Means for solving problems]

The present invention involves diluting blood with water or a hemolytic agent such as a hemolytic agent containing a hemolytic toxin such as saponin to hemolyze blood cell components, and then adding the diluted hemolysed sample to an immunoassay reaction system. A hemolytic assay method characterized by measuring antigens or antibodies;
And a micro sampling tube for sampling 10μ, hemolytic agent 100μ! Ampoule containing diluent 800μ! This invention relates to a hemolytic assay reagent kit characterized by comprising an ampoule containing ampoules, a sensitized blood cell reagent, control blood cells, and a reaction device.

Here, as the blood, it is also possible to use blood immediately after blood collection, instead of using a blood scale for transfusion. Non-coagulated blood is preferred. Blood for transfusion usually contains anticoagulants such as sodium citrate, ethylenediaminetetraacetic acid, heparin, etc., but even if these anticoagulants are added, they have little effect.

As the hemolytic agent, water or a hemolytic agent containing a hemolytic toxin can be used.

The water may be fresh water, but ion-exchanged water or distilled water is preferable in consideration of non-specific effects on the measurement reaction. However, if blood is diluted with water to a high dilution ratio, the ionic strength will drop too much and sensitized blood cells will tend to aggregate non-specifically. Therefore, supplement the salt concentration in the measurement reaction system. Do it like this.

As the hemolytic toxin, chemical agents such as saponin, animal hemolytic toxins such as snake venom, plant hemolytic toxins such as lithin, bacterial hemolytic toxins such as staphytetralysine, etc. can be used. The solvents for these hemolytic toxins include water, physiological saline, and phosphorus, which is approximately isotonic with blood.
! ! #Buffers such as buffer can be used. The concentration varies depending on the type of hemolytic toxin, but in the case of saponin, a concentration of about 1 is appropriate.When using a hemolytic toxin, it is convenient because it can be hemolyzed in a hemolytic state that is isotonic with blood.

The ratio of blood to hemolytic agent is preferably 1:10 or more. If the dilution factor is smaller than 10 times, for example, 2 times or 5 times, hemolysis will be incomplete and blood cells that have not been hemolysed will sediment, which may make it difficult to determine weakly positive samples in the case of the R-PHA method. be. Furthermore, when the dilution factor is 10 times or more, for example 20 times, as mentioned above, when water is used as a hemolytic agent, the sedimentation of sensitized blood cells decreases due to the decrease in salt concentration and protein concentration, resulting in non-specific aggregation. These trends need to be compensated for.

Hemolysis is almost complete in about 10 seconds, but shaking is preferred. Add the 10-fold diluted hemolysed sample to buffer components as needed.
It is also possible to perform multiple dilution using a diluent consisting of a protein component or the like.

Immunological measurement reaction systems include blood antigen or antibody measurement reaction systems, such as antigen measurement systems for HB virus-related antigens, α-fetoprotein, ORP, anti-Treponemal antibodies, anti-Streptococcal antibodies, and anti-HB virus. Related antigen antibody, anti-D
Antibody measurement systems such as NA antibodies can be used. It is particularly suitable for a measurement reaction system using a reagent in which red blood cells or latex are sensitized with an antibody or antigen corresponding to the antigen or antibody to be measured.

These measurement reaction systems may be independently prepared, or
A commercially available measurement reagent kit can also be used.

If the blood is diluted 10 times or more with the hemolytic agent, there is a concern about the stability of the antigen or antibody to be measured, but in the case of HB virus-related antigens, hemolysis with distilled water and hemolysis containing hemolytic toxins such as saponin etc. Stable against hemolysis caused by agents. Even when measuring a substance that is unstable in distilled water, stable measurement can be achieved by appropriately selecting the hemolytic toxin and buffer system.

Each of the above compositions can be filled into ampoules, vials, etc., and a measurement reagent kit can be prepared by attaching test tubes, round-bottom ampoules, reaction trays, etc. as reaction instruments.

[For production]

Since hemocyte components in blood are hemolyzed in water or a hemolytic agent containing lytic toxin, the agglutination reaction of sensitized red blood cells can be prevented even when added to the immunoassay reaction system as is or diluted as necessary. Antigens or antibodies in blood can be measured without interfering with sedimentation or affecting the agglutination or non-agglutination images of the sensitized latex.

Since the sensitized red blood cells are stabilized by fixation and the sensitized latex is stable, a slight decrease in ionic strength or the presence of a trace amount of hemolytic toxin will not interfere with the reaction.

In addition, since a diluted hemolyzed sample is added to the immunoassay reaction thread, a sample with a low dilution ratio will not exhibit a strong red color of hemoglobin, which will not interfere with the determination.

Using a hemolytic measurement reagent kit requires minimal preparation for measurement, making it easy to measure.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example I 500 μl of 50 μl of HB antigen positive and negative whole blood samples
of distilled water, shake to lyse, add diluent, and perform multiple dilution series (X10, X20, X40, X80).
, X160, X820, X640, X1280) and use them as samples for the R-PHA method.

For comparison, 50 μl of each of the above whole blood samples was diluted 10 times by adding 500 μl of each field containing a diluent, and then a series of similar multiple dilutions was made with the diluent to obtain a non-hemolyzed diluted sample.

These samples were added to a blood cell reagent sensitized with an anti-HBS antibody, stirred well, and left to stand for 2 hours, and determined by tube bottom image. Anti-HBs to check for non-specific agglutination of blood cell reagents
Similar reaction procedures were performed using control blood cells that had not been sensitized to the 8 antibody for comparison.

As the diluent, blood cell reagent, and control blood cells, a commercially available R-PHA reagent for measuring HBB antigen (Shichi Rhodia HBBi, manufactured by Fujirebio) was used. Judgment was made by observing the tube bottom image with the naked eye, and an agglutination image was evaluated as +, and a sedimentation image was evaluated as - (the same applies hereinafter).

The results are shown in Table 1 below.

Table 1 @ Last name When the sample is measured by the dissolved IfIl measurement method, X10
While all of Xi 280 had agglomeration (+),
In the comparative example using a non-hemolyzed sample, blood cell components derived from the test whole blood sample precipitate, so unless the non-hemolyzed sample is diluted 160 times or more, a positive sample may be mistaken as a negative sample. Therefore, the non-hemolyzed whole blood measurement method, which requires dilution of serum specimen by 160 times or more, has a detection sensitivity of 178 or less, which is disadvantageous compared to the normal R-PHA method, which measures from a 20-fold dilution of the serum sample. On the other hand, the hemolytic measurement method of the present invention can be expected to have a detection sensitivity comparable to that of normal serum measurement.

Example 2 HB antigen positive and negative whole blood specimens 5 used in Example 1
0μ was diluted and hemolyzed using 500μ of a saponin-containing hemolytic agent (saponin concentration 1%), and the measurement was performed in the same manner as in Example 1.

The results were similar to those of the Examples shown in Table 1 above.

Example 8 Five whole blood samples were selected, and sodium citrate, ethylenediaminetetraacetic acid, and heparin were added to each sample at concentrations commonly used, and measurements were performed in the same manner as in Example 1.

The measurement results were not affected by the presence or absence of these anticoagulants or their names.

Example 4 28 whole blood samples were diluted 10 times with distilled water to cause hemolysis, and further diluted 4 times with a diluent to obtain diluted hemolyzed samples. This diluted hemolyzed sample was measured using an anti-HBs monoclonal antibody-sensitized blood cell reagent, and serum was separated from the whole blood sample and measured using a conventional R-PHA method using commercially available HB and measurement reagents for comparison.

The anti-HBs monoclonal antibody-sensitized blood cell reagent used was a commercially available anti-HB6 monoclonal antibody (Hyplitic Co., Ltd.: Subclub ml, and Medical and Biological Research Institute: Subgroup a), and It was prepared by sensitizing chicken red blood cells using the tannic acid method.

The results are shown in Table 2 below, and the results obtained by the hemolytic method of the present invention using anti-HBB monoclonal antibody sensitized hemocyte reagent No. and the results obtained by the conventional method were in 100 degree agreement.

Table 2 Example 5 Seventy-one whole blood samples were diluted in the same manner as in Example 4 and diluted 40 times and 80 times in terms of original blood to obtain diluted hemolyzed samples. This diluted hemolyzed sample is measured using a commercially available HBs measurement reagent kit, and serum is separated from the whole blood sample and commercially available HBs
It was measured and compared using a B6 measurement reagent kit.

The results are shown in Tables 3 and 4 in 1B below.

Table 8 Table 4 With the hemolytic measurement method of the present invention, 16 cases were determined to be positive for the 40-fold diluted sample, 55 cases were determined to be negative, and 16 cases were determined to be negative for the 80-fold diluted sample. One example,
Seventy cases were determined to be negative. In addition, 4 out of 16 samples (25%) that were determined to be positive after 40-fold dilution were found to be positive using the HB8 antigen measurement reagent kit (manufactured by Abbott) using the enzyme immunoassay method. Therefore, although the anti-HBs antibody-sensitized m-cells of the commercially available R-PHA method HB8 antigen measurement reagent kit are slightly inferior in specificity and sensitivity compared to the anti-HB8 monoclonal antibody-sensitized blood cells used in Example 4, they are not practical. The above is sufficient.

Example 6 An example of the composition of a reagent kit for carrying out the hemolytic measurement method of the present invention is as follows: a micro sampling tube for collecting 10 μl, an ampoule containing 100 μl of distilled water, and 800 μl of diluent! ampoule, antibody-sensitized blood cells, and control blood cells.

Also, as another example of the composition of the reagent kit, 10μ! Microsampling tube for collection, buffer containing hemolytic agent 4
00μ! ampoule, antibody-sensitized blood cells, and control blood cells.

In the above cases, antibody-sensitized blood cells and control blood cells are packed into vials or ampoules; in the case of vials, reaction test tubes or reaction trays are used as reaction equipment; in the case of ampoules, round-bottom ampoules are used. It is advisable to use this method so that it can be used as is for the reaction.

〔effect〕

As described above, the hemolytic measurement method of the present invention and the reagent kit used therein produce various excellent effects as described below.

(r) Since the whole blood is directly diluted and hemolyzed and added to the immunoassay reaction system, the separation and removal of blood cell components from the whole blood, which was conventionally required, is no longer necessary. Therefore,
Since a centrifuge and many other instruments are not required, and time-consuming operations can be omitted, a large amount of measurement specimens can be measured in a short time with simple operations.

(1 [) Since it is not affected by anticoagulants, blood for transfusion,
Antigens or antibodies in blood test samples can be easily measured. It is particularly useful for measuring HB-related antigens, which have become a problem in recent years.

(1) Whole blood is directly diluted and hemolyzed and added to the reaction system, so the amount of blood sample is extremely small.

(G) Since only a small amount of blood sample is required, blood can be collected from the ear, for example, without the need for conventional venous blood collection, and blood can be easily collected from children, the elderly, anemic patients, etc., and it can be done frequently. It also becomes possible to collect and measure blood. Therefore, syringes, needles, test tubes, etc. for venous blood sampling become unnecessary.

Blood sampling from the ear is as shown in Figures (a) and (b).
Injure the ear flap (11) with a scalpel and let it bleed, collect 10μ of blood with a microsampling tube (2), and in the case of Figure (a) dilute it in 100μ of distilled water (Sr). After hemolyzing by shaking, transfer the entire volume to a diluent (800μ) vial (4), and add R-P)IA.
Measured by method. In the case of Figure (b), the hemolytic agent-containing buffer is diluted into a 400 μm vial (5) and measured by the R-PHA method. In this way, the measurement operation does not require any complicated operations and is extremely simple.
Since only a small amount of blood sample is required, blood samples collected from the gums in the dental field can also be measured.

This is important at a time when the spread of hepatitis B is attracting attention as a social issue, even for dentists who often come into contact with patients' saliva and blood with their bare hands.

In other words, in the dental field, patients often go to the hospital for long-term treatment, and dentists are exposed to repeated infections.Therefore, there is a need for a simple and rapid measurement system for HB8 antigen, and the hemolysis method of the present invention The formula measurement method and the reagent kit used therefor can meet these demands.

[Brief explanation of the drawing]

Figure (-r) is a diagram showing an example of the operating method of the hemolytic measurement method of the present invention (→ is a diagram showing an example of the same value. Revial, (4) shows a vial containing 800 μl of diluent, and (5) shows a vial containing 400 μl of buffer containing hemolytic agent. Patent applicant Masakatsu Hashimoto, engraving on page 1 (no change in content) ○■ ○ ■ (10) (-) ke) (-) Procedure Amendment (Method) % formula % 1 Display of incident 1982 Patent Application No. 99704 2 Name of invention Hemolytic measurement method and reagent used therein Kit 3 Person making the amendment Patent applicant No. 15-80-1, Saiwai-cho, Itabashi-ku, Tokyo 4 Date of the amendment order (shipment date) August 8, 1980 (59, 8, 28) 5 Kusunoki in the brief explanation of the drawings in the specification to be amended, content of the amendment to drawing 6 (1) Amendment in the column of the brief explanation of the drawings in the specification, “Figure (A)” in lines 19 and 20 on page 18 1 (a) and (b) are explanatory diagrams of the hemolytic measuring method of the present invention, and FIG. 1 (a) is a diagram showing an example of the measuring method.
FIG. 1(b) is a diagram showing another example of the measuring method. ” he corrected. (II) Correction of the drawing The drawing plan is deleted and FIG. 1 is newly added. 7 List of attached documents (1) 1 copy of drawings

Claims (1)

[Claims] l) Hemolysis characterized by diluting blood with a hemolytic agent to hemolyze blood cell components, and then adding the diluted hemolysed sample to an immunoassay reaction system to measure antigens or antibodies. formula measurement method. S) The hemolytic measuring method according to claim 1, wherein the hemolytic agent is water. 8) The hemolytic measuring method according to claim 1, wherein the hemolytic agent is a hemolytic agent containing a hemolytic toxin. 4) The hemolytic measuring method according to claim 8, wherein the hemolytic toxin is saponin. 6) 10μ! 4 sampling microphones, 100μ hemolytic agent! A hemolytic measurement reagent kit comprising an ampule containing 800 μm of diluent, a sensitized blood cell reagent, control blood cells, and a reaction device. 6) The hemolytic assay reagent kit according to claim 6, wherein the hemolytic agent is water. f) The hemolytic assay reagent kit according to claim 6, wherein the hemolytic agent is a hemolytic agent containing a hemolytic toxin. 8) The hemolytic assay reagent kit according to claim 7, wherein the hemolytic toxin is saponin.