JPH0420858A - Detecting method of anti-platelet antibody - Google Patents

Abstract

PURPOSE:To make it possible to detect only an anti-platelet antibody efficiently by using a soluble platelet film antigen which is extracted from a platelet and treated with chloroquine and from which HAL antigen is removed, and performing antigen antibody reaction. CONSTITUTION:A film antigen component is extracted from a platelet. The antigen component of the platelet is obtained as solubilized dissolution material by applying physical force such as ultrasonic waves on the platelet. Then, the obtained platelet film antigen component is provided at the inner wall of the well of a microplate in a solid phase. For an obtaining method of the solid phase of the antigen component, a coupling agent of tannic acid, glutaraldehyde or the like can be used. Then, the antigen component is made to be the solid state. Thereafter, each well is treated with chloroquine. HAL antigen is removed from the antigen component in the solid phase. Antigen antibody reaction is performed by using the soluble platelet film antigen from which the HAL antigen is removed. Thus, only the anti-platelet antibody can be efficiently detected.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for detecting anti-platelet antibodies.

[Conventional technology]

Platelets are nonnucleated cells with a diameter of 2 to 3 mm present in the blood, and are produced by fragmentation of megakaryocytes present in the bone marrow. When platelets in the bloodstream come into contact with injured vascular endothelial cells, they undergo an adhesive aggregation reaction, which plays an important role in hemostasis.

In recent years, platelet transfusion therapy has been performed for patients with a bleeding tendency due to thrombocytopenia or platelet dysfunction for the purpose of hemostasis and prevention. This has enabled many patients to avoid death from hemorrhage and extend their lives. Since the introduction of platelet transfusion therapy, the amount of platelet preparations used has increased rapidly.

However, in patients with acute leukemia and aplastic anemia who require frequent and large amounts of platelet transfusion over a long period of time, antibodies are produced against antigens present on the surface membranes of transfused platelets. Such patients no longer respond to platelet transfusions from random donors and their platelet counts no longer increase after transfusion. When platelet transfusion becomes ineffective as described above, it is necessary to transfuse platelets having an antigen type that does not react with the patient's anti-platelet antibodies.

Anti-platelet antibodies are broadly classified into intervening antibodies and autoantibodies.

Anti-platelet antibodies Antibodies are antibodies produced by the above-mentioned platelet transfusion or pregnancy. This alloantibody not only influences the effectiveness of platelet transfusion as described above, but also causes posttransfusion purpura and neonatal thrombocytopenic purpura.

On the other hand, anti-platelet autoantibodies are produced by patients with autoimmune diseases such as idiopathic thrombocytopenic purpura. These autoantibodies are considered to be the causative agent of autoimmune diseases.

Detection of anti-platelet antibodies has conventionally been carried out using a number of methods. The main ones are as follows.

(a) Aggregation method A method first used to detect platelet antibodies, originally
This method was used to detect the LA antigen system and KO antigen system. In this method, mainly Igkl antibodies are detected, and Igkl antibodies are mainly detected.
gG antibodies are rarely detected. In addition, non-specific aggregation due to natural aggregation of platelets tends to occur, and sensitivity is low.

(b) Complement fixation reaction After the test serum, platelets, and complement are reacted, hemolysin-sensitized sheep red blood cells are added thereto. Complement consumption is determined from the degree of hemolysis of the sheep red blood cells. This method can also detect HLA antigens, ABO blood group antigens, etc. on the platelet membrane surface. However, the sensitivity is low.

Furthermore, it is difficult to determine if the test serum has anti-complement activity.

(c) Antiglobulin consumption test In this test, platelets are first reacted with immobilized test serum, and then washed. Antiglobulin serum is added to this, reacted, and centrifuged. The antiglobulinka titer of the obtained supernatant is determined by adding anti-antibody-sensitized red blood cells and causing a reaction. This method is prone to false positives and has low sensitivity.

(d) RIA (radioimmunoass)
ay) This is an antiglobulin test using anti-human IgG labeled with a radioactive isotope. In this method, the patient's anti-platelet antibodies bound to platelets are combined with radioisotope-labeled anti-human
After binding the IgG, the radioisotope bound to the platelets is counted.

(e) MPHA (mixed passive haemagglut)
In this method, platelets are first immobilized on the inner wall of a well of a U-shaped microblate, and a test serum is added to the well and reacted. After washing the wells, anti-human IgG immobilized sheep red blood cells are added and reacted. After the reaction, platelet antibodies are detected by determining that an image in which sheep red blood cells have gathered in the center of the well is negative, and an image in which they have spread throughout the well is positive.

[Problem to be solved by the invention]

The conventional methods (a) to (e) above use platelets themselves as antigens. By the way, not only platelet antigens are expressed on platelet cells (HLA antigens are also expressed. Therefore, not only antibodies that react with platelet antigens but also antibodies that react with HLA antigens are detected, and the detected antibodies It was not possible to distinguish whether the antibody was an anti-platelet antibody or an anti-HLA antibody.

This invention was made focusing on the above problem,
The purpose of the present invention is to provide a method that can easily and efficiently detect only anti-platelet antibodies without including anti-HLA antibodies.

[Means and effects for solving the problem] The inventors,
In view of the above circumstances, we conducted extensive research to develop a method that can easily and quickly remove HLA antigens from platelets and detect only anti-platelet antibodies. As a result, HLA antigens can be removed by treating soluble platelet membrane antigen extracted from platelets with chloroquine, and by using this chloroquine-treated soluble platelet membrane antigen, only platelet-specific alloantibodies can be efficiently detected. I discovered that.

The method for detecting anti-platelet antibodies of the present invention usually includes the above-mentioned MP.
Do this in accordance with the HA method. That is, instead of fixing the platelets themselves to the inner walls of the wells of the microplate, the anti-platelet antibodies treated with chloroquine to remove HLA antigens are immobilized. Hereinafter, a method for preparing a microplate used in the detection method of the present invention will be explained.

First, membrane antigen components are extracted from platelets. Antigen components of platelets can be obtained as solubilized solutes by applying physical force such as ultrasound to platelets. It can also be obtained by solubilizing platelets by treating them with a surfactant. Furthermore, it can also be obtained by applying physical force to platelets to crush them, and then treating the platelets with a surfactant to solubilize the platelets.

Next, the obtained platelet membrane antigen component is immobilized on the inner wall of the well of the microplate. A known method can be used to immobilize the antigen component. For example, a method using a coupling agent such as tannic acid, glutaraldehyde, carbodiimide, chromium chloride, etc. can be mentioned. Furthermore, the antigen component can also be immobilized by physical adsorption.

After immobilizing the antigen component, each well is treated with chloroquine. That is, chloroquine is added to each well, stirred well, incubated, and then washed. This treatment removes the HLA antigen from the immobilized antigen component.

Only platelet antigens are immobilized on the inner walls of the wells of the microplate thus prepared. Therefore, by performing the MPHA method using this microplate, it becomes possible to efficiently detect only anti-platelet antibodies.

〔Example〕

Removal of HLA Antigen from Platelet Extracted Antigen CPD blood collected was centrifuged, and the platelet sediment obtained was resuspended in physiological saline. After storing this at 4°C for 3 days, the supernatant was separated and used as an extracted antigen. Determine the optimal concentration of this extracted antigen, drop 25 μj into each well of a 96-well U-shaped microplate (manufactured by Nunk, Maxisorp), and store it overnight at 4°C to create a platelet-extracted antigen plate. did.

HLA antigens were removed from this platelet-extracted antigen plate using the following procedure. First, the top of the extracted antigen was discarded from the plate, and 50 μj of 0.8 M chloroquine was dropped into each well and mixed vigorously. Each well was then washed 10 times with 0.05% Tween 20-PBS after incubation for 1 hour at room temperature.

Twenty-five wafers of 0.05% Tween 20 was dropped into each well of the platelet-extracted antigen plate treated with chloroquine according to the above procedure, and the antibody was allowed to react at room temperature for 1 hour.

The antibody used here was a platelet monoclonal antibody Yu3.
-51-16 (specificity: GpHb/Ha),
HLA monoclonal antibody TOK39-1 (specificity:
HLA class Emonomorphic) Anti-YuK” Anti-Nak’ Anti-Bak’ as platelet-extracted antigen
and anti-)HLA-A11+ as HLA alloantibody
A31+Av33.

As a result, platelet monoclonal antibody Yu3-51-1
6, and platelet alloantibodies anti-YuK, anti-Nak” anti-B
ak'', no change in reactivity was observed between chloroquine-untreated plates and chloroquine-treated plates.In contrast, HLA monoclonal antibody TOK39
-1 and HLA alloantibody anti-HLA-A11+A31+
The reaction of Av33 was no longer observed after treatment with ookin. Anti-HLA-A11+A31+Av33 (LCT Ti
target cells with AI using serum containing ter x2)
? Figure 1a shows the results when A (HLA-AIl, A2B) platelets were used as the extracted antigen;
The results when platelets and extracted antigens were used are shown in Figure 1b. In FIG. 1a and FIG. 1b, the vertical axis indicates the dilution factor of serum. Furthermore, the horizontal axes in FIGS. 1a and 1b both indicate, from the left, untreated platelets and extracted antigens, and chloroquine-treated platelets and extracted antigens. Figure 1a shows that both platelets and extracted antigens showed a reaction even to 16-fold diluted serum in the untreated case, whereas those treated with chloroquine showed no reaction even to the undiluted serum. It represents something. Figure 1b shows that both untreated and treated with chloroquine, platelets were reactive up to a 2-fold dilution of serum, and extracted antigens were reactive up to a 4-fold dilution of serum.

Detection of anti-platelet antibodies (1) Preparation of HLA-depleted platelet extraction antigen plate A
Seven volumes of type O human whole blood are collected for each volume of CD-A solution or other anticoagulant. Usually, the volume of ACD-A solution is 31, and 21 tiI whole blood is collected. ACD
- Centrifuge whole blood to which solution A has been added at 1400 rpII for 10 minutes to separate platelet-rich plasma (PRP).

At this time, in order to avoid contamination with red blood cells as much as possible, it is preferable to start centrifugation after leaving the blood for about 30 minutes after blood collection. In order to improve the yield of platelets, after adding 10% of the obtained PRP ACD-A solution to the PRP, 250
Centrifuge in Orpm for 15 minutes, discard the supernatant, and collect the platelet concentrate (
P, C,) are obtained. Sterile physiological saline (manufactured by Terumo Corporation) 5- is added to these p and c, and washing is repeated twice by centrifugation at 250 rpm for 10 minutes. The washed platelets are suspended in 0.51 saline and left at 4°C for 3 to 5 days to extract the antigen.

The above saline suspension is centrifuged at 100oc for 5 minutes, and the resulting supernatant is diluted 50 times with physiological saline to obtain an extracted antigen.

25 mJ of the above-mentioned extracted antigen is placed in each well of a styrene 966 microplate (Nunk: Maxisorp) from which static electricity has been removed in advance, and the plate is left overnight at 4°C to prepare an antigen-attached plate.

After washing this antigen-attached plate three times with PBS, 50 μg of 0.8M chloroquine is dropped into each well, mixed vigorously, and incubated at room temperature for 1 hour. After incubation, discard the chloroquine solution and wash 10 times with 0.05% Tween 20-PBS.

(it) Sensitization of HLA-depleted platelet-extracted antigen plate Into each well of the above HLA-depleted platelet-extracted antigen plate, add 0.
Add 325 μg of 05% Tween 2O-PB, and add platelet-specific antiserum (Nak' antiserum/fi) and HL.
A antiserum (33 containing All+A31+) is added in 25 d portions according to the following protocol and sensitized for 1 hour under humid conditions at room temperature.

(Iii) Preparation of sensitized particles Tannic acid-fixed sheep red blood cells are prepared as follows.

Fresh sheep red blood cells (5RBC) within 2 days after blood collection
Wash 4 times with B S (p)I 7.2). Next, centrifugation is performed at 2500 rpm for 5 minutes to completely remove the resulting white film-like substance, followed by washing and dilution with PBS to give 5% red blood cells. To 100 g of this 5% red blood cells, 20 g of 2.5% geltaraldehyde solution is added as a fixative, and the mixture is fixed by stirring at room temperature using a turntable for 3 hours. Note that formalin can also be used as the fixative. After 3 hours, P
Wash by adding BS and centrifuging at 250 rpm for 5 minutes, and repeat this 4 times. After washing, dilute with PBS to obtain 5% fixed sheep red blood cells, and add 0 as preservative.
．． Add 01% NaN3 and save.

The above 5% fixed sheep red blood cells were washed once with PBS, and the red blood cells were mixed with 10 (lal) and 0.02% tannic acid solution.
00- for 15 minutes at 37°C. Then, P.B.
After washing once with S, rabbit-derived anti-human IgG is added, and the mixture is stirred at room temperature for 2 hours to react.

After the reaction, wash by adding PBS and centrifuging at 2500 rpm for 5 minutes, and repeat this 4 times. The obtained red blood cells were treated with 326 normal serum (NR5: Pel f'r
(manufactured by Eeze)-PBS solution to obtain a 0.2% blood cell suspension.

(1v) Detection of anti-platelet antibodies The HLA-depleted platelet extraction antigen plate sensitized as above was mixed with 0.05% Tween 20-P B 5 (pH 7,
Wash 5 times with 2). Then, Tobean 2O-PBS
, the above 0.2% blood cell suspension as indicator blood cells and 25tII naive blood cells are added to designated wells. Let this plate stand still at room temperature in a humid place without vibrations, and
Judgment will be made after 4 hours.

Judgment is made according to the criteria for passive hemagglutination reaction shown below.

++: Blood aggregates on the entire bottom of the well to form a --like film.

10: A small collection of large, thin blood cells can be seen in the center of the well ±: Blood cells can be seen in a ring shape, but a slight scattering of blood cells can be seen around the ring: All blood cells are gathered in the center of the well , forming a beautiful ring.

Based on the above judgment, (++) and (+) are positive, (
soil) and (-) are considered negative.

〔Effect of the invention〕

As described above, according to the method of the present invention, only HLA antigens can be removed from platelet-extracted antigens, and only anti-platelet antibodies can be efficiently detected by antigen-antibody reaction using this antigen.

[Brief explanation of drawings]

Figure 1a is a graph showing the reactivity of anti-HLA antibodies to untreated and chloroquine-treated platelets and platelet-extracted antigens. Figure 1b is a graph showing the reactivity of anti-platelet antibodies to untreated and chloroquine-treated platelets and platelet-extracted antigens. It is a graph. Applicant's representative Patent attorney Jun Tsuboi Figure 1a Figure 1b

Claims (1)

[Claims] HLA extracted from platelets and treated with chloroquine
A method for detecting anti-platelet antibodies, which comprises performing an antigen-antibody reaction using a soluble platelet membrane antigen from which the antigen has been removed.