JP4470014B2 - Test method for erythrocyte antigen - Google Patents

Description

  The present invention relates to a serological test method, particularly to a test method used for identification of irregular antibodies.

  Anti-erythrocyte antibodies are often produced when alloimmune stimulation such as blood transfusion and pregnancy is applied. This anti-erythrocyte antibody is called an irregular antibody. When antigen-positive blood enters the body during the next blood transfusion, not only the blood transfusion effect but also serious side effects may be caused. When blood transfusion is performed, in order to prevent this side effect, so-called antibody screening is performed on patients who receive blood transfusion for examining the presence of irregular antibodies against several types of O-type blood cells (screening blood cells). Positive if the patient has antibodies that react to these screening cells. Next, the type of antibody held by the patient is determined, and blood that does not react with the antibody is transfused. This determination of the type of antibody is called antibody identification. For antibody identification, more (typically 10 or more) type O blood cells (panel blood cells) than screening are used, and the specificity of the antibody is determined. Panel blood cells consist of a variety of different antigenic blood cells. Normally, antibodies can be identified only with panel blood cells, but when a plurality of antibodies are possessed, determination may not be possible with panel blood cells alone, and there is a need for different antigenic blood cells.

On the other hand, antigens on erythrocytes are treated with certain enzymes (for example, those commonly used in serology such as papain, ficin, bromelin, pronase, chymotrypsin) and sulfhydryl reagents (dithiothreitol and 2-mercaptoethanol). It is well known that the specific antigenicity disappears or the reaction with the antibody is enhanced by performing (Non-patent Document 1). For example, although Fy ^a antigens would reactivity disappeared in blood cells subjected to papain treatment, D antigen is stronger rather reactive. As described above, even in the same blood cell, a blood cell in which a specific antigen is negative by enzyme treatment can be prepared.

Furthermore, the effect on the antigen on blood cells varies depending on the type of enzyme. For example Fy ^a antigen not deactivated in but deactivated trypsin with papain. In serology, antibody identification is often performed by looking at changes in reactivity with blood cells treated with different types of enzymes and chemicals (Non-patent Document 2).

  By using this technique and changing the type of enzyme or chemical substance, it is possible to produce blood cells with several different specificities with respect to the original blood cells. However, each treatment method is extremely strict, and if an excessive treatment is performed, a false positive reaction occurs, and if the treatment is insufficient, the antigenicity that should be deactivated remains. Accordingly, it is necessary to set optimum conditions for each and perform processing correctly. Moreover, since the optimum conditions differ depending on the enzyme and chemical substance, it is not easy to treat several types of blood cells. Moreover, in order to store the treated blood cells, it is necessary to freeze them with liquid nitrogen or the like, and the maintenance and the freezing and thawing processing of the blood cells are very complicated.

On the other hand, as described in Patent Document 1 and Patent Document 2, it is known that erythrocytes can be stored for a long time by drying and fixing. However, there are many cases in which blood cells treated with enzymes or chemicals are not successful even if they are dried and fixed as they are. The reason is thought to be that the peptide chain of erythrocytes is decomposed by the enzyme treatment, so that the blood cells are peeled off from the solid phase surface or the structure of the blood cells themselves is destroyed by the hemolysis + drying operation. Until now, as described in Non-Patent Document 3, the enzyme treatment conditions of blood cells were mainly examined only for inactivation of Duffy antigen on blood cells by papain treatment. In addition, it has not been studied in detail that the action of the enzyme on the antigen on the blood cell is not uniform and the sensitivity varies depending on the antigen. Furthermore, enzyme treatment conditions suitable for dry fixation have not been studied.
JP 2000-32269 A Japanese Patent No. 3030710 American Red Cross (1993) Immunohematology Methods and Procedures, American Red Cross National Reference Laboratory, Rockville, MD JR Storry. Immunohematology 2000; 16: 101-104 AABB Technical Mannual 13ed p.673-679

  Accordingly, an object of the present invention is to provide a serological test container capable of stably using a red blood cell, in which a specific antigen is selectively inactivated in advance, for a long period of time, and a method for producing the same. It is another object of the present invention to provide a method capable of stably performing a test on surface antigens having different sensitivities without peeling or antigen damage.

Means for solving the above problems are as follows. That is,
(1) By reacting red blood cells that have been inactivated according to sensitivity to irregular antigens on surfaces with different sensitivities with a specimen in a solid phase in a predetermined container, different irregular antibodies can be obtained. A method for examining erythrocyte antigens, characterized by identification in a stable solid phase;
(2) The test method according to claim 1, wherein the identification is any one of an enzyme immunoassay, a fluorescent label immunoassay, and a chemiluminescence immunoassay;
(3) The inspection method according to claim 1, wherein the identification is a method in which indicator particles sensitized with an anti-globulin antibody are further reacted to generate a negative image or a positive image;
(4) The test method according to claim 1 or 2, wherein the inactivated red blood cells are further reacted with a specimen after the contents are removed by hemolysis.
(5) The method according to any one of claims 1 to 4, wherein the specimen is serum.
It is.

  As described above in detail, according to the present invention, there is provided a method capable of stably performing a test on surface antigens having different sensitivities without peeling or antigen damage during the reaction.

  The serological test container according to one embodiment of the present invention has a plurality of wells as a serum sample container on the same plate, and selectively applies untreated red blood cells and a specific antigen to the wall surface of each well. Any one of a plurality of red blood cells selected from the group consisting of inactivated red blood cells is dried and fixed. In addition, it is preferable that the red blood cells in which a specific antigen is selectively inactivated and the untreated red blood cells are derived from the same red blood cells.

  The method for producing a serological test container according to another aspect of the present invention includes: (a) a step of solid-phase untreated red blood cells on the wall surface of the serum specimen storage unit; A step of selectively deactivating antigen by enzyme treatment or chemical treatment, and a step of (c) contacting the treated erythrocyte with an isotonic or higher liquid containing a surfactant to elute the contents of the erythrocyte, (D) adding a drying solution to the red blood cells from which the contents have been eluted, and then performing a drying operation.

Step (b), for example, by optimal time reaction at room temperature to 37 ° C. in an alkaline isotonic buffer containing more trypsin 320U, on erythrocytes Lutheran, M, and at least one of Xg ^a antigen This is a step of selectively deactivating.

Step (b), for example, by optimal time reaction at room temperature to 37 ° C. at pH6 isotonic buffer containing papain concentration 0.05U～3U, on erythrocytes Duffy, M, S, ^Xg a, And a step of selectively inactivating at least one of Ge antigens.

Step (b) is, for example, by reacting Duffy, M, S on erythrocytes with an isotonic buffer of pH 6-8 containing bromelin at a concentration of 0.05 U to 1 U at room temperature to 37 ° C. for an optimal time. a step of selectively deactivating at least one of Xg ^a antigen.

  In the step (b), for example, at least one of Kell antigens on erythrocytes is reacted with an alkaline isotonic buffer containing 0.1 to 0.006 M dithiothreitol at room temperature to 37 ° C. for an optimal time. This is a step of selectively deactivating seeds.

  The method for producing a serological test container according to another aspect of the present invention comprises: (a) a step of selectively deactivating specific antigens on erythrocytes by enzymatic treatment or chemical treatment; and (b) treated erythrocytes. A step of solid-phased on the wall surface of the serum sample container, (c) a step of contacting the solid-phase red blood cells with an isotonic or higher liquid containing a surfactant to elute the contents of red blood cells, and (d) contents And a step of performing a drying operation after adding a drying solution to red blood cells from which the product has been eluted.

Step (a), for example, by optimal time reaction at room temperature to 37 ° C. in an alkaline isotonic buffer containing more trypsin 20U against blood cell concentration of 3% on erythrocytes Lutheran, M, and Xg ^a This is a step of selectively inactivating at least one of the antigens.

In the step (a), for example, Duffy, M on the erythrocyte is reacted with an isotonic buffer having a pH of 6 containing papain at a concentration of 0.4 U or less with respect to a blood cell concentration of 3% at room temperature to 37 ° C. for an optimal time. , S, Xg ^a , and Ge antigens are selectively inactivated.

In step (a), for example, Duffy, M, and R on erythrocytes are reacted with an isotonic buffer at pH 6 containing 0.4 U or less bromelin for a blood cell concentration of 3% for an optimal time at room temperature to 37 ° C. a step of selectively deactivating at least one of Xg ^a antigen.

  Hereinafter, the present invention will be described in more detail.

The serological laboratory container according to the present invention dry-fixes red blood cells selectively inactivated by treating a specific antigen present on red blood cells with an enzyme or a chemical substance on the wall surface of the serum specimen container. Thus, red blood cells having a desired antigenicity can be used stably over a long period of time. Here, in the present invention, the specific antigen may be any antigen that can usually exist on erythrocytes. For example, Lutheran, M, and at least one of Xg ^a antigen, Duffy, M, S, Xg ^a, and at least one of Ge antigen, Duffy, M, S, and Xg ^a least one of an antigen, Or it is preferable that it is at least 1 sort (s) among Kell type | system | group antigens.

  The serological test container of the present invention is preferably a microplate having a plurality of wells as a serum sample storage part on the same plate. The cuvette is preferably made of plastic, and the shape of the bottom surface is preferably U or V, but is not limited thereto.

  In addition, the serological test container of the present invention includes untreated erythrocytes that have not been treated for antigen and various erythrocytes that have selectively inactivated specific antigens on the wall surface of each well of the microplate. Any of a plurality of red blood cells selected from the group consisting of may be dry-fixed. The red blood cells and non-treated red blood cells in which a specific antigen is selectively inactivated may be derived from the same red blood cells.

  Optimum conditions are essential for drying erythrocytes treated with enzymes and chemicals. Therefore, the present inventors have clarified what reaction behavior each antigen takes on different concentrations of enzyme, rather than simply drying the erythrocytes by enzymatic treatment or chemical treatment, and for dry immobilization. Appropriate conditions were studied. Hereafter, the manufacturing method of the serological test container concerning this invention is shown. In the following description, a method of drying and fixing red blood cells in each well of the microplate is shown, but the present invention is not limited to this.

  In the first embodiment of the production method of the present invention, erythrocytes are solid-phased in a well, then treated with an enzyme or a chemical substance, and dried and fixed. Specifically, for example, according to the method of Ogama et al. Using lectin (J Ohgama et al., Transfusion Medicine 1996; 6: 351-359), erythrocytes are removed from the well bottom via lectins, anti-erythrocyte antibodies, or polycations. After the solid phase is washed, excess erythrocytes are removed by washing. The lectin is preferably a wheat germ lectin.

  The solid phase erythrocytes are reacted by adding enzyme solutions or chemical substances having various concentrations and pHs. As the enzyme used here, trypsin, papain, bromelin, ficin, chymotrypsin, pronase and the like are preferable. Moreover, as a chemical substance, sulfhydryl reagents, such as dithiothreitol (DTT) and 2-mercaptoethanol (2-Me), are preferable.

  Enzyme treatment is strongly influenced by its enzyme activity value and pH. In addition, each antigen has different sensitivities to chemical treatment or enzyme treatment, and some antigens will not disappear unless the treatment is strong. Therefore, it is necessary to clarify the optimum conditions for each antigen to be inactivated and dry the erythrocytes. In the present invention, conditions were established so that the reactivity of the antigen to be inactivated with the antibody disappeared and a false positive reaction did not occur in the negative sample. That is, the following treatment is performed on erythrocytes solid-phased on the inner wall of the well with the antigen to be inactivated.

Lutheran on erythrocytes, M, and deactivate at least one of Xg ^a antigen, alkaline containing more trypsin 320U (preferably pH8 or higher) were added to erythrocytes solid phase isotonic buffer, The reaction is carried out at room temperature to 37 ° C for an optimum time.

Duffy on erythrocytes, M, S, Xg ^a, and the deactivating at least one of Ge antigen, pH6 isotonic buffer containing papain concentration 0.05U~3U the erythrocytes solid phase Add and react at room temperature to 37 ° C for an optimal time.

Duffy on erythrocytes, M, S, and the deactivating at least one of Xg ^a antigen, isotonic buffer pH6~8 containing bromelain concentration of 0.05U~1U the erythrocytes solid phase Add and react at room temperature to 37 ° C for an optimal time.

  In order to inactivate at least one of the Kell antigens on erythrocytes, an alkaline (preferably pH 8 or higher) isotonic buffer containing 0.1 to 0.006 M DTT is added to the solid erythrocytes. The reaction is carried out at room temperature to 37 ° C for an optimum time.

  In addition, although reaction time changes with conditions, about 10 to 30 minutes are preferable.

  The erythrocytes treated under the conditions as described above are dried and fixed according to the method of Tamai et al. (Japanese Patent Laid-Open No. 2000-32269). That is, after washing the treated red blood cells, the contents are gently discharged by contacting with an isotonic or higher liquid containing a low-concentration surfactant, and after washing, a drying solution containing sugars and proteins is prepared. Add, lightly drain, and dry by vacuum drying, natural drying, or air drying. As the surfactant, Triton X-100 or the like can be used. Moreover, it is preferable to use animal serum proteins such as saccharose as the saccharide and BSA as the protein. As a method for lysing red blood cells, in addition to using a surfactant, it is possible to use a method of rupturing red blood cells using a hypotonic solution, but in order to keep the red blood cells in good condition after dry fixation. Most preferably, a surfactant is used.

  In the second embodiment of the present invention, floating red blood cells are treated with an enzyme or a chemical in advance in a test tube or the like before the red blood cells are solid-phased on the microplate. The treated erythrocytes are washed, then solid-phased on the bottom surface of the well and dried and fixed in the same manner as described in the first embodiment. Here, preferred enzymes or chemicals for treating red blood cells are the same as those described in the first embodiment. However, since the red blood cell treatment before the solid phase adds other factors such as the concentration of red blood cells, the scale to be treated, and the stirring conditions, the case where the red blood cells are enzymatically treated after the solid phase in the first embodiment described above, The appropriate concentration is different. When the treatment is performed before the red blood cells are solid-phased on the microplate, the following treatment is performed on the red blood cells before the solid phase.

Lutheran on erythrocytes, M, and the deactivating the Xg ^a antigen, alkaline isotonic buffer containing more trypsin 20U against 3% blood concentration was added to the red blood cells, optimal at room temperature to 37 ° C. Let react for hours.

Duffy on erythrocytes, M, S, Xg ^a, and the deactivating at least one of Ge antigen, pH 6 isotonic buffer containing the following concentrations of papain 0.4U against blood cell concentration of 3% Is added to erythrocytes and allowed to react at room temperature to 37 ° C. for an optimal time.

Duffy on erythrocytes, M, and deactivate at least one of Xg ^a antigen, was added to pH6 isotonic buffer containing the following bromelain 0.4U against 3% blood concentration in erythrocytes, The reaction is carried out at room temperature to 37 ° C for an optimum time.

  In addition, although reaction time changes with conditions, about 10 to 30 minutes are preferable.

  In addition, identification of antibodies by drying and fixing at least two kinds of red blood cells selected from the group consisting of various red blood cells treated under the above-mentioned conditions and untreated red blood cells, respectively, to separate wells of the same microplate, An erythrocyte panel that can be easily confirmed can be produced. Further, all of these red blood cells may be made from the same red blood cells.

  Usually, erythrocytes having the desired antigenicity do not necessarily exist in nature. Therefore, conventionally, when a desired red blood cell is not found, it has been necessary to perform various treatments on the existing red blood cell each time. Moreover, the treated blood cells could not be stored for a long time. According to the present invention, various erythrocytes having the target antigenicity can be prepared by using a desired treatment method. Furthermore, it is possible to produce a plurality of types of red blood cells having different antigenicity from the same red blood cells. By providing a test container in which these are dried and fixed in advance in each well of the microplate, it is not necessary to carry out the enzyme treatment which has been troublesome until now.

  In order to identify irregular antibodies using the test container of the present invention, EIA method (enzyme immunoassay), RIA method (radioimmunoassay), FIA method (fluorescence-labeled immunoassay), or CIA method (chemiluminescence) Immunoassay method) can be used. Alternatively, the method of Tamai et al. (T Tamai. Et al., Transfusion Medicine 1999; 9: 351-359.) Using a magnetic substance-containing indicator particle sensitized with an anti-human globulin antibody can be used. That is, glycine LISS (low ionic strength solution) is added to the container of the present invention in which red blood cells are dried and fixed, and a sample such as serum is added thereto, incubated at 37 ° C. for 10 minutes, and then washed with a saline solution. The indicator particle | grains containing the magnetic body in which the anti-human IgG antibody was sensitized are added here, and a container is left to stand on a magnet for a fixed time. When this is observed from the top or bottom of the container, an image in which the indicator particles spread is obtained in the case of positive, and an image centered in the case of negative. According to this method, a pattern can be formed by a magnetic force without using a centrifugal operation. The substance previously sensitized to the indicator particles used here is not limited to the anti-human IgG antibody, and any substance that specifically binds to immunoglobulin, such as protein G or protein A, may be used.

  Hereinafter, the present invention will be described by way of examples, but the present invention is not limited thereto.

<Example 1: Dry fixation of trypsin-treated blood cells>
Wheat germ lectin microplate solid phase, O-type screening blood ^{(Lu a,} M, Fy a or Fy ^b positive) were solid-phase. 50 μL of 0-2720 U trypsin solution dissolved in pH 6-8 PBS (phosphate buffered saline) was dispensed into each well and treated at 37 ° C. or room temperature for 10-30 minutes. Thereafter, a saline solution containing 0.0125% Triton X-100 (manufactured by Sigma) was dispensed to lyse the blood cells. After washing and removing the contents of blood cells and Triton X-100, the saccharose solution added with BSA was dispensed, drained lightly, and dried at room temperature under reduced pressure. Thereafter, assay was performed according to the method of Tamai et al. (T Tamai. Et al., Transfusion Medicine 1999; 9: 351-359.). That is, after drying glycine LISS (low ionic strength solution) was dispensed anti Lu ^a antibodies, anti-M antibody, dispensed anti Fy ^a antibody, anti-Fy ^b antibody, and a negative specimen min, after incubation 37 ° C. 10 minutes And washed 5 times with saline solution. Finally dispensed antihuman IgG antibody sensitized magnetic particles min by standing to patterning it on a magnet, an anti-Lu ^a antibodies, and anti-M antibody, anti Fy ^a antibodies, and anti-Fy ^b antibody And the occurrence of false positive reactions in negative samples were observed.

As a result, the trypsinization erythrocytes solid phase, the anti-M antibody has pH7 or less, did not disappear reactivity of anti-Lu ^a antibodies. pH8 with anti-M antibody or 320U by incubating 30 minutes at room temperature to 37 ° C., reactivity with anti-Lu ^a antibodies disappear, remaining reactivity with anti-Fy ^a antibody, occurrence of false positive reactions 2560U I did not accept until.

  In addition, erythrocytes were prepared with 0-2720 U trypsin-containing PBS (pH 8) solution so that the concentration of erythrocytes was 3% before solid phase, and treated with wheat germ at 37 ° C. or room temperature for 10-30 minutes. A test was performed in the same manner on a solid-coated microplate with lectin and dried.

Solid phase before the blood treatment, for 30 minutes at room temperature to 37 ° C., by treatment with 20 U, reactivity with anti-M, anti Lu ^a antibodies disappeared, there was no occurrence of false positive reactions to 2560U.

<Example 2: Dry fixation of papain-treated blood cells>
In the same manner as in Example 1, type O screening blood cells (S, s, M, Fy ^a , or Fy ^b positive) were immobilized on a microplate on which wheat germ lectin was immobilized. The papain solution whose activity value was determined by the casein decomposition method was diluted with PBS of pH 6-8 to adjust to 0-16U. 50 μL of papain solution was dispensed into each well and treated at room temperature for 10 to 30 minutes. Thereafter, similar processing to anti Fy ^a antibodies as in Example 1, and observed anti-Fy ^b antibody, anti-S antibody, anti-s antibodies, and generation of a false-positive reaction of the reactive and negative samples with negative samples.

As a result, the reactivity with anti-M, s antibody was not lost at pH 7 or higher. At pH 6, since a false positive reaction occurred at 3.75 U or more when incubated at room temperature for 10 to 30 minutes, 0.05 to 3 U was considered to be the optimum concentration in the papain treatment. At 1 U, reactivity with anti-Fy ^a , Fy ^b , M, and S antibodies was negative, but reactivity with anti-s antibody remained. At 0.05 U, reactivity with anti-Fy ^a and Fy ^b antibodies became negative, but reactivity with anti-M, S, and s antibodies remained.

  In addition, wheat germ lectin was prepared by adjusting blood cells with a PBS (pH 6) solution containing 0-30 U of papain so that the concentration of blood cells was 3% before solid phase and treating them at 37 ° C. or room temperature for 10-30 minutes. The same test was performed on the solid phase microplate and dried.

When blood cells are treated before solid phase, a false positive reaction occurs in those treated with 0.4 U or more of papain for 30 minutes at room temperature. In the treatment with 0.4 U, anti-Fy ^a , Fy ^b , M, S The reactivity with the antibody disappeared.

<Example 3: Dry fixation of bromelin-treated blood cells>
In the same manner as in Example 1, O-type screening blood cells (S, s, M, Fy ^a or Fy ^b positive) were immobilized on a microplate on which wheat germ lectin was immobilized. The bromelin solution whose activity value was previously determined by the casein decomposition method was diluted with PBS having a pH of 6 to 8 and adjusted to a concentration of 0 to 16 U. 50 μL of bromelin solution was dispensed into each well and treated at room temperature for 10 minutes. Thereafter, anti-Fy ^a antibody treated in the same manner as in Example 1, anti-Fy ^b antibody, anti-M antibody, anti-S antibodies, the generation of false positive reaction of the reactive and negative samples with anti-s antibody and negative samples observed did.

As a result, the reactivity with anti-Fy ^a , Fy ^b , and M antibodies disappeared in the range of 0.05 U to 2 U at pH 6 or less, but the reactivity with anti-S and s antibodies did not disappear, and negative at 2 U or more. A false positive reaction of the specimen occurred. At pH 7 and 8, reactivity with anti-Fy ^a , Fy ^b , M and S antibodies disappeared at 0.05 U to 2 U, but reactivity with anti-s antibody remained. Since false positive reaction occurred at 2 U or more, 0.05 U to 1 U was considered to be the optimum concentration. At 1 U, reactivity with anti-Fy ^a , Fy ^b , M, and s antibodies became negative, but reactivity with anti-s antibodies remained.

  In addition, a wheat germ lectin solid phase microplate prepared by adjusting with 0-16 U bromelin-containing PBS (pH 6) solution at a concentration of 3% before solid phase and treated at 37 ° C. for 10-30 minutes. The same test was performed on the solid phase and dried.

When blood cells were treated before solid phase, a false positive reaction occurred after treatment at 0.4 U or more for 10 minutes at room temperature. Further, in the treatment with 0.4 U, only the reactivity with anti-Fy ^a , Fy ^b , and M antibody disappeared.

<Example 4: Dry fixation of dithiothreitol (DTT) -treated blood cells>
In the same manner as in Example 1, type O screening blood cells (K, k, Kp ^b , or Js ^b positive) were immobilized on a microplate on which wheat germ lectin was immobilized. 50 μL of 0-0.2 M DTT dissolved in pH 6-8 PBS was dispensed into each well and treated at room temperature or 37 ° C. for 10-30 minutes. Then observed anti K antibody were treated in the same manner as in Example 1, anti-k antibody, anti Kp ^b antibody, the occurrence of false positive reactions of the reactive and negative samples with anti-Js ^b antibodies and negative specimens.

As a result, pH 7 anti K be treated with 37 ° C. 30 minutes or less, k, reactivity with Kp ^b antibody did not disappear. When incubated at pH 8 and 37 ° C. for 30 minutes, reactivity with anti-K, k, Kp ^b , and Js ^b antibodies disappeared at 0.006 M or more. Since a false positive reaction occurred at 0.2 M or more, 0.006 to 0.1 M was considered optimal.

  In addition, the blood cells were adjusted with PBS (pH 8) containing 0 to 0.2 M DTT so that the concentration of blood cells was 3% before solid phase, and treated with wheat germ lectin solidified at 37 ° C. for 10 to 30 minutes. The same test was performed on the solid phase dried microphase plate.

  However, the DTT treatment before the solid phase was considered to be unfavorable because the DTT treatment before the solid phase did not completely eliminate these reactivity even at 0.2M.

<Example 5: Preservability of erythrocytes dried and fixed after antigen treatment>
After solidifying O type erythrocytes on a microplate, after treatment with 320 U trypsin (pH 8), 1 U papain (pH 6), and a solution containing 0.0125 M DTT (pH 8) in the same manner as in Examples 1 to 3, About what was dry-fixed and stored for 6 months in the refrigerator, the reaction with each antibody was observed. The results are shown in Table 1.

After drying saved DTT treatment blood cells K, k, have been deactivated antigen of Kell system such Kp ^b, other antigens was retained. Papain treatment blood cells Duffy, M, S, Ge, Xg a antigen deactivated, other antigens was retained. Regarding the s antigen, although the reactivity decreased, it was not inactivated. Trypsinization blood cells Lutheran, M, although Xg ^a antigen had been deactivated, the other antigens were retained.

Claims (8)

After immobilization on the wall surface of the specimen container, Lutheran, M on the surface is reacted with an alkaline isotonic buffer containing trypsin of 320 U / 50 μL or more and 2560 U / 50 μL or less at room temperature to 37 ° C. for an optimal time. and at least one is selectively quenched of Xg ^a antigen against erythrocytes was dried by further elution of the contents, by reacting the analyte, identifying irregular antibody in said sample A method for examining erythrocyte antigens. After immobilization on the wall surface of the specimen container, Duffy on the surface is reacted with an isotonic buffer of pH 6 containing papain at a concentration of 0.05 to 3 U / 50 μL at room temperature to 37 ° C. for an optimal time. M, S, to Xg at least one of ^a and Ge antigen selectively quenched, erythrocytes dried by further elution of the contents, by reacting the specimen, irregularities in the sample A method for examining erythrocyte antigens, characterized by identifying an antibody. After immobilization on the wall surface of the specimen container, reaction is carried out at room temperature to 37 ° C. for an optimal time with an isotonic buffer of pH 6-8 containing bromelin at a concentration of 0.05-1 U / 50 μL. Duffy, M, S, and Xg ^a selectively inactivate at least one of an antigen, against erythrocytes was dried by further elution of the contents, by reacting the specimen, irregularities in the sample A method for examining erythrocyte antigens, comprising identifying a sex antibody. After immobilization on the wall of the specimen container, the Kell system on the surface is reacted with an alkaline isotonic buffer containing 0.1-0.006M dithiothreitol at room temperature to 37 ° C. for an optimal time. It is characterized by identifying irregular antibodies in the specimen by selectively inactivating at least one of the antigens and further reacting the specimen with red blood cells from which the contents have been eluted and dried. To test for erythrocyte antigen. By optimal time reaction at room temperature to 37 ° C. in an alkaline isotonic buffer containing following trypsin 20 U / 50 [mu] L or 2560U / 50μL against blood concentration of 3% on the surface Lutheran, M, and Xg ^a antigen After selectively deactivating at least one of them, the sample is reacted with erythrocytes which are solid-phased on the wall surface of the specimen container and the contents are eluted and dried, thereby A method for examining erythrocyte antigens, characterized by identifying irregular antibodies. By optimal time reaction at room temperature to 37 ° C. at pH6 isotonic buffer containing 0.4 U / 50 [mu] L or less of the concentration of papain against blood cell concentration of 3% on the surface Duffy, M, S, Xg ^a , And after selectively inactivating at least one of the Ge antigens, solidified on the wall surface of the specimen container, further elution of the contents, and reacting the specimen against the dried red blood cells, A method for examining erythrocyte antigens, comprising identifying irregular antibodies in the specimen . By optimal time reaction at room temperature to 37 ° C. at pH6 isotonic buffer containing the following bromelain 0.4 U / 50 [mu] L with respect to the blood cell concentration 3%, Duffy on the surface, M, and among the Xg ^a antigen After selectively deactivating at least one species, the sample is reacted with erythrocytes which are solid-phased on the wall surface of the sample container and the contents are eluted and dried, thereby causing irregularities in the sample. A method for examining erythrocyte antigens, comprising identifying a sex antibody. The method according to any one of claims 1 to 7, characterized in that said sample is serum.