CN110174519B - A kind of pool-checking red blood cell blood group irregular antibody detection kit based on solid-phase agglutination technology and preparation method - Google Patents

Abstract

The invention relates to a confluent type erythrocyte blood group irregular antibody detection kit based on a solid phase agglutination technology and a preparation method thereof, belonging to the technical field of kit preparation, wherein the kit comprises: (1) a confluent solid phase agglutination reaction microplate; (2) a low ionic strength solution; (3) an indication system; (4) negative control serum; (5) a positive control serum; the preparation method comprises the following steps: (1) preparing a confluent solid-phase agglutination reaction microplate; (2) preparing a low ionic strength solution; (3) preparing an indicating system; (4) preparing negative control serum; (5) preparing positive control serum; the technical scheme is that a two-step method indicating system which creatively utilizes the high specificity adsorption of the monoclonal antibody to the erythrocytes, the freeze drying preservation technology and the high sensitivity is utilized, so that the problems of low sensitivity, difficult preservation of the erythrocytes, easy omission and the like in the existing clinical irregular antibody detection method are practically solved, and a detection kit with high cost performance is provided for the first-line clinical detection work.

Description

A kind of detection kit and preparation method of erythrocyte irregular blood group antibody detection kit based on solid phase agglutination technology

technical field

The invention relates to a collection-type erythrocyte blood group irregular antibody detection kit based on solid-phase agglutination technology and a preparation method, and belongs to the technical field of kit preparation.

Background technique

Irregular antibodies of the red blood cell blood group system refer to blood group antibodies other than anti-A and anti-B in the human body. In addition to ABO antigens in human erythrocytes, common antigens include D, C, E, c, e, JK ^a , JK ^b , M, N, S, s, Fy ^a , Fy ^b , Lu ^b , etc. Accordingly, antibodies raised against these antigens belong to the category of irregular antibodies.

Irregular antibodies are mostly immune antibodies, which are produced mainly by blood transfusion, pregnancy, and exposure to natural immune antigens. These antibodies are mainly of the IgG type. For the blood recipient, when there are irregular antibodies in the body, once the red blood cells positive for the corresponding antigen are transfused, it will cause a hemolytic transfusion reaction in light, and seriously endanger the life safety of the patient; for blood donors, if there are irregular antibodies in the body Antibodies, once the antibody is transfused into the blood recipient containing the corresponding antigen, it is also easy to cause blood transfusion reaction and even life-threatening; for pregnant women, IgG-type irregular antibodies enter the fetus through the placenta, which can cause fetus/newborn. Hemolytic disease. Therefore, in the "Technical Specifications for Clinical Blood Transfusion", the National Health and Family Planning Commission clearly stipulates that the pre-transfusion examination must include an irregular antibody screening test to reduce the risk of blood transfusion reactions in blood recipients; in clinical obstetric practice, it is also constantly Strengthen the screening of irregular antibodies in pregnant women with multiple pregnancies as a routine test in pregnancy care to reduce the risk of hemolytic disease in the fetus/newborn.

At present, the methods of detecting irregular antibodies in domestic medical institutions and blood collection and supply institutions are mainly test tube method and anti-human globulin microcolumn gel card method. The test tube method is mainly based on the reaction of IgM-type antibodies with the corresponding antigens in saline medium, but cannot detect IgG-type irregular antibodies. In addition, the test tube method requires manual shaking and observation, which is easily affected by the operator's personal experience, resulting in large differences in the interpretation of results. Therefore, in recent years, anti-human globulin microcolumn gel card method has been widely used. The principle is mainly to use the molecular sieve effect of the gel medium to suspend the gel particles in the buffer solution and pour them into the micro-column card. During the experiment, anti-sieve cells and serum to be tested were added to the reaction chamber respectively, and after incubation for a certain period of time, centrifugation was performed to observe the results. If the test sample contains irregular antibodies, the formed agglutinates cannot pass through the molecular sieve of the gel column and stay in the upper layer of the gel column or disperse in the column; if there are no irregular antibodies in the test serum, the free reagent red blood cells after centrifugation Can be deposited on the bottom of the microcolumns by molecular sieves. This method is affected by factors such as reagent red blood cell concentration, centrifugal force and centrifugation time, gel particle size, anticoagulant concentration in the suspension medium and other factors, and the detection sensitivity is easily affected, such as Kidd blood group system antibodies and high-frequency irregular antibodies in the Chinese population Anti-Mur and other dose-responsive antibodies are easily missed when tested by microcolumn gel card method, resulting in transfusion reactions [Brian Kay, Jessica L.Poisson, Chirstoppher W.Tuma, et al. Anti-JK ^a that are detected by solid-phase red blood cell adherence but missed by gel testing can cause hemolytic transfusion reactions. Transfusion, 2016, 56: 2973-2979.]. On the other hand, the price of microcolumn gel card is relatively high, and it requires matching incubation and centrifugation equipment, which limits its application in most medical institutions.

The solid phase method is a technique invented by Plapp FV et al. in the early 1980s [Plapp FV, Sinor LT, Rachel JM, et al. A solid phase antibody screen. Am J Clin Pathol, 1984, 82: 179.]. A specific organic adsorbent is coated on a 96-well microplate, and the antigenic substances to be adsorbed are coated in the microplate wells by charge adsorption, and finally used to detect the relevant antibody substances. On the one hand, due to the long coating time of traditional adsorbents, and steps such as fixation and moisturizing are required after the cells are plated, it is relatively time-consuming and labor-intensive; 24 hours. Therefore, the above-mentioned defects affect its practical application in clinic. However, the method has high sensitivity, and its effect will be greatly improved after a series of innovations in the coating matrix, the long-term preservation technology of the coated cells, and the indicator system.

Existing conventional detection methods all need to screen cells with antibodies suspended in red blood cell preservation solution to complete the experiment. On the one hand, subject to the lifespan of the red blood cells and the quality of the preservation solution, the shelf life of reagent red blood cells is generally less than 3 months. If it is not used in time, it is easy to cause waste; With aging, antigen loss and hemolysis occur continuously, which may lead to false negative test results.

To sum up, in order to overcome the defects of the current irregular antibody detection technology, there is an urgent need for a red blood cell irregular antibody detection kit that is easy to operate, can store antigenic substances for a long time, reduce the missed detection of antibodies and has a reasonable cost in clinical practice.

As a special cell, red blood cells have a large number of blood group antigen molecules distributed on their surface. These antigen molecules are highly specific, that is, they can only react with corresponding antibodies. When the surface of a solid phase carrier is coated with specific antibody molecules, the antigen molecules carried on the surface of red blood cells will be specifically immunologically bound, and the binding between the antigen and antibody can strongly fix the red blood cells on the surface. Solid support surface. Under the action of the red blood cell cryoprotective solution, the red blood cells immobilized on the surface of the solid phase carrier can break through the three-month period of conventional liquid preservation solution for long-term preservation, and the membrane antigen can be kept stable for a long time.

The present invention intends to coat the monoclonal antibody against erythrocyte antigen on the inner surface of the 96-well microplate, utilize its high specificity to adsorb the erythrocytes to the bottom of the microplate well, and realize long-term storage after being lysed and freeze-dried by adding a cell protection solution. ; Add the plasma to be detected and the low ionic liquid that promotes the reaction to the microplate wells after freeze drying, and the immune binding between the red blood cell antigen and the plasma antibody to be detected can be quickly realized, and finally indicated by a high-sensitivity indicator system. , the positive reaction will present a uniform indicator red blood cell monolayer on the bottom surface of the microplate well; the negative reaction will present a circular indicator red blood cell button on the bottom surface of the center of the microplate well. The invention innovatively utilizes the specific reaction of monoclonal antibodies to erythrocyte antigens to fix erythrocytes on the inner surface of the solid phase carrier, utilizes freeze-drying technology to replace the maintenance solution of conventional erythrocytes, and utilizes a high-sensitivity two-step indicator system, which can effectively It solves the problems of low sensitivity, difficult to store red blood cells, and missed detection of antibodies by conventional methods in the current clinical irregular antibody detection methods, and provides a cost-effective red blood cell blood group irregular antibody detection kit for clinical first-line detection work.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a collection-type red blood cell blood group irregular antibody detection kit based on solid phase agglutination technology, which can solve the problems of short red blood cell storage period, missed antibody detection, cumbersome operation and cost in the existing detection methods. higher issues.

Another object of the present invention is to provide a preparation method of a red blood cell irregular blood group antibody detection kit based on solid phase agglutination technology.

The present invention provides a collection-type erythrocyte blood group irregular antibody detection kit based on solid-phase agglutination technology.

The above-mentioned Hui-check solid-phase agglutination reaction microplate is composed of a 96-well U-bottom detachable micro-strip, and the material of the micro-strip is preferably polystyrene. Membrane, containing test plasma, low ionic liquid and indicator system, etc., do not participate in chemical reactions themselves; on the basis of monoclonal coating, the pooled anti-sieve cells are fixed on the bottom of the U-shaped well of the micro-strip, and then lysed. , After washing, add the cell membrane cryoprotective solution, and after freeze-drying, it is a 96-well U-bottom solid-phase reaction microplate that has been coated with anti-sieve cell membrane, which can be used for solid-phase reaction experiments; The invention uses a monoclonal antibody with high specificity to erythrocytes as a coating matrix, and the organic coating medium (such as methylene blue) used in non-traditional solid phases has a high adsorption effect on the anti-sieve erythrocytes plated on the plate, thereby fixing the erythrocytes on the surface. The bottom of the U-shaped microplate well forms a uniform red blood cell molecular monolayer, which is freeze-dried and stored without moisturizing overnight, which saves the coating time and improves the plating efficiency of red blood cells;

The collection-type anti-screening erythrocyte membrane in the above-mentioned microplate means that three single O-type erythrocytes are pooled and coated on the inner surface of a single well bottom of the U-shaped plate, and their complementary antigen spectrum at least includes: D, C, E , c, e, JK ^a , JK ^b , Lu ^b , k, M, N, S, s, Fy ^a , P1, Mur, Dia antigen, the cell membrane formed by the lysis of the pooled cells in ^a single well is the sink Detection type anti-sieve cell membrane monolayer can be used to detect irregular antibodies corresponding to the above antigens; the Rh antigen phenotypes of the three single red blood cells should be: CCDee, ccDEE, CcDEe;

The above-mentioned low ionic strength solution is a buffer containing glycine and 0.10% sodium azide;

The above-mentioned indicator system includes indicator erythrocytes and anti-human globulin reagents, specifically, indicator erythrocytes refer to IgG anti-D sensitized O-type RhD(+) erythrocyte suspension with a concentration of 0.2-0.6%; anti-human globulin reagents Refers to goat anti-human or rabbit anti-human IgG antibodies diluted 1:1 to 1:128 times;

The above-mentioned negative control serum refers to the human serum that has no agglutination reaction with the above-mentioned anti-sieve red blood cells and adds a certain amount of preservatives. Specifically, the negative control serum is the healthy human serum that does not contain irregular antibodies and adds 0.10% sodium azide. ;

The above-mentioned positive control serum refers to the human serum that has agglutination reaction with the above-mentioned screening red blood cells and adds a certain amount of preservatives. Specifically, the positive control serum is a healthy person containing IgG anti-D and adding 0.10% sodium azide. serum.

The present invention provides a method for preparing a test kit for detecting irregular antibodies of the red blood cell blood group system based on solid-phase agglutination technology, comprising the following steps:

1. Formation of irregular antibody screening lineages: Monoclonal antibodies are used to screen O-type red blood cells. According to the principle of antigen complementation, an antibody-screening cell line composed of 3 O-type red blood cells is formed and pooled in equal amounts to form a pooled detection formula. The anti-sieve cell cluster, the specific antigen typing profile is shown in Table 1:

Table 1. Irregular antibody screening cell antigen spectrum to be pooled

2. Preparation of the solid-phase agglutination reaction microplate of the pooled detection type The blank microplate material is preferably polystyrene, and its function is to coat the anti-erythrocyte monoclonal antibody, fix the anti-sieve erythrocyte membrane, accommodate the tested plasma, low ionic liquid and indicator The system, etc., does not participate in the chemical reaction itself. More specifically, the strips and strips of the microplate are detachable; after the anti-erythrocyte monoclonal antibody is diluted with carbonate buffer, 100 μL is added to each well, and it is coated at room temperature for 16 hours. Here On the basis, the anti-sieve red blood cells in the above step (1) were pooled in equal amounts to prepare a suspension of 0.1% concentration, 100 μL was added to each microwell, centrifuged for 5 minutes, washed 5 times, and added red blood cell lysate for lysis. ; After washing the strips, add cryoprotective solution containing 8% sucrose, 50 μL/well, then remove the liquid in the wells, and dry the residual liquid with absorbent paper, put it in a freeze dryer for 2 hours, and put it in aluminum foil after taking it out. In a bag, heat and seal;

3. Preparation of low ionic strength solution Weigh 18 grams of glycine and 1 gram of sodium azide and dissolve them in purified water, make up to 1 L, and then add an appropriate amount of bromocresol violet solution until the color of the solution changes from transparent and colorless to dark Purple, adjust the pH of the solution to 6.8;

4. Preparation of indicator system The indicator system includes indicator erythrocytes and rabbit or goat anti-human globulin reagents. The preparation process of indicator erythrocytes is as follows: after washing, make packed O-type erythrocytes, add equal volume of IgG anti-D, 37°C Incubate for 50 minutes to prepare IgG anti-D-sensitized red blood cells, wash 5 times with normal saline, and store with red blood cell preservation solution. The preparation process of anti-human globulin reagent is as follows: Dilute rabbit or goat anti-human globulin reagent with antibody The solution is diluted and reacted with sensitized red blood cells. After centrifugation, the non-agglutinating tube is observed under a microscope as the dilution factor of the anti-human globulin reagent. invented indicating system;

5. Preparation of negative control serum Screening out the human serum that has no agglutination reaction with the anti-sieve cells of the present invention, adding sodium azide, the final concentration is 0.10%;

6. Positive control serum preparation refers to human serum containing IgG anti-D and added with 0.10% sodium azide.

The kit for the detection of erythrocyte blood group irregular antibodies according to the present invention provides a new detection concept and technology; its advantages are as follows:

1. The present invention adopts the monoclonal antibody against O-type erythrocyte membrane antigen to carry out the coating of the slats, and replaces the method of coating the slats with non-specific traditional adsorption reagents in the imported similar products. With a high degree of specificity, the coated antibody reacts with the antigen against erythrocytes, thereby fixing the erythrocytes at the bottom of the U-shaped microplate well, forming a uniform erythrocyte molecular monolayer, which greatly improves the coating efficiency of erythrocytes. The traditional low-performance coating matrix (such as methylene blue, etc.) is easy to cause the phenomenon of missing edge and centering of the plated red blood cells, resulting in uneven thickness of the red blood cell molecular monolayer, which interferes with the subsequent experimental results (Figure 1);

2. The anti-sieving cells of Ⅰc, Ⅱc and Ⅲc are pooled and then plated, which realizes the detection of various irregular antibodies contained in a single plasma in a single detection well, which is convenient to reduce the detection cost and improve the detection throughput;

3. Designed for the Chinese population with relatively high frequency of anti-Mur antibodies, and specially screened Mur(+) red blood cells (Ic spectrum cells) as one of the selected screening cells to avoid the lack of the above-mentioned antigens in the anti-screening cells currently on the market. The problem that led to the missed detection of the corresponding anti-Mur antibody (Figure 4);

4. The storage period of the micro-strip coated with the red blood cell membrane described in the invention is as long as 6 months, which is twice the storage time of conventional red blood cells, which is convenient for storage and avoids reagents caused by short red blood cell storage time. waste; prepare a special two-step method indicating system for end-point indication, which improves the detection sensitivity and avoids the missed detection of low-titer antibodies (Figure 5, Figure 6); at the same time, the detection method of the present invention has a simple operation process and No special equipment is required, and the micro-slabs can be flexibly disassembled, which can realize single-unit detection and large-scale detection. Whether it is sensitivity, stability or detection of low-titer irregular antibodies, it is superior to the currently commonly used antibodies. The human spherical microcolumn gel card method has good clinical application value.

In the present invention, the anti-erythrocyte monoclonal antibody is coated in a 96-well microplate, and the specific red blood cells are adsorbed to the bottom of the microplate by its highly specific reaction to the red blood cell antigen, and the long-term storage is realized after being lysed and freeze-dried; Plasma to be tested and a low ionic liquid that promotes the reaction are added to the microplate wells after lyophilization, and the immunological binding between the red blood cell antigen and the plasma antibody to be tested can be quickly realized. The reaction will show a uniform monolayer of indicator red blood cells on the bottom surface of the microplate well; the negative reaction will present a circular indicator red blood cell button on the bottom surface of the center of the microplate well. The invention can effectively solve the problem by innovatively using the specific immune reaction between antigens and antibodies to produce high-strength adsorption and fixation on red blood cells, using freeze-drying technology to replace the maintenance solution preservation of conventional red blood cells, and using a high-sensitivity two-step method indicating system. At present, the coating and plating steps in the clinical irregular antibody detection method are cumbersome, the sensitivity is low, the red blood cells are difficult to preserve, and the irregular antibodies are missed by conventional methods. .

Description of drawings

Figure 1. Comparison of the effects of preparing erythrocyte monolayers with different matrix materials by solid-phase agglutination method. No cracks or missing edges; the thickness of the cell monolayer prepared by lath ② is uneven, with missing edges and cell centroid;

Figure 2 illustrates the standard diagram of the agglutination strength judgment standard of solid-phase agglutination method and micro-column gel card method;

Figure 3. The test chart of red blood cell membrane antigenicity before and after freeze-drying of the solid-phase agglutination reaction microplate of the Hui-examination type. In the figure, ① and ② are the microplate strips before (before H) and after (after H) freeze-drying, respectively; from top to bottom Anti-D, -E, ^{-Jka, -Jk b , -Fya} antibodies, Normal (normal plasma), Neg (negative control), Pos (positive control) were used to detect red blood cell membrane monolayer antigens in turn; Post-antigen detection showed 4+ agglutination, normal plasma and negative control were negative, and positive control showed 4+ agglutination;

Figure 4. Comparison of the effect of the Huijian RBC Irregular Antibody Detection Kit and the conventional commercial anti-screening cells in the detection of high-frequency antibody anti-Mur in Chinese population. Plasma), anti-Mur, anti-D, Normal (normal plasma), anti-Mur, anti-D, Neg (negative control), Pos (positive control); conventional commercially available anti-sieve cells were pooled on a microcolumn gel The plasma detected from left to right in the card is Normal (normal plasma), anti-Mur, anti-D, Normal (normal plasma), anti-Mur, and anti-D. The pooled solid-phase agglutination kit can detect low-titer anti-Mur, and the agglutination strength reaches 1+ to 2+, while conventional commercial anti-screening cells cannot detect anti-Mur;

Figure 5. The test chart of erythrocyte membrane antigen stability during the storage period of the Huizheng RBC Irregular Antibody Detection Kit (compared with the micro-column gel card method), Figure 1: The low-titer human antibody test was used for freeze-drying and storage 2 Month's kit for erythrocyte membrane antigen. In the figure, anti-Jk ^b , -Jk ^a , and ^-Fya antibodies with titers of 4 and 2 were used to test the corresponding antigens in sequence from top to bottom on the microplate strip of the kit, and the agglutination strengths all reached 4+. The micro-column gel card uses anti- ^Jkb , ^-Jka , ^-Fya antibodies with titers of 4 and 2 to test the corresponding antigens of fresh red blood cells from left to right, and the agglutination strength is between W+～2+ ^s ; ②Picture: The erythrocyte membrane antigen of the kit, which was stored for 4 months after lyophilization, was tested with low-titer human antibody. In the figure, the kit microplate strips use anti-Jk ^b , ^-Jka , and ^-Fya antibodies with titers of 2 and 1 to test the corresponding antigens from top to bottom, and the agglutination strengths all reach 4+. The micro-column gel card uses anti- ^Jkb , ^-Jka , ^-Fya antibodies with titers of 2 and 1 to test the corresponding antigens of fresh red blood cells from left to right, and the agglutination strength is between W+～4+; ③ Figure: The kit erythrocyte membrane antigen stored for 6 months after freeze-drying was tested with low-titer human antibody. In the figure, anti-Jk ^b , -Jk ^a , and ^-Fya antibodies with titers of 2 and 1 were used to test the corresponding antigens in sequence from top to bottom on the microplate strip of the kit, and the agglutination strengths all reached 4+. The micro-column gel card uses anti- ^Jkb , ^-Jka , ^-Fya antibodies with titers of 2 and 1 to test the corresponding antigens of fresh red blood cells from left to right, and the agglutination strength is between W+～3+;

Figure 6. Sensitivity test chart during the storage period of the Huizheng RBC Irregular Antibody Detection Kit (compared with the micro-column gel card method); ①Figure: Use the double-diluted human IgG anti-D test to freeze-dry and store 2 month kit sensitivity. In the figure, the kit micro-strips tested human anti-D with dilutions of 2, 4, 8, 16, 32, and 64 times from top to bottom, and the agglutination strengths reached 3+ to 4+. Tube anti-D dilution is 64. The microcolumn gel card uses fresh O-type RhD(+) red blood cells from left to right to test human anti-D with dilutions of 2, 4, 8, 16, 32, and 64 times, and the agglutination strength is between 2+ and 2 Between + ^s , the measurable dilution of the last tube of IgG anti-D is 32; ②Figure: The sensitivity of the kit stored for 4 months after lyophilization was tested by using the double-diluted human IgG anti-D. In the figure, the kit microplate strips test human anti-D with dilutions of 2, 4, 8, 16, 32, and 64 times from top to bottom, and the agglutination strengths all reach 4+. The last tube of IgG anti-D that can be detected -D dilution is 64. The micro-column gel card uses fresh O-type RhD(+) red blood cells to test human anti-D at dilutions of 2, 4, 8, 16, 32, and 64 times from left to right, and the agglutination strength is between 2+ and 1 Between +, the measurable dilution of the last tube of IgG anti-D is 16; ③Picture: The sensitivity of the kit stored for 6 months after lyophilization was tested by using the double-diluted human IgG anti-D. In the figure, the kit micro-strips tested human anti-D with dilutions of 2, 4, 8, 16, 32, and 64 times from top to bottom, and the agglutination strengths reached 3+ to 4+. Tube IgG anti-D dilution is 64. The microcolumn gel card uses fresh O-type RhD(+) red blood cells from left to right to test human anti-D with dilutions of 2, 4, 8, 16, 32, and 64 times, and the agglutination strength is between 2+ and 1 Between +, the measurable end-tube IgG anti-D dilution is 16;

Fig. 7 Sensitivity comparison between the Huijian type red blood cell blood group irregular antibody detection kit and imported products of the same kind; ① and ② in the figure are the microslabs of the kit of the present invention and the imported kits, respectively, and are tested in sequence from top to bottom Human anti- ^Jka with titers 2 and 1, human anti-D with titers 32 and 16, Neg (negative control), Pos (positive control). Both can detect the above-mentioned human antibody, and the agglutination strength is the same; in the figure ③ and ④ micro-strips are respectively the kit of the present invention and the imported kit micro-strips, from top to bottom, test Normal (normal plasma), Human anti-Mur at titers of 8 and 4, Neg (negative control), Pos (positive control). The kit of the invention can strongly detect human-derived anti-Mur with titers of 8 and 4, the agglutination strengths are 4+ and 3+s respectively, and the agglutination strength of imported similar products is w+, and the detection efficiency of the two is obvious. difference.

Detailed ways

The present invention will be further explained below in conjunction with the accompanying drawings and implementation examples. It should be understood that the above-mentioned embodiments are only used to explain the present invention, but not to limit the protection scope of the present invention.

Example 1 Preparation of Hui-check-type erythrocyte blood group irregular antibody detection kit

1. Formation of irregular antibody screening lineages: Monoclonal antibodies are used to screen O-type red blood cells. According to the principle of antigen complementation, an antibody-screening cell line composed of 3 O-type red blood cells is established. The specific antigen typing spectrum is shown in Table 1;

2. Preparation of the pooled solid-phase agglutination reaction microplate

(1) Dilute the anti-erythrocyte monoclonal antibody with carbonate coating solution to 100ug/mL, take out a blank 96-well reaction microplate, add 100 μL to each well, coat at room temperature for 16 hours, and dry the liquid in the microwell After washing with purified water for 3 times, the residue in the micropores was dried with absorbent paper for the last time;

(2) The anti-sieve cells numbered Ⅰc, Пc, and Шc were washed three times with normal saline and then pooled in equal volumes to prepare a 0.1% anti-sieve red blood cell suspension, which was added to the reaction wells of the microplate strips coated with monoclonal antibodies. Add 100 μL of the above-mentioned pooled anti-sieve cells to each microwell, centrifuge for 5 minutes in a plate centrifuge, wash 5 times with normal saline, spin dry the liquid in the strips, and dry the residual liquid in the microwells with absorbent paper for the last time ;

(3) Add 0.45% hypotonic saline, 50 μL/well to the micro-strip coated with the erythrocyte monolayer, to lyse the erythrocyte monolayer, spin dry the liquid in the micro-well, and wash the strip with normal saline for 5 times, Dry the liquid in the slats, and dry the residual liquid in the micropores with absorbent paper for the last time;

(4) Add erythrocyte antigen membrane protection solution (8% sucrose solution) to the above-mentioned micro-strips, 50 μL/well, spin dry, and dry the residual liquid in the micro-well with absorbent paper, put it in a freeze-drying machine to freeze-dry 2 After taking it out, put it in an aluminum foil bag, heat the mouth of the sealed bag, and put it in a 4°C refrigerator for later use;

2. Preparation of Low Ionic Solution

Weigh 18 grams of glycine and 1 gram of sodium azide and dissolve them in purified water, make up to 1L, and then add an appropriate amount of bromocresol violet solution until the color of the solution changes from transparent and colorless to dark purple, and adjust the pH of the solution to 6.8;

3. Preparation of the Indicating System

(1) Preparation of sensitized erythrocytes: add an equal volume of IgG anti-D to the washed O-packed erythrocytes, incubate at 37°C for 50 minutes to prepare IgG anti-D sensitized erythrocytes, and wash 5 times with normal saline Then, prepare a 0..2-0.6% suspension with red blood cell preservation solution, which is sensitized red blood cells;

(2) Anti-human globulin reagents: Rabbit or goat anti-human globulin reagents are diluted with antibody diluents in a gradient ratio (eg: 1:1, 1:16, 1:32, 1:64, 1:128, etc.) , react with the above-mentioned sensitized erythrocytes, and observe the non-agglutinating tube after centrifugation under a microscope as the dilution ratio of the anti-human globulin reagent, and the anti-human globulin reagent of the dilution ratio and the sensitized O-type erythrocytes constitute the indication of the present invention system;

4. Preparation of negative control serum: screen out healthy human serum that has no agglutination reaction with the anti-sieve cells of the present invention, add sodium azide, and the final concentration is 0.10%;

5. Preparation of positive control serum: healthy human serum containing IgG anti-D was screened out, and 0.10% sodium azide was added.

Example 2 Erythrocyte membrane antigenicity test before and after lyophilization of solid-phase agglutination reaction microplates

Select the D, E, ^Jka , ^Jkb , ^Fya antigens with dose effect as the target antigen, and use the kit of the present invention to detect the human plasma containing the above-mentioned antibodies to evaluate whether the antigenicity of the erythrocyte membrane after freeze-drying is used. change. The experimental results showed that the detection results of the above-mentioned antibodies on the microplates before and after freeze-drying were consistent, all of which were 4+, indicating that the antigenicity of the red blood cell membrane did not change before and after freeze-drying. The results are shown in Figure 3. The specific operation method is as follows:

1. After taking out one microplate strip from the kit prepared in Example 1, equilibrate to room temperature, and mark the 1st to 8th wells as -D, -E, -Jk ^a , -Jk ^b , from top to bottom, respectively. -Fy ^a , Normal, Neg (negative control), Pos (positive control), this lyophilized strip is an experimental strip. Another microplate was prepared and carried out according to step (2) of Example 1, except that the fourth step was removed, that is, the freeze-drying step was not performed, and the rest of the labels were the same as the experimental strips;

2. Add 2 drops (100 μL) of low-ion solution to each of the above wells, and add 1 drop (50 μL) of the sample to be tested and negative and positive control serum to the corresponding wells respectively. The low ionic strength solution turns purple into turquoise or light green, if it is still purple, the sample to be tested may be missed;

3. Seal the microplate with sealing glue, mix gently, and incubate in a 37°C water bath for 15 minutes;

4. Take out the incubated micro-strip, tear off the sealing glue, dry the liquid in the well, and wash with normal saline for 5 times. After the last wash, place the microstrip upside down on absorbent paper to blot up the residual liquid;

5. Immediately after adding 1 drop (50 μL) of goat/rabbit anti-human IgG, add 1 drop (50 μL) of indicator red blood cells, and gently shake to mix;

6. Put the micro-strip into the plate centrifuge, centrifuge at 200g for 5 minutes, and judge the result.

Example 3 Comparison of anti-Mur and conventional anti-screening cells for the detection of high-frequency antibodies in Chinese population

The high-frequency antibody anti-Mur in the Chinese population was used as the target to detect antibodies, and compared with conventional anti-screening cells. As a result, the kit of the present invention can better detect anti-Mur antibodies, while conventional anti-sieve cells cannot be detected, as shown in FIG. 4 . The specific operation method is as follows:

1. After taking out one microplate strip from the kit prepared in Example 1, equilibrate to room temperature, and label the 1st to 8th wells as Normal (normal plasma), -Mur, -D, Normal ( normal plasma), -Mur, -D, Neg (negative control), Pos (positive control);

2. Add 2 drops (100 μL) of low-ion solution to each of the above wells, and add 1 drop (50 μL) of the test specimen and negative and positive control serum corresponding to the label to the corresponding wells. The low ionic strength solution turns purple into turquoise or light green, if it is still purple, the sample to be tested may be missed;

3. Seal the microplate with sealing glue, mix gently, and incubate in a 37°C water bath for 15 minutes;

4. Take out the incubated micro-strip, tear off the sealing glue, dry the liquid in the well, wash 5 times with normal saline, and after the last wash, put the micro-strip upside down on absorbent paper to absorb the residual liquid;

5. Immediately after adding 1 drop (50 μL) of goat/rabbit anti-human IgG, add 1 drop (50 μL) of indicator red blood cells, and gently shake to mix;

6. Put the micro-strip into the plate centrifuge, centrifuge at 200g for 5 minutes, and judge the result.

Example 4 Contrast test of stability and sensitivity of cell membrane antigens of Hui-check type erythrocyte blood group irregular antibody detection kit

1. Erythrocyte Membrane Antigen Stability Test: The kit prepared in Example 1 was stored in a refrigerator at 2-8°C, and samples were taken during the storage period of 2 months, 4 months, and 6 months for antigen stabilization of the kit. Antigen stability test: ^Jka , ^Jkb , Fya antigens were selected as target antigens, and low-titer human anti- ^Jka , ^-Jkb , ^-Fy were easily missed by conventional microcolumn gel card method. ^a antibody is tested, and the results show that the kit prepared by the present invention can detect the corresponding Jka by the humanized anti-Jka, ^{-Jk b} and ^-Fya antibodies with ^a minimum titer of 1 within 6 months of storage. , Jk ^b , Fy ^a antigen, the agglutination strength is 4+, which is better than the conventional micro-column gel card method, which fully shows the good stability of the red blood cell membrane antigen of the kit. The specific results are shown in Figure 5;

The specific operation of solid phase agglutination method:

(1) After taking out one microplate strip from the kit within the storage period, equilibrate to room temperature, and mark the 1st to 8th wells as ^-JKb , ^-JKa , ^-Fya , Neg (negative) from top to bottom. Control), Pos (positive control), and indicate the titer of the corresponding antibody;

(2) Add 2 drops (100 μL) of low-ion solution to each of the above wells, and add 1 drop (50 μL) of the sample to be tested and negative and positive control serum corresponding to the label to the corresponding wells. The low ionic strength solution turns purple into turquoise or light green, if it is still purple, the sample to be tested may be missed;

(3) Seal the microplate with sealing glue, mix gently, and incubate in a 37°C water bath for 15 minutes;

(4) Take out the incubated micro-strip, tear off the sealing glue, dry the liquid in the well, wash 5 times with normal saline, and after the last wash, put the micro-strip upside down on absorbent paper to absorb the residual liquid;

(5) After adding 1 drop (50 μL) of goat/rabbit anti-human IgG, immediately add 1 drop (50 μL) of indicator erythrocytes, and mix with gentle shaking;

(6) Put the micro-strip into the plate centrifuge, centrifuge at 200g for 5 minutes, and judge the result.

The specific operation of micro-column gel card method:

(1) Take out the micro-column gel card, and visually observe whether the gel in the card has dry cracks, bubbles or foreign objects before use. If there is, it will be regarded as an invalid card; , must be centrifuged for 2 minutes before use;

(2) Mark -Jk ^b , Jka ^a , -Fy ^a and the titer of the antibody used (such as 2 and 1) under the micro-column card in order, and add 0.8% O-type to the above-marked micro-column tube respectively Red blood cells 50uL;

(3) Add 25uL of human antibody corresponding to the label to each of the above tubes;

(4) Incubate in a special incubator at 37°C for 15 minutes, take it out, put it in a special centrifuge, and centrifuge at 1030 rpm for 10 minutes;

(5) After centrifugation, take out the micro-column gel card for observation, determine the result and record;

2. Sensitivity test: The kit prepared in Example 1 was stored in a refrigerator at 2-8°C, and samples were taken at 2 months, 4 months, and 6 months during the storage period for the sensitivity comparison test of the kit; The source anti-D was subjected to gradient dilution for detection; the results found that the sensitivity of the kit prepared by the present invention was no different in each test period within 6 months of storage, and was superior to the conventional micro-column gel card method by 2 Gradient, the comparison test results are shown in Figure 6; the specific operation method is as follows:

Solid phase coagulation method:

(1) Take 6 test tubes, marked as 2, 4, 8, 16, 32, 64, and add 200 μL of antibody diluent to each test tube. Pipette 200 μL of centrifuged human IgG anti-D into the test tube marked 2, gently pipette and mix, then transfer 200 μL of the dilution in the test tube to the test tube marked 4, repeat the above operation, transfer in turn until The last tube (that is, the test tube marked 64); the liquid prepared after the above operation is completed is the human anti-D diluted in gradient times, and the dilution times are 2, 4, 8, 16, 32, and 64 times respectively;

(2) After taking out a microplate strip from the kit within the storage period, equilibrate to room temperature, and mark the 1st to 8th wells as -D 2, -D 4, -D 8, -D 16 from top to bottom. , -D 32, -D 64, Neg (negative control), Pos (positive control);

(3) Add 2 drops (100 μL) of low-ion solution to each of the above wells, and add 1 drop (50 μL) of the corresponding sample to be tested and negative and positive control serum to the corresponding wells. The low ionic strength solution turns purple into turquoise or light green, if it is still purple, the sample to be tested may be missed;

(4) Seal the microplate with sealing glue, mix gently, and incubate in a 37°C water bath for 15 minutes;

(5) Take out the incubated micro-strip, tear off the sealing glue, dry the liquid in the well, and wash with normal saline for 5 times. After the last wash, place the microstrip upside down on absorbent paper to blot up the residual liquid;

(6) After adding 1 drop (50 μL) of goat/rabbit anti-human IgG, immediately add 1 drop (50 μL) of indicator erythrocytes, and gently shake to mix;

(7) Put the micro-slab into the plate centrifuge, centrifuge at 200g for 5 minutes, and judge the result;

The specific operation of the microcolumn gel card method is as follows:

(1) Take out the micro-column gel card, and visually observe whether the gel in the card has dry cracks, bubbles or foreign objects before use. If there is, it will be regarded as an invalid card; , must be centrifuged for 2 minutes before use;

(2) Mark 2, 4, 8, 16, 32, 64 below the card micro-column in sequence, and add 50 uL of 0.8% O-type RhD(+) red blood cells to the above-marked micro-column tubes;

(3) Add 25uL of IgG anti-D dilution solution in the test tube corresponding to the label to each of the above-mentioned tubes;

(4) Incubate in a special incubator at 37°C for 15 minutes, take it out, put it in a special centrifuge, and centrifuge at 1030 rpm for 10 minutes;

(5) After the centrifugation, take out the micro-column gel card for observation, determine the result and record it.

Example 5 Comparison of the detection results between the Huixian type erythrocyte blood group irregular antibody detection kit and imported products of the same kind

Irregular antibodies anti- ^Jka , -D, -Mur, etc., which are common in the Chinese population and are easily missed, are selected as the target detection antibodies, and the solid-phase agglutination detection kit invented in this project and similar imported products are used. Parallel comparative detection is carried out to test whether there is a difference in the detection efficiency between the two; the results show that the two-step end-point indicator system of the present invention is better than the one-step end-point indicator system of similar imported products in detecting low-titer irregular antibodies , see Figure 7; the specific operation method is as follows:

1. Solid-phase agglutination method for detecting red blood cell irregular antibody detection kit

(1) Take out 2 microplate strips from the kit within the storage period, equilibrate to room temperature, and mark the 1st to 8th wells of one of them as -Jk ^a 2, -Jk ^a 1, Neg (negative control) , Pos (positive control), -D 32, -D 16, Neg (negative control), Pos (positive control). Wells 1-8 of the other strip are marked as Normal (normal plasma), Normal (normal plasma), Neg (negative control), Pos (positive control), -Mur 8, -Mur 4, Neg (negative control), Pos (positive control);

(2) Add 2 drops (100 μL) of low ionic solution to each of the above wells, and add 1 drop (50 μL) of the sample to be tested and negative and positive control serum corresponding to the label to the corresponding wells. The low ionic strength The solution turns purple into turquoise or light green, if it is still purple, the sample to be tested may be missed;

(3) Seal the microplate with sealing glue, mix gently, and incubate in a 37°C water bath for 15 minutes;

(4) Take out the incubated micro-strip, tear off the sealing glue, dry the liquid in the well, wash 5 times with normal saline, and after the last wash, put the micro-strip upside down on absorbent paper to absorb the residual liquid;

(5) After adding 1 drop (50 μL) of goat/rabbit anti-human IgG, immediately add 1 drop (50 μL) of indicator erythrocytes, and mix with gentle shaking;

(6) Put the micro-strip into the plate centrifuge, centrifuge at 200g for 5 minutes, and judge the result.

2. Import kits

(1) After taking out 2 microplate strips from the imported kit stored in the refrigerator, equilibrate to room temperature, and mark the 1st to 8th wells of one of them as -Jk ^a 2, -Jk ^a 1, Neg (negative control) ), Pos (positive control), -D 32, -D 16, Neg (negative control), Pos (positive control); the 1-8 wells of the other strip are marked as Normal (normal plasma), Normal (normal plasma) , Neg (negative control), Pos (positive control), -Mur 8, -Mur 4, Neg (negative control), Pos (positive control);

(2) Add 2 drops (100 μL) of low ionic solution to each of the above wells, and add 1 drop (50 μL) of the sample to be tested and negative and positive control serum corresponding to the label to the corresponding wells. The low ionic strength The solution turns purple into turquoise or light green, if it is still purple, the sample to be tested may be missed;

(3) Seal the microplate with sealing glue, mix gently, and incubate in a 37°C water bath for 15 minutes;

(4) Take out the incubated micro-strip, tear off the sealing glue, dry the liquid in the well, wash 5 times with normal saline, and after the last wash, put the micro-strip upside down on absorbent paper to absorb the residual liquid;

(5) Add 1 drop of indicator red blood cells, gently shake and mix;

(6) Put the micro-strip into the plate centrifuge, centrifuge at 300g for 3 minutes, and judge the result.

Claims (5)

1. A collection-check type erythrocyte blood group irregular antibody detection kit based on solid-phase agglutination technology, is characterized in that: this collection check-type erythrocyte blood group irregular antibody detection kit comprises: (1) Hui-check solid-phase agglutination reaction microplate, the Hui-check solid-phase agglutination reaction microplate is composed of 96-well U-shaped bottom detachable micro-strip, and the inner surface of the U-shaped hole bottom of the micro-strip is coated There are highly specific anti-erythrocyte monoclonal antibodies, and the qualified Ⅰc, Ⅱc, Ⅲc anti-sieve erythrocytes are washed 3 times with normal saline and then pooled in equal volume to prepare a 0.1% anti-sieve erythrocyte suspension. Add 100uL, through the highly specific immune response of anti-erythrocyte monoclonal antibody to erythrocytes, the erythrocytes are evenly coated on the inner surface of the well bottom, and after lysis, a confluent anti-sieve erythrocyte membrane fixed on the inner surface of the U-shaped well bottom is formed. , on this basis, the cell membrane cryoprotectant is added, and the microplate prepared after freeze-drying; (2) low ionic strength solution, described low ionic strength solution contains glycine and 0.1% sodium azide; (3) an indicator system, the indicator system includes indicator red blood cells and an anti-human globulin reagent; Among them, indicator red blood cells refers to O-type RhD(+) red blood cell suspension sensitized by IgG anti-D, with a concentration of 0.2-0.6%; anti-human globulin reagent refers to goat anti-human diluted 1:1-1:128 times Or rabbit anti-human IgG antibody; (4) Negative control serum, which is healthy human serum without irregular antibodies and added with 0.10% sodium azide; (5) Positive control serum, which is healthy human serum containing IgG anti-D and adding 0.10% sodium azide; The said collection type anti-sieve red blood cell membrane means that three single O-type red blood cells are pooled and coated on the inner surface of a single well bottom of the U-shaped plate, and the complementary antigen spectrum at least includes: D, C, E, c, e, JKa, JKb, Lub, k, M, N, S, s, Fya, P1, Mur, Dia antigens, the cell membrane formed by the lysis of the cells pooled in a single well is the anti-sieve cell membrane monolayer, It can be used to detect irregular antibodies corresponding to the above antigens; the Rh antigen phenotypes of 3 single red blood cells should be: CCDee, ccDEE, CcDEe; The cell membrane cryoprotective solution is a sucrose solution. 2 . The detection kit for detecting irregular red blood cell blood group antibodies based on solid-phase agglutination technology according to claim 1 , wherein the inner surface of the U-shaped hole bottom of the micro-strip has been coated with anti-erythrocytes. 3 . Monoclonal antibody, highly specific for the adsorption of pooled Ic, IIc, IIIc anti-sieve red blood cells. 3. A method for preparing a red blood cell blood group irregular antibody detection kit based on solid-phase agglutination technology, characterized in that: the method comprises the following steps: (1) Preparation of the pool-check solid-phase agglutination reaction microplate; (2) Preparation of low ionic strength solution; (3) Preparation of the indicating system; (4) preparation of negative control serum; (5) preparation of positive control serum; Specific steps are as follows: (1) Formation of an irregular antibody screening lineage cell line Using monoclonal antibodies to screen O-type red blood cells, according to the principle of antigen complementation, an antibody screening cell line consisting of 3 O-type red blood cells was established. The specific antigen typing spectrum is as follows:

(2) Preparation of the solid-phase agglutination reaction microplate of the pool-checking type. The blank microplate is made of polystyrene. , itself does not participate in the chemical reaction, and the strips of the microplate are detachable; after the anti-erythrocyte monoclonal antibody is diluted with carbonate buffer, add 100 μL to each well, and coat at room temperature for 16 hours. Anti-sieve erythrocytes were pooled in equal amounts and prepared into a 0.1% suspension, 100 μL was added to each microwell, centrifuged for 5 minutes, washed 5 times, and lysed by adding erythrocyte lysate; after washing the strips, add 8% sucrose 50μL/well of cryoprotective solution, then remove the liquid in the well, and dry the residual liquid with absorbent paper, put it in a freeze dryer for 2 hours, put it in an aluminum foil bag, and heat and seal it; (3) Preparation of low ionic strength solution: Weigh 18 grams of glycine and 1 gram of sodium azide and dissolve them in purified water, make up to 1L, and then add an appropriate amount of bromocresol violet solution until the color of the solution changes from transparent and colorless to dark purple, and adjust the pH of the solution to 6.8; (4) Preparation of indicator system: the indicator system includes indicator red blood cells and rabbit or goat anti-human globulin reagents. The preparation process of indicator red blood cells is as follows: after washing, packed O-type red blood cells are made, and an equal volume of IgG anti-D is added. Incubate at 37°C for 50 minutes to prepare IgG anti-D sensitized red blood cells. After washing 5 times with normal saline, store them in red blood cell preservation solution. The preparation process of anti-human globulin reagent is as follows: use rabbit or goat anti-human globulin reagent with The antibody diluent is diluted and reacted with sensitized red blood cells. After centrifugation, observe under a microscope to a non-agglutinating tube as the dilution ratio of the anti-human globulin reagent. constitute an indicating system; (5) Preparation of negative control serum: screen out the human serum that has no agglutination reaction with the anti-sieve red blood cells, add sodium azide, and the final concentration is 0.10%; (6) Preparation of positive control serum: refers to human serum containing IgG anti-D and added with 0.10% sodium azide. 4 . The method for preparing a red blood cell abnormal blood group antibody detection kit based on solid-phase agglutination technology according to claim 3 , wherein the material of the microplate is polystyrene. 5 . 5 . The method for preparing a red blood cell blood group irregular antibody detection kit based on solid-phase agglutination technology according to claim 3 , wherein the strips of the microplate are detachable. 6 .

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