CN110174519B - Confluent-detection type erythrocyte blood type irregular antibody detection kit based on solid-phase agglutination technology and preparation method thereof - Google Patents
Confluent-detection type erythrocyte blood type irregular antibody detection kit based on solid-phase agglutination technology and preparation method thereof Download PDFInfo
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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
Technical Field
The invention relates to a confluent type erythrocyte blood type irregular antibody detection kit based on a solid phase agglutination technology and a preparation method thereof, belonging to the technical field of kit preparation.
Background
Antibodies to irregularities of the erythrocyte blood group system are blood group antibodies other than anti-A and anti-B in humans. In addition to ABO antigens, common antigens of human erythrocytes are D, C, E, C, E, JKa、JKb、M、N、S、s、Fya、Fyb、LubAnd so on. Accordingly, antibodies raised against these antigens fall into the category of irregular antibodies.
The irregular antibodies mostly belong to immune antibodies, and the generation routes are mainly as follows: blood transfusion, pregnancy, exposure to natural immune antigenic substances, etc. These antibodies are predominantly of the IgG type. For a blood recipient, when irregular antibodies exist in the body, once red blood cells positive to corresponding antigens are input, hemolytic transfusion reaction is caused slightly, and the life safety of the patient is seriously threatened; in the case of donors who contain irregular antibodies in their body, such antibodies, once transfused into recipients containing the corresponding antigens, are also susceptible to transfusion reactions and even life-threatening; for pregnant and lying-in women, irregular antibodies of the IgG type penetrate the placenta into the fetus and can trigger fetal/neonatal hemolytic disease. Therefore, in "clinical transfusion technical Specification" by the national health council, it is specifically stated that pre-transfusion testing must include an irregular antibody screening test to reduce the risk of transfusion reactions in recipients; in clinical obstetrical practice, the screening of irregular antibodies is also being continuously intensified for pregnant women with multiple pregnancies as a routine test for pregnancy care to reduce the risk of developing hemolytic disease in the fetus/neonate.
At present, the methods for detecting irregular antibodies by various medical institutions and blood collection institutions in China mainly comprise a test tube method and an anti-human globulin microcolumn gel card method. The test tube method is mainly based on the reaction of antibodies of the IgM type with the corresponding antigen in a saline medium, but does not detect irregular antibodies of the IgG type. And the test tube method needs manual shaking observation and is easily influenced by personal experience of an operator, so that the result interpretation difference is large. Therefore, in recent years, the anti-human globulin microcolumn gel cartridge has been widely used. The principle of the gel is mainly that gel particles are suspended in a buffer solution by utilizing the molecular sieve action of a gel medium and poured into a micro-column card to prepare the gel. In the experiment, anti-sieve cells and serum to be detected are respectively added into a reaction cavity, incubated for a certain time, and then centrifuged to observe the result. If the tested sample contains irregular antibodies, the formed agglomerations can not pass through the molecular sieve of the gel column and can not be retained in the upper layer of the gel column or dispersed in the column; if the serum to be tested has no irregular antibodies, the free reagent red blood cells can be deposited on the bottom of the micro-column through the molecular sieve after centrifugation. The method is influenced by factors such as reagent erythrocyte concentration, centrifugal force, centrifugal time, gel particle size, anticoagulant concentration in suspension medium and the like, and the detection sensitivity is easily influenced, for example, antibodies with dosage effect such as antibodies of Kidd blood group system and high-frequency irregular antibodies of Chinese population-Mur and the like are easy to leak and cause transfusion reaction [ Brian Kay, Jessica L.Poisson, Chirstopher W.Tuma, and the likea 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 the micro-column gel card is relatively high, and matched incubation and centrifugation equipment is required, so that the application of the micro-column gel card in most medical institutions is limited.
The solid phase method is a technique invented by Plapp FV et al in the early 80 s [ Plapp FV, Sinor LT, Rachel JM, et al. A solid phase antibody screen. am J Clin Pathol,1984,82:179 ]. Coating a 96-well microplate with a specific organic adsorbent, coating antigen substances to be adsorbed in the microplate wells by using charge adsorption, and finally detecting related antibody substances by using the antigen substances. On one hand, because the traditional adsorbent is coated for a long time, and the steps of fixing, moisturizing and the like are needed after the cells are plated, the time and the labor are relatively consumed; on the other hand, the coated red blood cells were not subjected to the lyophilization step and had a service life of only 24 hours. Therefore, the above-mentioned drawbacks affect its practical application in clinics. However, the method has high sensitivity, and the effect of the method is greatly improved by carrying out a series of innovations on a coating substrate, a long-term preservation technology of coated cells, an indication system and the like.
The conventional detection methods all require the screening of cells with antibody suspended in red blood cell preservation solution to complete the experiment. On one hand, the preservation period of the reagent red blood cells is generally less than 3 months due to the life of the red blood cells and the quality of the preservation solution, and if the reagent red blood cells are not used in time, waste is easily caused; on the other hand, in the process of preserving the reagent red blood cells, along with the aging of the red blood cells, the antigen loss and hemolysis can continuously occur, and the false negative experiment result can possibly occur.
In summary, in order to overcome the defects of the conventional irregular antibody detection technology, a kit for detecting an irregular antibody in erythrocytes, which is simple and convenient to operate, can store antigen substances for a long time, reduces antibody omission and has a proper cost, is urgently needed in clinical practical detection work.
Erythrocytes are a special kind of cell, on the surface of which are distributed a large number of blood group antigen molecules, which are highly specific, i.e. capable of immunoreacting only with the corresponding antibodies. When a solid phase carrier is coated with specific antibody molecules, the antigen molecules carried on the surface of the red blood cells are specifically and immunologically combined, and the combination of the antigen and the antibody can strongly fix the red blood cells on the surface of the solid phase carrier. Under the action of the erythrocyte freezing protection solution, the erythrocyte fixed on the surface of the solid phase carrier can break through the three-month period of the conventional liquid preservation solution for long-term preservation, and the membrane antigen can be kept stable for a long time.
The invention aims to coat monoclonal antibody aiming at erythrocyte antigen on the inner surface of a 96-well microplate, adsorb erythrocyte to the bottom of the microplate well by utilizing the high specificity of the monoclonal antibody, and realize long-term storage after cracking, adding cell protection solution and freeze drying; adding plasma to be detected and low-ion liquid for promoting reaction into the freeze-dried microplate well, so that immunological combination between erythrocyte antigen and plasma antibody to be detected can be quickly realized, finally, indication is carried out through a high-sensitivity indication system, and a uniform and consistent indication erythrocyte monolayer is shown on the bottom surface of the microplate well due to positive reaction; negative reactions will present a rounded indicator red blood cell button on the bottom surface of the microplate well center. The invention innovatively fixes the erythrocytes on the inner surface of a solid phase carrier by utilizing the specific reaction of a monoclonal antibody to erythrocyte antigens, replaces the preservation of maintenance solution of conventional erythrocytes by utilizing a freeze drying technology, utilizes a high-sensitivity two-step method indicating system, can practically solve the problems of low sensitivity, difficult preservation of erythrocytes, antibody omission of the conventional method and the like in the conventional clinical irregular antibody detection method, and provides a high-cost-performance erythrocyte blood type irregular antibody detection kit for the first-line clinical detection work.
Disclosure of Invention
The invention aims to provide a confluent type erythrocyte blood type irregular antibody detection kit based on a solid phase agglutination technology, which can solve the problems of short preservation period of erythrocytes, missed detection of antibodies, complex operation, higher cost and the like in the existing detection method.
The invention also aims to provide a preparation method of the confluent type erythrocyte blood group irregular antibody detection kit based on the solid phase agglutination technology.
The invention provides a confluent type erythrocyte blood group irregular antibody detection kit based on a solid phase agglutination technology, which comprises: solid phase agglutination reaction microplate, low ion solution, negative control serum, positive control serum and indication system.
The confluent solid phase agglutination reaction microplate consists of a 96-hole U-shaped bottom detachable microplate strip, the microplate strip is preferably polystyrene, and has the functions of coating an anti-erythrocyte monoclonal antibody, fixing a confluent erythrocyte antigen membrane, accommodating detected blood plasma, low ionic liquid, an indicating system and the like, and does not participate in chemical reaction per se; on the basis of coating monoclone, fixing the collected anti-sieve cells at the bottom of a U-shaped hole of the microplate, adding a cell membrane freezing protection solution after cracking and washing, and freeze-drying to obtain a 96-hole U-shaped bottom solid-phase reaction microplate coated with the anti-sieve cell membranes, which can be used for a solid-phase reaction experiment; different from the traditional solid phase method, the invention adopts the monoclonal antibody with high specificity to the red blood cells as the coating substrate, and the organic coating medium (such as methylene blue) used by the non-traditional solid phase has high adsorption effect on the anti-screening red blood cells of the plating, so that the red blood cells are fixed at the bottom of the holes of the U-shaped microplate to form a uniform red blood cell molecular monolayer, and the uniform red blood cell molecular monolayer is stored after freeze drying without moisturizing, overnight and the like, thereby saving the coating time and improving the plating efficiency of the red blood cells;
the confluent anti-sieve erythrocyte membrane in the microplate is that 3 single O-type erythrocytes are gathered in equal quantity and then coated on the inner surface of the bottom of a single hole of the U-shaped microplate, and the complementary antigen spectrum at least comprises: d, C, E, C, E, JKa，JKb，Lub，k，M，N，S，s， Fya，P1，Mur，DiaThe cell membrane formed by cracking the cells collected in the single hole is a collection detection type anti-sieve cell membrane single layer and can be used for detecting the irregular antibody corresponding to the antigen; the Rh antigen phenotype of 3 single individual erythrocytes should be: CCDee, CCDee;
the low ionic strength solution is a buffer containing glycine and 0.10% sodium azide;
the indicating system comprises indicating red blood cells and an anti-human globulin reagent, wherein the indicating red blood cells are IgG anti-D sensitized O-type RhD (+) red blood cell suspension, and the concentration of the indicating red blood cells is 0.2-0.6%; the anti-human globulin reagent is a goat anti-human or rabbit anti-human IgG antibody diluted by 1: 1-1: 128 times;
the negative control serum is human serum which has no agglutination reaction with the anti-screening erythrocyte and is added with a certain amount of preservative, and concretely, the negative control serum is healthy human serum which does not contain irregular antibody and is added with 0.10% of sodium azide;
the positive control serum is human serum which has agglutination reaction with the screened red blood cells and is added with a certain amount of preservative, and concretely, the positive control serum is healthy human serum which contains IgG anti-D and is added with 0.10% of sodium azide.
The invention provides a preparation method of a confluent type erythrocyte blood group system irregular antibody detection kit based on a solid phase agglutination technology, which comprises the following steps:
1. an irregular antibody screening spectrum cell line is established, a monoclonal antibody is adopted to screen O-type red blood cells, an antibody screening cell line consisting of 3O-type red blood cells is established according to an antigen complementation principle and is collected in equal amount to form a collection-check type anti-screening cell cluster, and a specific antigen typing spectrum is shown in a table 1:
TABLE 1 irregular antibody screening cell antigen Profile to pool
2. The blank microplate prepared by the confluent solid phase agglutination reaction microplate is preferably polystyrene, has the functions of coating an anti-erythrocyte monoclonal antibody, fixing an anti-sieve erythrocyte membrane, accommodating detected blood plasma, low ionic liquid, an indicating system and the like, does not participate in chemical reaction, and more particularly, the strips of the microplate are detachable; diluting the anti-erythrocyte monoclonal antibody with a carbonate buffer solution, adding 100 mu L of anti-screening erythrocyte in each hole, coating for 16 hours at room temperature, on the basis, collecting the anti-screening erythrocyte in the step (1) in an equivalent manner to prepare a suspension with the concentration of 0.1%, adding 100 mu L of anti-screening erythrocyte in each micropore, centrifuging for 5 minutes, washing for 5 times, and adding erythrocyte lysate for lysis; after washing the lath, adding a freezing protection solution containing 8% of sucrose into the lath, wherein the volume of each hole is 50 mu L, removing liquid in each hole, sucking and drying residual liquid by using absorbent paper, putting the residual liquid into a freeze dryer for freeze drying for 2 hours, taking out the residual liquid, putting the residual liquid into an aluminum foil bag, and heating and sealing the aluminum foil bag;
3. preparing a low ionic strength solution, weighing 18 g of glycine and 1 g of sodium azide, dissolving with purified water, metering to 1L, adding a proper amount of bromocresol purple solution until the color of the solution is changed from transparent colorless to deep purple, and adjusting the pH value of the solution to 6.8;
4. preparation of indicator system the indicator system comprises indicator red blood cells and rabbit or sheep anti-human globulin reagent, and the indicator red blood cells are prepared as follows: washing to obtain packed O-type red blood cells, adding IgG anti-D with the same volume, incubating at 37 ℃ for 50 minutes to obtain IgG anti-D sensitized red blood cells, washing with normal saline for 5 times, and preserving with red blood cell preserving fluid, wherein the anti-human globulin reagent is prepared by the following steps: diluting a rabbit or sheep anti-human globulin reagent by using an antibody diluent, reacting with sensitized erythrocytes, observing a non-agglutination tube under a microscope after centrifugation as the dilution multiple of the anti-human globulin reagent, wherein the anti-human globulin reagent with the dilution multiple and sensitized O-type erythrocyte suspension form the indicating system;
5. preparing negative control serum to screen out human serum which has no agglutination reaction with the anti-sieve cells, and adding sodium azide to the human serum to obtain a final concentration of 0.10%;
6. positive control serum preparation refers to human serum containing IgG anti-D with 0.10% sodium azide added.
The kit for detecting the erythrocyte blood group irregular antibody provides a novel detection concept and technology; its advantages are as follows:
1. the invention adopts monoclonal antibody aiming at O-type erythrocyte membrane antigen to coat the laths, replaces the method of coating the laths by using non-specific traditional adsorption reagent in the prior imported like products, has high specificity to the anti-sieve erythrocyte of the planking, and leads the erythrocyte to be fixed at the bottom of the hole of the U-shaped microplate through immunoreaction of the coated antibody and the antigen of the anti-sieve erythrocyte, thereby forming a uniform erythrocyte molecular monolayer, greatly improving the coating efficiency of the erythrocyte, and the traditional low-performance coating matrix (such as methylene blue and the like) is easy to cause the phenomena of lacking edges and approach of the planking erythrocyte, thus leading the thickness of the erythrocyte molecular monolayer to be uneven and interfering the subsequent experimental results (figure 1);
2. the anti-sieve cells I c, II c and III c are collected and then paved, so that the detection of various irregular antibodies contained in single plasma by a single detection hole is realized, the detection cost is convenient to reduce, and the detection flux is improved;
3. aiming at the high-frequency anti-Mur antibody in Chinese population, red blood cells (i.e. No. ic spectral cells) of Mur (+) are specially screened to be used as one of the selected cells, so that the problem that the corresponding anti-Mur antibody is missed to be detected due to the lack of the antigen of the anti-Mur cells on the market at present is solved (figure 4);
4. the storage period of the microplate coated with the confluent erythrocyte membrane is as long as 6 months, which is 2 times of the storage time of the conventional erythrocyte, the storage is convenient, and the reagent waste caused by short storage time of the erythrocyte is avoided; a special two-step indicating system is prepared for end point indication, so that the detection sensitivity is improved, and the omission of low-titer antibodies is avoided (fig. 5 and 6); meanwhile, the detection method provided by the invention is simple in operation process and does not need special equipment, the microplate strips can be flexibly disassembled, single-person detection can be realized, large-scale detection can also be realized, the detection of the antibody with low titer and irregularity is superior to that of the conventional anti-human-ball microcolumn gel card method in sensitivity, stability and detection of the antibody with low titer and irregularity, and the clinical application value is good.
The invention coats the anti-erythrocyte monoclonal antibody in a 96-hole microplate, absorbs specific erythrocytes to the bottom of the microplate by utilizing the highly specific reaction of the anti-erythrocyte monoclonal antibody to erythrocyte antigens, and realizes long-term storage after cracking and freeze drying; adding plasma to be detected and low-ion liquid for promoting reaction into the freeze-dried microplate well, so that immunological combination between erythrocyte antigen and plasma antibody to be detected can be quickly realized, finally, indication is carried out through a high-sensitivity indication system, and a uniform and consistent indication erythrocyte monolayer is shown on the bottom surface of the microplate well due to positive reaction; negative reactions will present a rounded indicator red blood cell button on the bottom surface of the microplate well center. The invention creatively utilizes the specific immunoreaction among antigen antibodies to generate high-strength adsorption and fixation to erythrocytes, utilizes a freeze drying technology to replace the conventional maintenance liquid storage of erythrocytes and utilizes a high-sensitivity two-step method indication system, can practically solve the problems of complicated coating and plate paving steps, low sensitivity, difficult preservation of erythrocytes, omission of irregular antibodies in the conventional method and the like in the conventional clinical irregular antibody detection method, and provides a high-cost-performance sorting type irregular antibody detection kit for clinical detection work.
Drawings
FIG. 1 is a comparison graph of the effect of different matrix materials for preparing erythrocyte monolayers by solid phase agglutination, wherein the microplate I and II are coated with anti-erythrocyte monoclonal antibody matrix and Melan matrix respectively, and the cell monolayers prepared by the microplate I are uniform and consistent and have no cracks or edges; the thickness of the cell monolayer prepared by the lath II is not uniform, and the phenomena of edge deletion and cell centering exist;
FIG. 2 is a schematic diagram of the solid phase agglutination method and the microcolumn gel cartridge agglutination intensity judgment standard;
FIG. 3 shows the antigenicity test of erythrocyte membranes before and after freeze-drying of a confluent solid-phase agglutination reaction microplate, wherein the first and second images are the microplate before (before H) and after (after H) freeze-drying respectively; sequentially using anti-D, -E and-Jk from top to bottoma、-Jkb、-FyaAntibody, Normal plasma, Neg (negative control), Pos (positive control) detecting erythrocyte membrane monolayer antigen; antigen detection before and after freeze-drying shows 4+ agglutination, normal blood plasma and negative control show negative, and positive control shows 4+ agglutination;
FIG. 4 is a comparison graph showing the effect of the confluent red blood cell blood group irregular antibody detection kit and the conventional commercially available anti-sieve cell detection high frequency antibody anti-Mur effect of Chinese population, wherein the plasma detected by the kit micro-strip from top to bottom is Normal (Normal plasma), anti-Mur, anti-D, Normal (Normal plasma), anti-Mur, anti-D, Neg (negative control), and Pos (positive control); plasma detected in the microcolumn gel card from left to right after pooling of conventional commercially available anti-sieve cells was Normal (Normal plasma), anti-Mur, anti-D, Normal (Normal plasma), anti-Mur, anti-D in that order. The confluence detection type solid phase agglutination kit can detect the anti-Mur with low titer, the agglutination strength reaches 1+ to 2+, and the anti-Mur is not detected by the anti-sieve cell sold in the conventional market;
FIG. 5 is a test chart of erythrocyte membrane antigen stability within the storage period of the confluent erythrocyte blood group irregular antibody test kit (compared with the microcolumn gel card method), which is shown in the following steps: and (3) testing the erythrocyte membrane antigen of the kit which is stored for 2 months after freeze-drying by adopting a low-titer humanized antibody. In the figure, the micro-laths of the reagent box adopt anti-Jk with the titer of 4 and 2 from top to bottom in sequenceb、-Jka、-FyaThe agglutination strength of the antibody tested by the corresponding antigen reaches 4 +. The microcolumn gel card sequentially adopts anti-Jk with the titer of 4 and 2 from left to rightb、-Jka、-FyaThe antibody tests the corresponding antigen of the fresh red blood cell, the agglutination strength is between W + and 2+sTo (c) to (d); secondly, drawing: and (3) testing the erythrocyte membrane antigen of the kit which is stored for 4 months after freeze-drying by adopting a low-titer humanized antibody. In the figure, the micro-laths of the reagent box adopt anti-Jk with the titer of 2 and 1 from top to bottom in sequenceb、-Jka、-FyaThe agglutination strength of the antibody tested for the corresponding antigen reaches 4 +. The micro-column gel card adopts anti-Jk with the titer of 2 and 1 from left to right in sequenceb、-Jka、-FyaThe antibody tests the corresponding antigen of the fresh red blood cell, and the agglutination strength is between W + and 4 +; diagram three: and (3) testing the erythrocyte membrane antigen of the kit which is stored for 6 months after freeze-drying by adopting a low-titer human antibody. In the figure, the micro-laths of the reagent box adopt anti-Jk with the titer of 2 and 1 from top to bottom in sequenceb、-Jka、-FyaThe agglutination strength of the antibody tested by the corresponding antigen reaches 4 +. The micro-column gel card adopts anti-Jk with the titer of 2 and 1 from left to right in sequenceb、-Jka、-FyaThe antibody tests the corresponding antigen of the fresh red blood cell, and the agglutination strength is between W + and 3 +;
FIG. 6 is a graph showing the sensitivity of the assembled erythrocyte blood group irregular antibody detection kit during the storage period (compared with the micro-column gel card method); the method comprises the following steps:the sensitivity of the kit stored for 2 months after freeze-drying is tested by adopting human IgG anti-D diluted by multiple times. In the figure, the micro-laths of the kit sequentially test the human anti-D with 2, 4,8, 16, 32 and 64 times of dilution from top to bottom, the agglutination strength reaches 3+ to 4+, and the final tube anti-D dilution is 64. The microcolumn gel card adopts fresh O-type RhD (+) red blood cells from left to right to test human anti-D with dilution of 2, 4,8, 16, 32 and 64 times, and the agglutination strength is between 2+ and 2+sIn between, the end tube IgG anti-D dilution was measured to be 32; secondly, drawing: the sensitivity of the kit stored for 4 months after freeze-drying is tested by adopting human IgG anti-D diluted by multiple times. In the figure, the micro-lath of the kit sequentially tests the humanized anti-D with the dilution of 2, 4,8, 16, 32 and 64 times from top to bottom, the agglutination strength reaches 4+, and the measurable final tube IgG anti-D dilution is 64. The microcolumn gel card adopts fresh O-type RhD (+) red blood cells to test human anti-D with dilution of 2, 4,8, 16, 32 and 64 times from left to right in sequence, the agglutination strength is between 2+ and 1+, and the measurable end tube IgG anti-D dilution is 16; diagram three: the sensitivity of the kit stored for 6 months after freeze-drying is tested by adopting human IgG anti-D diluted by multiple times. In the figure, the micro-strip of the kit sequentially tests human anti-D with 2, 4,8, 16, 32 and 64 times of dilution from top to bottom, the agglutination strength reaches 3+ to 4+, and the final tube IgG anti-D dilution is 64. The microcolumn gel card adopts fresh O-type RhD (+) red blood cells to test human anti-D with dilution of 2, 4,8, 16, 32 and 64 times from left to right in sequence, the agglutination strength is between 2+ and 1+, and the measurable end tube IgG anti-D dilution is 16;
FIG. 7 is a graph showing the sensitivity comparison between the confluent red blood cell blood group irregular antibody detection kit and imported similar products; in the figure, the first micro-lath and the second micro-lath are respectively a kit of the invention and an imported kit micro-lath, and the humanized anti-Jk with the titer of 2 and the titer of 1 are sequentially tested from top to bottomaHuman anti-D, Neg (negative control), Pos (positive control) with titers of 32 and 16. The two antibodies can detect the humanized antibody and have consistent agglutination strength; in the figure, the microplate strips are respectively the microplate strip of the kit and the imported kit, and the Normal (Normal plasma), the humanized anti-Mur, Neg (negative control) and Pos (positive pair) with the titers of 8 and 4 are tested from top to bottom in sequenceLight). The kit can detect the humanized anti-Mur with the titer of 8 and 4 strongly, the agglutination strength is respectively 4+ and 3+ s, the agglutination strength of imported like products is w +, and the detection efficiency of the humanized anti-Mur and the agglutination strength of imported like products is obviously different.
Detailed Description
The invention is further explained below with reference to the drawings and the examples of implementation. It should be understood that: the examples are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
EXAMPLE 1 preparation of pooled erythrocyte blood group-irregular antibody detection kit
1. Establishing an irregular antibody screening spectrum cell line, screening O-type red blood cells by adopting a monoclonal antibody, and establishing an antibody screening cell line consisting of 3O-type red blood cells according to an antigen complementation principle, wherein a specific antigen typing spectrum is shown in a table 1;
2. preparation of confluent solid phase agglutination reaction microplate
(1) Diluting an anti-erythrocyte monoclonal antibody to 100ug/mL by using a carbonate coating solution, taking out a blank 96-hole reaction microplate, adding 100 mu L into each hole, coating for 16 hours at room temperature, drying liquid in the micropores by spin-drying, washing for 3 times by using purified water, and finally sucking residual liquid in the micropores by using absorbent paper;
(2) washing the anti-sieve cells numbered I c, II c and III c with physiological saline for 3 times, then collecting the cells in equal volume to prepare 0.1% anti-sieve erythrocyte suspension, adding the collected anti-sieve cells into the reaction holes of the microplate coated with the monoclonal antibody, adding 100 mu L of the collected anti-sieve cells into each micropore, centrifuging the microplate by a plate centrifuge for 5 minutes, washing the microplate with physiological saline for 5 times, spin-drying the liquid in the laths, and finally sucking the residual liquid in the micropores with absorbent paper for the last time;
(3) adding 0.45% hypotonic saline into the microplate coated with the erythrocyte monolayer, 50 mu L/hole, cracking the erythrocyte monolayer, drying the liquid in the micropores, washing the microplate with physiological saline for 5 times, drying the liquid in the microplate, and finally sucking the residual liquid in the micropores with absorbent paper;
(4) adding erythrocyte antigen membrane protective solution (8% sucrose solution) into the microplate, drying at 50 μ L/hole, drying, removing residual liquid in the micropore by using absorbent paper, freeze-drying in a freeze dryer for 2 hours, taking out, filling in an aluminum foil bag, heating to seal the opening of the bag, and placing in a refrigerator at 4 deg.C for later use;
2. preparation of Low-ion solutions
Weighing 18 g of glycine and 1 g of sodium azide, dissolving with purified water, metering to 1L, adding a proper amount of bromocresol purple solution until the color of the solution is changed from transparent colorless to deep purple, and adjusting the pH value of the solution to 6.8;
3. preparation of the indicator System
(1) Preparation of sensitized erythrocytes: adding IgG anti-D with the same volume into the washed O-type packed red blood cells, incubating for 50 minutes at 37 ℃ to prepare IgG anti-D sensitized red blood cells, washing for 5 times by using normal saline, and preparing a 0.2-0.6% suspension by using a red blood cell preservation solution, namely sensitized red blood cells;
(2) anti-human globulin reagent: the rabbit or sheep anti-human globulin reagent is diluted by an antibody diluent in a gradient fold ratio (such as: 1:1, 1:16, 1:32, 1:64, 1:128 and the like), reacts with the sensitized red blood cells, and is observed under a microscope after centrifugation as a dilution multiple of the anti-human globulin reagent, wherein the anti-human globulin reagent with the dilution multiple and the sensitized O-type red blood cells form the indicating system of the invention;
4. preparation of negative control serum: screening out the serum of healthy human which has no agglutination reaction with the anti-sieve cells, adding sodium azide to obtain a final concentration of 0.10%;
5. preparation of positive control serum: screening out the serum containing IgG anti-D, and adding 0.10% sodium azide.
Example 2 Convergence test of erythrocyte membrane antigenicity before and after lyophilization of solid-phase agglutination reaction microplate
Selecting D, E, Jk with dose effecta，Jkb、FyaAntigen is used as target antigen, human plasma containing the antibody is detected by using the kit of the invention, and whether the antigenicity of erythrocyte membrane is changed after freeze drying is evaluated. The experimental results show that the detection results of the antibody by the micro-lath before and after freeze-drying are consistent and are 4+, which indicates that the erythrocyte membraneThe antigenicity of (A) did not change any before and after lyophilization, and the results are shown in FIG. 3. The specific operation method comprises the following steps:
1. the kit prepared in example 1 was taken out of 1 microplate strip, equilibrated to room temperature, and labeled from top to bottom with the 1 st to 8 th wells as-D, -E, -Jk, respectivelya，-Jkb、-FyaNormal, Neg (negative control), Pos (positive control), this lyophilized strip is the experimental strip. Preparing 1 microplate, performing the step (2) of example 1, removing the step 4, namely, not performing the freeze-drying step, and marking the rest of the microplates with the same experimental strips;
2. 2 drops (100. mu.L) of low-ion solution are added into each well, and 1 drop (50. mu.L) of specimen to be tested and negative and positive control serum are added into the corresponding well. The purple of the low ionic strength solution is changed into cyan or light green, and if the solution is still purple, the sample to be detected may be missed;
3. sealing the micro-strip with sealing glue, gently mixing uniformly, and placing in a 37 ℃ water bath box for incubation for 15 minutes;
4. and taking out the incubated microplate strips, tearing off the sealing glue, throwing off the liquid in the holes, and washing for 5 times by using normal saline. After the last washing, the micro-strips are placed on absorbent paper upside down to absorb residual liquid;
5. adding 1 drop (50 μ L) of goat/rabbit anti-human IgG, immediately adding 1 drop (50 μ L) of indicator red blood cells, and gently shaking and mixing;
6. and (5) putting the micro-strip into a plate centrifuge, centrifuging for 5 minutes at 200g, and judging the result.
Example 3 Combined detection type erythrocyte blood group irregular antibody detection kit for detecting high-frequency antibody anti-Mur of Chinese population compared with conventional anti-sieve cell
High-frequency antibody anti-Mur in Chinese population is taken as a target detection antibody, and is compared with conventional anti-sieve cells. As a result, the kit of the present invention can well detect the anti-Mur antibody, which is not detected by the conventional anti-sieve cell, as shown in FIG. 4. The specific operation method comprises the following steps:
1. 1 microplate strip was taken out from the kit prepared in example 1, equilibrated to room temperature, and wells 1 to 8 were labeled with Normal (Normal plasma), -Mur, -D, Normal (Normal plasma), -Mur, -D, Neg (negative control), and Pos (positive control), respectively, from top to bottom;
2. 2 drops (100 mu L) of low-ion solution are respectively added into each hole, and 1 drop (50 mu L) of specimen to be detected and negative and positive control serum corresponding to the mark are respectively added into the corresponding holes. The purple of the low ionic strength solution is changed into cyan or light green, and if the solution is still purple, the sample to be detected may be missed;
3. sealing the micro-strip with sealing glue, gently mixing uniformly, and placing in a 37 ℃ water bath box for incubation for 15 minutes;
4. taking out the incubated microplate strips, tearing off the sealing glue, throwing off liquid in the holes, washing for 5 times by using normal saline, and after the last washing, inversely placing the microplate strips on absorbent paper to suck residual liquid;
5. adding 1 drop (50 μ L) of goat/rabbit anti-human IgG, immediately adding 1 drop (50 μ L) of indicator red blood cells, and gently shaking and mixing;
6. and (5) putting the micro-strip into a plate centrifuge, centrifuging for 5 minutes at 200g, and judging the result.
Example 4 stability and sensitivity comparison test of cell membrane antigen in confluent erythrocyte blood group irregular antibody detection kit
1. Erythrocyte membrane antigen stability test: the kit prepared in the embodiment 1 is placed in a refrigerator at 2-8 ℃ for storage, and samples are taken for 2 months, 4 months and 6 months in the storage period respectively to carry out antigen stability test on the kit; antigen stability test selection Jka、Jkb、FyaThe antigen is used as a target antigen, and the conventional microcolumn gel card method is adopted to easily miss detection of the low-titer human anti-Jka、 -Jkb、-FyaThe antibody was tested and the results showed: the kit prepared by the invention can be stored for 6 months by human anti-Jk with the minimum titer of 1a、-Jkb、-FyaAntibody detection of corresponding Jka、Jkb、FyaThe antigen and the agglutination strength are both 4+, which is superior to the conventional micro-column gel card method, and the good stability of the kit erythrocyte membrane antigen is fully displayed. The specific results are shown in FIG. 5;
the solid phase agglutination method comprises the following specific operations:
(1) taking out 1 microplate strip from the kit in shelf life, balancing to room temperature, and labeling 1 st-8 th wells from top to bottom as-JKb、-JKa、-FyaNeg (negative control), Pos (positive control) and the titer of the corresponding antibody is noted;
(2) 2 drops (100 mu L) of low-ion solution are respectively added into each hole, and 1 drop (50 mu L) of specimen to be detected and negative and positive control serum corresponding to the mark are respectively added into the corresponding holes. The purple of the low ionic strength solution is changed into cyan or light green, and if the solution is still purple, the sample to be detected may be missed;
(3) sealing the micro-strip with sealing glue, gently mixing uniformly, and placing in a 37 ℃ water bath box for incubation for 15 minutes;
(4) taking out the incubated microplate strips, tearing off the sealing glue, throwing off liquid in the holes, washing for 5 times by using normal saline, and after the last washing, inversely placing the microplate strips on absorbent paper to suck residual liquid;
(5) adding 1 drop (50 μ L) of goat/rabbit anti-human IgG, immediately adding 1 drop (50 μ L) of indicator red blood cells, and gently shaking and mixing;
(6) and (5) putting the micro-strip into a plate centrifuge, centrifuging for 5 minutes at 200g, and judging the result.
The micro-column gel card method comprises the following specific operations:
(1) taking out the micro-column gel card, and observing whether the gel in the card has the phenomena of drying crack, air bubbles or foreign matters by naked eyes before use, and if the gel in the card has the phenomena, determining the card as an invalid card; if bubbles or liquid drops exist in the part or the sealing of the reaction chamber, the reaction chamber must be centrifuged for 2 minutes before use;
(2) marked as-Jk in sequence under the micro-column cardb、Jka、-FyaAnd the titer of the antibody used (such as 2 and 1), 0.8% O type red blood cell 50uL is added into the labeled micro-column tube respectively;
(3) respectively adding 25uL of human antibody corresponding to the markers into each tube;
(4) placing the mixture in a special incubator to incubate for 15 minutes at 37 ℃, taking out the mixture, placing the mixture in a special centrifuge, and centrifuging the mixture for 10 minutes at 1030 rpm;
(5) after the centrifugation is finished, taking out the microcolumn gel card for observation, and judging and recording the result;
2. and (3) testing the sensitivity: the kit prepared in the embodiment 1 is placed in a refrigerator at 2-8 ℃ for storage, and samples are taken for 2 months, 4 months and 6 months in the storage period respectively to carry out sensitivity comparison test on the kit; carrying out gradient multiple dilution by adopting human anti-D and then detecting; the results show that the sensitivity of the kit prepared by the invention in each test time period has no difference when the kit is stored for 6 months, and is superior to the conventional microcolumn gel card method by 2 gradients, and the comparative detection results are shown in FIG. 6; the specific operation method is as follows:
solid phase agglutination method:
(1) 6 tubes were labeled 2, 4,8, 16, 32, 64, respectively, and 200. mu.L of antibody dilution was added to each tube. Adding 200 mu L of centrifuged human IgG anti-D extract into a test tube marked with 2, gently blowing and mixing uniformly, then transferring 200 mu L of diluent in the test tube into a test tube marked with 4, repeating the operation, and sequentially transferring until the last tube (namely the test tube marked with 64); the liquid prepared after the operation is the human anti-D diluted by gradient multiple ratios, wherein the dilution multiple is respectively 2, 4,8, 16, 32 and 64 times;
(2) taking out 1 microplate strip from the kit within the preservation period, balancing to room temperature, and respectively marking the 1 st to 8 th holes as-D2, -D4, -D8, -D16, -D32, -D64, Neg (negative control) and Pos (positive control) from top to bottom;
(3) 2 drops (100 mu L) of low-ion solution are respectively added into each hole, and 1 drop (50 mu L) of corresponding specimen to be detected and negative and positive control serum are respectively added into the corresponding holes. The purple of the low ionic strength solution is changed into cyan or light green, and if the solution is still purple, the sample to be detected may be missed;
(4) sealing the micro-strip with sealing glue, gently mixing uniformly, and placing in a water bath tank at 37 ℃ for incubation for 15 minutes;
(5) and taking out the incubated microplate strips, tearing off the sealing glue, throwing off the liquid in the holes, and washing for 5 times by using normal saline. After the last washing, the micro-strips are placed on absorbent paper upside down to absorb residual liquid;
(6) adding 1 drop (50 μ L) of goat/rabbit anti-human IgG, immediately adding 1 drop (50 μ L) of indicator red blood cells, and lightly shaking and mixing;
(7) putting the micro-slab into a flat centrifuge, centrifuging for 5 minutes at 200g, and judging the result;
the micro-column gel card method specifically operates as follows:
(1) taking out the micro-column gel card, and observing whether the gel in the card has the phenomena of drying crack, air bubbles or foreign matters by naked eyes before use, and if the gel in the card has the phenomena, determining the card as an invalid card; if bubbles or liquid drops exist in the reaction chamber part or the sealing part, the reaction chamber part or the sealing part needs to be centrifuged for 2 minutes before use;
(2) marking 2, 4,8, 16, 32 and 64 parts below the kappa microcolumn in sequence, and adding 0.8% of O-type RhD (+) red blood cell 50uL into the marked microcolumn tubes respectively;
(3) adding 25uL of IgG anti-D diluent in the test tube corresponding to the label into each tube;
(4) placing the mixture in a special incubator to incubate for 15 minutes at 37 ℃, taking out the mixture, placing the mixture in a special centrifuge, and centrifuging the mixture for 10 minutes at 1030 rpm;
(5) and after the centrifugation is finished, taking out the microcolumn gel card for observation, and judging and recording the result.
Example 5 comparison of the test results of the confluent erythrocyte blood group irregular antibody test kit and imported like products
Selecting irregular antibody anti-Jk which is common in Chinese population and is easy to miss detectionaThe solid phase agglutination method confluent detection kit is used for carrying out parallel comparison detection with similar imported products to test whether the detection efficiency is different or not by using the-D, -Mur and the like as target detection antibodies; the result shows that the detection of the low-titer irregular antibody by adopting the two-step method end point indicating system is superior to the one-step method end point indicating system of the similar imported product, and the figure is 7; the specific operation method comprises the following steps:
1. solid phase agglutination method confluent detection type erythrocyte blood group irregular antibody detection kit
(1) After 2 microplates were removed from the shelf life kit, the temperature was allowed to equilibrate to room temperature, and wells 1-8 of 1 of these were labeled-Jk a 2、-Jk a1. Neg (negative pair)Control), Pos (positive control), -D32, -D16, Neg (negative control), Pos (positive control). The other wells 1-8 were labeled Normal, Neg, Pos, Mur 8, -Mur 4, -Neg, and Pos, respectively;
(2) respectively adding 2 drops (100 mu L) of low-ion solution into each hole, respectively adding 1 drop (50 mu L) of sample to be detected corresponding to the mark and negative and positive control serum into the corresponding hole, wherein the low-ion strength solution changes purple into cyan or pale green, and the sample to be detected may be leaked if the low-ion strength solution is still purple;
(3) sealing the micro-strip with sealing glue, gently mixing uniformly, and placing in a 37 ℃ water bath box for incubation for 15 minutes;
(4) taking out the incubated microplate strips, tearing off the sealing glue, throwing off liquid in the holes, washing for 5 times by using normal saline, and after the last washing, inversely placing the microplate strips on absorbent paper to suck residual liquid;
(5) adding 1 drop (50 μ L) of goat/rabbit anti-human IgG, immediately adding 1 drop (50 μ L) of indicator red blood cells, and lightly shaking and mixing;
(6) and (5) putting the micro-strip into a plate centrifuge, centrifuging for 5 minutes at 200g, and judging the result.
2. Import kit
(1) Taking out 2 micro-strips from the imported kit stored in the refrigerator, balancing to room temperature, and marking the 1 st to 8 th holes as-Jk a 2、-Jk a1. Neg (negative control), Pos (positive control), -D32, -D16, Neg (negative control), Pos (positive control); the 1 st to 8 th wells of the other are labeled Normal (Normal plasma), Neg (negative control), Pos (positive control), -Mur 8, -Mur 4, Neg (negative control), Pos (positive control), respectively;
(2) respectively adding 2 drops (100 mu L) of low-ion solution into each hole, respectively adding 1 drop (50 mu L) of sample to be detected corresponding to the mark and negative and positive control serum into the corresponding hole, wherein the low-ion strength solution changes purple into cyan or pale green, and the sample to be detected may be leaked if the low-ion strength solution is still purple;
(3) sealing the micro-strip with sealing glue, gently mixing uniformly, and placing in a 37 ℃ water bath box for incubation for 15 minutes;
(4) taking out the incubated microplate strips, tearing off the sealing glue, throwing off liquid in the holes, washing for 5 times by using normal saline, and after the last washing, inversely placing the microplate strips on absorbent paper to suck residual liquid;
(5) adding 1 drop of indicating red blood cells, and lightly shaking and uniformly mixing;
(6) and (4) putting the micro-strip into a plate centrifuge, centrifuging for 3 minutes at 300g, and judging the result.
Claims (5)
1. The utility model provides a collection examines formula erythrocyte blood group irregular antibody detect reagent box based on solid phase agglutination technique which characterized in that: this collection examines irregular antibody detect reagent box of formula erythrocyte blood group includes:
(1) the confluent solid phase agglutination reaction microplate is composed of a 96-hole U-shaped bottom detachable microplate, the inner surface of the U-shaped bottom of the microplate is coated with a high specificity anti-erythrocyte monoclonal antibody, screened qualified No. I, II, III c anti-sieve erythrocytes are washed for 3 times by normal saline and then are converged in equal volume to prepare 0.1% anti-sieve erythrocyte suspension, 100uL is added into each micropore, erythrocytes are uniformly coated on the inner surface of the bottom of the microplate through the high specificity immunoreaction the erythrocytes by the anti-erythrocyte monoclonal antibody, confluent anti-sieve erythrocyte membranes fixed on the inner surface of the U-shaped bottom of the microplate are formed after cracking, cell membrane freezing protection liquid is added on the basis, and the microplate is prepared after freezing and drying;
(2) a low ionic strength solution comprising glycine and 0.1% sodium azide;
(3) an indicator system comprising indicator red blood cells and an anti-human globulin reagent;
wherein the indicating red blood cells refer to IgG anti-D sensitized O-type RhD (+) red blood cell suspension, and the concentration is 0.2-0.6%; the anti-human globulin reagent is a goat anti-human or rabbit anti-human IgG antibody diluted by 1: 1-1: 128 times;
(4) negative control serum, which is healthy human serum without irregular antibody and added with 0.10% sodium azide;
(5) positive control serum, which is healthy human serum containing IgG anti-D and 0.10% sodium azide added;
the confluent anti-sieve erythrocyte membrane is that 3 single O-shaped erythrocytes are equally gathered and then coated on the inner surface of the bottom of a single hole of a U-shaped plate, and the complementary antigen spectrum at least comprises: d, C, E, C, E, JKa, JKb, Lub, k, M, N, S, S, Fya, P1, Mur and Dia antigens, wherein cell membranes formed by splitting cells converged in a single hole are convergent anti-sieve cell membrane monolayers which can be used for detecting irregular antibodies corresponding to the antigens; the Rh antigen phenotype of 3 single individual erythrocytes should be: CCDee, CCDee;
the cell membrane freezing protection solution is sucrose solution.
2. The kit for detecting the blood group type irregular antibody of red blood cells based on the solid-phase agglutination technology according to claim 1, wherein: the inner surface of the U-shaped hole bottom of the microplate strip is coated with an anti-erythrocyte monoclonal antibody, and the microplate strip has high specificity on the adsorption of collected IC, IIc and IIIc anti-screening erythrocytes.
3. A preparation method of a confluent erythrocyte blood group irregular antibody detection kit based on solid phase agglutination technology is characterized by comprising the following steps: the method comprises the following steps:
(1) preparing a confluent solid-phase agglutination reaction microplate;
(2) preparing a low ionic strength solution;
(3) preparation of an indication system;
(4) preparing negative control serum;
(5) preparing positive control serum;
the method comprises the following specific steps:
(1) an irregular antibody screening spectrum cell line is established, a monoclonal antibody is adopted to screen O-type red blood cells, an antibody screening cell line consisting of 3O-type red blood cells is established according to an antigen complementation principle, and the specific antigen typing spectrum is as follows:
(2) the blank microplate prepared by the confluent solid phase agglutination reaction microplate is made of polystyrene, has the functions of coating an anti-erythrocyte monoclonal antibody, fixing an anti-sieving erythrocyte membrane, accommodating detected blood plasma, low-ion liquid and an indicating system, does not participate in chemical reaction, and can be detached from strips; diluting an anti-erythrocyte monoclonal antibody with a carbonate buffer solution, adding 100 mu L of anti-erythrocyte monoclonal antibody into each hole, coating for 16 hours at room temperature, collecting the anti-sieving erythrocytes with equal quantity to prepare a suspension with the concentration of 0.1 percent on the basis, adding 100 mu L of anti-sieving erythrocytes into each micropore, centrifuging for 5 minutes, washing for 5 times, and adding erythrocyte lysate for lysis; after washing the lath, adding a freezing protection solution containing 8% of sucrose into the lath, wherein the volume of each hole is 50 mu L, removing liquid in each hole, sucking and drying residual liquid by using absorbent paper, putting the residual liquid into a freeze dryer for freeze drying for 2 hours, taking out the residual liquid, putting the residual liquid into an aluminum foil bag, and heating and sealing the aluminum foil bag;
(3) preparation of low ionic strength solution:
weighing 18 g of glycine and 1 g of sodium azide, dissolving with purified water, metering the volume to 1L, adding a proper amount of bromocresol purple solution until the color of the solution is changed from transparent colorless to deep purple, and adjusting the pH value of the solution to 6.8;
(4) preparation of the indicator system: the indicating system comprises indicating red blood cells and rabbit or sheep anti-human globulin reagent, and the indicating red blood cells are prepared by the following steps: washing to obtain packed O-type red blood cells, adding IgG anti-D with the same volume, incubating at 37 ℃ for 50 minutes to obtain IgG anti-D sensitized red blood cells, washing with normal saline for 5 times, and preserving with red blood cell preserving fluid, wherein the anti-human globulin reagent is prepared by the following steps: diluting rabbit or sheep anti-human globulin reagent with antibody diluent, reacting with sensitized erythrocyte, observing non-agglutination tube under microscope after centrifugation as dilution multiple of anti-human globulin reagent, wherein the anti-human globulin reagent with the dilution multiple and sensitized O-type erythrocyte suspension form an indication system;
(5) preparation of negative control serum: screening out human serum which has no agglutination reaction with the anti-screening erythrocyte, adding sodium azide to obtain a final concentration of 0.10%;
(6) preparation of positive control serum: refers to human serum containing IgG anti-D plus 0.10% sodium azide.
4. The method for preparing the confluent red blood cell blood group irregular antibody detection kit based on the solid phase agglutination technology according to claim 3, wherein: the microplate is made of polystyrene.
5. The method for preparing the confluent red blood cell blood group irregular antibody detection kit based on the solid phase agglutination technology according to claim 3, wherein: the strips of the microplate are detachable from one another.
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