CN107643409B - Blood type antigen chip and application thereof in erythrocyte accidental antibody detection - Google Patents
Blood type antigen chip and application thereof in erythrocyte accidental antibody detection Download PDFInfo
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Abstract
The invention discloses a blood group antigen chip and application thereof in detecting an accidental antibody of red blood cells, wherein the detection comprises (1) coating a blood group red blood cell membrane antigen to a carrier; (2) contacting the test sample with an antigen coated onto a carrier; (3) adding a secondary antibody labeled with a marker; (4) signal detection is performed for the label. The blood group antigen chip and the erythrocyte accidental antibody detection method have high sensitivity and good accuracy, do not depend on erythrocytes, and can detect the binding antibodies of a plurality of rare blood group antigens.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a serological detection technology of blood group antibodies, and particularly relates to a blood group antigen chip, a preparation method thereof and application thereof in detection of erythrocyte accidental antibodies.
Background
Transfusion safety has become an important issue in health care work and has attracted a high degree of attention throughout the society. The World Health Organization (WHO) has consistently paid attention to the blood transfusion safety work, the strength of the blood safety work is further strengthened in recent years, and the blood safety is listed as one of seven key works of the global health work by the WHO. Transfusion safety issues include the problem of adverse reactions from infectious transfusions and the problem of adverse reactions from non-infectious transfusions. The uk, usa and france all started a transfusion serious hazard reporting system to collect and report major adverse events associated with blood component infusions that were voluntarily reported by each hospital. As can be seen from the statistical data in Europe and America, the incidence rate of non-infectious transfusion adverse reactions is high, and once the adverse reactions occur, the consequences are extremely serious.
The detection rate of the erythrocyte homologous accidental antibody in Chinese Han patients is 0.38-2.38%, and the erythrocyte homologous accidental antibody is a main reason causing non-infectious transfusion adverse reaction, neonatal hemolytic disease, difficult blood type identification, anemia with difficult matching and unknown reasons, ineffective erythrocyte infusion and the like. The accurate detection of the erythrocyte allogeneic irregular blood group antibody is carried out before blood transfusion, and the method is of great importance for the safety and curative effect of blood transfusion. In order to ensure the safety and curative effect of blood transfusion, the detection of the antibody of the same kind of accident of the red blood cells becomes a routine detection item before blood transfusion. If the unexpected antibody of the erythrocyte exists in the patient, once the corresponding antigen exists on the infused erythrocyte of the blood donor, the hemolytic transfusion adverse reaction can occur, and the serious patient even dies.
Since the precise structure of the antigens used to immunize monoclonal antibodies is unknown, derived from a variety of foreign materials including ovarian cyst fluid and soluble antigens of salivary glycoproteins and human cells, these clones used in conventional serological testing react more extensively in order to reduce the risk of missed detection due to antigenic variation, which also increases the risk of cross-reactivity between structurally similar antigens. It has been reported that cross-reactivity of ABO monoclonal antibodies with non-ABO antigens, although rarely seen in serological reactions, often occurs in non-serological reactions such as enzyme immunoassays and inhibition assays, and therefore cross-reactivity must be considered when antibodies are used in such assays. The conventional serology test has low detection sensitivity, easily causes the omission of weak antigens of blood donors, causes hemolytic transfusion adverse reaction after being infused into the body of a patient, threatens the life of the patient, has developed trend of detecting blood group antigens of the blood donors by using a non-serology test with high sensitivity, can cause wrong typing results if a rare blood group antibody reagent with weak specificity is used for typing the blood group antigens of the blood donors, and further cannot ensure safe and effective blood transfusion. The quality assessment of the rare blood group antibody reagents for blood group antigen testing of blood donors is therefore of particular importance. The method for evaluating the quality of the rare blood group antibody reagent currently uses the reagent red blood cells of known antigens to characterize the specificity of the antibody based on the agglutination reaction principle, but because the concentration of the red blood cell reagent is low, the antigenicity is weak, and the cells of different manufacturers are different before different batches; meanwhile, the agglutination reaction has low sensitivity and low specificity, and multiple detection cannot be realized, so that the quality of the rare blood type antibody reagent has certain defects.
The detection of accidental antibodies to erythrocytes has always been the focus of blood transfusion safety concerns, for which a number of methods for detecting accidental antibodies to erythrocytes have been developed, such as: classical antiglobulin method, column agglutination method, Capture method, etc. In patent CN00105438.4, platelet blood group antigen antibodies are detected by microcolumn gel method to match platelets. Patent CN200710049305.1 provides a kit for detecting blood type by using microfluidic technology, a preparation method and a detection method. However, the detection of the unexpected antibody of the red blood cells in the clinical blood in the above patents generally has the technical problems of poor sensitivity, narrow dynamic linear range, low throughput screening capability, serious false negative problem, inconvenient storage of detection reagents and the like.
Patent CN200880019301.1 realizes in vitro identification of anti-erythrocyte antibodies of individuals by fixing erythrocytes or erythrocyte membrane fragments on beads, however, the condition parameters described in the patent are not repeatable, and when the method is applied to detection of accidental antibodies of erythrocytes, the coating mode of the antigen, the adding concentration of the secondary antibody and the change of the dilution concentration of the sample all affect the accuracy of the detection, so the method needs to be redesigned and evaluated.
Disclosure of Invention
In order to solve the problems, the inventor of the invention provides a blood type antigen chip with good sensitivity and accuracy and application thereof in detecting the unexpected erythrocyte antibody or blood transfusion matching type by optimizing a detection method and detection conditions of the unexpected erythrocyte antibody.
The first aspect of the invention provides a blood group antigen chip, which comprises blood group erythrocyte membrane antigens and a carrier, wherein the blood group erythrocyte membrane antigens are coated on the carrier and can be combined with erythrocyte accidental antibodies.
The blood group red cell membrane antigen of the invention is selected from the group consisting of: purifying blood group antigen, recombining and expressing erythrocyte membrane antigen with specific glycosyl modification, chemically synthesizing erythrocyte membrane antigen with specific glycosyl modification, and separating erythrocyte membrane from erythrocyte. More preferably, the blood group erythrocyte membrane antigen is an erythrocyte membrane separated from erythrocytes, and the separation method is selected from a freezing thawing method, a solvent extraction method, and the like, and is, for example, a method described in patent CN106754692A, patent CN101109755A, and non-patent "comparison of two methods for extracting erythrocyte membrane proteins" (journal of cellular and molecular immunology, 1997 (2): 44-45). Particularly preferably, the blood group erythrocyte membrane antigen is prepared by a freezing and thawing method, and for example, the method comprises the following steps: (1) adding physiological saline into packed red blood cells, freezing at-20 deg.C to-200 deg.C, and thawing at 20 deg.C to 40 deg.C; (2) centrifuging, removing supernatant, and washing to obtain white precipitate. The invention isThe blood group erythrocyte membrane antigen comprises D, C, E, C, E and JKa、JKb、M、N、S、s、Kpa、Kpb、Lua、Lub、Fya、Fyb、K、k、Lea，LebAnd P1.
The carrier is a microsphere, the microsphere is one or a combination of more than two of silicon dioxide microspheres, polystyrene microspheres, magnetic microspheres and biomacromolecule polymer microspheres, preferably, the carrier is polystyrene microspheres, particularly preferably, the carrier is a polystyrene microsphere combined with fluorescent dye.
Preferably, the amount of the blood group red cell membrane antigen coated on the carrier is 1-90 mug/1.25X 105And (3) microspheres. More preferably, when the blood group erythrocyte membrane antigen is an erythrocyte membrane separated from erythrocytes, the amount of the erythrocyte membrane separated from erythrocytes coated to the carrier is 45-90 mug/1.25X 105Microspheres, particularly preferably, the erythrocyte membrane separated from erythrocytes is coated to the carrier in an amount of 45 μ g/1.25X 105And (3) microspheres. It will be understood by those skilled in the art that when the blood group erythrocyte membrane antigen is a purified blood group antigen, the amount of the blood group erythrocyte membrane antigen coated onto the carrier may be less than the amount of the erythrocyte membrane separated from the erythrocytes coated onto the carrier, and may be, for example, 1-10. mu.g/1.25X 105Microspheres, especially 1-2. mu.g/1.25X 105。
In a specific embodiment of the present invention, the blood group antigen chip is a liquid chip, and the microspheres coated with blood group erythrocyte membrane antigens in the blood group antigen chip are suspended in a preservation solution.
In a second aspect, the present invention provides a blood group antigen chip for use in: (1) screening and identifying the erythrocyte accidental antibody; (2) quantitative detection of erythrocyte accidental antibodies; (3) blood transfusion and matching.
Preferably, the blood group antibody of the present invention is an antibody against erythrocyte accident, and the antibody against erythrocyte accident is selected from Rh blood group system, MNS blood group system, P blood group system, Kell blood group system, Kidd blood group system, Lewis blood group system, Duffy blood group system, Luth blood group system, etc.
In a third aspect of the present invention, there is provided a method for preparing a blood group antigen chip, the method comprising:
(1) pretreatment of microspheres: carrying out ultrasonic resuspension on the microspheres;
(2) activation of the microspheres: sequentially adding N-hydroxy thiosuccinimide (Sulfo-NHS) and carbodiimide (EDC) into the microspheres for activation;
(3) and adding blood group erythrocyte membrane antigen into the microsphere solution for coupling, wherein the blood group erythrocyte membrane antigen can be combined with erythrocyte accidental antibodies.
Preferably, the amount of the blood group red cell membrane antigen coated on the carrier is 1-90 mug/1.25X 105More preferably, the blood group erythrocyte membrane antigen is an erythrocyte membrane separated from erythrocytes, and the erythrocyte membrane separated from erythrocytes is coated on the carrier in an amount of 45-90 mug/1.25X 105Microspheres, particularly preferably, the erythrocyte membrane separated from erythrocytes is coated to the carrier in an amount of 45 μ g/1.25X 105And (3) microspheres.
Preferably, in the step (2), the final concentration of the Sulfo-NHS and the EDC is 5 mg/mL.
In one embodiment of the present invention, the method for preparing the blood group antigen chip comprises:
after the microspheres are subjected to ultrasonic resuspension, removing supernatant; shaking the heavy suspension microspheres by using deionized water, and removing supernatant; shaking the resuspended microspheres with a disodium hydrogen phosphate buffer solution, adding a sulfoo-NHS solution, shaking and mixing uniformly, adding the mixture into an EDC solution, shaking and mixing uniformly, and incubating at room temperature for 5-30 minutes, preferably for 20 minutes; removing supernatant, using MES solution, resuspending microspheres, and removing supernatant; adding blood group erythrocyte membrane antigen into the suspended microsphere solution for coupling reaction, and incubating for 0.5-4 hours, preferably 2 hours; the supernatant was removed and the coupled microspheres were resuspended in PBS-TBN (1% BSA, 0.05% tween-20).
In a fourth aspect, the present invention provides a method for identifying or detecting a blood group antibody, comprising:
(1) coating a carrier with blood group erythrocyte membrane antigen, wherein the blood group erythrocyte membrane antigen can be combined with erythrocyte accidental antibody;
(2) contacting the test sample with an antigen coated onto a carrier;
(3) adding a secondary antibody labeled with a marker;
(4) signal detection is performed for the label.
The blood group red cell membrane antigen of the invention is selected from the group consisting of: purifying antigen, recombining and expressing the erythrocyte membrane antigen with specific glycosyl modification, chemically synthesizing the erythrocyte membrane antigen with specific glycosyl modification, and separating the erythrocyte membrane from the erythrocyte. More preferably, the blood group erythrocyte membrane antigen is prepared by a freezing and thawing method, for example, comprising the following steps: (1) adding physiological saline into packed red blood cells, freezing at-20 deg.C to-200 deg.C, and thawing at 20 deg.C to 40 deg.C; (2) centrifuging, removing supernatant, and washing to obtain white precipitate.
The carrier is a microsphere, the microsphere is one or a combination of more than two of silicon dioxide microspheres, polystyrene microspheres, magnetic microspheres and biomacromolecule polymer microspheres, preferably, the carrier is polystyrene microspheres, particularly preferably, the carrier is a polystyrene microsphere combined with fluorescent dye.
Preferably, the amount of the blood group red cell membrane antigen coated on the carrier is 1-90 mug/1.25X 105More preferably, the blood group erythrocyte membrane antigen is an erythrocyte membrane separated from erythrocytes, and the erythrocyte membrane separated from erythrocytes is coated on the carrier in an amount of 45-90 mug/1.25X 105Microspheres, particularly preferably, the erythrocyte membrane separated from erythrocytes is coated to the carrier in an amount of 45 μ g/1.25X 105And (3) microspheres.
The detection sample can be whole blood, serum, plasma or antibody, preferably, the detection sample is plasma. Preferably, the test sample is diluted and then contacted with the antigen coated on the carrier, wherein the dilution ratio of the test sample is 1:10-1:90, more preferably, the dilution ratio of the test sample is 1:20-40, and particularly preferably, the dilution ratio of the test sample is 1: 30.
The second antibody marked with the marker is selected from a protein or an antibody marked with fluorescein or enzyme and a conjugate thereof, the protein or the antibody and the conjugate thereof are selected from avidin, streptavidin, a digoxin antibody, a histidine antibody, a Ni-containing affinity molecule and a fluorescent molecule antibody, the fluorescein is selected from anthocyanin Cy series fluorescein, Alexa Fluor series fluorescein or fluorescein isothiocyanate, and the enzyme is alkaline phosphatase or peroxidase. Preferably, the secondary antibody labeled with a label is a Cy 3-labeled anti-human IgG antibody. Preferably, the secondary antibody of the present invention is added at a concentration of 1-8ug/ml, more preferably, the secondary antibody of the present invention is added at a concentration of 2-4ug/ml, and particularly preferably, the secondary antibody of the present invention is added at a concentration of 2 ug/ml.
The signal detection of the invention comprises fluorescence detection, chromogenic detection, electrochemical detection, mechanical detection and the like. Preferably, the signal detection of the present invention is fluorescence detection, and more preferably, the signal detection of the present invention is fluorescence detection at a wavelength of 532nm and 635 nm.
In one embodiment of the present invention, the method for detecting blood group antibodies comprises: adding PBS-TBN solution into each well of a 96-well microporous plate, incubating at 37 ℃ for 0.5-3h, and sealing; adding a carrier coated by a detection sample and blood group erythrocyte membrane antigen; centrifuging and removing supernatant; washing the 96-well plate by using a PBS-TBN solution; adding a fluorescence-labeled secondary antibody; the microspheres were resuspended in PBS-TBN and assayed using a flow cytometer.
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) high sensitivity and wide linear range. The sensitivity of the microsphere, chip and blood type antibody detection method is far higher than that of the atypical lectin screening method (AAS) and the microcolumn gel method commonly used in the prior art, the detection limit is far lower than that of the microcolumn gel method, and the sensitivity is improved by more than 1000 times; in addition, the invention has wide linear range and improved dynamic range by 2-3 orders of magnitude, and is particularly suitable for detecting low-concentration rare blood type antibodies and erythrocyte accidental antibodies.
(2) Accurate result and high coincidence rate. Compared with the microcolumn gel method, the blood group antibody detection method has 40% inconsistency in the detection of rare blood group characterization antibodies, and the agglutination inhibition test verification shows that the detection method is more accurate, so that the complex operation steps of the agglutination inhibition test are avoided, and the technical problem of false negative of the microcolumn gel method is solved.
(3) Is independent of erythrocyte, and is convenient for storage. The blood type antibody detection method disclosed by the invention does not need to use red blood cells, the microspheres coupled with the antigen can be stably stored for a long time, and the technical problems of insufficient sources of red blood cells carrying rare antigens, difficulty in storage and easiness in hemolysis are solved.
(4) The integration level is high, and high-flux comprehensive screening can be realized. The microsphere, the chip and the blood type antibody detection method can be properly integrated according to the requirements and the cost. Various rare blood group antigens can be integrated, and the purpose of comprehensively analyzing all blood group antigens by one-time detection is realized; and the same rare blood group antigen classification markers can be integrated to realize large-scale screening of a certain rare blood group antigen.
Drawings
Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings.
FIG. 1 is a flow chart of the blood group antigen chip for detecting blood group antibodies. Adding an antigen-coated microsphere into a 96-well plate, adding serum to form a microsphere-antigen-antibody compound, then adding a fluorescence-labeled secondary antibody to read a signal, and detecting by a flow cytometer, wherein a signal result is read by a 532nm fluorescence detection value and a 635nm microsphere detection together.
FIG. 2 shows the effect of the amount of erythrocyte membrane antigen coated microspheres on the detection result.
The vertical axis represents the coupling ratio, and the horizontal axis represents the antibody dilution. The five histograms from left to right at each antibody dilution represent the antigen loading of 5.625. mu.g, 11.25. mu.g, 22.5. mu.g, 45. mu.g, 90. mu.g, respectively.
FIG. 3 shows the effect of dilution factor of the sample on the test results.
P is a positive sample; n is a negative sample; i, II and III are three-line membrane antigens; and R is the mean of positive results/the mean of negative results.
FIG. 4 shows the effect of the concentration of the secondary antibody added on the test results.
P is a positive sample; n is a negative sample; i, II and III are three-line membrane antigens; and R is the mean of positive results/the mean of negative results.
FIG. 5 shows the results of the blood group antigen chip and the microcolumn gel method for detecting antibodies with different dilutions.
A is the detection result of the anti-s antibody; b is the detection result of the anti-B antibody.
The abscissa is the antibody dilution. The graph is the antigen chip detection result, and the corresponding ordinate is MFI (mean fluorescence intensity of flow cytometry); the histogram is the result of the microcolumn gel detection, and the ordinate is Score (agglutination strength by microcolumn gel method).
FIG. 6 shows the results of eighteen rare blood group-characterizing antibodies detected by a conventional commercial microcolumn gel assay.
Fig. 7 and 8 are the detection results of the blood group antigen chip for fifteen rare blood group characterization antibodies.
The "+" and "-" are the detection results of the micro-column gel method on the rare blood type characterization antibody; the bar chart is the detection result of the blood group antigen chip method to the rare blood group characterization antibody.
FIG. 7 is a graph showing the consistent results of the two methods for Anti-E, Anti-K, Anti-Lea、Anti-FyaThe detection results of the Anti-N, Anti-P1, Anti-k, Anti-s and Anti-M antibodies of the nine blood types are consistent.
FIG. 8 is a graph of antibodies characterized by inconsistent assay results for two methods, Anti-c and Anti-JKa、Anti-e、Anti-C、Anti-S、Anti-JKbThe detection results of the six blood group antibodies are inconsistent. The "-" in the 6 bar charts represents that the detection result of the microcolumn gel method is negative, and the detection result of the blood group antigen chip method is positive.
FIG. 9 is a representation of antibodies using agglutination inhibition to detect inconsistencies between microcolumn gel and protein chips. The results show that the pairs of Anti-c and Anti-JKa、Anti-e、Anti-C、Anti-S、Anti-JKbThe detection results of the six blood group antibodies are consistent with the protein chip.
FIG. 10 shows the results of the detection of accidental antibodies against erythrocytes in clinical serum. The horizontal axis is the detection result of the clinical microcolumn agglutination method of the serum and the corresponding blood group antigen; the vertical axis is the name of the different antigens coated onto the microspheres. The serum antibody binding signal of the protein chip method is from low to high represented by 0 to 3000.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
EXAMPLE 1 Effect of microsphere coupling conditions on accidental antibody detection of erythrocytes
Three uncoupled microspheres were selected for 2 min ultrasonic resuspension, 50. mu.l of 2.5X 106Putting the microspheres per ml into an EP tube, placing the EP tube on a magnetic plate for 60 seconds, and removing supernatant; removing the magnetic separator, shaking the resuspended microspheres with 100 microliters of deionized water, placing the EP tube on the magnetic plate for 60 seconds, and removing the supernatant; removing the magnetic separator and shaking the resuspended microspheres with 80. mu.l of 100mM, pH6.2 disodium phosphate buffer, adding 10. mu.l of 50mg/ml Sulfo-NHS solution (dissolved in water), shaking gently for mixing, adding 10. mu.l of 50mg/ml EDC solution (dissolved in water), shaking gently for mixing, incubating at room temperature for 20 minutes, and shaking gently once every 10 minutes; the EP tube was placed on a magnetic plate for 60 seconds, and the supernatant was removed; removing magnetismForce separator and shake resuspension of microspheres with 250. mu.l of 50mM MES, pH5.0 solution, place EP tube on magnetic plate for 60 seconds, remove supernatant; removing the magnetic separator and resuspending the activated and washed microspheres in 100. mu.l of 50mM MES, pH5.0 solution with shaking; adding 90 mug, 45 mug, 22.5 mug, 11.25 mug and 5.625 mug erythrocyte membrane antigen (obtained by taking peripheral blood for centrifugation, taking packed erythrocytes, repeatedly freezing and thawing and crushing, and then homogenizing) into the suspended microsphere solution, increasing the total volume to 500 microliter by using a solution of 50mM MES and pH5.0, shaking and uniformly mixing for coupling reaction, and incubating for 2 hours while turning and mixing at room temperature; the EP tube was placed on a magnetic plate for 60 seconds, and the supernatant was removed; removing the magnetic separator, shaking the magnetic separator with 500 microliters of PBS-TBN solution to resuspend the coupled microspheres, and performing turnover mixing incubation for 30 minutes at room temperature; the EP tube was placed on a magnetic plate for 60 seconds, and the supernatant was removed; the microspheres were washed 2 times with 1ml PBS-TBN solution, the magnetic separator was removed and the coupled and washed microspheres were resuspended in 500 microliters of PBS-TBN solution. Storing the coupled microspheres in dark environment at 2-8 ℃. Reacting the microspheres with different coupling ratios with the antibodies with concentrations of 0.1, 0.01 and 0.001 respectively.
The coupling ratio of erythrocyte membrane antigen to microsphere can affect the sensitivity of the test, so we have screened the optimal condition. The results are shown in FIG. 2. The results show that the signal value is in an increasing trend along with the increase of the antibody concentration, the signal values of different coupling ratios are different when the antibody concentration is 0.01 and 0.1, and the signal value is strongest when the erythrocyte membrane antigen is 45 mug, so that the optimal coupling amount is obtained when 45 mug of erythrocyte membrane antigen is added into a 500 microliter reaction system.
Example 2 testing the Effect of dilution factor of samples on accidental antibody detection of erythrocytes
Taking 10 positive samples (detected by a microcolumn gel method) and 10 negative samples (detected by the microcolumn gel method), respectively diluting 20 samples by 10 times, 30 times and 90 times, and reacting with the coupled microspheres prepared in example 1, wherein the specific steps are as follows: a96-well plate is taken, 200 microliter PBS-TBN solution is added into each well, and the mixture is incubated at 37 ℃ for 1h and sealed. Adding 50 microliters of antibody with different dilution times and 2500 coupled microspheres, and complementing the volume of the system to 100 microliters for reaction; centrifuging the microplate at 2000rpm for 2 minutes, then placing the 96-well plate on a magnetic plate for 2 minutes, and removing the supernatant; washing the 96-well plate with 100 μ l PBS-TBN solution for 2 times; adding 50 microliter of Cy3 labeled anti-human IgG/IgM, adding 50 microliter of PBS-TBN, and oscillating and incubating for 0.5 hour at room temperature; washing the 96-well plate with 100 μ l PBS-TBN solution for 2 times; the microspheres were resuspended in 200. mu.l PBS-TBN and assayed, as shown in FIG. 3.
As can be seen from the results, when the dilution factor of the test sample is different, the signal values are different, and the ratio of the positive result to the negative result is different, the positive result and the negative result are differentiated to the maximum degree when the test sample is diluted by 30 times, and the dilution factor is not suitable for being low or high. And when the dilution multiple is low, the overall signal is high, the negative background is high, a false positive result is easily caused when a cut off value is established, and when the dilution multiple is high, the overall signal is low, the positive signal is also low, and the weak antibody is easily identified as false negative. It can be seen that the test sample dilution factor has a great influence on the test.
EXAMPLE 3 Effect of the concentration of Secondary antibody added on accidental antibody detection of erythrocytes
10 positive samples (detected by a microcolumn gel method) and negative samples (detected by a microcolumn gel method) were taken and reacted with the coupled microspheres prepared in example 1, wherein 8ug/ml of Cy 3-labeled anti-human IgG and 2ug/ml of Cy 3-labeled anti-human IgG were used as secondary antibodies, respectively, and the method specifically comprises the following steps: a96-well plate is taken, 200 microliter PBS-TBN solution is added into each well, and the mixture is incubated at 37 ℃ for 1h and sealed. Adding 50 microliters of antibody with the same dilution factor and 2500 coupled microspheres, and complementing the volume of the system to 100 microliters for reaction; centrifuging the microplate at 2000rpm for 2 minutes, then placing the 96-well plate on a magnetic plate for 2 minutes, and removing the supernatant; washing the 96-well plate with 100 μ l PBS-TBN solution for 2 times; adding 50 microliters of Cy 3-labeled anti-human IgG/IgM with different concentrations, adding 50 microliters of PBS-TBN, and oscillating and incubating for 0.5 hour at room temperature; washing the 96-well plate with 100 μ l PBS-TBN solution for 2 times; the microspheres were resuspended in 200. mu.l PBS-TBN and assayed, as shown in FIG. 4.
As can be seen from the results, when an anti-human IgG labeled with Cy3 at 8ug/ml was used, the ratio between the positive and negative results was only about 1.3, and the negative and positive results were indistinguishable, whereas when an anti-human IgG labeled with Cy3 at 2ug/ml was used, the ratio between the positive and negative results reached about 7.8,
the negative and positive specimens can be clearly distinguished, and the effect of the added concentration of the secondary antibody on the test is large.
EXAMPLE 4 sensitivity test of blood group antigen chip for blood group antibody detection
As shown in the detection scheme of FIG. 1, a 96-well plate is taken, 200 microliters of PBS-TBN solution is added into each well, and the incubation is carried out for 1h at 37 ℃ for blocking. Adding 50 microliters of diluted antibody and 2500 coupled microspheres, and complementing the volume of the system to 100 microliters for reaction; centrifuging the microplate at 2000rpm for 2 minutes, then placing the 96-well plate on a magnetic plate for 2 minutes, and removing the supernatant; washing the 96-well plate with 100 μ l PBS-TBN solution for 2 times; adding 50 microliter of Cy3 labeled anti-human IgG/IgM, adding 50 microliter of PBS-TBN, and oscillating and incubating for 0.5 hour at room temperature; washing the 96-well plate with 100 μ l PBS-TBN solution for 2 times; the microspheres were resuspended in 200. mu.l PBS-TBN and assayed by flow cytometry.
The erythrocyte blood type antibodies are mainly IgM and IgG antibodies, the optimal reaction temperature of the IgM antibody is 4 ℃, and the optimal reaction temperature of the IgG antibody is 37 ℃, so in order to ensure that a erythrocyte antigen chip reaction system is suitable for both the two antibodies, one representative antibody (IgG antibody s and IgM antibody B) is respectively selected for detection. And the two antibodies were tested in parallel using the microcolumn gel method simultaneously, and the lowest detection limit and the kinetic range of the two methods were compared (fig. 5). Table 1 summarizes the minimum detection limits and kinetic range values for both methods.
TABLE 1 lowest detection limit and kinetic Range values
The results show that the lowest detection limits of the anti-s antibody detected by the erythrocyte antigen chip and the microcolumn gel method are 1: 1.59X 10 respectively5And 1:90 (Ratio value of 1763 for chip/gel method), and dynamic ranges of 1: 1.59X 10, respectively5-1:10 and 1:90-1:10 (Rat for chip/gel method)io value 1763); the lowest detection limit of the anti-B antibody detected by the erythrocyte antigen chip and the microcolumn gel method is 1: 1.56X 106And 1:810 (Ratio value of 1931 for chip/gel method), and dynamic ranges of 1: 1.56X 1061:90 and 1:81-1:10 (Ratio value of 215 for chip/gel method); therefore, the blood group antigen chip has high sensitivity to the detection of IgG and IgM antibodies. The liquid phase chip utilizes a biotin avidin signal amplification system, the affinity of the liquid phase chip is higher than that of a pure antibody by more than 10,000 times, meanwhile, the concentration of erythrocyte antigens is improved by several times, so that the detection result is more sensitive, less interfered by the environment and high in stability, and two beams of laser detected by the liquid phase chip do not analyze the information of the whole microsphere but only analyze the information within a certain radius of the particle, so that the background value of the detection result is low, the specificity is good, and the reliability of the detection result is high.
EXAMPLE 5 testing of the reliability of blood group antigen chips for the detection of rare blood group antibody reagents
The microcolumn gel technology is widely applied to clinical examination before blood transfusion at present so as to ensure the safety and effectiveness of clinical blood. It is thought that it can solve most problems of classical blood group serology simply, directly and effectively, but when the binding strength of erythrocyte antigen and antibody is weak and the shearing stress generated by gel card centrifugation exceeds the affinity, the antibody-dependent agglutination of erythrocytes is separated and false negative result appears. The blood group antigen chip has high sensitivity and can avoid the occurrence of false negative results, so the blood group antigen chip is used for representing clinical rare blood group antibodies and comparing with the conventional microcolumn gel method (figure 6 and figure 7). The results showed that 15 rare blood group antibody reagents (C, E, C, E, Jk)a、Jkb、M、N、S、s、Fya、K、k、LeaAnd P1), 9 (60%) of them E, K, Lea、FyaThe results of the two methods, N, P1, k, s and M, were consistent, with 6 (40%) Jka、c、e、JKbC, S do not match. Because the sensitivity of the blood group antigen chip is far higher than that of the microcolumn gel method, and the microcolumn gel method causes common reports of antibody missed detection, the inconsistent result can be false negative caused by the microcolumn gel method,therefore, the results of the disagreement were verified by the agglutination inhibition test.
In the research, the blood group antigen chip and the microcolumn gel method are adopted to carry out 40% inconsistency on the characterization result of the rare blood group antibody reagent, and the blood group antigen chip is probably far higher in sensitivity than the microcolumn gel method to further detect the cross reaction of the monoclonal antibody, so that the third test method, namely the agglutination inhibition test, is adopted to verify the inconsistency result. The results are shown in FIG. 6. The results show that after the 6 inconsistent results are verified by an agglutination inhibition test, the agglutination inhibition test result is consistent with the result of the erythrocyte antigen chip, and the results show that part of the rare blood type antibody reagents have cross reaction and can be detected only in a test method with higher sensitivity. The result shows that the agglutination inhibition test result is consistent with the blood group antigen chip result, which indicates that cross reaction exists in part of the rare blood group characterization antibodies, and the detection cannot be performed by using the conventional microcolumn gel method with low sensitivity, so that the quality control result performed by using the rare blood group characterization antibodies of the blood group antigen chip is more reliable.
EXAMPLE 6 clinical application of blood group antigen chip to detection of accidental erythrocyte antibodies
Selecting six kinds of uncoupled microspheres, and mixing Fya、Fyb、Lua、LubAnd K, k purified antigens are respectively coated on six different microspheres, and 20 clinical samples are detected. Wherein, the detection result of 20 clinical samples by the conventional microcolumn gel method is 3 anti-Fy samplesb1 example against Fya1 anti-K, 1 anti-D, 1 anti-M antibody, 3 anti-E in combination with other antibodies, 3 unknown antibodies, 7 negative samples. The results of the detection using the blood group antigen chip are shown in FIG. 9, in which 3 cases of Fyb1 example FyaAnd 1 case K and 6 cases are negative, and the result is consistent with the result of the conventional microcolumn gel method, wherein 1 case of the negative sample detected by the microcolumn gel method is positive through the blood group antigen chip, and 3 cases of unknown antibodies and 3 cases of anti-E combined with other antibodies have positive reactions of other antigens except the corresponding antigens through the blood group antigen chip. The result shows that the blood group antigen chip has high sensitivity, so that weak positive reactions which cannot be detected in the conventional detection method can be foundAt the same time, because the coating is a purified antigen, the type of the contained antibody can be clearly identified without presumption on the basis of a cell spectrum, and the accuracy is extremely high.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (3)
1. A method for identifying or detecting an antibody to an accident of erythrocytes, comprising:
(1) coating a carrier with blood group erythrocyte membrane antigen, wherein the blood group erythrocyte membrane antigen can be combined with erythrocyte accidental antibodies, the blood group erythrocyte membrane antigen is an erythrocyte membrane separated from erythrocytes, and the blood group erythrocyte membrane antigen is prepared by a method comprising the following steps: (1) adding physiological saline into packed red blood cells, freezing at-20 deg.C to-200 deg.C, and thawing at 20 deg.C to 40 deg.C; (2) centrifuging, removing supernatant, and washing to obtain white precipitate; the carrier is polystyrene microsphere, and the coating method comprises the following steps: carrying out ultrasonic re-suspension on the microspheres, and sequentially adding N-hydroxy thiosuccinimide and carbodiimide to activate the microspheres; adding blood group erythrocyte membrane antigen into the microsphere solution for coupling, wherein the amount of the blood group erythrocyte membrane antigen coated to the carrier is 45 mu g/1.25 multiplied by 105A plurality of microspheres;
(2) contacting a detection sample with an antigen coated on a carrier, wherein the detection sample is diluted and then contacted with the antigen coated on the carrier, and the dilution multiple of the detection sample is 1: 30;
(3) adding a secondary antibody labeled with a marker, wherein the secondary antibody labeled with the marker is an anti-human IgG antibody labeled with Cy3, and the concentration of the secondary antibody is 2 mu g/ml;
(4) detecting a signal for the label;
the blood type erythrocyte membrane antigen comprises one or the combination of more than two of D, C, E, C, E, JKa, JKb, M, N, S, S, Kpa, Kpb, Lua, Lub, Fya, Fyb, K, K, Lea, Leb and P1.
2. The method of claim 1, wherein the signal is detected by fluorescence.
3. A method of identifying or detecting an antibody to an erythrocyte accident as defined in claim 1, wherein the signal detection is fluorescence detection at a wavelength of 532nm and 635 nm.
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| CN201710845503.2A CN107643409B (en) | 2017-09-19 | 2017-09-19 | Blood type antigen chip and application thereof in erythrocyte accidental antibody detection |
| PCT/CN2018/072935 WO2019056673A1 (en) | 2017-09-19 | 2018-01-17 | Blood group antigen chip and use thereof in detection of erythrocyte unexpected antibody |
| PCT/CN2018/106040 WO2019057014A1 (en) | 2017-09-19 | 2018-09-17 | Blood type antigen chip and use of same in detection of accidental antibody in red blood cell |
| CN201880003173.5A CN110121649A (en) | 2017-09-19 | 2018-09-17 | A kind of blood group antigens chip and its application in red blood cell accident antibody test |
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| CN107643409B (en) * | 2017-09-19 | 2021-05-04 | 中国人民解放军总医院 | Blood type antigen chip and application thereof in erythrocyte accidental antibody detection |
| KR20210003721A (en) * | 2018-02-16 | 2021-01-12 | 디아개스트 | In vitro diagnostic device containing beads and use thereof |
| CN109725145A (en) * | 2019-01-03 | 2019-05-07 | 北京蛋白质组研究中心 | A kind of Inb blood group antigens chip and its application in red blood cell accident antibody test |
| CN109725144A (en) * | 2019-01-03 | 2019-05-07 | 北京蛋白质组研究中心 | A kind of JMH blood group antigens chip and its application in red blood cell accident antibody test |
| CN113092793A (en) * | 2021-04-16 | 2021-07-09 | 江苏力博医药生物技术股份有限公司 | Method and system for detecting Rh blood group antigen and application thereof |
| CN113640528A (en) * | 2021-06-28 | 2021-11-12 | 青岛益柏生物科技有限公司 | Detection method of full-automatic RH system blood type detector |
| CN114137231B (en) * | 2022-01-29 | 2022-04-29 | 北京大有天弘科技有限公司 | Detection kit for blood group irregular antibody and application thereof |
| CN114113639B (en) * | 2022-01-29 | 2022-04-19 | 北京大有天弘科技有限公司 | Blood type antibody detection method and application thereof |
| CN114397464B (en) * | 2022-03-24 | 2022-06-10 | 天津德祥生物技术有限公司 | Coupling compound of erythrocyte membrane fragments and carrier, coupling method and application thereof |
| CN114660305A (en) * | 2022-03-31 | 2022-06-24 | 中国人民解放军军事科学院军事医学研究院 | Reagent for rapidly detecting blood type antibody titer, preparation method, test strip and application |
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| NL1003570C2 (en) * | 1996-07-11 | 1998-01-15 | Stichting Centraal Lab | Method for antigen and antibody determination in blood group serology. |
| US5776711A (en) * | 1996-11-12 | 1998-07-07 | The Regents Of The University Of California | Simultaneous human ABO and RH(D) blood typing or antibody screening by flow cytometry |
| CN1282871A (en) * | 2000-03-29 | 2001-02-07 | 长春生物制品研究所 | Technique and reagent kit for investigating blood platelet, blood type, antigen and antibody by microcolumn gel method |
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