CN115753652A - Method for quantitatively detecting RhD antigen by using erythrocyte endogenous peroxidase - Google Patents
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Abstract
The invention relates to a method for quantitatively detecting RhD antigen by using erythrocyte endogenous peroxidase, belonging to the technical field of biological detection. Adding the red blood cells to be detected into a container coated with an anti-D antibody, centrifuging, washing to remove the red blood cells which are not adsorbed, adding a peroxidase substrate for color development, adding a stop solution to stop the reaction, detecting the absorbance value by an enzyme-labeling instrument under a light filter with a certain wavelength, and judging whether the red blood cells to be detected have the D antigen or not according to the OD value. The method is easy to operate automatically and has high stability; the dosage of the coating antibody is less, the sample red blood cells are also trace after dilution, the reaction result can be quantified by an enzyme-labeling instrument, and the kit has high sensitivity and strong specificity. Different from the prior erythrocyte D antigen identification method which only can carry out qualitative analysis, the method can carry out quantitative analysis on positive results by detecting OD values, the magnitude of the OD values indirectly reflects the quantity of the D antigens on the detected erythrocytes, and whether the D antigens belong to weak D and the zygotes thereof can be indirectly analyzed.
Description
Technical Field
The invention relates to a method for quantitatively detecting RhD antigen by using erythrocyte endogenous peroxidase, belonging to the field of biological detection.
Background
The Rh blood group system is the most complex blood group system of 43 blood group systems, is the blood group system with important clinical significance next to the ABO blood group system, is the most important Rh blood group system with RhD, and belongs to the necessary examination item for clinical pre-transfusion examination. According to the existence of the RhD antigen, the red blood cells are clinically divided into two main categories of RhD positive and RhD negative, wherein the RhD negative accounts for 3-5% of Han people. The RhD negative blood recipient transfuses RhD positive blood, so that the recipient can generate an anti-D antibody, and hemolytic transfusion reaction can be generated during secondary transfusion; when a RhD-positive fetus is delivered, a RhD-negative mother may also cause immunization of the mother with RhD-positive blood of the fetus, and when the RhD-positive fetus is again pregnant, a fetal neonatal hemolytic disease may occur. Therefore, the method has important clinical significance for correctly judging the RhD blood type, and belongs to the necessary examination item for clinical pre-transfusion examination.
The current RhD blood typing method generally uses the principle that anti-D antibodies and erythrocytes undergo agglutination reaction in vitro, and commonly used methods include a slide method (paper sheet method), a test tube method, a microplate method, a microcolumn gel method and the like (the slide method, the test tube method, the microplate agglutination method and the microcolumn gel method are all described in national clinical test operating procedures). The blood grouping on the 96-well microplate by using the agglutination reaction principle has the similar points with the scheme from the reaction carrier, but the blood grouping principle is completely different from the blood grouping principle of the scheme. RhD blood type identification on a microplate by utilizing the principle of agglutination reaction judges the agglutination strength according to the size of an agglutination block formed by an anti-D antibody and red blood cells, thereby achieving the purpose of distinguishing RhD positive and RhD negative. However, these methods all have drawbacks, such as:
the slide method and the test tube method need manual operation, are time-consuming and labor-consuming, and are easy to generate human errors;
the stability of results can be influenced by the sample adding amount of the antibody and the red blood cells and the difference between antibody batches in the microplate agglutination method;
the micro-column gel method is easy to deform and generate bubbles in the transportation process, and the detection result is influenced.
CN113092793A provides a positive typing method for detecting Rh blood group antigen based on solid phase adsorption, which is to add the red blood cells to be typed and the Rh blood group antibody into a container coated with a second antibody of another species for centrifugation, observe whether the red blood cells to be typed are adsorbed at the bottom of the container coated with the second antibody of the other species, if the red blood cells to be typed are adsorbed at the bottom of the container coated with the second antibody of the other species, the red blood cells to be typed and the Rh blood group antigen corresponding to the Rh blood group antibody are positive, and if the red blood cells to be typed are not adsorbed at the bottom of the container coated with the second antibody of the other species, the red blood cells to be typed and the Rh blood group antigen corresponding to the Rh blood group antibody are negative; the method has the advantages of high sensitivity, strong specificity and low detection cost, is easy to automatically operate, and can avoid human errors in the operation process. However, the method still needs to judge the adsorption condition through naked eyes or CCD, and is a qualitative detection method, different people may generate discrimination differences for the same result, and machine CCD discrimination is easy to generate errors.
Disclosure of Invention
In order to solve the above problems, the present invention provides a method for quantitatively detecting RhD antigen using endogenous peroxidase of erythrocytes, comprising an identification step; the identification steps are as follows: adding the red blood cells to be detected into a container coated with an anti-D antibody for centrifugation, washing the microporous plate for 3-6 times by using normal saline after the centrifugation is finished, sucking residual liquid in the microporous plate by using absorbent paper after the last washing is finished, adding a peroxidase substrate, developing for 3-20 minutes by using endogenous peroxidase of the red blood cells, stopping the reaction for 2-10 minutes by using an acid solution, measuring an OD value by using an enzyme-labeling instrument at a specific wavelength, and determining the RhD blood type of the detected sample according to the OD value;
if the detected OD values of the red blood cells to be detected in the container coated with the anti-D antibody are all smaller than the Cutoff value, the RhD blood type of the red blood cells to be detected is negative;
if the detected OD values of the red blood cells to be detected in the container coated with the anti-D antibody are all larger than the Cutoff value, the RhD blood type of the red blood cells to be detected is positive.
Furthermore, the rotating speed of the centrifugation is 90-360g, and the time is 1-30min.
Further, before the identification step, a dilution step is also included; the dilution step comprises the following steps: diluting the red blood cells to be detected to 0.1-1% of volume concentration.
Further, the dilution step is: the erythrocytes to be typed are diluted with Liss (low ionic strength solution) or physiological saline to a volume concentration of 0.1-1%.
In one embodiment of the present invention, the diluting step is: after washing the red blood cells to be tested 3-5 times with normal saline, diluting the red blood cells to be typed with Liss or normal saline to the volume concentration of 0.1-1%.
Furthermore, the addition amount of the red blood cells to be detected in the container coated with the anti-D antibody is 10-100 mu L.
Further, the preparation method of the container coated with the anti-D antibody comprises the following steps: properly diluting an anti-D antibody with a buffer solution A, adding the diluted anti-D antibody into a container, and coating for 8-16h at 2-8 ℃ to obtain a coated container; adding buffer solution B containing skimmed milk powder, sucrose, tween20 (Tween-20) into the coated container, and sealing at 37 deg.C for 0.5-3 hr to obtain anti-D antibody coated container.
Further, the buffer solution A is carbonate buffer solution with the pH value of 7.2-9.8.
Further, the buffer B is PBS buffer solution with pH value of 6.5-8.5; the mass concentration of the skimmed milk powder in the buffer solution B is 0.1-3%; the mass concentration of the sucrose in the buffer solution is 1-10%; the volume concentration of the Tween20 in the buffer B is 0.01-0.5%.
Further, the buffer C is PBS buffer with pH6.5-8.5 and containing Tween 20; the volume concentration of the Tween20 in the buffer C is 0.01-0.5%.
Further, the method for preparing the anti-D antibody-coated container comprises the following steps:
coating: diluting the anti-D antibody by 16-2048 times by using a carbonate buffer solution A with the pH of 7.2-9.8 to obtain a diluent; adding the diluent into a container, and coating at 2-8 deg.C for 8-16h to obtain a coated container;
a first plate washing step: adding PBS buffer solution C with pH of 6.5-8.5 into the coated container for washing, and repeating washing for 1-5 times to obtain a washed container;
and (3) sealing: adding 0.1-3% skimmed milk powder, 1-10% sucrose, 0.01-0.5% Tween20, PBS buffer solution B with pH of 6.5-8.5 into the washed container, and sealing at 37 deg.C for 0.5-3 hr to obtain sealed container;
and a second plate washing step: adding PBS buffer solution C (containing Tween20 with a certain volume concentration) with pH of 6.5-8.5 into the sealed container for washing, and repeating the washing for 0-5 times to obtain the container coated with the anti-D antibody.
Further, in the coating step, the anti-D antibody is diluted to 16 to 2048 times with a carbonate buffer solution A having a pH of 7.2 to 9.8 to obtain a diluted solution, and the diluted solution is added to a container.
Further, the washing step is: the washing was performed by adding PBS buffer C (containing Tween20 at a certain volume concentration) at pH6.5-8.5, and the washing was repeated 1-5 times.
The invention also provides application of the method or the system in detection of RhD blood group antigens.
The technical scheme of the invention has the following advantages:
the invention provides a method for quantitatively detecting RhD antigen by using endogenous peroxidase of red blood cells, which comprises the steps of adding the red blood cells to be detected into a container coated with an anti-D antibody, centrifuging, washing to remove the red blood cells which are not adsorbed, adding a peroxidase substrate for color development, adding a stop solution for stopping reaction, detecting an absorbance value by using an enzyme labeling instrument under a light filter with a certain wavelength, and judging whether the D antigen exists on the red blood cells to be detected according to the OD value.
The method has the advantages of easiness in automatic operation and high stability, the amount of the coated antibody is small, the red blood cells of the sample are also trace after dilution, the judgment of a reaction result is objective, and the method has the advantages of high sensitivity and strong specificity. In addition, the method gets rid of the dependence on higher-price gel, and the RhD blood type of a sample erythrocyte can be identified by one micropore coated with an anti-D antibody and a trace amount of sample erythrocyte, so that the method has the advantage of low cost. In addition, the method is different from the conventional erythrocyte D antigen identification method only in qualitative analysis, positive results can be quantitatively analyzed by detecting OD values, the magnitude of the OD values indirectly reflects the quantity of the D antigens on the detected erythrocytes, and whether the D antigens belong to weak D and the zygotes thereof can be indirectly analyzed.
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FIG. 1 is a detection schematic diagram of the method for quantitatively detecting RhD antigen by using erythrocyte endogenous peroxidase.
Detailed Description
The following examples are provided to better understand the present invention, not to limit the best mode, and not to limit the content and protection scope of the present invention, and any product that is the same or similar to the present invention and is obtained by combining the present invention with other features of the prior art and the present invention falls within the protection scope of the present invention.
The following examples, where specific experimental procedures or conditions are not indicated, can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the art. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
A method for quantitatively detecting RhD antigen by utilizing erythrocyte endogenous peroxidase is disclosed, and is shown in figure 1, a U-shaped microporous plate is coated with anti-D antibody, erythrocytes to be detected are added for centrifugation, erythrocytes positive to the D antigen are combined with the antibody and are uniformly distributed at the bottom of the U-shaped microporous plate, erythrocytes negative to the D antigen are converged at the bottom of the U-shaped microporous plate to form a cell buckle after centrifugation, the erythrocytes positive to the D antigen, which are uniformly adsorbed at the bottom of the hole, are not washed away after the plate is washed by normal saline, the erythrocytes negative to the D antigen are washed away, a peroxidase substrate is added, the absorbance value (OD value) is determined after the reaction is stopped by utilizing the endogenous peroxidase for color development of the erythrocytes, the OD value is in direct proportion to the amount of erythrocytes adsorbed at the bottom of the microporous plate, and whether the corresponding erythrocytes contain the D antigen or not is judged according to the OD value.
Example 1: method for quantitatively detecting RhD antigen by using erythrocyte endogenous peroxidase
The method comprises the following steps:
(1) Coating: diluting the anti-D antibody with the titer of 512 by 512 times by using a carbonate buffer solution with the pH of 9.6 to obtain a diluent containing the anti-D antibody, adding the diluent into a U-shaped microporous plate by the addition amount of 100 mu L per hole, and coating for 12h at 4 ℃ to obtain a coated microporous plate;
(2) Washing the plate for the first time: adding PBS buffer solution C (containing 0.02% Tween 20) with pH of 7.2 into the coated microplate at an addition amount of 250 μ L per well for washing for 1 time, and sucking off the residual liquid in the microplate after washing is completed to obtain a washed microplate;
(3) And (3) sealing: adding Tween20 with volume concentration of 0.02%, skimmed milk powder with mass concentration of 0.2% and sucrose with mass concentration of 5% into the washed microplate by the addition amount of 250 μ L per well, respectively, and sealing at 37 deg.C for 2h to obtain a sealed microplate;
(4) Diluting: washing the red blood cells to be classified for 3 times by using normal saline, and diluting the red blood cells to be classified to the concentration of 0.2% by using Liss or normal saline to obtain red blood cell suspension to be detected;
(5) And (3) detection: respectively adding the suspension of the red blood cells to be detected into a washed pore plate according to the addition amount of 40 mu L of each pore, and centrifuging for 1min at the rotating speed of 190g to obtain a centrifuged microporous plate;
(6) Washing: adding 250 mu L of normal saline into each hole of the microporous plate for washing for 5 times, and sucking residual liquid by using absorbent paper after the last washing is finished;
(7) Color development: adding 100 mu L of peroxidase substrate TMB into each hole, and developing for 10 minutes in a dark place;
(8) And (4) terminating: 0.16M H per well 2 SO 4 The reaction was terminated by leaving 50. Mu.L of the resulting solution to stand for 5 minutes in the dark.
(9) And (3) detection: and (5) detecting the OD value by using a microplate reader at 450nm light wave.
(10) The OD was determined in 8 known different samples, see Table 1 for details.
TABLE 1
Note: + represents Rh positive; -represents Rh-negativity
In the table, the detection result of the OD value is compared with the Cutoff value =0.2, and the negative and positive determination results of the RhD blood type are consistent with the known results of the sample.
Example 2
A method for quantitatively detecting RhD antigen using peroxidase endogenous to erythrocytes (see fig. 1 for the principle), comprising the steps of:
(1) Coating: diluting the anti-D antibody with the titer of 512 by 1024 times by using a carbonate buffer solution with the pH of 9.3 to obtain a diluent containing the anti-D antibody, adding the diluent into a microporous plate (U type) by the addition amount of 100 mu L per hole, and then coating for 16h at the temperature of 2 ℃ to obtain a coated microporous plate;
(2) Washing the plate for the first time: washing the coated microplate 1 time with PBS buffer C (containing 0.05%;
(3) And (3) sealing: adding Tween20 with volume concentration of 0.2%, skimmed milk powder with mass concentration of 0.9% and sucrose with mass concentration of 8% into the washed microplate at the addition amount of 250 μ L per well, respectively, and sealing at 37 deg.C for 1h to obtain sealed microplate;
(4) Diluting: washing the red blood cells to be classified for 4 times by using normal saline, and diluting the red blood cells to be classified to the concentration of 0.5% by using Liss or normal saline to obtain red blood cell suspension to be detected;
(5) And (3) detection: respectively adding the suspension of the red blood cells to be detected into a washed pore plate according to the addition amount of 40 mu L of each pore, and centrifuging for 1.5min at the rotating speed of 100g to obtain a centrifuged microporous plate;
(6) Washing: adding 250 microliter of normal saline into each hole of the microporous plate for washing for 4 times, and sucking residual liquid by using absorbent paper after the last washing is finished;
(7) Color development: adding 100 mu L of peroxidase substrate TMB into each hole, and developing for 8 minutes in a dark place;
(8) And (4) terminating: 0.18M H per well 2 SO 4 50. Mu.L of the reaction mixture was left standing for 3 minutes in the dark to terminate the reaction.
(9) And (3) detection: and (5) placing the microplate on an enzyme-labeling instrument to detect the OD value by using 450nm light waves.
(10) OD values were determined on 8 different known samples, as detailed in table 1.
TABLE 2
| Sample number and Rh blood type | OD value | Cutoff value | Results |
| 568(O+) | 2.848 | 0.2 | Positive for RhD |
| 569(B+) | 3.14 | 0.2 | RhD YangProperty of (2) |
| 570(B+) | 2.395 | 0.2 | Positive for RhD |
| 571(O+) | 2.795 | 0.2 | RhD positive |
| 572(O+) | 2.552 | 0.2 | RhD positive |
| 573(O+) | 3.035 | 0.2 | RhD positive |
| 574(AB+) | 2.139 | 0.2 | RhD positive |
| 575(AB+) | 3.146 | 0.2 | RhD positive |
Note: + is Rh positive; -represents Rh-negativity
In the table, the detection result of the OD value is compared with the Cutoff value =0.2, and the negative and positive determination results of the RhD blood type are consistent with the known results of the sample.
Example 3
A method for quantitative detection of RhD antigen using red blood cell endogenous peroxidase (see fig. 1 for the principle), said method comprising the steps of:
(1) Coating: diluting the anti-D antibody with the titer of 512 by 128 times by using a carbonate buffer solution with the pH of 9.5 to obtain a diluent containing the anti-D antibody, adding the diluent into a microporous plate (flat bottom) according to the addition of 100 mu L of the diluent into each hole, and coating for 8h at 8 ℃ to obtain a coated microporous plate;
(2) Washing the plate for the first time: adding PBS buffer solution C (containing 0.3% Tween 20) with pH of 7.5 into the coated microplate at an addition amount of 250 μ L per well for washing for 1 time, and blotting the residual liquid in the microplate after washing to obtain a washed microplate;
(3) And (3) sealing: adding Tween20 with volume concentration of 0.3%, skimmed milk powder with mass concentration of 2% and sucrose with mass concentration of 10% into the washed microplate at an addition amount of 250 μ L per well, respectively, and sealing at 37 deg.C for 0.5h to obtain sealed microplate;
(4) Diluting: washing the red blood cells to be classified for 5 times by using normal saline, and diluting the red blood cells to be classified to 1% by using Liss or normal saline to obtain red blood cell suspension to be detected;
(5) And (3) detection: respectively adding the suspension of the red blood cells to be detected into a washed pore plate according to the addition amount of 40 mu L of each pore, and centrifuging for 5min at the rotating speed of 360g to obtain a centrifuged microporous plate;
(6) Washing: adding 250 mu L of normal saline into each hole of the microporous plate for washing for 5 times, and sucking residual liquid by using absorbent paper after the last washing is finished;
(7) Color development: adding 100 mu L of peroxidase substrate TMB into each hole, and developing for 12 minutes in a dark place;
(8) And (4) terminating: 0.16M H per well 2 SO 4 The reaction was terminated by leaving 50. Mu.L of the resulting solution to stand for 9 minutes in the dark.
(9) And (3) detection: and (5) placing the microplate on an enzyme-labeling instrument to detect the OD value by using 450nm light waves.
(10) The OD was determined in 8 known different samples, see Table 1 for details.
TABLE 3
| Sample number and Rh blood type | OD value | Cutoff value | Results |
| 576(B+) | 3.707 | 0.2 | RhD positive |
| 577(B+) | 3.519 | 0.2 | Positive for RhD |
| 578(A+) | 3.664 | 0.2 | Positive for RhD |
| 579(A+) | 3.631 | 0.2 | Positive for RhD |
| 580(AB+) | 3.593 | 0.2 | Positive for RhD |
| 581(A+) | 3.512 | 0.2 | Positive for RhD |
| 582(B-) | 0.047 | 0.2 | RhD negative |
| 583(O-) | 0.056 | 0.2 | RhD negative |
Note: + is Rh positive; -represents Rh-negativity
In the table, the OD value detection result is compared with the Cutoff value =0.2, and the RhD blood type negative and positive judgment results are consistent with the known results of the sample.
Claims (10)
1. A method for quantitatively detecting RhD antigen using peroxidase endogenous to erythrocytes, comprising: adding the red blood cells to be detected into a container coated with an anti-D antibody for centrifugation, washing with normal saline after the centrifugation is finished, removing residual liquid, adding a peroxidase substrate, developing by using endogenous peroxidase of the red blood cells, adding an acidic stop solution to terminate the reaction, placing the microporous plate into an enzyme-labeling instrument, and detecting an absorbance value, namely an OD value, under a proper light wave;
if the detected OD values of the red blood cells to be detected in the container coated with the anti-D antibody are all smaller than the Cutoff value, the RhD blood type of the red blood cells to be detected is negative;
if the detected OD values of the red blood cells to be detected in the container coated with the anti-D antibody are all larger than the Cutoff value, the RhD blood type of the red blood cells to be detected is positive.
2. The method for quantitatively detecting RhD antigen using endogenous peroxidase of red blood cells according to claim 1, wherein the centrifugation is performed at a rotation speed of 90 to 360g for 1 to 30min; washing the centrifuged microplate 3-6 times with physiological saline; adding peroxidase substrate into the washed container of the microporous plate, and developing for 3-20 min.
3. The method for quantitatively detecting RhD antigen using peroxidase endogenous to erythrocytes according to claim 1, wherein the Cutoff value =0.2.
4. The method for quantitatively detecting RhD antigen using erythrocytic endogenous peroxidase according to claim 1, further comprising a dilution step before the typing step; the dilution step comprises the following steps: diluting the red blood cells to be classified to 0.1-1% by volume concentration; and diluting the red blood cells to be detected by using Liss or normal saline.
5. The system for detecting RhD antigen by the method for quantitatively detecting RhD antigen using peroxidase endogenous to red blood cells according to any one of claims 1 to 4, wherein the system comprises a container coated with an anti-D antibody.
6. The system of claim 5, wherein the container is a microwell plate.
7. A method of making the system of claim 5, comprising the steps of:
coating: adding the anti-D antibody diluted by the buffer solution A into a container, coating for 8-16h at 2-8 ℃, and washing to obtain a coated container; the buffer solution A is a carbonate buffer solution;
and (3) sealing: sealing buffer solution B in the coated container at 37 ℃ for 0.5-3h, and washing to obtain a container coated with anti-D antibody; the buffer B is PBS buffer containing skimmed milk powder, sucrose and Tween 20.
8. The method of claim 7, wherein the concentration of the skim milk powder in the buffer B is 0.1-3% by mass, the concentration of the sucrose in the buffer is 1-10% by mass, and the concentration of the Tween20 in the buffer is 0.01-0.5% by volume.
9. The method according to claim 7 or 8, wherein in the coating step, the anti-D antibody is diluted 16-2048 times with a carbonate buffer A having a pH of 7.2-9.8 to obtain a diluted solution, and the diluted solution is added to a container;
the washing steps are as follows: adding a buffer solution C for washing, and repeatedly washing for 1-5 times; the buffer C is PBS buffer solution containing Tween20 and having pH value of 6.5-8.5; the volume concentration of the Tween20 in the buffer solution is 0.01-0.5%.
10. Use of the method for the quantitative determination of RhD antigens by means of the peroxidase endogenous to erythrocytes according to any one of claims 1 to 4, for the detection of RhD blood group antigens.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US4608246A (en) * | 1983-03-10 | 1986-08-26 | Immucor, Inc. | Testing for a blood group immunological reaction |
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