CN112067827A - Antibody diluent and blood type test card comprising same - Google Patents
Antibody diluent and blood type test card comprising same Download PDFInfo
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Abstract
Provided herein are antibody dilutions for blood group test cards comprising: 5-15g/L of sodium chloride, 0.1-1g/L of potassium chloride, 0.5-1.5g/L of disodium hydrogen phosphate, 0.1-1g/L of monopotassium phosphate, 10-30g/L of bovine serum albumin, 5-15g/L of cane sugar, 0.1-1g/L of beta-cyclodextrin and 0.1-1g/L of sodium azide. The blood type test card prepared by the antibody diluent can be stored at normal temperature for 18 months, and is beneficial to storage and transportation of the blood type test card.
Description
Technical Field
The application relates to a blood type detection card, in particular to an ABO blood type positive typing and Rh (D) blood type detection card which can be stored at normal temperature.
Background
There are many blood group antigen systems on the human erythrocyte membrane, of which the ABO blood group antigen system is the most important and the Rh blood group antigen system is the next. Correct identification of ABO blood groups is a guarantee of safe transfusion, and if ABO blood groups are incompatible, severe immediate hemolytic transfusion reaction symptoms are likely to occur during transfusion. The most common phenotypes for ABO blood groups are four: A. b, AB and O-shaped. The ABO blood typing test comprises positive typing and negative typing, and the detection card provided by the invention can be used for positive typing detection of ABO blood types.
The D antigen is the most important antigen in the Rh blood group antigen system. The existence of D antigen on human erythrocyte membrane is Rh positive, and the absence of D antigen is Rh negative. If Rh-negative patients are transfused with blood containing the corresponding antigen (e.g., D antigen), antibodies against the antigen are produced and cause severe immune hemolytic transfusion reactions when transfused again. For pregnant women, due to the strong ability of Rh system antibodies of IgG nature to pass through the placenta, the erythrocytes of the fetus with the corresponding antigens can be destroyed, causing severe hemolytic disease of the newborn. Therefore, accurate Rh typing is of great clinical significance for reducing immune hemolytic transfusion reaction and diagnosing, preventing and treating hemolytic disease of newborn.
At present, some manufacturers at home and abroad have produced human ABO blood type positive typing and Rh (D) blood type detection cards, the storage conditions are that the cards are generally stored for 12 months at the temperature of 2-8 ℃, but the cards are still inconvenient in the specific use process because the cards are kept at the temperature of 2-8 ℃ during transportation and storage.
Disclosure of Invention
In one aspect, provided herein is an antibody diluent for a blood typing card comprising: 5-15g/L of sodium chloride, 0.1-1g/L of potassium chloride, 0.5-1.5g/L of disodium hydrogen phosphate, 0.1-1g/L of monopotassium phosphate, 10-30g/L of bovine serum albumin, 5-15g/L of cane sugar, 0.1-1g/L of beta-cyclodextrin and 0.1-1g/L of sodium azide.
In some embodiments, the antibody dilution comprises:
5g/L of sodium chloride, 0.1g/L of potassium chloride, 0.5g/L of disodium hydrogen phosphate, 0.1g/L of monopotassium phosphate, 10g/L of bovine serum albumin, 5g/L of cane sugar, 0.1g/L of beta-cyclodextrin and 0.1g/L of sodium azide;
8.2g/L of sodium chloride, 0.22g/L of potassium chloride, 0.86g/L of disodium hydrogen phosphate, 0.24g/L of monopotassium phosphate, 15g/L of bovine serum albumin, 8g/L of sucrose, 0.2g/L of beta-cyclodextrin and 0.2g/L of sodium azide;
10.1g/L of sodium chloride, 0.54g/L of potassium chloride, 0.98g/L of disodium hydrogen phosphate, 0.52g/L of monopotassium phosphate, 20g/L of bovine serum albumin, 10g/L of cane sugar, 0.5g/L of beta-cyclodextrin and 0.5g/L of sodium azide;
12.2g/L of sodium chloride, 0.87g/L of potassium chloride, 1.25g/L of disodium hydrogen phosphate, 0.88g/L of monopotassium phosphate, 25g/L of bovine serum albumin, 12g/L of cane sugar, 0.8g/L of beta-cyclodextrin and 0.8g/L of sodium azide; or
15g/L of sodium chloride, 1g/L of potassium chloride, 1.5g/L of disodium hydrogen phosphate, 1g/L of monopotassium phosphate, 30g/L of bovine serum albumin, 15g/L of sucrose, 1g/L of beta-cyclodextrin and 1g/L of sodium azide.
In some embodiments, the pH of the antibody dilution is 7.2-7.4.
In another aspect, provided herein is an antibody working fluid prepared using the above antibody dilutions.
In another aspect, provided herein is a blood typing card comprising the above antibody diluent and/or the above antibody working solution.
In some embodiments, the blood typing card is an ABO blood group orthotyping and rh (d) blood group typing card.
In some embodiments, the blood type test card comprises at least 3 microcolumns, which are filled with an antibody working solution against the a antigen, an antibody working solution against the B antigen, and an antibody working solution against the D antigen, respectively.
In some embodiments, the blood type test card comprises 6 micro-columns, wherein 3 micro-columns are respectively filled with an antibody working solution for A antigen, an antibody working solution for B antigen and an antibody working solution for D antigen, and the other 3 micro-columns are filled with the above antibody diluent as a control.
In some embodiments, the microcolumn is filled with glass beads.
In some embodiments, the blood type test card itself or its packaging box is marked with a cryopreservation pattern.
The antibody diluent provided by the invention is suitable for diluting or storing various blood type related antibodies, and the preparation process of the blood type detection card is simplified. The glass bead blood group test card provided by the invention can be stored at normal temperature, and the storage time can be up to 18 months, thereby being beneficial to the storage and transportation of the glass bead blood group test card.
Drawings
FIG. 1 shows the interpretation criteria for the results of blood type tests.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
The "antibody diluent" herein refers to a solution used for the preparation of an antibody working solution, i.e., a solution used for diluting a blood group-related antibody (e.g., anti-a antibody, anti-B antibody, and anti-D antibody) concentrate for use in a blood group test card, and may also be referred to as an "antibody stock solution". The antibody in the antibody dilution solution can retain its binding activity to the corresponding antigen.
Herein, "antibody working solution" means a solution containing an appropriate concentration of antibody capable of undergoing an agglutination reaction with blood cells carrying the corresponding antigen when it is placed in a blood group test card microcolumn.
When referring to a blood type test card, the term "microcolumn" is used to refer to a generally cylindrical recess formed in the test card to provide a receiving space for reagents used in performing blood type tests. These micro-pillars may be formed integrally with the test card. A blood typing card may typically have a plurality of micro-posts, such as 4, 6, or more micro-posts disposed thereon.
As used herein, "ambient temperature" refers to ordinary laboratory temperatures, e.g., about 20-25 deg.C, 22-25 deg.C, etc.
The term "storage at room temperature" means "storage at room temperature" or the like, for example, storage at room temperature, storage temperature of not higher than 25 ℃, storage temperature of 18-20 ℃, 18-25 ℃ or 20-25 ℃ or the like, and is used to indicate that the blood type test card is not necessarily stored at low temperature (e.g., 2-8 ℃).
Some embodiments of the invention are based, at least in part, on the unexpected discovery that: when the antibody diluent containing certain components is adopted, the blood type card can be stored for a long time (18 months) at normal temperature.
In some embodiments, the antibody diluent comprises sodium chloride 5-15g/L, potassium chloride 0.1-1g/L, disodium hydrogen phosphate 0.5-1.5g/L, potassium dihydrogen phosphate 0.1-1g/L, bovine serum albumin 10-30g/L, sucrose 5-15g/L, beta-cyclodextrin 0.1-1g/L, and sodium azide 0.1-1 g/L. Preferably, the pH of the antibody dilution is 7.2-7.4.
And diluting the antibodies aiming at various blood group antigens by using the antibody diluent to obtain an antibody working solution. Adding the antibody working solution and the glass beads into a microcolumn of the blood type card to prepare the blood type detection card.
The glass bead blood type detection card provided by the invention organically combines the blood type serology technology and the glass bead molecular sieve technology by utilizing the principles of the molecular sieve technology, the centrifugal technology and the specific immunoreaction technology, and can sensitively detect the possible weak blood type antigen-antibody reaction.
The invention is further illustrated by the following specific examples.
EXAMPLE 1 preparation of antibody dilutions and antibody working solutions
Respectively preparing 5 kinds of antibody diluents, wherein the serial numbers are respectively 1# to 5#, and the antibody diluents comprise the following components in concentration (the pH value is adjusted to 7.2-7.4):
1 #: 5g/L of sodium chloride, 0.1g/L of potassium chloride, 0.5g/L of disodium hydrogen phosphate, 0.1g/L of monopotassium phosphate, 10g/L of bovine serum albumin, 5g/L of cane sugar, 0.1g/L of beta-cyclodextrin and 0.1g/L of sodium azide;
2 #: 8.2g/L of sodium chloride, 0.22g/L of potassium chloride, 0.86g/L of disodium hydrogen phosphate, 0.24g/L of monopotassium phosphate, 15g/L of bovine serum albumin, 8g/L of sucrose, 0.2g/L of beta-cyclodextrin and 0.2g/L of sodium azide;
3 #: 10.1g/L of sodium chloride, 0.54g/L of potassium chloride, 0.98g/L of disodium hydrogen phosphate, 0.52g/L of monopotassium phosphate, 20g/L of bovine serum albumin, 10g/L of cane sugar, 0.5g/L of beta-cyclodextrin and 0.5g/L of sodium azide;
4 #: 12.2g/L of sodium chloride, 0.87g/L of potassium chloride, 1.25g/L of disodium hydrogen phosphate, 0.88g/L of monopotassium phosphate, 25g/L of bovine serum albumin, 12g/L of cane sugar, 0.8g/L of beta-cyclodextrin and 0.8g/L of sodium azide;
5 #: 15g/L of sodium chloride, 1g/L of potassium chloride, 1.5g/L of disodium hydrogen phosphate, 1g/L of monopotassium phosphate, 30g/L of bovine serum albumin, 15g/L of sucrose, 1g/L of beta-cyclodextrin and 1g/L of sodium azide.
Each antibody dilution was also used as a control reagent without antibody in the blood group card.
Preparing each antibody working solution:
mixing an anti-A monoclonal antibody concentrated solution (anti-A mouse monoclonal (IgM) antibody (clone BIRMA-1) from Merck Millipore Corporation, product number JH-1L-BK) with each antibody diluent respectively to ensure that the titer of the obtained anti-A monoclonal antibody working solution (hereinafter referred to as anti-A working solution) is more than or equal to 128;
mixing an anti-B monoclonal antibody concentrated solution (anti-B mouse monoclonal (IgM) antibody (clone LB-2) from Merck Millipore Corporation, item number JM-1L-BK) with each antibody diluent respectively to ensure that the titer of the obtained anti-B monoclonal antibody working solution (hereinafter referred to as anti-B working solution) is more than or equal to 128;
an anti-D monoclonal antibody concentrated solution (anti-Rh (D) human monoclonal (IgM) antibody (clone RUM-1) from Merck Millipore Corporation, product number JV-1L-BK) is taken and mixed with each antibody diluent respectively, so that the titer of the obtained anti-D monoclonal antibody working solution (hereinafter referred to as anti-D working solution) is more than or equal to 64.
EXAMPLE 2 blood type card filling
An empty blood type detection card (Hangzhou Jiacheng mold manufacturing Co., Ltd.) with 6 micro-columns is taken, anti-A working solution, anti-B working solution and anti-D working solution which are prepared by the 1# antibody diluent of the embodiment 1 are respectively filled into the 1 st, 2 nd and 3 rd micro-columns according to 23uL of each micro-column, and the 1# antibody diluent of the embodiment 1 is filled into the 4 th, 5 th and 6 th micro-columns to serve as a contrast reagent. Then, filling glass beads (particle size 70-90um, Wuxi Michi biomedical science and technology Co., Ltd.) into each microcolumn to make the total height of the solid-liquid mixed phase be 8-10mm, and the liquid phase be not less than 1mm higher than the solid phase, and finally sealing with aluminum foil. After the above steps are completed, an ABO blood group orthotyping card and an Rh (D) blood group blood type detection card (hereinafter referred to as "detection card" or "blood type detection card") capable of detecting the ABO blood group and the Rh (D) blood group are formed.
Similarly, a glass bead blood type test card including the 2# -5# antibody dilution of example 1 was prepared.
The prepared detection card is stored at the laboratory temperature of 18-25 ℃.
Example 3 blood typing card expiration date comparison test
The blood type test cards prepared in example 2 were used to test blood samples of type A, type B and type RhD (the samples used in each test were from Tianjin blood center), and the test was performed every 3 months, and the test card was compared with the currently prepared test card (the preparation method is the same as that described in example 2) to see if the positive agglutination intensity was changed.
The detection method comprises the following steps:
1. before use, the detection card is centrifuged for 1min at 200g in a card centrifuge, and the main purpose is to centrifuge off the liquid volatilized to the aluminum foil, so that a sample can be detected more accurately.
2. Centrifuging the blood samples of the A type, the B type and the RhD type for 5min by a centrifugal force of 900-.
TABLE 1 erythrocyte suspension preparation
3. Carefully tearing off the aluminum foil for sealing on the detection card, adding 10 uL of erythrocyte suspension of the A-type sample into the 1 st microcolumns and the 4 th microcolumns, adding 10 uL of erythrocyte suspension of the B-type sample into the 2 nd microcolumns and the 5 th microcolumns, and adding 10 uL of erythrocyte suspension of the D-type sample into the 3 rd microcolumns and the 6 th microcolumns.
4. The mixture was immediately centrifuged for 5min in a cartridge centrifuge (two-phase centrifugation was used, the first phase was about 55 g.times.2 min and the second phase was about 200 g.times.3 min).
5. The interpretation standard interpretation results shown in fig. 1 are recorded and compared with the currently prepared test card.
The results of the measurements are shown in table 2.
TABLE 2 shelf life test results for test cards
The results of the above tests can be summarized in Table 3 below.
TABLE 3 results summary
From the results, the blood type detection card prepared by the 1# -5# antibody diluent can be stored at normal temperature for 18 months, the detection result is still normal at 18 months, the positive result has a weakening trend after exceeding 18 months, and the positive result is negative after 27 months, which indicates that the glass bead blood type detection card provided by the invention can be stored at normal temperature for as long as 18 months. Such a shelf life at ambient temperature has been sufficient to meet the requirements of production, sale, transport and laboratory storage of test cards.
The antibody dilutions provided herein are suitable for use with glass bead blood typing cards, but it is also contemplated that such antibody dilutions may also be used with gel-type (e.g., sephadex) blood typing cards. In addition, it is also contemplated that storage of the test cards provided herein at lower temperatures, such as 2-8 ℃, is also feasible, and that storage times can be further extended.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these changes and modifications are all within the scope of the present invention.
Claims (9)
1. An antibody diluent for a blood group test card comprising: 5-15g/L of sodium chloride, 0.1-1g/L of potassium chloride, 0.5-1.5g/L of disodium hydrogen phosphate, 0.1-1g/L of monopotassium phosphate, 10-30g/L of bovine serum albumin, 5-15g/L of cane sugar, 0.1-1g/L of beta-cyclodextrin and 0.1-1g/L of sodium azide.
2. The antibody dilution of claim 1, comprising:
5g/L of sodium chloride, 0.1g/L of potassium chloride, 0.5g/L of disodium hydrogen phosphate, 0.1g/L of monopotassium phosphate, 10g/L of bovine serum albumin, 5g/L of cane sugar, 0.1g/L of beta-cyclodextrin and 0.1g/L of sodium azide;
8.2g/L of sodium chloride, 0.22g/L of potassium chloride, 0.86g/L of disodium hydrogen phosphate, 0.24g/L of monopotassium phosphate, 15g/L of bovine serum albumin, 8g/L of sucrose, 0.2g/L of beta-cyclodextrin and 0.2g/L of sodium azide;
10.1g/L of sodium chloride, 0.54g/L of potassium chloride, 0.98g/L of disodium hydrogen phosphate, 0.52g/L of monopotassium phosphate, 20g/L of bovine serum albumin, 10g/L of cane sugar, 0.5g/L of beta-cyclodextrin and 0.5g/L of sodium azide;
12.2g/L of sodium chloride, 0.87g/L of potassium chloride, 1.25g/L of disodium hydrogen phosphate, 0.88g/L of monopotassium phosphate, 25g/L of bovine serum albumin, 12g/L of cane sugar, 0.8g/L of beta-cyclodextrin and 0.8g/L of sodium azide; or
15g/L of sodium chloride, 1g/L of potassium chloride, 1.5g/L of disodium hydrogen phosphate, 1g/L of monopotassium phosphate, 30g/L of bovine serum albumin, 15g/L of sucrose, 1g/L of beta-cyclodextrin and 1g/L of sodium azide.
3. The antibody dilution according to claim 1 or 2, having a pH of 7.2 to 7.4.
4. An antibody working solution prepared using the antibody dilution solution of any one of claims 1 to 3.
5. Blood typing card comprising an antibody dilution according to any one of claims 1 to 3 and/or an antibody working solution according to claim 4.
6. The blood typing card according to claim 5, which is an ABO blood group orthotyping and Rh (D) blood group typing card.
7. The blood type test card of claim 6, which comprises at least 3 microcolumns, respectively filled with an antibody working solution against the A antigen, an antibody working solution against the B antigen and an antibody working solution against the D antigen.
8. The blood type test card of claim 7, which comprises 6 micro-columns, wherein 3 micro-columns are filled with the working solution of antibody against A antigen, the working solution of antibody against B antigen and the working solution of antibody against D antigen, respectively, and the other 3 micro-columns are filled with the antibody diluent as a control.
9. The blood type test card of claim 7 or 8, wherein the microcolumns are filled with glass beads.
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| CN113238038A (en) * | 2021-05-11 | 2021-08-10 | 图凌(杭州)生物医药有限公司 | Antibody diluent for immunohistochemical detection and preparation method and application thereof |
| CN113252914A (en) * | 2021-06-23 | 2021-08-13 | 天津德祥生物技术有限公司 | Antibody diluent for Rh system parting detection card and detection card |
| CN115428783A (en) * | 2022-09-16 | 2022-12-06 | 天津科技大学 | Erythrocyte membrane fragment freeze-drying protective solution, use method and application |
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| CN113252914A (en) * | 2021-06-23 | 2021-08-13 | 天津德祥生物技术有限公司 | Antibody diluent for Rh system parting detection card and detection card |
| CN115428783A (en) * | 2022-09-16 | 2022-12-06 | 天津科技大学 | Erythrocyte membrane fragment freeze-drying protective solution, use method and application |
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Application publication date: 20201211 |