Reagent and kit for rapidly detecting blood type and application of reagent and kit

Abstract

The invention relates to a reagent and a kit for rapidly detecting blood types and application of the reagent and the kit, and belongs to the field of blood type detection. The reagent comprises a nano antibody of an anti-A antigen, a nano antibody of an anti-B antigen and a nano antibody of an anti-RhD antigen. The blood type detection reagent card prepared on the basis of the nano antibody detects the blood type in a sample to be detected by using blood type agglutination reaction, the detection result can be observed only in 30 seconds, and the blood type detection reagent card has the advantages of high sensitivity, low cost and storage resistance.

Description

Technical Field

[0001] The present invention belongs to the field of blood type detection, and particularly relates to a reagent and a kit for rapid blood type detection and their uses. Background of the Invention

[0003] Red blood cell blood types are classified according to the antigens on the surface of red blood cell membranes in the blood. According to different antigen types, more than 20 common classification systems can be divided, including the ABO blood type system and the Rh blood type system. In the ABO blood type system, there are A antigens (type A), B antigens (type B), both (type AB), or neither (type O) on the surface of red blood cell membranes; moreover, there are anti-B antibodies in type A blood serum, anti-A antibodies in type B blood serum, no anti-A and anti-B antibodies in type AB blood serum, and anti-A and anti-B antibodies in type O blood serum. In blood transfusion and organ transplantation, the matching of ABO blood types is very important to avoid serious immune reactions. In the Rh blood type system, it mainly includes antigens such as D, C, c, E, e, etc., among which the D antigen is the most important, determining Rh positive (Rh+) and Rh negative (Rh-).

[0004] At present, blood type identification is mainly divided into two categories: the agglutination method and the gene method. The agglutination method is a traditional blood type identification method, mainly based on the principle of antigen-antibody reaction, and determines the blood type by observing whether agglutination reaction occurs after red blood cells contact with the corresponding antibodies, including the paper strip method, the test tube method, the microcolumn method, etc. Its advantages are simple operation, rapidity, low cost, and the ability to obtain results in a short time without complex equipment, suitable for rapid screening and routine blood type identification, with relatively low costs of reagents and equipment, suitable for large-scale applications. However, its disadvantage is that the detection effect is restricted by antibodies. For some weak antigens or low-titer antibodies, the agglutination reaction may not be obvious or accurate. If not properly stored, the antibodies may also become ineffective, resulting in relatively large errors. The gene method is a blood type identification method based on molecular biology technology, which determines the blood type by detecting the polymorphism of blood type-related genes, such as PCR technology, gene chip technology, sequencing technology, etc. Due to its advantages of high sensitivity and high specificity, and its disadvantages of complex operation, high cost, and long time consumption, the gene method is more suitable for the identification of complex rare blood types. Therefore, it is still necessary to develop a blood type detection tool with simple operation, rapid detection, and accurate detection results, such as a kit for detecting human blood type by migration chromatography.

[0005] The migration chromatography blood type detection card uses chromatography technology to react red blood cells in a blood sample with antibodies on the detection card. The detection card is usually coated with specific antibodies, such as anti-A, anti-B, and anti-D antibodies. When the corresponding antigen exists on the surface of red blood cells in the sample, it will react with the antibodies on the detection card to cause agglutination. The agglutinated red blood cells will be retained in a specific area of the detection card, showing a positive reaction. By observing the color change in different areas of the detection card or the deposition of red blood cells, the blood type of the sample can be determined. Due to its simple operation and easy result interpretation, the migration chromatography blood type detection card is also suitable for on-site rapid screening, such as preliminary blood type detection at blood donation points.

[0006] However, it is still a type of agglutination method, relying on antigen-antibody reactions. The detection effect is restricted by antibodies, and is also affected by factors such as the migration rate of the sample in the chromatography medium and the immobilization amount of antibodies in the chromatography medium, thereby affecting the accuracy of the detection results. Summary of the Invention

[0007] The object of the present invention is to solve the above problems in blood type detection and provide a rapid, convenient, and low-cost blood type detection reagent and detection kit. In the first aspect, the present invention provides a reagent for rapid blood type detection, the reagent comprising anti-A antibody, anti-B antibody, anti-D antibody, and anti-human red blood cell antibody, wherein the anti-A antibody is a nanobody NbA1 that specifically binds to red blood cell A antigen, the amino acid sequence of the nanobody NbA1 is SEQ ID NO.1, the anti-B antibody is a nanobody NbB1 that specifically binds to red blood cell B antigen, the amino acid sequence of the nanobody NbB1 is SEQ ID NO.2, the anti-D antibody is a nanobody NbD1 that specifically binds to red blood cell RhD antigen, and the amino acid sequence of the nanobody NbD1 is SEQ ID NO.3.

[0008] Further, the nanobody NbA1 is obtained by camelizing the VH sequence of anti-A monoclonal antibody mAb-A, and the amino acid sequence of mAb-A is as shown in SEQ ID NO.4.

[0009] Further, the nanobody NbB1 is obtained by camelizing the VH sequence of anti-B monoclonal antibody mAb-B, and the amino acid sequence of mAb-B is as shown in SEQ ID NO.5.

[0010] Further, the nanobody NbD1 is obtained by camelizing the VH sequence of anti-RhD monoclonal antibody mAb-D, and the amino acid sequence of mAb-D is as shown in SEQ ID NO.6.

[0011] In the second aspect, the present invention provides a nucleic acid encoding the nanobodies NbA1, NbB1, and NbD1 in the reagent for rapid blood type detection as described in the first aspect.

[0012] In a third aspect, the present invention provides a recombinant vector or recombinant cell containing the nucleic acid described in the second aspect.

[0013] In a fourth aspect, the present invention provides a method for preparing a reagent for rapid blood type detection, the method comprising: (1) preparing monoclonal antibodies against red blood cell A antigen, B antigen and RhD antigen; (2) obtaining the VH sequence information of the monoclonal antibodies; (3) camelizing the VH sequence by transplanting the CDRs of the monoclonal antibody VH sequence onto a common nanobody framework to prepare nanobodies; (4) recombinantly expressing and purifying the nanobodies, and diluting the nanobodies into an antibody preservation solution.

[0014] In a fifth aspect, the present invention provides a blood type detection kit, the detection kit comprising a blood type detection card, the blood type detection card comprising a plastic outer shell, a multi-layer porous membrane and a backing, the plastic outer shell comprising 1 central sample addition hole and 4 detection windows, the 4 detection windows being detection window A, detection window B, detection window D and detection window QC respectively, the multi-layer porous membrane being a nitrocellulose membrane, and anti-A antibody NbA1, anti-B antibody NbB1, anti-D antibody NbD1 and anti-human red blood cell antibody being immobilized at positions corresponding to the detection windows respectively, and the backing being attached under the multi-layer porous membrane.

[0015] Further, the detection kit may further comprise a blank detection card, anti-A antibody, anti-B antibody, anti-D antibody and anti-human red blood cell antibody reagents and an antibody preservation solution, and the blank detection card has the same structure as the blood type detection card.

[0016] In a sixth aspect, the present invention provides a use of the reagent in blood type detection, which is not for diagnostic purposes. When in use, a blood sample is dropped into the central sample addition hole, the climbing speed of the nitrocellulose membrane is 65 - 115 s / 40 mm, and after 30 seconds, an agglutination reaction can be observed at the detection window, and the blood type of the sample is judged according to the result.

[0017] Further, if the result measured by the blood type detection card is blurred or in doubt, the blank detection card can be used to measure again. The tester drops 2 μL of the corresponding antibody onto the corresponding detection window, dries it at 37 degrees or dries it naturally, then adds the sample to be tested into the central sample addition hole again, and after 30 seconds, an agglutination reaction can be observed at the detection window, and the blood type of the sample is judged according to the result.

[0018] Advantageous effects: The blood type detection reagent of the present invention includes nanobodies NbA1, NbB1 and NbD1 that can specifically recognize red blood cell A antigen, B antigen and RhD antigen. The nanobodies have high affinity for the corresponding antigens and strong specific recognition ability, and their molecular sizes are It matches the CN95 pore phase, has a large immobilization amount on the nitrocellulose membrane, so the antibody titer is higher. Compared with the gold standard gel cassette method, the detection accuracy of the ultra-rapid blood type detection card can reach 100%, and it can be maintained for 24 months at room temperature. The detection time only takes 30 seconds, which is significantly lower than the 10 to 30 minutes of clinical detection. Brief Description of the Drawings

[0019] Figure 1 It is the blood type agglutination reaction of the nanobody prepared in Example 2 with various blood samples;

[0020] Figure 2 It is a schematic diagram of the blood type detection reagent card in Example 3. Detailed Embodiments

[0021] Hereinafter, examples are given to illustrate the specific embodiments of the present invention. However, the embodiments of the present invention are not limited by these examples, and any selection and change can be made within the scope not affecting the technical effects to be achieved by the present invention. The technical terms and abbreviations used in the present invention have the conventional meanings known to those skilled in the art; unless otherwise specified, all materials in the following examples are obtained through commercial channels.

[0022] Example 1 Preparation of anti-A antibody, anti-B antibody, and anti-RhD antibody.

[0023] (I) Obtaining the VH sequence of monoclonal antibody

[0024] The preparation of monoclonal antibodies against red blood cell A antigen, B antigen, and RhD antigen was entrusted to Nanjing Genscript Biotech Co., Ltd., and the delivery result was monoclonal antibodies stored in cell culture medium.

[0025] The screening process can be briefly described as follows: Mice were immunized with red blood cells with A antigen on the surface, red blood cells with B antigen on the surface, and red blood cells with RhD antigen on the surface respectively. After several weeks of immunization cycle, booster immunization was carried out to increase the number of B lymphocytes producing anti-A antigen antibodies in the mice. The spleen cells of these immunized mice were collected, and these cells contained immunized B lymphocytes. These spleen cells were fused with myeloma cells to form hybridoma cells that could proliferate infinitely, and polyethylene glycol (PEG) was used as a fusion agent to promote the fusion of hybridoma cells. The fused cells were cultured in HAT selection medium to screen out successfully fused and viable hybridoma cells.

[0026] In order to ensure the acquisition of monoclonal antibodies with strong specificity and high yield, the selected hybridoma cells need to be cloned and processed for monoclonality by limiting dilution method: count the cells to determine the cell concentration, dilute it to a concentration of less than 1 cell per well, and continue to culture in a 96-well plate. During the 7-14-day culture process, the wells containing single cells will gradually form cell clusters, while the wells without cells remain blank. The wells containing cell clusters are monoclonal cells, and the culture medium contains monoclonal antibodies. Take 10μL of the culture supernatant to perform a blood agglutination test to test the antibody characteristics. The monoclonal antibody that can specifically produce an agglutination reaction is the desired monoclonal antibody.

[0027] The monoclonal cell wells with the fastest monoclonal antibody in each group of blood coagulation were selected, and the culture was continued to be expanded, and the culture supernatant was taken for monoclonal antibody sequencing. The sequencing of monoclonal antibodies was entrusted to the antibody discovery platform of Shanghai Kuaisuo Biotechnology Co., Ltd., and the VH fragment was intercepted to obtain the anti-A monoclonal antibody VH sequence mAb-A (SEQ ID NO.4), the anti-B monoclonal antibody VH sequence mAb-B (SEQ ID NO.5), and the anti-RhD monoclonal antibody VH sequence mAb-D (SEQ ID NO.6).

[0028] (II) Preparation of Nanobodies

[0029] The VH fragments of conventional antibodies and nanobody VHH have a homology of more than 80%, and have the same structure, both consisting of four framework regions (FR) and three complementary determining regions (CDR). Because the VH of conventional antibodies needs to be combined with VL, the structure of the VH region alone is unstable, so we prepare nanobodies by camelizing the mouse VH fragment to obtain a stable VHH structure to form nanobodies.

[0030] Compared with conventional antibodies, four hydrophobic amino acids in FR2 of nanobodies are replaced by hydrophilic amino acids: F37V, E44G, R45L and G47W, which make it have the advantages of strong hydrophilicity and high solubility. At the same time, the structural characteristics of nanobodies also include 1 to 2 disulfide bonds, one of which is a conservative disulfide bond H22-H92. Nanobodies from camel and llama tribes also have another disulfide bond at H33-CDR3 and H50-CDR3, respectively. The inter-ring disulfide bond can stabilize CDR3, resulting in stronger affinity activity, and increase the stability and stress resistance of nanobodies.

[0031] Based on the above two points, the modification methods of mAb-A (SEQ ID NO.4), mAb-B (SEQ ID NO.5) and mAb-D (SEQ ID NO.6) in the present invention are as follows: transplant their CDR regions onto the common nanobody framework (Dumoulin M, et.al. Single-domain antibody fragments with high conformational stability. Protein Sci. 2002 Mar;11(3):500-15.), and introduce 2 disulfide bonds. The sequences of the modified nanobodies are shown in Table 1. Among them, NbA1 corresponds to mAb-A, NbB1 corresponds to mAb-B, and NbD1 corresponds to mAb-D.

[0032] Table 1 Sequences of camelized nanobodies (VHH)

[0033]

[0034] (III) Preparation of nanobodies

[0035] The preparation process is as follows:

[0036] Outsource the total synthesis of the recombinant protein sequence, ligate the constructed gene sequence to the pET23a vector, and transfer it into Escherichia coli.

[0037] Culture of recombinant bacteria and expression of recombinant protein:

[0038] The basal medium for the recombinant protein is TB medium. Inoculate according to an inoculum size of 5%, culture at 37°C for 3 - 5 h, add the inducer isopropyl β-D-thiogalactoside (IPTG) (final concentration 0.25 mM, the same below) for overnight induction; after the induction is completed, centrifuge at 4000 rpm for 20 min to obtain the bacterial cells containing the recombinant protein.

[0039] Purification of recombinant protein: Add lysis buffer (10 mM imidazole, 500 mM NaCl, pH 7.4, 0.02 M PB) to the obtained bacterial cells at a ratio of 1:10, and use a high-pressure homogenizer at 700 bar to disrupt the cells; centrifuge at 4°C and 10000 rpm for 20 min, and take the supernatant; filter the supernatant through a 0.45 μm filter, and then separate and purify the recombinant protein through an affinity chromatography column (GE Healthcare, US), where the packing material of the affinity chromatography column is Ni Sepharose High Performance. The purified recombinant protein is obtained.

[0040] Example 2 Blood group agglutination reaction

[0041] Dilute the nanobody in the antibody preservation solution, which is DMEM medium containing 15 mM sodium azide, and adjust the working concentration of the nanobody to 0.1 mg / mL.

[0042] Take 3 μL of nanobody (NbA1, NbB1, and NbD1) solutions and coat them on the bottom of a 96-well plate. Coat NbA1 on L1, NbB1 on L2, coat BSA on L3 and L4 as a control, and coat NbD1 on L5. Dry at 37 °C or room temperature. Subsequently, add 50 μL of blood sample solution (20 μL of blood dissolved in 30 μL of PBS), and observe the red blood cell agglutination result after 30 s. As Figure 1 shown, the blood sample added to R1 is known type O blood. It can be seen that no red blood cell agglutination reaction occurred in L1R1, L2R1, and L3R1; the blood sample added to R2 is known type AB blood. It can be seen that red blood cell agglutination reactions occurred in L1R2 and L2R2, and no red blood cell agglutination reaction occurred in L3R2; the blood sample added to R3 is known type A blood. It can be seen that a red blood cell agglutination reaction occurred in L1R3, and no red blood cell agglutination reactions occurred in L2R3 and L3R3; the blood sample added to R4 is known type B blood. It can be seen that no red blood cell agglutination reactions occurred in L1R4 and L3R4, and a red blood cell agglutination reaction occurred in L2R4; and agglutination reactions occurred in R1 - R5 in L5, indicating that they are all Rh positive blood types. An unknown blood sample was added to R5, and the result was the same as that of R3. The test result can be determined as type A Rh positive.

[0043] Example 3 Preparation of Blood Group Detection Reagent Card

[0044] Fix the antibodies on the nitrocellulose membrane at the positions as Figure 2 shown.

[0045] The nitrocellulose membrane used in the present invention is CN95 membrane. The total thickness of the membrane is 240 - 270 μm, with a 100 - μm - thick transparent polyester backing. The membrane pore size is about 15 μm. The membrane surface is anionic, and it can adsorb 20 μg of protein (such as IgG) per square centimeter. Its capillary climbing speed is 65 - 115 s / 40 mm, which is an ideal choice for lateral flow rapid diagnosis.

[0046] Fix the antibodies on the nitrocellulose membrane at the positions as Figure 2Fix it at the indicated position on the nitrocellulose membrane (light blue). Specifically, dilute the nanobody to 0.1 mg / mL, and evenly coat 3 μL at the square position. Detection window A corresponds to the nanobody NbA1 against antigen A, detection window B corresponds to the nanobody NbB1 against antigen B, detection window D corresponds to the nanobody NbD1 against antigen RhD, and detection window QC corresponds to the commercial anti-human red blood cell antibody (CAT: FB1031). The reagent concentration is 1.0 mg / mL and needs to be diluted to 0.1 mg / mL, and evenly coat 3 μL at the corresponding position. Wait for the antibody to dry naturally or dry it at 37°C.

[0047] Note that the preferred method is to coat NbA1 and NbB1 on the opposite side, and coat NbD1 and the commercial anti-human red blood cell antibody on the opposite side. The red center position is the blood sample loading hole.

[0048] Sampling: After dropping the blood sample or red blood cell suspension (5 - 10 μL) onto the nitrocellulose membrane, it will be quickly adsorbed by the membrane and undergo capillary crawling, reacting with the antibodies coated on the membrane.

[0049] Example 4 Preparation of Blank Detection Reagent Card and Detection Reagent

[0050] As another embodiment of the present invention, a method of using a blank detection reagent card and a detection reagent in combination can also be selected for blood type detection.

[0051] Due to Due to the pore structure and surface characteristics of the CN95 membrane, the nanobody and the antibody can be coated without chemical coupling, but can be directly dropped and dried by the direct coating method. Therefore, the experimenter can complete the antibody coating work by himself. Directly drop 2 μL of the corresponding detection reagent into the detection window. After the antibody dries naturally or is dried at 37°C, drop the blood sample or red blood cell suspension (5 - 10 μL) from the blood sample loading hole, and observe the result after 30 s.

[0052] Detection reagent A: Dilute the NbA1 nanobody into the antibody preservation solution. The antibody preservation solution is DMEM medium containing 15 mM sodium azide, and the antibody concentration is 0.1 mg / mL.

[0053] Detection reagent B: Dilute the NbB1 nanobody into the antibody preservation solution. The antibody preservation solution is DMEM medium containing 15 mM sodium azide, and the antibody concentration is 0.1 mg / mL.

[0054] Detection reagent D: Dilute the NbD1 nanobody into the antibody preservation solution. The antibody preservation solution is DMEM medium containing 15 mM sodium azide, and the antibody concentration is 0.1 mg / mL.

[0055] In summary, the detection reagent prepared by the present invention is a nanobody composition, which has strong specificity, high sensitivity, stable structure and low cost; the blood group detection reagent and blood group detection kit prepared with the nanobody of the present invention also have the advantages of high sensitivity, low cost and good storage stability. The blood group detection method based on the above kit has accurate and reliable results and high sensitivity.

[0056] Obviously, those skilled in the art can make various changes and modifications to this application without departing from the spirit and scope of this application. Thus, if these modifications and variations of this application fall within the scope of the claims of this application and their equivalent technologies, this application is also intended to include these modifications and variations.

Claims

1. A reagent for rapid blood type detection, characterized in that, The reagents include: a nanobody NbA1 that specifically binds to red blood cell A antigen, with its amino acid sequence being SEQ ID NO.1; a nanobody NbB1 that specifically binds to red blood cell B antigen, with its amino acid sequence being SEQ ID NO.2; and a nanobody NbD1 that specifically binds to red blood cell RhD antigen, with its amino acid sequence being SEQ ID NO.

3.

2. The reagent for rapid blood type detection according to claim 1, characterized in that, The nanobody NbA1 is obtained by camelizing the VH sequence mAb-A of an anti-A monoclonal antibody, and the amino acid sequence of the mAb-A is as shown in SEQ ID NO.

4.

3. The reagent for rapid blood type detection according to claim 1, wherein, The nanobody NbB1 is obtained by camelizing the VH sequence mAb-B of an anti-B monoclonal antibody, and the amino acid sequence of the mAb-B is as shown in SEQ ID NO.

5.

4. The reagent for rapid blood type detection according to claim 1, wherein The nanobody NbD1 is obtained by camelizing the VH sequence mAb-D of an anti-RhD monoclonal antibody, and the amino acid sequence of the mAb-D is as shown in SEQ ID NO.

6.

5. A nucleic acid encoding the nanobodies NbA1, NbB1, and NbD1 in the reagent for rapid blood type detection as claimed in claim 1.

6. A recombinant vector or recombinant cell containing the nucleic acid as claimed in claim 5.

7. The preparation method of the reagent for rapid blood type detection according to claim 1, wherein, The method includes: (1) preparing monoclonal antibodies against red blood cell A antigen, B antigen, and RhD antigen; (2) obtaining the VH sequence information of the monoclonal antibodies; (3) camelizing the VH sequence by transplanting the CDR of the VH sequence of the monoclonal antibody onto a common nanobody framework to prepare nanobodies; and (4) recombinantly expressing and purifying the nanobodies, and diluting the nanobodies into an antibody preservation solution.

8. A blood type detection kit, characterized in that, The kit consists of a blood type detection reagent card, and the detection reagent card includes a plastic outer shell, a multi-layer pore membrane, and a backing. The plastic outer shell includes 1 central sample addition hole and 4 detection windows, namely detection window A, detection window B, detection window D, and detection window QC. Among them, detection window A corresponds to the nanobody NbA1 against A antigen, detection window B corresponds to the nanobody NbB1 against B antigen, detection window D corresponds to the nanobody NbD1 against RhD antigen, and detection window QC corresponds to a commercial anti-human red blood cell antibody. The multi-layer pore membrane is a nitrocellulose membrane, and the backing is attached under the multi-layer pore membrane.

9. The blood type detection kit according to claim 8, wherein, The kit further includes a blank detection card, on which no antibody is pre-coated, and the kit contains the detection reagents anti-A antibody NbA1, anti-B antibody NbB1, and anti-D antibody NbD1.

10. Use of the blood group detection reagent according to claim 1 in blood group detection, which is not for the purpose of diagnosis, characterized in that, During use, a blood sample is dropped into the central sample addition hole, the climbing speed of the nitrocellulose membrane is 65 - 115 s / 40 mm. After 30 seconds, an agglutination reaction can be observed at the detection window, and the blood type of the sample is judged according to the result.

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