Reagent and kit for rapid detection of blood type and use thereof

Abstract

The present application relates to a kind of reagent and kit for rapid detection of blood group and purposes thereof, belong to blood group detection field.The reagent includes anti-A antigen nanobody, anti-B antigen nanobody and anti-RhD antigen nanobody.Based on the blood group detection reagent card prepared using the nanobody, blood group agglutination reaction is used to detect blood group in the sample to be measured, and the detection result can be observed in only 30s, and has the advantages of high sensitivity, low cost, storage resistance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of blood type detection, and particularly relates to a reagent and kit for rapidly detecting blood type and use thereof. BACKGROUND

[0002] Red blood cell blood type is classified according to the antigens on the surface of red blood cell membranes. According to the different types of antigens, 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, the surface of red blood cell membranes has A-type antigens (type A), B-type antigens (type B), both (type AB), or neither (type O); and the serum of type A blood has anti-B antibodies, the serum of type B blood has anti-A antibodies, the serum of type AB blood has no anti-A and anti-B antibodies, and the serum of type O blood has anti-A and anti-B antibodies. In blood transfusion and organ transplantation, the matching of the ABO blood type is very important to avoid serious immune reactions. In the Rh blood type system, the main antigens include D, C, c, E, e, etc., among which the D antigen is the most important and determines Rh positive (Rh+) and Rh negative (Rh-).

[0003] Currently, blood type identification mainly includes agglutination method and gene method. The agglutination method is a traditional blood type identification method, mainly based on the principle of antigen-antibody reaction, which determines the blood type by observing whether the red blood cells and the corresponding antibodies will cause agglutination after contact. The agglutination method includes paper method, test tube method, microcolumn method, etc. The advantages of the agglutination method are simple operation, rapidness, low cost, and no need for complex equipment to obtain results in a short time, which is suitable for rapid screening and routine blood type identification, and the cost of reagents and equipment is relatively low, which is suitable for large-scale application. However, the detection effect of the agglutination method is restricted by antibodies, and for some weak antigens or low titer antibodies, the agglutination reaction may not be obvious or accurate, and if not properly stored, the antibodies may also be ineffective, resulting in 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 high sensitivity and high specificity, the gene method is more suitable for the identification of complex and 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 reagent kit for detecting human blood type by migration chromatography.

[0004] The migration chromatography blood type detection card utilizes 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 corresponding antigens exist on the surface of red blood cells in the sample, agglutination reactions occur with the antibodies on the detection card. Agglutinated red blood cells are trapped in specific areas of the detection card, showing a positive reaction. By observing the color changes or red blood cell deposition in different areas of the detection card, the blood type of the sample can be determined. Due to its simple operation and easy-to-read results, the migration chromatography blood type detection card is also suitable for on-site rapid screening, such as preliminary blood type detection at blood donation sites.

[0005] However, it is still a kind of agglutination method, relying on antigen-antibody reactions, and the detection effect is affected by the antibody preparation, as well as factors such as the migration rate of the sample in the chromatography medium and the amount of antibody immobilized in the chromatography medium, thereby affecting the accuracy of the detection results. SUMMARY

[0006] The purpose of the present application is to solve the above problems in blood type detection, and to provide a rapid, convenient, and low-cost blood type detection reagent and kit. In a first aspect, the present application provides a reagent for rapid detection of blood type, which comprises anti-A antibody, anti-B antibody, anti-D antibody and anti-human red blood cell antibody, the anti-A antibody is a nanobody NbA1 which 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 which 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 which specifically binds to red blood cell RhD antigen, the amino acid sequence of the nanobody NbD1 is SEQ ID NO. 3.

[0007] Further, the nanobody NbA1 is camelized from the VH sequence of anti-A monoclonal antibody mAb-A, and the amino acid sequence of the mAb-A is shown as SEQ ID NO. 4.

[0008] Further, the nanobody NbB1 is camelized from the VH sequence of anti-B monoclonal antibody mAb-B, and the amino acid sequence of the mAb-B is shown as SEQ ID NO. 5.

[0009] Further, the nanobody NbD1 is camelized from the VH sequence of anti-RhD monoclonal antibody mAb-D, and the amino acid sequence of the mAb-D is shown as SEQ ID NO. 6.

[0010] In a second aspect, the present application provides a nucleic acid encoding the nanobodies NbA1, NbB1 and NbD1 in the reagent for rapid detection of blood type as described in the first aspect.

[0011] In a third aspect, the present application provides a recombinant vector or a recombinant cell containing the nucleic acid of the second aspect.

[0012] In a fourth aspect, the present application provides a preparation method of a reagent for rapid blood group detection, the method comprising: (1) preparing monoclonal antibodies of red blood cell A antigen, B antigen and RhD antigen; (2) obtaining VH sequence information of the monoclonal antibodies; (3) camelizing the VH sequence by grafting the CDR of the VH sequence of the monoclonal antibodies onto a universal nanobody skeleton to prepare a nanobody; and (4) recombinantly expressing and purifying the nanobody, and diluting the nanobody into an antibody storage solution.

[0013] In a fifth aspect, the present application provides a blood group detection kit, which comprises a blood group detection card, the blood group detection card comprising a plastic shell, a multi-layer porous membrane and a backing, the plastic shell comprising one central sample addition hole and four detection windows, the four 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 the positions corresponding to the detection windows being respectively immobilized with anti-A antibody NbA1, anti-B antibody NbB1, anti-D antibody NbD1 and anti-human red blood cell antibody, and the backing being attached to the multi-layer porous membrane.

[0014] Further, the detection kit can 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 storage solution, the blank detection card having the same structure as the blood group detection card.

[0015] In a sixth aspect, the present application provides a use of the reagent in blood group detection, which is not for diagnostic purposes, and in use, a blood sample is added dropwise to the central sample addition hole, the crawling speed of the nitrocellulose membrane is 65-115 s / 40 mm, and after 30 seconds, agglutination reaction can be observed in the detection window, and the blood group of the sample is determined according to the result.

[0016] Further, if the result measured by the blood group detection card is ambiguous or questionable, the blank detection card can be used for re-measurement. The tester adds 2 μL of the corresponding antibody to the corresponding detection window, and after drying at 37 degrees or natural drying, the sample to be measured is added to the central sample addition hole again, and after 30 seconds, agglutination reaction can be observed in the detection window, and the blood group of the sample is determined according to the result.

[0017] Beneficial effects: the blood group detection reagent of the present application comprises nanobodies NbA1, NbB1 and NbD1 which can specifically recognize red blood cell A antigen, B antigen and RhD antigen, the nanobodies have high affinity to the corresponding antigens and strong specific recognition ability, the molecular size of the nanobodies is close to that of the corresponding antigens, and the nanobodies have high sensitivity and specificity. CN95 has a large amount of immobilization on the nitrocellulose membrane, and therefore has a higher antibody titer. Compared with the gold standard gel cassette method, the detection accuracy of the ultra-fast blood type detection card can reach 100%, and can be maintained for 24 months at room temperature. The detection time is only 30 seconds, which is significantly lower than the 10 to 30 minutes of clinical detection. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the agglutination reaction of the nanobody prepared in Example 2 with various blood samples;

[0019] Figure 2 is a schematic diagram of the blood type detection reagent card in Example 3. DETAILED DESCRIPTION

[0020] Hereinafter, specific embodiments of the present application will be described by way of examples, but the embodiments of the present application are not limited to the following examples, and any selection and modification can be made within the scope that does not affect the technical effects to be achieved by the present application. The technical terms and abbreviations used in the present application have the conventional meanings known to those skilled in the art; except for special descriptions, all materials in the following examples are obtained from commercial channels.

[0021] Preparation of anti-A antibody, anti-B antibody, and anti-RhD antibody

[0022] (I) Obtaining of monoclonal antibody VH sequence

[0023] The preparation of monoclonal antibodies against red blood cell A antigen, B antigen, and RhD antigen was entrusted to Nanjing Kingsrui Biotechnology Co., Ltd., and the delivery result was a monoclonal antibody preserved in a cell culture solution.

[0024] The screening process can be briefly described as follows: 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 were used to immunize mice. After several weeks of immunization cycle, the number of B lymphocytes producing anti-A antigen antibodies in the mice was increased by means of booster immunization. The spleen cells of these immunized mice were collected, and these cells contained the immunized B lymphocytes. These spleen cells were fused with myeloma cells to form hybridoma cells capable of unlimited proliferation, 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 hybridoma cells that successfully fused and could survive.

[0025] In order to ensure that the specific strong, high yield of monoclonal antibodies, the need to screen hybridoma cells for cloning, and by limiting dilution method for monoclonal treatment: cell counting to determine the concentration of cells, and then diluted to less than 1 cell per hole concentration, placed in 96-well plates for further culture, 7-14 days of culture, the hole containing a single cell will gradually form a cell population, and the hole does not contain cells remain blank. The hole containing the cell population is a monoclonal cell, the culture medium containing monoclonal antibodies. Take 10 μL of culture supernatant for blood agglutination test to test the properties of the antibody, which can specifically produce agglutination reaction is the desired monoclonal antibody.

[0026] Select the fastest blood agglutination rate of each group of monoclonal antibody-containing monoclonal cell holes, continue to expand the culture, and take the culture supernatant for monoclonal antibody sequencing. The sequencing of the monoclonal antibody is entrusted to the antibody discovery platform of Shanghai Fast Sequence Biotechnology Co., Ltd. The VH fragment is 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).

[0027] (ii) Nanobody preparation

[0028] The VH fragment of the conventional antibody and the nanobody VHH have a homology of more than 80%, and the structures are the same, both of which are composed of four framework regions (FR) and three complementarity determining regions (CDR). Because the VH of the conventional antibody needs to be combined with VL, the structure of the single VH region is unstable, so we prepare the nanobody by camelizing the murine VH fragment to obtain a stable VHH structure to form the nanobody.

[0029] Compared with the conventional antibody, the FR2 of the nanobody has four hydrophobic amino acids replaced by hydrophilic amino acids: F37V, E44G, R45L and G47W, which have the advantages of strong hydrophilicity and high solubility. At the same time, the structural characteristics of the nanobody also contain 1-2 disulfide bonds, one of which is a conservative disulfide bond H22-H92, and the other one is present in H33-CDR3 and H50-CDR3 of the nanobody from the Camelidae and the Camelidae, respectively. The inter-loop disulfide bond can stabilize CDR3, thereby resulting in stronger affinity activity, and increasing the stability and resistance of the nanobody.

[0030] 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) are as follows: transplanting the CDR regions of the three antibodies to a universal nanobody skeleton (Dumoulin M, et. al. Single-domain antibody fragments with high conformational stability. Protein Sci. 2002 Mar; 11(3): 500-15.) and introducing two 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.

[0031] Table 1 Sequences of camelized and modified nanobodies (VHH)

[0032]

[0033] (III) Preparation of nanobodies

[0034] The preparation process is as follows:

[0035] The recombinant protein sequence is outsourced for full synthesis, and the constructed gene sequence is connected to the pET23a vector and then transformed into E. coli.

[0036] Recombinant bacteria culture and recombinant protein expression:

[0037] The basic culture medium of the recombinant protein is TB medium, which is inoculated at a 5% inoculation amount, cultured at 37°C for 3-5 h, and then induced overnight by adding inducer galactoside (IPTG) (final concentration 0.25 mM, same below); after induction, centrifugation is performed at 4000 rpm for 20 min to obtain the bacterial body containing the recombinant protein.

[0038] Recombinant protein purification: the obtained bacterial body is added with lysis solution (10 mM imidazole, 500 mM NaCl, pH 7.4, 0.02 M PB) at a ratio of 1:10, and cell disruption is performed using a 700 bar high-pressure homogenizer; centrifugation is performed at 4°C and 10000 rpm for 20 min, and the supernatant is taken; the supernatant is filtered through a 0.45 μm filter, and then subjected to separation and purification of the recombinant protein through an affinity chromatography column (GE Healthcare, US), wherein the filler of the affinity chromatography column is Ni Sepharose High Performance. The purified recombinant protein is obtained.

[0039] Example 2 Blood agglutination reaction

[0040] The nanobodies were diluted into antibody storage solution, which was DMEM medium containing 15 mM sodium azide, to adjust the working concentration of the nanobodies to 0.1 mg / mL.

[0041] 3 μL of the nanobody (NbA1, NbB1 and NbD1) solution was taken and coated on the bottom of a 96-well plate, NbA1 was coated on L1, NbB1 was coated on L2, BSA was coated on L3 and L4 as a control, and NbD1 was coated on L5. The plate was dried at 37°C or room temperature. Then, 50 μL of the blood sample solution (20 μL of blood was dissolved in 30 μL of PBS) was added, and the result of red blood cell agglutination was observed after 30 s. As shown in Figure 1 , the blood sample added by R1 was known O blood, and no red blood cell agglutination reaction occurred in L1R1, L2R1 and L3R1; the blood sample added by R2 was known AB blood, and red blood cell agglutination reaction occurred in L1R2 and L2R2, and no red blood cell agglutination reaction occurred in L3R2; the blood sample added by R3 was known A blood, and red blood cell agglutination reaction occurred in L1R3, and no red blood cell agglutination reaction occurred in L2R3 and L3R3; the blood sample added by R4 was known B blood, and no red blood cell agglutination reaction occurred in L1R4 and L3R4, and red blood cell agglutination reaction occurred in L2R4; and red blood cell agglutination reaction occurred in L5R1-L5R5, indicating that they were Rh-positive blood. The unknown blood sample added by R5 was consistent with the result of R3, and the detection result could be determined as A Rh-positive.

[0042] Preparation of blood group detection reagent card

[0043] The antibodies were immobilized on a nitrocellulose membrane at positions as shown in Figure 2 .

[0044] The nitrocellulose membrane used in the present application is CN95 membrane, the total thickness of the membrane is 240-270 μm, the membrane has a 100 μm thick transparent polyester backing, the pore size of the membrane is about 15 μm, the surface of the membrane is anionic, 20 μg of protein (such as IgG) can be adsorbed per square centimeter, and the capillary wicking speed is 65-115 s / 40 mm, which is an ideal choice for lateral flow rapid diagnosis.

[0045] The antibodies were immobilized on a nitrocellulose membrane at positions as shown in Figure 2The positions are immobilized on a nitrocellulose membrane (light blue). Specifically, the nanobodies are diluted to 0.1 mg / mL, 3 μL of which is uniformly coated on the square positions, detection window A corresponds to the anti-A antigen nanobody NbAl, detection window B corresponds to the anti-B antigen nanobody NbBl, detection window D corresponds to the anti-RhD antigen nanobody NbDl, and detection window QC corresponds to the commercial anti-human red blood cell antibody (CAT: FB1031) with a reagent concentration of 1.0 mg / mL, which needs to be diluted to 0.1 mg / mL and uniformly coated on the corresponding positions. The antibodies are naturally air-dried or oven-dried at 37°C.

[0046] Note that the preferred mode is that NbAl and NbBl are coated on the opposite side, NbDl and the commercial anti-human red blood cell antibody are coated on the opposite side, and the red circle center position is the blood sample addition hole.

[0047] Addition: After the blood sample or red blood cell suspension (5-10 μL) is added to the nitrocellulose membrane, it is quickly absorbed by the membrane and capillary crawls to react with the coated antibodies on the membrane.

[0048] Preparation of a blank detection reagent card and detection reagents

[0049] As another embodiment of the present application, a blank detection reagent card and detection reagents can also be used in combination for blood type detection.

[0050] Because Due to the pore size structure and surface properties of the CN95 membrane, the nanobodies and antibodies can be coated without chemical coupling, but can be directly coated by directly adding and air-drying, so that the antibody coating work can be completed by the experimenter. 2 μL of the corresponding detection reagent is directly added to the detection window, and after the antibody is naturally air-dried or oven-dried at 37°C, the blood sample or red blood cell suspension (5-10 μL) is added from the blood sample addition hole, and the results are observed after 30 s.

[0051] Detection reagent A: NbAl nanobodies are diluted in an antibody storage solution, the antibody storage solution is a DMEM medium containing 15 mM sodium azide, and the antibody concentration is 0.1 mg / mL.

[0052] Detection reagent B: NbBl nanobodies are diluted in an antibody storage solution, the antibody storage solution is a DMEM medium containing 15 mM sodium azide, and the antibody concentration is 0.1 mg / mL.

[0053] Detection reagent D: NbDl nanobodies are diluted in an antibody storage solution, the antibody storage solution is a DMEM medium containing 15 mM sodium azide, and the antibody concentration is 0.1 mg / mL.

[0054] In summary, the prepared detection reagent is a nanobody composition, which has high specificity, high sensitivity, stable structure and low cost; the blood type detection reagent and the blood type detection kit prepared from the nanobody have the advantages of high sensitivity, low cost and storage resistance, and the blood type detection method based on the kit has accurate and reliable results and high sensitivity.

[0055] Obviously, various modifications and changes can be made to the present application by those skilled in the art without departing from the spirit and scope of the present application. Thus, it is intended that the present application cover the modifications and changes as they come within the scope of the claims, and their equivalents.

Claims

1. A reagent for rapid blood typing, characterized in that, The reagents include: NbA1, a nanobody specifically binding to erythrocyte A antigen, with the amino acid sequence SEQ ID NO.1; NbB1, a nanobody specifically binding to erythrocyte B antigen, with the amino acid sequence SEQ ID NO.2; and NbD1, a nanobody specifically binding to erythrocyte RhD antigen, with the amino acid sequence SEQ ID NO.

3.

2. A nucleic acid encoding nanobodies NbA1, NbB1, and NbD1 in a reagent for rapid blood typing as described in claim 1.

3. A recombinant vector or recombinant cell containing the nucleic acid of claim 2.

4. A blood typing test kit prepared according to the blood typing reagent as described in claim 1, characterized in that, The kit consists of blood typing reagents and a blood typing reagent card. The reagent card includes a plastic shell, a multilayer porous membrane, and a backing. The plastic shell includes a central sample application well and four detection windows: detection window A, detection window B, detection window D, and detection window QC. Detection window A corresponds to the anti-A antigen nanobody NbA1, detection window B corresponds to the anti-B antigen nanobody NbB1, detection window D corresponds to the anti-RhD antigen nanobody NbD1, and detection window QC corresponds to a commercially available anti-human erythrocyte antibody. The multilayer porous membrane is a nitrocellulose membrane, and the backing is attached beneath the multilayer porous membrane.

5. The blood typing kit as described in claim 4, characterized in that, The kit also includes a blank test card that is not pre-coated with antibodies. The kit contains detection reagents: anti-A antibody NbA1, anti-B antibody NbB1, and anti-D antibody NbD1.

6. The use of the rapid blood typing reagent as described in claim 1 in blood typing, wherein the use is not for diagnostic purposes, characterized in that, When using this product, drop a blood sample into the central well. The nitrocellulose membrane will spread at a rate of 65-115 s / 40 mm. After 30 seconds, an agglutination reaction can be observed in the detection window. The blood type of the sample can be determined based on the results.

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