Application of a blood group antigen trisaccharide A analog protein conjugate in blood group antibody detection

Abstract

The present invention belongs to the field of blood type antibody detection, and relates to the application of a blood type antigen trisaccharide A analog protein conjugate in blood type antibody detection. The blood type antigen trisaccharide A analog is A2 type; it is coupled with hemocyanin or bovine serum albumin to form a blood type antigen trisaccharide A analog protein conjugate; the coupling ratio between it and the hemocyanin or bovine serum albumin is 10:1-80:1. The blood type antigen trisaccharide conjugate of the present invention has good specificity, can identify corresponding blood type antibodies, has high detection accuracy, and can be applied to various types of blood type antibody detection.

Description

Technical Field

[0001] The invention belongs to the field of blood type antibody detection and relates to the application of a blood type antigen trisaccharide A analog protein conjugate in blood type antibody detection. Background Art

[0002] Blood typing is the primary task before clinical blood transfusion, because when blood of different blood types is transfused to each other, hemolysis will occur due to the coagulation of antigens and antibodies, which may endanger human life safety. Therefore, correct blood typing is a prerequisite for ensuring the safety of blood transfusion. At present, the methods of blood typing include positive typing and reverse typing. The positive typing method detects red blood cell antigens, and the reverse typing method detects antibodies in the serum. Among them, the detection of human A, B, and O blood type antigens / antibodies is the most important. According to the different surface antigens on the red blood cell membrane in human blood, different blood types can be divided. The red blood cells of type A blood carry blood type antigen A (hereinafter referred to as "A antigen"), and there is blood type antibody B (hereinafter referred to as "B antibody") in the serum; the red blood cells of type B blood carry blood type antigen B (hereinafter referred to as "B antigen"), and there is blood type antibody A (hereinafter referred to as "A antibody") in the serum; the red blood cells of type AB blood have both A antigen and B antigen, and there are no A antibody and B antibody in the serum; the red blood cells of type O blood have neither A antigen nor B antigen, and there are both A antibody and B antibody in the serum.

[0003] At present, the common positive and negative typing of blood types generally adopts the agglutination method, and its principle is to use the blood type antigen and antibody reaction to cause visible red blood cell agglutination to judge the result. During positive typing, IgM anti-A or anti-B reagents are used to detect the red blood cell antigens of the sample; during negative typing, a red blood cell reagent of known A or B type is used to determine the IgM blood type antibodies in the sample serum. However, given that fresh red blood cells are not easy to preserve (the antigenicity of blood type antigens on the surface of red blood cells gradually decreases with the extension of storage time), the A type or B type red blood cell reagents used for negative typing have high requirements for storage conditions and transportation conditions, which increases the cost of negative typing and limits the application of negative typing. Some technical solutions in the prior art attempt to extract natural blood type antigens (for example, extracting natural A antigens and B antigens through red blood cell membranes) to replace complete red blood cells in negative typing, but the relevant extraction and preparation processes are often more complicated and difficult to mass produce, and the extracted natural blood type antigens still have high requirements for storage conditions.

[0004] In summary, artificial synthesis of blood group antigens is one of the research directions to solve the above technical problems, but the currently known artificial synthesis of blood group antigens have technical problems in specificity, affinity, stability, etc., and cannot be effectively applied to reverse determination detection. It is necessary to propose new methods or strategies. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide an application of an artificially synthesized blood group antigen trisaccharide A analog protein conjugate in blood group antibody detection, and the specific technical scheme is as follows.

[0006] The invention discloses an application of a blood group antigen trisaccharide analog protein conjugate in preparing a blood group antibody detection reagent. The blood group antigen trisaccharide analog is a blood group antigen trisaccharide A analog, which is type A2. The blood group antigen trisaccharide A analog is coupled with hemocyanin or bovine serum albumin to form a blood group antigen trisaccharide A analog protein conjugate, and the coupling ratio of the blood group antigen trisaccharide A analog protein conjugate to the hemocyanin or bovine serum albumin is 10:1-80:1.

[0007] In some embodiments, the coupling ratio of the blood group antigen trisaccharide A analog to the hemocyanin (Keyhole Limpet Hemocyanin, KLH) is 10:1, 20:1, 40:1, 60:1 and 80:1.

[0008] In some embodiments, the coupling ratio of the blood group antigen trisaccharide A analog to bovine serum albumin (BSA) is 10:1, 20:1, 40:1, 60:1 and 80:1.

[0009] In some embodiments, the blood type antibody detection reagent is used for immunochromatography detection, optical density detection and column agglutination detection.

[0010] In some embodiments, the blood type antibody detection reagent is used for immunochromatographic detection, and when the blood type antigen trisaccharide A analog is coupled to hemocyanin, the coupling ratio is 40:1; when the blood type antigen trisaccharide A analog is coupled to bovine serum albumin, the coupling ratio is 40:1.

[0011] An immunochromatographic detection kit comprises a detection card, wherein the detection card adopts a nitrocellulose membrane; the detection card is coated with a conjugate of a blood group antigen trisaccharide A analogue and hemocyanin or bovine serum albumin, and is used to detect blood group antibodies in a serum sample; the blood group antigen trisaccharide A analogue is A2 type, and its coupling ratio with hemocyanin is 10:1, 20:1, 40:1, 60:1 and 80:1, and its coupling ratio with bovine serum albumin is 10:1, 20:1, 40:1, 60:1 and 80:1.

[0012] Preferably, when the blood group antigen trisaccharide A analog is coupled to hemocyanin, the coupling ratio is 40:1; when the blood group antigen trisaccharide A analog is coupled to bovine serum albumin, the coupling ratio is 40:1.

[0013] A column agglutination kit comprises an ABO blood type reverse typing test card, a conjugate of a blood type antigen trisaccharide A analogue and hemocyanin or bovine serum albumin, wherein the blood type antigen trisaccharide A analogue is A2 type, and the conjugation ratios of the blood type antigen trisaccharide A analogue with hemocyanin are 10:1, 20:1, 40:1, 60:1 and 80:1; and the conjugation ratios of the blood type antigen trisaccharide A analogue with bovine serum albumin are 10:1, 20:1, 40:1, 60:1 and 80:1.

[0014] Preferably, when the blood group antigen trisaccharide A analog is coupled to hemocyanin, the coupling ratio is 40:1; when the blood group antigen trisaccharide A analog is coupled to bovine serum albumin, the coupling ratio is 40:1.

[0015] Beneficial technical effects

[0016] 1) Experiments have shown that the above-mentioned blood type antigen trisaccharide A2 analog protein conjugate (coupled with BSA or KLH) provided by the present invention has good specificity. As an artificially synthesized blood type antigen, it can distinguish and identify the corresponding blood type antibodies, with high detection accuracy. It can be used for various types of blood type antibody detection and has great potential clinical application value. Especially in the application of immunochromatographic detection, some of the blood type antigen trisaccharide analog protein conjugates adopted by the technical solution of the present invention can be stably fixed on the test paper and efficiently capture the corresponding blood type antibodies. The color development effect (for example, by colloidal gold labeling) is easy to identify and visible to the naked eye. Therefore, it has the potential to be applied to rapid blood type screening, POCT detection, ordinary people's blood type self-examination and other scenarios. The experiment of the present invention confirmed that the best effect is achieved when the coupling ratio of A2 to KLH and BSA is 40:1.

[0017] 2) The blood type antigen trisaccharide analog protein conjugate used in the present invention is synthesized by chemical process, which is relatively easy to achieve industrial-grade mass production. It can be used as a blood type antigen raw material in various experimental systems, thereby replacing red blood cell reagents or natural blood type antigens in, for example, reverse determination detection, avoiding the cumbersome process of natural blood type antigen extraction and preparation, and reducing the cost of blood type detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. Obviously, the drawings described below are some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0019] Figure 1 This is a color development diagram of the conjugate of B1-B2 and BSA on a nitrocellulose membrane (NC membrane) in an embodiment of the present invention;

[0020] Figure 2 This is a color development diagram of the conjugate of B3-B4 and BSA on the NC membrane in the embodiment of the present invention;

[0021] Figure 3 This is a color development effect diagram of the conjugate of B1-B2 and KLH on the NC membrane in the embodiment of the present invention;

[0022] Figure 4 This is a color development diagram of the conjugate of B3-B4 and KLH on the NC membrane in the embodiment of the present invention;

[0023] Figure 5 This is a color development diagram of the conjugate of A1-A2 and BSA on the NC membrane in the embodiment of the present invention;

[0024] Figure 6 This is a color development diagram of the conjugate of A3-A4 and BSA on the NC membrane in the embodiment of the present invention;

[0025] Figure 7 This is a color development effect diagram of the conjugate of A1-A2 and KLH on the NC membrane in the embodiment of the present invention;

[0026] Figure 8 This is a color development effect diagram of the conjugate of A3-A4 and KLH on the NC membrane in the embodiment of the present invention;

[0027] Fig. 9 The effect diagram of the blood type reverse typing test card for A1-KLH detection of A and B antibodies;

[0028] Fig.10 The effect diagram of the blood type reverse typing test card for A2-KLH to detect A and B antibodies;

[0029] Fig.11 This is a rendering of the blood type reverse typing test card for A3-KLH to detect A and B antibodies;

[0030] Fig.12 The effect diagram of the blood type reverse typing test card for A4-KLH to detect A and B antibodies;

[0031] Fig.13 This is the effect picture of the blood type reverse typing test card for A1-BSA to detect A and B antibodies;

[0032] Fig.14 This is the effect picture of the blood type reverse typing test card for A2-BSA to detect A and B antibodies;

[0033] Fig.15 This is the effect picture of the blood type reverse typing test card for A3-BSA to detect A and B antibodies;

[0034] Fig.16This is the effect picture of the blood type reverse typing test card for A4-BSA to detect A and B antibodies;

[0035] Fig.17 This is the effect diagram of the blood type reverse typing test card for B1-BSA to detect A and B antibodies;

[0036] Fig.18 This is the effect picture of the blood type reverse typing test card for B2-BSA to detect A and B antibodies;

[0037] Fig.19 This is the effect picture of the blood type reverse typing test card for B3-BSA to detect A and B antibodies;

[0038] Fig. 20 This is the effect picture of the blood type reverse typing test card for B4-BSA to detect A and B antibodies;

[0039] Fig.21 This is a rendering of the blood type reverse typing test card for B1-KLH to detect A and B antibodies;

[0040] Fig. 22 The effect diagram of the blood type reverse typing test card for B2-KLH to detect A and B antibodies;

[0041] Fig.23 This is a rendering of the blood type reverse typing test card for B3-KLH to detect A and B antibodies;

[0042] Fig.24 This is a rendering of the blood type reverse typing test card for B4-KLH to detect A and B antibodies;

[0043] Fig.25 The synthetic route of the blood group antigen trisaccharide B analog type I of the present invention;

[0044] Fig.26 The synthetic route of the blood group antigen trisaccharide B analog type II of the present invention;

[0045] Fig. 27 The synthetic route of the blood group antigen trisaccharide B analog type III of the present invention;

[0046] Fig.28 The present invention is a synthetic route for the blood group antigen trisaccharide B analog type IV. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0048] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0049] In this specification, certain embodiments may be disclosed in a format of being in a certain range. It should be understood that such description of "being in a certain range" is merely for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0050] The "blood group antigen trisaccharide A analogues" described in the present invention refer to four compounds and their derivatives as shown in the basic chemical formulas such as Chemical Formulas A1-A4, which are type I A antigen compound (chemical formula A1, hereinafter referred to as "A1" or "A1 type"), type II A antigen compound (chemical formula A2, hereinafter referred to as "A2" or "A2 type"), type III A antigen compound (chemical formula A3, hereinafter referred to as "A3" or "A3 type") and type IV A4 antigen compound (chemical formula A4, hereinafter referred to as "A4" or "A4 type").

[0051]

[0052]

[0053]

[0054]

[0055] The "blood group antigen trisaccharide analog protein conjugate" described in the present invention refers to a protein conjugate formed by coupling the above-mentioned blood group antigen trisaccharide A analog with BSA or KLH protein (hereinafter referred to as "A-BSA", "A-KLH").

[0056] Example 1. Preparation of blood group antigen trisaccharide analog protein conjugate

[0057] 1.1 Preparation of blood group antigen trisaccharide A analog protein conjugate

[0058] In some embodiments, the modular assembly strategy of the Bovin group (Drouillar S, et al., Large-scale synthesis of H-antigen oligosaccharides by expressing helicobacter pyloria1,2-fucosyltransferase in metabolically engineered Escherichia coli cells [J]. Angew Chem, 2006, 118 (11): 1810-1812) can be used to first synthesize two target sugar building blocks, and then synthesize the target sugar chain through glycosylation reaction. The main reaction steps are shown as follows:

[0059]

[0060] Then, the terminal of the target sugar chain generated by the above method reacts with the butyramide connected to the carboxyl group to generate the antigen trisaccharide A analog of the present invention, with a total of four types (A1-A4), and the chemical structural formulas are shown in chemical formulas A1-A4. Finally, a linear connecting arm with low immunogenicity, good reactivity and high coupling rate is used to couple the blood group antigen trisaccharide A analog with the amino group on the lysine residue of the BSA protein or KLH protein (for example, a segment of alkane chain connected to an azide group pre-installed on the starting substrate during sugar chain synthesis is used as a connecting arm, and then the blood group antigen trisaccharide analog is coupled to the carrier protein through a double activated ester reagent), thereby synthesizing the blood group antigen trisaccharide A analog protein conjugate of the present invention (A1-BSA, A2-BSA, A3-BSA, A4-BSA; A1-KLH, A2-KLH, A3-KLH, A4-KLH), whose chemical formula is shown below:

[0061] A1-BSA

[0062]

[0063] A2-BSA

[0064]

[0065] A3-BSA

[0066]

[0067] A4-BSA

[0068]

[0069] A1-KLH:

[0070]

[0071] A2-KLH:

[0072]

[0073] A3-KLH

[0074]

[0075] A4-KLH

[0076]

[0077] Among them, n=10, 20, 40, 60, 80, that is, the ratio of the blood group antigen trisaccharide A analog to the protein (BSA or KLH) is in the range of 10:1 to 80:1, see Table 1 for details.

[0078] 1.2 Preparation of blood group antigen trisaccharide B analog protein conjugate

[0079] First, four disaccharide precursors were synthesized. The strategy adopted was to introduce a linker arm containing an azide group at the reducing end of the starting monosaccharide (GlcNAc, GlaNAc) by chemical method, and then the chemically synthesized GlcNAcβProN3, GalNAcαProN3, and GalNAcβProN3 monosaccharides were used as acceptors to complete the synthesis of the four types of precursors through a one-pot multi-enzyme method. The chemical formula is as follows:

[0080] Blood group antigen trisaccharide B analogue disaccharide precursor type 1:

[0081]

[0082] Blood group antigen trisaccharide B analog disaccharide precursor type II:

[0083]

[0084] Blood group antigen trisaccharide B analog disaccharide precursor type III:

[0085]

[0086] Blood group antigen trisaccharide B analog disaccharide precursor type IV:

[0087]

[0088] Then, an α1-3 galactosyltransferase GTB was introduced into the non-reducing end of the above four disaccharide precursors to synthesize four blood group antigen trisaccharide B analogs B1-B4 (molecular structures are shown in chemical formulas B1-B4). The specific synthesis route is as follows: Figure 25-28 shown.

[0089] Finally, a linear linker with low immunogenicity, good reactivity and high coupling rate is used to couple the blood group antigen trisaccharide B analog (B1-B4) with the amino group on the lysine residue of the BSA protein or KLH protein (for example, an alkane chain connected to an azide group pre-installed on the starting substrate during sugar chain synthesis is used as a linker, and then the blood group antigen trisaccharide analog is coupled to the carrier protein through a double activated ester reagent) to synthesize antigen trisaccharide B analog protein conjugates (B1-BSA, B2-BSA, B3-BSA, B4-BSA; B1-KLH, B2-KLH, B3-KLH, B4-KLH), the chemical formula of which is as follows:

[0090] B1-BSA

[0091]

[0092] B2-BSA

[0093]

[0094] B3-BSA

[0095]

[0096] B4—BSA

[0097]

[0098] B1-KLH

[0099]

[0100] B2-KLH

[0101]

[0102] B3-KLH

[0103]

[0104] B4-KLH

[0105]

[0106] Among them, n=5, 10, 20, 40, that is, the ratio of the blood group antigen trisaccharide B analog to KLH coupling ranges from 5:1 to 40:1, see Table 1 for details.

[0107] Table 1 Blood group antigen trisaccharide analogs and protein coupling ratio

[0108]

[0109]

[0110] Embodiment 2

[0111] Qualitative verification of blood group antigen trisaccharide analog protein conjugates (antigen immobilization immunochromatography technique)

[0112] In order to study whether the blood group antigen trisaccharide analog protein conjugate synthesized by the present invention can be used for simple and rapid qualitative blood group detection, the present invention adopts antigen immobilization immunochromatography technology for verification. In order to detect antibody A and antibody B in serum, a test card assembled with Sartorius nitrocellulose (NC) membrane CN140 (see Figure 1-Figure 8 ), the NC membrane includes a detection line (i.e., T line) and a quality control line (i.e., C line). The T line is solidified and coated with a blood group antigen trisaccharide A analog protein conjugate or a blood group antigen trisaccharide B analog protein conjugate, and the C line is coated with anti-chicken IgY antibody. The colloidal gold / microsphere pad contains labeled anti-human μ chain antibody and chicken IgY antibody.

[0113] During the test, the sample is added from the loading hole (marked as S on the test card). The blood type antibody A or antibody B contained in the sample can bind to the anti-human μ chain antibody labeled with colloidal gold to form a complex and chromatograph on the NC membrane. When the complex is chromatographed to the T line, the blood type antibody A or B specifically binds to the corresponding blood type antigen trisaccharide A analog protein conjugate or blood type antigen trisaccharide B analog protein conjugate to form a color line visible to the naked eye (that is, blood type antibody A binds to artificial synthetic antigen A; blood type antibody B binds to artificial synthetic antigen B). If there is no corresponding antibody A or antibody B in the sample, a complex cannot be formed and color cannot be developed at the T line. Based on this, when the sample contains antibody A but not antibody B, it is type A blood; the sample contains antibody B but not antibody A, it is type B blood; the sample does not contain antibody A and antibody B, it is type AB blood; the sample contains both antibody A and antibody B, it is type O blood. The sample is first chromatographed to the T line and then to the C line. The C line should show color when testing all samples, otherwise the test is invalid.

[0114] In the present application, the test card coated with blood group antigen trisaccharide A analog protein conjugate (A-BSA, A-KLH) is referred to as A test card in the present invention; the test card coated with blood group antigen trisaccharide B analog protein conjugate (B-BSA, B-KLH) is referred to as B test card in the present invention.

[0115] Add 1 μl of standard blood type antibody A (brand: Millipore, batch number: JHE2103, item number: JH-1L-BK) to each A test card, and then add 80 μl of sample diluent (0.01M PBS). After standing for about 15-20 minutes, observe the color development at the C line and T line with the naked eye and take photos for record.

[0116] Add 1 μl of standard blood type antibody B (brand: Millipore, batch number: JMC2103, item number: JM-1L-BK) to each B test card, and then add 80 μl of sample diluent (0.01 M PBS). Let it stand for about 15 min-20 min, observe the color development at the C line and T line with the naked eye, and take photos for record.

[0117] The above test results are as follows Figure 1-Figure 8 As shown and summarized in Tables 2a–2d, colors visible to the naked eye are indicated by a “+” sign and colors invisible to the naked eye are indicated by a “-” sign.

[0118] The above test results show that for blood group antigen trisaccharide B analogs, B2 type can be effectively colored on the T line (visible to the naked eye) after coupling with BSA or KLH. However, the color development effect of B2-BSA is only obvious when the coupling ratio is 20:1, while B2-KLH shows a good color development effect in a wider range of coupling ratios (from 5:1 to 40:1). In addition, when the B-KLH coupling ratio is 40:1, the color development effects of B1-KLH, B3-KLH and B4-KLH can also be visible to the naked eye. For blood group antigen trisaccharide A analogs, A2-A4, whether coupled with BSA or KLH, the formed blood group antigen trisaccharide analog protein conjugates can be effectively colored on the T line (coupling ratio: 10:1, 40:1, 60:1, 80:1), but when the protein coupling ratio of A-BSA and A-KLH is 20:1, only A2-BSA / A2-KLH can be effectively colored, and A3-BSA / A3-KLH and A4-BSA / A4-KLH cannot be effectively colored. In addition, A1, whether coupled with BSA or KLH, failed to be effectively colored at the coupling ratios tested by the present invention.

[0119] The above experimental results show that when a specific blood group antigen trisaccharide analog is coupled to a specific protein (for example, B2-KLH, A2-BSA, A2-KLH), or at a specific coupling ratio (20:1 (B2-BSA); 40:1 (B1-KLH, B3-KLH, B4-KLH); 10:1, 40:1–80:1 (A3-BSA, A4-BSA, A3-KLH, A4-KLH)), when detecting blood group antibody standards, the T line This shows that the blood type antigen trisaccharide analogs can be effectively colored to a level visible to the naked eye, proving that they can be stably and effectively fixed on NC membranes or other types of solid media (e.g., ELISA plates, microspheres or magnetic beads made of various materials) after protein coupling, and can effectively and specifically capture the corresponding blood type antibodies. Therefore, they can be used for rapid and easy qualitative detection of blood type antibodies (e.g., because they are visible to the naked eye, ordinary people can read the results), allowing blood type testing to be completed at home or in small clinics.

[0120] For those skilled in the art, the blood group antigen trisaccharide analog protein conjugate of the present invention can also be used in experimental systems using other labeling methods (e.g., enzyme labeling, fluorescent labeling or other luminescent labeling). In addition, in addition to labeling antibodies, the blood group antigen trisaccharide analog protein conjugate can also be labeled with different labeling methods and used for various heterogeneous or homogeneous detections according to actual needs.

[0121] Table 2a

[0122]

[0123] Form 2b

[0124]

[0125] Form 2c

[0126]

[0127]

[0128] Table 2d

[0129]

[0130] Embodiment 3

[0131] Quantitative validation of blood group antigen trisaccharide B analog protein conjugate

[0132] 3.1 Experiment on coupling blood group antigen trisaccharide B with KLH protein

[0133] 1) Test results of diluting the standard antibody 100 times

[0134] The main experimental reagents used in this experiment include: latex microspheres (125 μL latex microspheres, labeled with 0.1 mg antigen in total); reagent R1 (1 g BSA, 0.24 g Tris, 0.1 g PC300, 5 g PEG6000, 100 mL water, pH 8.5).

[0135] The loading method of this experiment is as follows: first dilute the A and B antibody standards (brand Millipore, batch number JHE2103, item number JH-1L-BK; brand Millipore, batch number JMC2103, item number JM-1L-BK) 100 times with R1 to obtain a volume of 200 μL of diluted A and B antibody standards, and then add reagent R2 (i.e., dilute the above latex microspheres 100 times with TBS) 200 μL to obtain a total of 400 μL of the sample to be tested. In this experiment, the detection wavelength is 340nm.

[0136] The above experimental results are summarized in the following table:

[0137] Table 3a

[0138]

[0139]

[0140] The above experimental results show that after the antigen B used in the present invention is coupled with KLH, no matter which type of B1-B4 is used, and no matter which coupling ratio with KLH in the range of 5:1-40:1 is used, the OD value of antigen B combined with B antibody (greater than or equal to 0.334) is significantly higher than the OD value obtained when A antibody is added (the highest can only reach 0.261), and the P / N value (positive serum OD value / negative serum OD value) can reach 1.4 or more, that is, the antigen B used in the present invention can well distinguish A antibody from B antibody in the detection of blood type antibodies, and has good specificity. It is worth noting that for B1-KLH, B3-KLH and B4-KLH, the P / N value is the highest when the coupling ratio is 40:1, that is, it can best distinguish A antibody from B antibody. The P / N value of B2-KLH is relatively high within the coupling ratio range (5:1-40:1) adopted in the present invention, especially when the coupling ratio is 20:1, the P / N value is the best, reaching 2.459.

[0141] 2) Test results of diluting the standard antibody 200 times

[0142] In order to further verify the effect of the blood group antigen trisaccharide B analog protein conjugate (B-KLH) synthesized by the present invention in detecting at a low antibody concentration, the standard antibody was diluted 200 times for detection in this experiment. The main experimental reagents used in this experiment include: latex microspheres (125μL latex microspheres labeled 0.1mg antigen); reagent R1 (1g BSA, 0.24g Tris, 0.1g PC300, 5g PEG6000, 100mL water, pH8.5). The loading method of this experiment is as follows: dilute the A and B antibody standards 200 times with R1 (brand Millipore, batch number JHE2103, item number JH-1L-BK; brand Millipore, batch number JMC2103, item number JM-1L-BK) to obtain a volume of 200 μL of diluted antibody standards, and then add reagent R2 (the above latex microspheres are diluted 200 times with TBS) 200 μL, a total of 400 μL of the sample to be tested. In this experiment, the detection wavelength is 340nm.

[0143] The results of the above experiments are summarized in the following table:

[0144] Form 3b

[0145] Coupling ratio A Antibody OD value B antibody OD value P / N B1:KLH=5:1 0.251 0.307 1.224 B1:KLH=10:1 0.239 0.319 1.336 B1:KLH=20:1 0.253 0.347 1.371 B1:KLH=40:1 0.257 0.379 1.475 B2:KLH=5:1 0.237 0.346 1.458 B2:KLH=10:1 0.236 0.383 1.624 B2:KLH=20:1 0.261 0.557 2.133 B2:KLH=40:1 0.227 0.403 1.777 B3:KLH=5:1 0.243 0.311 1.281 B3:KLH=10:1 0.229 0.300 1.311 B3:KLH=20:1 0.234 0.316 1.352 B3:KLH=40:1 0.251 0.367 1.462 B4:KLH=5:1 0.237 0.330 1.393 B4:KLH=10:1 0.242 0.322 1.329 B4:KLH=20:1 0.248 0.361 1.456 B4:KLH=40:1 0.252 0.373 1.481

[0146] The above experimental results show that even if the standard antibody is diluted 200 times, the antigen B used in the present invention, no matter which type of B1-B4 is used, and no matter which coupling ratio with KLH is 5:1-40:1, the OD value of antigen B combined with B antibody (greater than or equal to 0.300) is significantly higher than the OD value obtained when adding A antibody (the highest can only reach 0.261), and the P / N value can almost reach more than 1.3 (except for B1-KLH and B3-KLH when the coupling ratio is 5:1). In other words, even in the case of low antibody concentration, the antigen B used in the present invention can well distinguish A antibody from B antibody in the detection of blood type antibodies.

[0147] 3.2 Blood group antigen trisaccharide B binding to BSA protein experiment

[0148] 1) Test results of diluting the standard antibody 100 times

[0149] The main experimental reagents used in this experiment include: latex microspheres (125 μL latex microspheres, labeled with 0.1 mg antigen in total); reagent R1 (1 g BSA, 0.24 g Tris, 0.1 g PC300, 5 g PEG6000, 100 mL water, pH 8.5).

[0150] The loading method of this experiment is as follows: dilute the A and B antibody standards (brand Millipore, batch number JHE2103, item number JH-1L-BK; brand Millipore, batch number JMC2103, item number JM-1L-BK) 100 times with R1 to obtain a volume of 200 μL of diluted antibody standards, and then add reagent R2 (the above latex microspheres are diluted 100 times with TBS) 200 μL to obtain a total of 400 μL of the sample to be tested. In this experiment, the detection wavelength is 340nm.

[0151] The above experimental results are summarized in the following table:

[0152] Form 3c

[0153] Coupling ratio A Antibody OD value B antibody OD value P / N B1:BSA=5:1 0.187 0.233 1.244 B1:BSA=10:1 0.188 0.241 1.279 B1:BSA=20:1 0.176 0.254 1.446 B1:BSA=40:1 0.182 0.239 1.314 B2:BSA=5:1 0.177 0.258 1.459 B2:BSA=10:1 0.161 0.239 1.483 B2:BSA=20:1 0.147 0.247 1.678 B2:BSA=40:1 0.171 0.261 1.522 B3:BSA=5:1 0.185 0.228 1.231 B3:BSA=10:1 0.200 0.253 1.267 B3:BSA=20:1 0.162 0.244 1.503 B3:BSA=40:1 0.180 0.238 1.325 B4:BSA=5:1 0.187 0.256 1.367 B4:BSA=10:1 0.177 0.239 1.352 B4:BSA=20:1 0.194 0.253 1.303 B4:BSA=40:1 0.181 0.262 1.451

[0154] The above experimental results show that the antigen B used in the present invention, regardless of which type of B1-B4 is used, and regardless of which coupling ratio with BSA in 5:1-40:1 is used, the OD value of antigen B combined with B antibody is different from the OD value obtained when A antibody is added, but it is not as good as the difference in the OD value when antigen B is coupled with KLH when identifying A antibody and B antibody, and the P / N value of multiple groups of data is between 1.231 and 1.678. The above results prove that blood group antigen trisaccharide B (B1-B4) analogs coupled with BSA protein can also detect B antibody and distinguish A antibody from B antibody in the above experiment, but its ability and effect are not as good as blood group antigen trisaccharide B (B1-B4) analogs coupled with KLH protein.

[0155] 2) Test results of diluting the standard antibody 200 times

[0156] In order to further verify the effect of the blood group antigen trisaccharide B analog protein conjugate (B-BSA) synthesized by the present invention in detecting at a low antibody concentration, the standard antibody was diluted 200 times for detection in this experiment. The main experimental reagents used in this experiment include: latex microspheres (125μL latex microspheres labeled 0.1mg antigen); reagent R1 (1g BSA, 0.24g Tris, 0.1g PC300, 5g PEG6000, 100mL water, pH8.5). The loading method of this experiment is as follows: dilute the A and B antibody standards 200 times with R1 (brand Millipore, batch number JHE2103, item number JH-1L-BK; brand Millipore, batch number JMC2103, item number JM-1L-BK) to obtain a volume of 200 μL of diluted antibody standards, and then add reagent R2 (dilute the above latex microspheres 200 times with TBS) to obtain a total of 400 μL of the sample to be tested. In this experiment, the detection wavelength is 340 nm.

[0157] The above experimental results are summarized in the following table:

[0158] Table 3d

[0159] Coupling ratio A Antibody OD value B antibody OD value P / N B1:BSA=5:1 0.243 0.295 1.214 B1:BSA=10:1 0.239 0.292 1.223 B1:BSA=20:1 0.253 0.353 1.396 B1:BSA=40:1 0.244 0.311 1.276 B2:BSA=5:1 0.233 0.321 1.376 B2:BSA=10:1 0.229 0.319 1.392 B2:BSA=20:1 0.252 0.358 1.421 B2:BSA=40:1 0.254 0.337 1.325 B3:BSA=5:1 0.228 0.274 1.201 B3:BSA=10:1 0.237 0.277 1.168 B3:BSA=20:1 0.245 0.342 1.394 B3:BSA=40:1 0.257 0.316 1.231 B4:BSA=5:1 0.251 0.304 1.211 B4:BSA=10:1 0.243 0.309 1.272 B4:BSA=20:1 0.231 0.280 1.214 B4:BSA=40:1 0.229 0.297 1.296

[0160] The above experimental results are similar to the test results of the standard antibody diluted 100 times. No matter which type of B1-B4 is used, or which coupling ratio with BSA in 5:1-40:1, the OD value of antigen B combined with B antibody is different from the OD value obtained when A antibody is added, but it is obviously not as good as the difference between the OD value when antigen B is coupled with KLH when identifying A antibody and B antibody. Only when B2:BSA=20:1, the P / N value can reach 1.421; when B3:BSA=20:1, the P / N value can reach 1.394. Combined with the NC membrane color development experiment (Table 2a), only when B2:BSA=20:1, the color development reaction can be seen on the test card with the naked eye. The above results prove that blood group antigen trisaccharide B (B1-B4) analogs coupled to BSA protein can also detect B antibodies to a certain extent and distinguish A antibodies from B antibodies, but its ability is obviously inferior to blood group antigen trisaccharide B (B1-B4) analogs coupled to KLH protein.

[0161] Embodiment 4

[0162] Quantitative verification of blood group antigen trisaccharide A analog protein conjugate (optical density experiment)

[0163] 4.1 Blood group antigen trisaccharide A binding to BSA protein experiment

[0164] 1) Test results of diluting the standard antibody 100 times

[0165] The main experimental reagents used in this experiment include: latex microspheres (125 μL latex microspheres, labeled with 0.1 mg antigen in total); reagent R1 (1 g BSA, 0.24 g Tris, 0.1 g PC300, 5 g PEG6000, 100 mL water, pH 8.5).

[0166] The loading method of this experiment is as follows: dilute the A and B antibody standards (brand Millipore, batch number JHE2103, item number JH-1L-BK; brand Millipore, batch number JMC2103, item number JM-1L-BK) 100 times with R1 to obtain a volume of 200 μL of diluted antibody standards, and then add reagent R2 (the above latex microspheres are diluted 100 times with TBS) 200 μL to obtain a total of 400 μL of the sample to be tested. In this experiment, the detection wavelength is 340nm.

[0167] The above experimental results are summarized in the following table:

[0168] Form 4a

[0169]

[0170]

[0171] The above experimental results show that when the antigen A used in the present invention is coupled to BSA, the vast majority of A2-A4 (except A3:BSA=20:1, A4:BSA=20:1) have a higher OD value of antigen A binding to antibody A (greater than or equal to 0.4) than the OD value of antigen A binding to antibody B (the highest is only 0.245) regardless of which coupling ratio with BSA in 10:1-80:1. Experiments have shown that the antigens A2-A4 used in the present invention can well distinguish between antibody A and antibody B in the detection of blood type antibodies, especially with A2:BSA=40:1. In contrast, the ability of A1-BSA coupling to distinguish and recognize antibody A and antibody B is poor, and the P / N value is only slightly higher than 1.

[0172] 2) Test results of diluting the standard antibody 200 times

[0173] In order to further verify the effect of the blood group antigen trisaccharide A analog protein conjugate (A-BSA) synthesized by the present invention in detecting at a low antibody concentration, the standard antibody was diluted 200 times for detection in this experiment. The main experimental reagents used in this experiment include: latex microspheres (125μL latex microspheres labeled 0.1mg antigen); reagent R1 (1g BSA, 0.24g Tris, 0.1g PC300, 5g PEG6000, 100mL water, pH8.5). The loading method of this experiment is as follows: dilute the A and B antibody standards 200 times with R1 (brand Millipore, batch number JHE2103, item number JH-1L-BK; brand Millipore, batch number JMC2103, item number JM-1L-BK) to obtain a volume of 200 μL of diluted antibody standards, and then add reagent R2 (the above latex microspheres are diluted 200 times with TBS) 200 μL, a total of 400 μL of the sample to be tested. In this experiment, the detection wavelength is 340nm.

[0174] The above experimental results are summarized in the following table:

[0175] Form 4b

[0176]

[0177]

[0178] The above experimental results show that even after the standard sample is diluted 200 times, when the antibody concentration is low, when the antigen A2-A4 used in the present invention is coupled to BSA, no matter which coupling ratio with BSA is used in 10:1-80:1 (except A3:BSA=20:1, A4:BSA=20:1), the P / N value is relatively high, that is, it can still distinguish A antibody and B antibody well in the detection of blood type antibodies, especially A2:BSA=40:1. The best distinction effect. In contrast, the ability of A1-BSA coupling to distinguish and recognize A antibody and B antibody is poor, and the P / N value is only slightly higher than 1.

[0179] 4.2 Blood group antigen trisaccharide A binding to KLH protein experiment

[0180] 1) Test results of diluting the standard antibody 100 times

[0181] The main experimental reagents used in this experiment include: latex microspheres (125 μL latex microspheres, labeled with 0.1 mg antigen in total); reagent R1 (1 g BSA, 0.24 g Tris, 0.1 g PC300, 5 g PEG6000, 100 mL water, pH 8.5).

[0182] The loading method of this experiment is as follows: dilute the A and B antibody standards (brand Millipore, batch number JHE2103, item number JH-1L-BK; brand Millipore, batch number JMC2103, item number JM-1L-BK) 100 times with R1 to obtain a volume of 200 μL of diluted antibody standards, and then add reagent R2 (the above latex microspheres are diluted 100 times with TBS) 200 μL to obtain a total of 400 μL of the sample to be tested. In this experiment, the detection wavelength is 340nm.

[0183] The above experimental results are summarized in the following table:

[0184] Form 4c

[0185] Coupling ratio A Antibody OD value B antibody OD value P / N A1:KLH=10:1 0.284 0.255 1.112 A1:KLH=20:1 0.342 0.264 1.297 A1:KLH=40:1 0.346 0.259 1.337 A1:KLH=60:1 0.317 0.251 1.263 A1:KLH=80:1 0.342 0.253 1.351 A2:KLH=10:1 0.479 0.266 1.801 A2:KLH=20:1 0.524 0.271 1.933 A2:KLH=40:1 0.629 0.283 2.223 A2:KLH=60:1 0.489 0.264 1.854 A2:KLH=80:1 0.489 0.269 1.819 A3:KLH=10:1 0.485 0.277 1.751 A3:KLH=20:1 0.494 0.283 1.744 A3:KLH=40:1 0.485 0.274 1.769 A3:KLH=60:1 0.464 0.285 1.627 A3:KLH=80:1 0.473 0.293 1.613 A4:KLH=10:1 0.473 0.288 1.641 A4:KLH=20:1 0.459 0.271 1.694 A4:KLH=40:1 0.484 0.284 1.704 A4:KLH=60:1 0.474 0.275 1.723 A4:KLH=80:1 0.472 0.272 1.737

[0186] The above experimental results show that after the standard sample is diluted 100 times, when the antigen A used in the present invention is coupled to KLH, the OD value of antigen A combined with A antibody (greater than or equal to 0.4) is higher than the OD value of combined with B antibody (the highest is only 0.293) in most of A2-A4, regardless of which coupling ratio with KLH in the range of 10:1-80:1. Experiments have shown that the antigens A2-A4 used in the present invention can well distinguish between A antibody and B antibody in the detection of blood type antibodies, especially A2:KLH=40:1 has the best distinction effect. In contrast, the ability of A1-KLH coupling to distinguish and recognize A antibody and B antibody is relatively poor, and the P / N value is only slightly higher than 1.

[0187] 2) Test results of diluting the standard antibody 200 times

[0188] In order to further verify the detection effect of the blood group antigen trisaccharide A analog protein conjugate (A-KLH) synthesized by the present invention at a low antibody concentration, the standard antibody was diluted 200 times for detection. The main experimental reagents used in this experiment include: latex microspheres (125μL latex microspheres labeled 0.1mg antigen); reagent R1 (1g BSA, 0.24g Tris, 0.1g PC300, 5g PEG6000, 100mL water, pH8.5). The loading method of this experiment is as follows: dilute the A and B antibody standards 200 times with R1 (brand Millipore, batch number JHE2103, item number JH-1L-BK; brand Millipore, batch number JMC2103, item number JM-1L-BK) to obtain a volume of 200 μL of diluted antibody standards, and then add reagent R2 (the above latex microspheres are diluted 200 times with TBS) 200 μL, a total of 400 μL of the sample to be tested. In this experiment, the detection wavelength is 340nm.

[0189] The above experimental results are summarized in the following table:

[0190] Table 4d

[0191] Coupling ratio A Antibody OD value B antibody OD value P / N A1:KLH=10:1 0.262 0.255 1.029 A1:KLH=20:1 0.324 0.264 1.227 A1:KLH=40:1 0.331 0.259 1.278 A1:KLH=60:1 0.287 0.251 1.145 A1:KLH=80:1 0.308 0.253 1.217 A2:KLH=10:1 0.447 0.266 1.679 A2:KLH=20:1 0.479 0.271 1.766 <h2 style=";text-align:left;direction:ltr">A2:KLH=40:1 0.614 0.283 2.170 <h2 style=";text-align:left;direction:ltr"> A2:KLH=60:1 0.447 0.264 1.694 <h2 style=";text-align:left;direction:ltr"> A2:KLH=80:1 0.446 0.269 1.657 <h2 style=";text-align:left;direction:ltr"> A3:KLH=10:1 0.443 0.277 1.601 <h2 style=";text-align:left;direction:ltr"> A3:KLH=20:1 0.449 0.283 1.585 <h2 style=";text-align:left;direction:ltr"> A3:KLH=40:1 0.435 0.274 1.588 <h2 style=";text-align:left;direction:ltr"> A3:KLH=60:1 0.416 0.285 1.461 <h2 style=";text-align:left;direction:ltr"> A3:KLH=80:1 0.439 0.293 1.498 <h2 style=";text-align:left;direction:ltr"> A4:KLH=10:1 0.422 0.288 1.464 <h2 style=";text-align:left;direction:ltr"> A4:KLH=20:1 0.407 0.271 1.503 <h2 style=";text-align:left;direction:ltr"> A4:KLH=40:1 0.431 0.284 1.519 <h2 style=";text-align:left;direction:ltr"> A4:KLH=60:1 0.420 0.275 1.527 <h2 style=";text-align:left;direction:ltr"> A4:KLH=80:1 0.414 0.272 1.523

[0192] The above experimental results show that even after the standard sample is diluted 200 times, when the antigen A2-A4 used in the present invention is coupled to KLH, no matter which coupling ratio with KLH is 10:1-80:1, the P / N value is relatively high, that is, it can still distinguish A antibody and B antibody well in the detection of blood type antibodies, especially A2:KLH=40:1. In contrast, the ability of A1-KLH coupling to distinguish and recognize A antibody and B antibody is relatively poor, and the P / N value is only slightly higher than 1.

[0193] Embodiment 5

[0194] Blood group antigen trisaccharide analog protein conjugate column agglutination neutralization test

[0195] This embodiment uses a human ABO blood type reverse typing test card (column agglutination method) to conduct a neutralization experiment, that is, a certain amount of antibodies are first neutralized with artificial antigens, and then indicator cells are added to check whether there are still antibodies that have not been neutralized by the artificial antigens. If the artificial antigen has strong specificity and high efficiency in binding with antibodies, the antibodies can be fully neutralized by the artificial antigen, and when the indicator cells (corresponding to the antibodies) are added, there are no antibodies that can react with the surface antigens of the indicator cells, so the experimental results show negative (no agglutination phenomenon); on the contrary, if the artificial antigen has weak specificity and low efficiency in binding with antibodies, the antibodies cannot be fully neutralized by the artificial antigen, then when the indicator cells (corresponding to the antibodies) are added, there are still antibodies that can react with the surface antigens of the indicator cells, so the experimental results show positive (with agglutination phenomenon). Through the above experimental design, the reaction performance of the artificially synthesized antigen (i.e., the blood group antigen trisaccharide B analog protein conjugate and the blood group antigen trisaccharide A analog protein conjugate described in the present invention) and the antibody can be effectively verified, that is, whether the artificially synthesized antigen has good specificity when recognizing its corresponding antibody.

[0196] The information of the A antibody standard used in this experiment is: brand Millipore, batch number JHE2103, item number JH-1L-BK. The information of the B antibody standard used in this experiment is: brand Millipore, batch number JMC2103, item number JM-1L-BK.

[0197] 5.1B-BSA antigen detection antibody

[0198] 1) First, dilute the B antibody standard with physiological saline by 10 times, 100 times, 1000 times, 10000 times, and 100000 times; then mix the diluted antibody (using physiological saline as a negative control) with B antigen (0.01 mg / mL) of different configurations and different BSA coupling ratios in equal volumes, and incubate at room temperature for about 15 minutes; then take 40 μL of the incubated mixture as the test object and add it to the above ABO blood type reverse typing test card; finally, add 10 μL of indicator cell B (with blood type antigen B), centrifuge and read the results. The results are as follows:

[0199] Table 5a

[0200]

[0201] 2) First, dilute the A antibody standard with physiological saline by 10 times, 100 times, 1000 times, 10000 times, and 100000 times; then mix the diluted antibody (using physiological saline as a negative control) with B antigen (0.01 mg / mL) of different configurations and different BSA coupling ratios in equal volumes, and incubate at room temperature for about 15 minutes; take 40 μL of the incubated mixture as the test object and add it to the above ABO blood type reverse typing test card, then add 10 μL of indicator cell B (with blood type antigen B), and read the results after centrifugation. The results are as follows:

[0202] Form 5b

[0203]

[0204]

[0205] Experimental results: Table 5a shows that the neutralization ability of B antigen (B1-B4) coupled with BSA to B antibody is weak overall. In terms of internal comparison of B-BSA, B2-BSA is relatively good (when the B antibody is diluted 10,000 times, the experimental results are negative at all coupling ratios), especially the neutralization ability when B2:BSA=20:1 is relatively strong (when the B antibody is diluted 1,000 times, it can be fully neutralized). When the B antibody is diluted 1,000 times, B1-BSA and B3-BSA show better B antibody neutralization ability at a coupling ratio of 20:1 relative to other coupling ratios of the same type of antigen; B4-BSA shows better B antibody neutralization ability at a coupling ratio of 40:1 relative to other coupling ratios of the same type of antigen. Table 5b is a control experiment using antibody A. The BSA protein coupled with antigen B (B1-B4) should not undergo a neutralization reaction, nor will it react with indicator cell B. Therefore, the experimental results are all negative, proving that this experimental system operates normally.

[0206] 5.2B-KLH antigen detection antibody

[0207] 1) First, dilute the B antibody standard with physiological saline by 10 times, 100 times, 1000 times, 10000 times, and 100000 times; then mix the diluted antibody (using physiological saline as a negative control) with B antigen (0.01 mg / mL) of different configurations and different KLH coupling ratios in equal volumes, and incubate at room temperature for about 15 minutes; then take 40 μL of the incubated mixture as the test object and add it to the above ABO blood type reverse typing test card; finally, add 10 μL of indicator cell B, centrifuge and read the results. The results are as follows:

[0208] Form 5c

[0209]

[0210] 2) First, dilute the A antibody standard with physiological saline by 10 times, 100 times, 1000 times, 10,000 times, and 100,000 times; then mix the diluted antibody (using physiological saline as a negative control) with B antigen (0.01 mg / mL) of different configurations and different KLH coupling ratios in equal volumes, and incubate at room temperature for about 15 minutes; then take 40 μL of the incubated mixture as the test object and add it to the above ABO blood type reverse typing test card; finally, add 10 μL of indicator cell B, centrifuge and read the results. The results are as follows:

[0211] Table 5d

[0212]

[0213] Experimental results: Table 5c shows that the neutralization ability of B antigen (B1-B4) coupled with KLH on B antibody is relatively good for B2-KLH, especially the neutralization ability is the strongest when B2:KLH=20:1 (even in a high concentration environment where B antibody is only diluted 10 times, B2:KLH=20:1 can still fully neutralize B antibody); B2:KLH=40:1 also shows good specificity, and can fully neutralize B antibody in a higher concentration environment where B antibody is diluted 100 times. B1-KLH, B3-KLH, and B4-KLH show better B antibody neutralization ability than other coupling ratios of the same type antigen when the coupling ratio is 40:1, and can fully neutralize B antibody in an environment where B antibody is diluted 1000 times. Table 5d is a control experiment using antibody A. KLH coupled with antigen B (B1-B4) should not undergo a neutralization reaction, nor will it react with indicator cell B. Therefore, the experimental results are all negative, proving that this experimental system operates normally.

[0214] 5.3A-BSA antigen detection antibody

[0215] 1) First, dilute the A antibody standard with physiological saline by 10 times, 100 times, 1000 times, 10000 times, and 100000 times; then mix the diluted antibody (physiological saline as a negative control) with A antigen (0.01 mg / mL) of different configurations and different BSA coupling ratios in equal volumes, and incubate at room temperature for about 15 minutes; then take 40 μL of the incubated mixture as the test object and add it to the above ABO blood type reverse typing test card; finally, add 10 μL of indicator cell A, centrifuge and read the results. The results are as follows:

[0216] Form 6a

[0217]

[0218]

[0219] 2) First, dilute the B antibody standard with physiological saline 10 times, 100 times, 1000 times, 10000 times, and 100000 times; then mix the diluted antibody (physiological saline as a negative control) with A antigen (0.01 mg / mL) of different configurations and different BSA coupling ratios in equal volumes, and incubate at room temperature for about 15 minutes; then take 40 μL of the incubated mixture as the test object and add it to the above ABO blood type reverse typing test card; finally, add 10 μL of indicator cell A, centrifuge and read the results. The results are as follows:

[0220] Form 6b

[0221]

[0222] Experimental results:

[0223] Table 6a shows that the neutralization ability of A antigen (A1-A4) coupled with BSA to antibody A is relatively good for A2-BSA, especially the strongest neutralization ability when A2:BSA=40:1 (even in a high concentration environment where antibody A is only diluted 10 times, A2:BSA=40:1 can still fully neutralize antibody A); A2-BSA also shows good specificity at other coupling ratios (10:1-20:1, 60:1–80:1), and can fully neutralize antibody A in a high concentration environment where antibody A is diluted 100 times. A3-BSA and A4-BSA show better neutralization ability against antibody A than other coupling ratios of the same type of antigen in the coupling ratio range of 40:1–80:1, and can fully neutralize antibody A in an environment where antibody A is diluted 1000 times. Table 6b is a control experiment using antibody B. KLH coupled with antigen A (A1-A4) should not undergo a neutralization reaction, nor will it react with indicator cell A. Therefore, the experimental results are all negative, proving that this experimental system operates normally.

[0224] 5.4A-KLH antigen detection antibody

[0225] 1) First, dilute the A antibody standard with physiological saline by 10 times, 100 times, 1000 times, 10000 times, and 100000 times; then mix the diluted antibody (physiological saline as a negative control) with A antigen (0.01 mg / mL) of different configurations and different KLH coupling ratios in equal volumes, and incubate at room temperature for about 15 minutes; then take 40 μL of the incubated mixture as the test object and add it to the above ABO blood type reverse typing test card; finally, add 10 μL of indicator cell A, centrifuge and read the results. The results are as follows:

[0226] Form 6c

[0227]

[0228] 2) First, dilute the B antibody standard with physiological saline by 10 times, 100 times, 1000 times, 10000 times, and 100000 times; then mix the diluted antibody (physiological saline as a negative control) with A antigen (0.01 mg / mL) of different configurations and different KLH coupling ratios in equal volumes, and incubate at room temperature for about 15 minutes; then take 40 μL of the incubated mixture as the test object and add it to the above ABO blood type reverse typing test card; finally, add 10 μL of indicator cell A, centrifuge and read the results. The results are as follows:

[0229] Form 6d

[0230]

[0231]

[0232] Experimental results: Table 6c shows that the neutralization ability of A antigen (A1-A4) coupled with KLH on antibody A is relatively good with A2-KLH, especially when A2:KLH=40:1, the neutralization ability is the strongest (even in a high concentration environment where antibody A is only diluted 10 times, A2:KLH=40:1 can still fully neutralize antibody A); A2-KLH also shows good specificity at other coupling ratios (10:1-20:1, 60:1-80:1), and can fully neutralize antibody A in a high concentration environment where antibody A is diluted 100 times. A3-KLH and A4-KLH also show a certain ability to neutralize antibody A at various coupling ratios (10:1–80:1), and can fully neutralize antibody A in an environment where antibody A is diluted 1000 times. Table 6d is a control experiment using antibody B. KLH coupled with antigen A (A1-A4) should not undergo a neutralization reaction, nor will it react with indicator cell A. Therefore, the experimental results are all negative, proving that this experimental system operates normally.

[0233] In the above experiments, blood group antigen trisaccharide A analog protein conjugates with obvious or relatively obvious experimental effects are summarized in Table 7a; blood group antigen trisaccharide A analog protein conjugates with obvious or relatively obvious experimental effects are summarized in Table 7b.

[0234] Form 7a

[0235]

[0236]

[0237] Form 7b

[0238]

[0239] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. Use of a blood type antigen trisaccharide analog protein conjugate in the preparation of a blood type antibody detection reagent, characterized in that: The blood group antigen trisaccharide analog in the blood group antigen trisaccharide analog protein conjugate is a blood group antigen trisaccharide A analog, which is type A2; the blood group antigen trisaccharide A analog is coupled with hemocyanin or bovine serum albumin to form a blood group antigen trisaccharide A analog protein conjugate, and the coupling ratio of the blood group antigen trisaccharide A analog to the hemocyanin or bovine serum albumin is 10:1-80:1; the A2 type chemical formula is as follows: Type A2.

2. The use according to claim 1, characterized in that The coupling ratio of the blood group antigen trisaccharide A analog to the hemocyanin is 10:1, 20:1, 40:1, 60:1 or 80:

1.

3. The use according to claim 1, characterized in that The coupling ratio of the blood group antigen trisaccharide A analog to bovine serum albumin is 10:1, 20:1, 40:1, 60:1 or 80:

1.

4. The use according to claim 1, characterized in that The blood type antibody detection reagent is used for immunochromatographic detection, optical density detection and column agglutination detection.

5. The use according to claim 4, characterized in that The blood type antibody detection reagent is used for immunochromatographic detection. When the blood type antigen trisaccharide A analog is coupled to hemocyanin, the coupling ratio is 40:1; when the blood type antigen trisaccharide A analog is coupled to bovine serum albumin, the coupling ratio is 40:

1.

6. An immunochromatographic detection kit, characterized in that: The invention comprises a test card, wherein the test card adopts a nitrocellulose membrane; the test card is coated with a conjugate of a blood group antigen trisaccharide A analogue and hemocyanin or bovine serum albumin, and is used to detect blood group antibodies in a serum sample; the blood group antigen trisaccharide A analogue is A2 type, and the coupling ratio of the blood group antigen trisaccharide A analogue to hemocyanin is 10:1, 20:1, 40:1, 60:1 or 80:1, and the coupling ratio of the blood group antigen trisaccharide A analogue to bovine serum albumin is 10:1, 20:1, 40:1, 60:1 or 80:1; the chemical formula of the A2 type is as follows: Type A2.

7. The immunochromatographic detection kit according to claim 6, characterized in that: When the blood group antigen trisaccharide A analog is coupled to hemocyanin, the coupling ratio is 40:1; when the blood group antigen trisaccharide A analog is coupled to bovine serum albumin, the coupling ratio is 40:

1.

8. A column agglutination kit, characterized in that: The invention comprises an ABO blood type reverse typing test card, a conjugate of a blood type antigen trisaccharide A analog and hemocyanin or bovine serum albumin, wherein the blood type antigen trisaccharide A analog is of type A2, and the conjugation ratio of the blood type antigen trisaccharide A analog to hemocyanin is 10:1, 20:1, 40:1, 60:1 or 80:1; the conjugation ratio of the blood type antigen trisaccharide A analog to bovine serum albumin is 10:1, 20:1, 40:1, 60:1 or 80:1; the chemical formula of type A2 is as follows: Type A2.

9. The column agglutination kit according to claim 8, characterized in that When the blood group antigen trisaccharide A analog is coupled to hemocyanin, the coupling ratio is 40:1; when the blood group antigen trisaccharide A analog is coupled to bovine serum albumin, the coupling ratio is 40:1.

Images