CN116868059A - Application of blood group antigen trisaccharide conjugate in blood group antibody detection - Google Patents
Application of blood group antigen trisaccharide conjugate in blood group antibody detection Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
Abstract
The invention belongs to the field of blood group antibody detection, relates to application of blood group antigen trisaccharide conjugate in blood group antibody detection, and in particular relates to application of blood group antigen trisaccharide A analogue protein conjugate and blood group antigen trisaccharide B analogue protein conjugate in blood group antibody detection. The blood group antigen trisaccharide B analog is coupled with hemocyanin or bovine serum albumin; when the blood group antigen trisaccharide B analogue is B2 type, the coupling ratio of the blood group antigen trisaccharide B analogue to the hemocyanin is 5:1-40:1, and the coupling ratio of the blood group antigen trisaccharide B analogue to the bovine serum albumin is 20:1; when the blood group antigen trisaccharide B analogue is B1 type, B3 type or B4 type, the coupling ratio of the blood group antigen trisaccharide B analogue to the hemocyanin is 40:1. The blood group antigen trisaccharide conjugate has good specificity, can identify corresponding blood group antibodies, has high detection accuracy, and can be applied to detection of blood group antibodies of various types.
Description
Technical Field
The invention belongs to the field of blood group antibody detection, relates to application of blood group antigen trisaccharide conjugate in blood group antibody detection, and in particular relates to application of blood group antigen trisaccharide A analogue protein conjugate and blood group antigen trisaccharide B analogue protein conjugate in blood group antibody detection.
Background
Blood typing is the primary work before clinical transfusion, because hemolysis phenomenon occurs due to the aggregation of antigens and antibodies when blood of different blood types is mutually transfused, and further life safety of people can be endangered, so that correct blood typing is a precondition for ensuring transfusion safety. Current blood typing methods include forward typing and reverse typing. The positive typing method detects erythrocyte antigens, and the negative typing method detects antibodies in serum, wherein the detection of human A, B, O blood group antigens/antibodies is the most important. According to the surface antigen of erythrocyte membrane in human blood, it can be divided into different blood types. The red blood cells of the blood type A have blood group antigen A (hereinafter referred to as "A antigen"), and the blood serum has blood group antibody B (hereinafter referred to as "B antibody"); the red blood cells of the B type blood have blood group antigen B (hereinafter referred to as B antigen), blood group antibody A (hereinafter referred to as A antibody) in serum, the red blood cells of the AB type blood have both A antigen and B antigen, and the serum has no A antibody and B antibody; the red blood cells of the O-type blood have no A antigen and B antigen, and the A antibody and the B antibody are simultaneously present in serum.
The current common blood type positive and negative typing generally adopts an agglutination method, and the principle is that the blood type antigen and antibody reaction is utilized to cause macroscopic red blood cell agglutination to judge the result. At positive timing, detecting erythrocyte antigens of the sample using IgM anti-a or anti-B reagents; reversely timed, igM blood group antibodies in the sample serum are determined using known red blood cell reagents of type a or B. However, since fresh erythrocytes are not easy to preserve (the antigenicity of erythrocyte surface blood group antigens gradually decreases with the prolongation of preservation time), the A or B type erythrocyte reagent for the anti-assay has higher requirements on preservation conditions and transportation conditions, increases the cost of the anti-assay and limits the application of the anti-assay. Some technical schemes in the prior art try to extract natural blood group antigens (for example, natural A antigen and B antigen are extracted through erythrocyte membranes) to replace complete erythrocytes in anti-setting detection, but related extraction and preparation processes are often complex and difficult to produce in mass, and the extracted natural blood group antigens still have higher requirements on preservation conditions. The artificial synthesis of blood group antigen is one of the research directions for solving the technical problems, but the currently known artificial synthesis blood group antigen has technical problems in the aspects of specificity, affinity, stability and the like, and cannot be effectively applied to the reverse-localization detection.
Disclosure of Invention
In view of this, the present invention provides the use of a blood group antigen trisaccharide conjugate, which is a blood group antigen trisaccharide analogue protein conjugate, comprising a blood group antigen trisaccharide B analogue, in the preparation of a blood group antibody detection reagent; the blood group antigen trisaccharide B analog is conjugated with hemocyanin (Keyhole Limpet Hemocyanin, KLH) or bovine serum albumin (Bovine Serum Albumin, BSA) to form a blood group antigen trisaccharide B analog protein conjugate; when the blood group antigen trisaccharide B analogue is B2 type, the coupling ratio of the blood group antigen trisaccharide B analogue to the hemocyanin is 5:1-40:1, and the coupling ratio of the blood group antigen trisaccharide B analogue to the bovine serum albumin is 20:1; when the blood group antigen trisaccharide B analogue is B1 type, B3 type or B4 type, the coupling ratio of the blood group antigen trisaccharide B analogue to the hemocyanin is 40:1.
In some embodiments, the blood group antigen trisaccharide B analog is of type B2 and the coupling ratio of the blood group antigen trisaccharide B analog to the hemocyanin coupling is 20:1.
In some embodiments, the blood group antigen trisaccharide analog further comprises a blood group antigen trisaccharide a analog; the blood group antigen trisaccharide A analogue is coupled with hemocyanin or bovine serum albumin to form a blood group antigen trisaccharide A analogue protein conjugate.
In some embodiments, when the blood group antigen trisaccharide a analog is of type A2, the coupling ratio of the blood group antigen trisaccharide a analog to the hemocyanin is 10:1-80:1; when the blood group antigen trisaccharide A analogue is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analogue to the hemocyanin is 10:1-80:1.
In some embodiments, the blood group antigen trisaccharide a analog is of type A2 and the coupling ratio of the blood group antigen trisaccharide a analog to the hemocyanin is 40:1.
In some embodiments, when the blood group antigen trisaccharide a analog is of type A2, the coupling ratio of the blood group antigen trisaccharide a analog to bovine serum albumin is 10:1 to 80:1; when the blood group antigen trisaccharide A analogue is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analogue to bovine serum albumin is 10:1 or 40:1-80:1.
In some embodiments, the blood group antigen trisaccharide a analog is of type A2 and the coupling ratio of the blood group antigen trisaccharide a analog to the bovine serum albumin is 40:1.
In some embodiments, the blood group antibody detection reagent is used for immunochromatographic detection, densitometric detection, or column agglutination detection.
In some embodiments, the blood group antibody detection reagent is used for immunochromatographic detection, and when the blood group antigen trisaccharide a analog is a type A3 or a type A4, the coupling ratio of the blood group antigen trisaccharide a analog to the hemocyanin is 10:1 or 40:1-80:1.
The invention also provides an immunochromatography detection kit, which comprises a detection card, wherein the detection card adopts a nitrocellulose membrane; the conjugate of the blood group antigen trisaccharide B analogue and hemocyanin or bovine serum albumin is coated on the detection card and is used for detecting blood group antibodies in a serum sample; when the blood group antigen trisaccharide B analogue is B2 type, the coupling ratio of the blood group antigen trisaccharide B analogue to the hemocyanin is 5:1-40:1, and the coupling ratio of the blood group antigen trisaccharide B analogue to the bovine serum albumin is 20:1; when the blood group antigen trisaccharide B analogue is B1 type, B3 type or B4 type, the coupling ratio of the blood group antigen trisaccharide B analogue to hemocyanin is 40:1.
In some embodiments, the test card is further coated with a conjugate of a blood group antigen trisaccharide a analog with hemocyanin or a conjugate with bovine serum albumin.
In some embodiments, when the blood group antigen trisaccharide a analog is of type A2, the coupling ratio of the blood group antigen trisaccharide a analog to hemocyanin is 10:1-80:1; when the blood group antigen trisaccharide A analogue is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analogue to hemocyanin is 10:1 or 40:1-80:1.
In some embodiments, when the blood group antigen trisaccharide a analog is of type A2, the coupling ratio of the blood group antigen trisaccharide a analog to bovine serum albumin is 10:1 to 80:1; when the blood group antigen trisaccharide A analogue is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analogue to bovine serum albumin is 10:1 or 40:1-80:1.
The invention also provides a column agglutination kit which is characterized by comprising an ABO blood group inverse typing detection card and a blood group antigen trisaccharide analogue protein conjugate; the blood group antigen trisaccharide analogue protein conjugate comprises coupling of blood group antigen trisaccharide B analogues with hemocyanin or bovine serum albumin; when the blood group antigen trisaccharide B analogue is B2 type, the coupling ratio of the blood group antigen trisaccharide B analogue to hemocyanin is 10:1-40:1, and the coupling ratio of the blood group antigen trisaccharide B analogue to bovine serum albumin is 20:1; when the blood group antigen trisaccharide B analogue is B1 type, B3 type or B4 type, the coupling ratio of the blood group antigen trisaccharide B analogue to hemocyanin coupling is 40:1.
In some embodiments, the blood group antigen trisaccharide analog protein conjugate further comprises a blood group antigen trisaccharide a analog conjugated to hemocyanin or bovine serum albumin; when the blood group antigen trisaccharide A analogue is A2 type, the coupling ratio of the blood group antigen trisaccharide A analogue to hemocyanin or bovine serum albumin is 10:1-80:1; when the blood group antigen trisaccharide A analogue is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analogue to hemocyanin is 10:1-80:1, and the coupling ratio of the blood group antigen trisaccharide A analogue to bovine serum albumin is 10:1 or 40:1-80:1.
Beneficial technical effects
1) Experiments prove that the blood group antigen trisaccharide A analogue (A1-A4) protein conjugate (coupled BSA or KLH) and the blood group antigen trisaccharide B analogue (B1-B4) protein conjugate (coupled BSA or KLH) adopted by the technical scheme of the invention have good specificity (see tables 7a and 7B), can be used as artificially synthesized blood group antigens, can distinguish and identify corresponding blood group antibodies, has high detection accuracy, can be used for detecting blood group antibodies of various types, and has huge potential clinical application value. Especially in the immunochromatography detection, the partial blood group antigen trisaccharide analogue protein conjugate adopted in the technical scheme of the invention can be stably fixed on detection test paper, and the corresponding blood group antibodies can be efficiently captured, and the color development effect (for example, color development through colloidal gold labeling) is easy to identify and visible to naked eyes, so that the partial blood group antigen trisaccharide analogue protein conjugate has the potential of being applied to the scenes of rapid blood group screening, POCT detection, general human blood group self-checking and the like.
2) The blood group antigen trisaccharide analogue protein conjugate adopted by the invention is synthesized by a chemical process, is easy to realize industrial mass production, can be used as blood group antigen raw materials in various experimental systems, and can replace erythrocyte reagents or natural blood group antigens in reverse determination detection, thereby avoiding complex processes of extracting and preparing the natural blood group antigens and reducing the cost of blood group detection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are of some embodiments of the invention and that other drawings may be derived from these drawings without inventive faculty.
FIG. 1 is a graph showing the effect of the B1-B2 conjugate with BSA on nitrocellulose membrane (NC membrane) in the examples of the present invention;
FIG. 2 is a graph showing the color development effect of B3-B4 and BSA conjugates on NC membrane in the examples of the present invention;
FIG. 3 is a graph showing the color development effect of the conjugates of B1-B2 and KLH on NC film according to the examples of the invention;
FIG. 4 is a graph showing the color development effect of the conjugates of B3-B4 and KLH on NC film according to the examples of the invention;
FIG. 5 is a graph showing the effect of the color development of the conjugates A1-A2 with BSA on NC membrane in the examples of the present invention;
FIG. 6 is a graph showing the effect of color development of the conjugates A3-A4 with BSA on NC membrane in the examples of the present invention;
FIG. 7 is a graph showing the effect of a conjugate of A1-A2 and KLH on NC film according to the examples of the invention;
FIG. 8 is a graph showing the effect of a conjugate of A3-A4 and KLH on NC film according to the examples of the invention;
FIG. 9 is a graph showing the effect of A1-KLH detection A, B antibody blood typing detection cards;
FIG. 10 is a graph showing the effect of A2-KLH detection A, B antibody on blood typing reversal test cards;
FIG. 11 is a graph showing the effect of A3-KLH detection A, B antibody on blood typing reversal test cards;
FIG. 12 is a graph showing the effect of A4-KLH detection A, B antibody on blood typing reversal test cards;
FIG. 13 is a graph showing the effect of A1-BSA detection A, B on a blood group reverse typing detection card;
FIG. 14 is a graph showing the effect of A2-BSA detection A, B on the blood typing reversal test card;
FIG. 15 is a graph showing the effect of A3-BSA detection A, B on a blood group reverse typing detection card;
FIG. 16 is a graph showing the effect of A4-BSA detection A, B on a blood group reverse typing detection card;
FIG. 17 is a graph showing the effect of the B1-BSA detection A, B antibody on the blood typing reversal test card;
FIG. 18 is a graph showing the effect of the B2-BSA detection A, B antibody on the blood typing test card;
FIG. 19 is a graph showing the effect of the B3-BSA detection A, B antibody on the blood typing reversal test card;
FIG. 20 is a graph showing the effect of the B4-BSA detection A, B antibody on the blood typing test card;
FIG. 21 is a graph showing the effect of the B1-KLH detection A, B antibody on blood typing reversal test cards;
FIG. 22 is a graph showing the effect of the B2-KLH detection A, B antibody on blood typing reversal test cards;
FIG. 23 is a graph showing the effect of the B3-KLH detection A, B antibody on blood typing reversal test cards;
FIG. 24 is a chart showing the effect of the B4-KLH detection A, B antibody on blood typing reversal test cards;
FIG. 25 is a synthetic route pattern for a trisaccharide B analog type I of the present invention;
FIG. 26 is a synthetic route pattern for the trisaccharide B analog type II of the present invention;
FIG. 27 is a synthetic route pattern of the blood group antigen trisaccharide B analog type III of the present invention;
FIG. 28 is a synthetic route pattern of the blood group antigen trisaccharide B analog type IV of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As used in this specification, the term "about" is typically expressed as +/-5% of the value, more typically +/-4% of the value, more typically +/-3% of the value, more typically +/-2% of the value, even more typically +/-1% of the value, and even more typically +/-0.5% of the value.
In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be appreciated that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a inflexible limitation on the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, a rangeThe description of (c) should be taken as having 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 such ranges, e.g., 1,2,3,4,5, and 6. The above rule applies regardless of the breadth of the range.
The blood group antigen trisaccharide A analogue disclosed by the invention refers to four compounds and derivatives thereof with basic chemical formulas shown as chemical formulas A1-A4, namely an I type A antigen compound (chemical formula A1, hereinafter referred to as "A1" or "A1 type"), an II type A antigen compound (chemical formula A2, hereinafter referred to as "A2" or "A2 type"), a III type A antigen compound (chemical formula A3, hereinafter referred to as "A3" or "A3 type") and an IV type A4 antigen compound (chemical formula A4, hereinafter referred to as "A4" or "A4 type").
The blood group antigen trisaccharide B analog refers to four compounds and derivatives thereof with basic chemical formulas shown as chemical formulas B1-B4, namely a type I B antigen compound (chemical formula B1, hereinafter referred to as "B1" or "B1"), a type II B antigen compound (chemical formula B2, hereinafter referred to as "B2" or "B2"), a type III B antigen compound (chemical formula B3, hereinafter referred to as "B3" or "B3") and a type IV B antigen compound (chemical formula B4, hereinafter referred to as "B4" or "B4").
The "blood group antigen trisaccharide analogue protein conjugate" as referred to in the present invention refers to a protein conjugate (hereinafter referred to as "A-BSA", "A-KLH") formed by coupling the above blood group antigen trisaccharide A analogue with BSA or KLH protein, and a protein conjugate (hereinafter referred to as "B-BSA", "B-KLH") formed by coupling the above blood group antigen trisaccharide B analogue with BSA or KLH protein.
Example one blood group antigen trisaccharide analog protein conjugate preparation
Preparation of 1.1 blood group antigen trisaccharide A analogue protein conjugate
In some embodiments, two target sugar blocks may be synthesized first, followed by a glycosylation reaction to synthesize target sugar chains, following a modular assembly strategy method of the Bovin subject group (druulolar S, et al, large-scale synthesis of H-antigen oligosaccharides by expressing helicobacter pyloria1,2-fucosyltransferase in metabolically engineered Escherichia coli cells [ J ]. Angelw Chem,2006,118 (11): 1810-1812), the main reaction steps being illustrated below:
Then, the target sugar chain terminal position generated by the method and butyramide connected with carboxyl undergo an addition reaction to generate the antigen trisaccharide A analogue of the invention, which is of a total of four types (A1-A4), and the chemical structural formula is shown as chemical formulas A1-A4. Finally, a linear connecting arm with low immunogenicity, good reactivity and high coupling rate is used for coupling the blood group antigen trisaccharide A analogue with amino groups on lysine residues of BSA protein or KLH protein (for example, an alkane chain which is pre-loaded on an initial substrate and is connected with azido is used as a connecting arm during sugar chain synthesis, and the coupling of the blood group antigen trisaccharide analogue and carrier protein is realized through a dual-activation ester reagent), so that the blood group antigen trisaccharide A analogue protein conjugate (A1-BSA, A2-BSA, A3-BSA, A4-BSA; A1-KLH, A2-KLH, A3-KLH and A4-KLH) is synthesized, and the chemical formula is shown as follows:
A1-BSA
A2-BSA
A3-BSA
A4-BSA
A1-KLH:
A2-KLH:
A3-KLH
A4-KLH
wherein n=10, 20, 40, 60, 80, i.e. the ratio of the blood group antigen trisaccharide a analogue to protein (BSA or KLH) coupled ranges from 10:1 to 80:1, see in particular table 1.
Preparation of 1.2 blood group antigen trisaccharide B analogue protein conjugate
First, 4 disaccharide precursors were synthesized using a strategy of chemically introducing an azide group-containing linker at the reducing end of the starting monosaccharide (GlcNAc, glaNAc), followed by chemically synthesizing GlcNAc. Beta. ProN 3 ，GalNAcαProN 3 ，GalNAcβProN 3 The monosaccharide is used as a receptor, and the synthesis of 4 types of precursors is completed by a one-pot multienzyme method, and the chemical formula is as follows:
blood group antigen trisaccharide B analog disaccharide precursor type one:
blood group antigen trisaccharide B analog disaccharide precursor type two:
blood group antigen trisaccharide B analog disaccharide precursor type three:
blood group antigen trisaccharide B analog disaccharide precursor type four:
then, an alpha 1-3 galactose glycosyltransferase GTB is introduced into the non-reducing end of the synthesized 4 disaccharide precursors to synthesize 4 blood group antigen trisaccharide B analogues B1-B4 (the molecular structure is shown as chemical formulas B1-B4), and the specific synthetic route is shown in figures 25-28.
Finally, a linear connecting arm with low immunogenicity, good reactivity and high coupling rate is used for coupling the blood group antigen trisaccharide B analogue (B1-B4) with amino groups on lysine residues of BSA protein or KLH protein (for example, an alkane chain which is pre-arranged on a starting substrate and is connected with azido groups is used as a connecting arm in the process of sugar chain synthesis, and the coupling of the blood group antigen trisaccharide analogue and carrier protein is realized through a double-activated ester reagent), so as to synthesize an antigen trisaccharide B analogue protein conjugate (B1-BSA, B2-BSA, B3-BSA, B4-BSA; B1-KLH, B2-KLH, B3-KLH, B4-KLH), wherein the chemical formula is as follows:
B1-BSA
B2-BSA
B3-BSA
B4—BSA
B1-KLH
B2-KLH
B3-KLH
B4-KLH
Wherein n=5, 10, 20, 40, i.e. the ratio of the blood group antigen trisaccharide B analogue to KLH coupling ranges from 5:1 to 40:1, see in particular table 1.
Table 1 blood group antigen trisaccharide analogues and protein coupling ratio
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Example two
Qualitative verification of blood group antigen trisaccharide analogue protein conjugate (antigen solidification immunochromatography technique)
In order to study whether the synthesized blood group antigen trisaccharide analogue protein conjugate can be used for simple and rapid qualitative blood group detection, the invention adopts an antigen curing method immunochromatography technology for verification. For detecting the antibodies a and B in serum, a detection card (see fig. 1 to 8) assembled by a certolius Nitrocellulose (NC) film CN140 is used, and the NC film includes a detection line (i.e., T line) and a quality control line (i.e., C line). And the T line is respectively coated with blood group antigen trisaccharide A analogue protein conjugate or blood group antigen trisaccharide B analogue protein conjugate in a solidifying way, and the C line is coated with an anti-chicken IgY antibody. The colloidal gold/microsphere pad contains labeled anti-human mu chain antibody and chicken IgY antibody.
At the time of detection, the sample is added from the loading well (labeled S on the detection card). Blood group antibody A or antibody B contained in the sample can be combined with anti-human mu chain antibody marked by colloidal gold to form a complex and is chromatographed on NC membrane, when the complex is chromatographed to a T line, the blood group antibody A or B is specifically combined with a corresponding blood group antigen trisaccharide A analogue protein conjugate or blood group antigen trisaccharide B analogue protein conjugate to form a macroscopic color development line (namely, the blood group antibody A is combined with the artificial synthetic antigen A, and the blood group antibody B is combined with the artificial synthetic antigen B). If the corresponding antibody a or antibody B is not present in the sample, a complex cannot be formed, and color cannot be developed at the T line. Based on this, when the sample contains the antibody a and does not contain the antibody B, it is blood type a; the sample contains the antibody B, does not contain the antibody A, and is B-type blood; the sample does not contain an antibody A and an antibody B, and is AB type blood; the sample contains antibody A and antibody B, which are O-type blood. The sample is chromatographed to the T line and then to the C line, and the C line is chromogenic when all the samples are detected, otherwise, the test is invalid.
Detection card in the present application, the detection card coated with the blood group antigen trisaccharide A analog protein conjugate (A-BSA, A-KLH) is referred to as an A detection card in the present application; the detection card coated with the blood group antigen trisaccharide B analog protein conjugate (B-BSA, B-KLH) is referred to as B detection card in the present application.
1 μl of standard blood group antibody A (brand Millipore, batch No. JHE2103, cat No. JH-1L-BK) is added to each A detection card, 80 μl of sample diluent (0.01 MPBS) is added, and after standing for about 15-20 min, the color development at the C line and the T line is observed with naked eyes, and the record is photographed.
1 μl of standard blood group antibody B (Millipore, batch JMC2103, JM-1L-BK) was added to each B test card, and 80 μl of sample diluent (0.01 MPBS) was added thereto, and the mixture was left for about 15min to 20min, and the color development at the C line and T line was observed with naked eyes, and recorded by photographing.
The above-described detection results are shown in FIGS. 1 to 8 and summarized in tables 2a to 2d, with the developed macroscopic being indicated by a "+" sign and the macroscopic being indicated by a "-" sign.
The detection result shows that: for the trisaccharide B analogues of the blood group antigen, both B2 type and BSA or KLH can be effectively visualized on the T-line after coupling. However, the color development effect of B2-BSA is obvious only when the coupling ratio is 20:1, and B2-KLH shows better color development effect in a wider coupling ratio range (from 5:1 to 40:1). In addition, when the coupling ratio of B-KLH is 40:1, the color development effect of B1-KLH, B3-KLH and B4-KLH can be seen with naked eyes. For the blood group antigen trisaccharide A analogues, the formed blood group antigen trisaccharide analogue protein conjugate can be effectively developed on a T line (coupling ratio: 10:1,40:1,60:1, 80:1) no matter the A2-A4 is coupled with BSA or coupled with KLH, but when the protein coupling ratio of the A-BSA to the A-KLH is 20:1, only the A2-BSA/A2-KLH can be effectively developed, and neither the A3-BSA/A3-KLH nor the A4-BSA/A4-KLH can be effectively developed. In addition, neither A1 was coupled to BSA nor KLH, and color was not developed efficiently at the coupling ratios tested in the present application.
The above experimental results show that when specific blood group antigen trisaccharide analogues are conjugated with specific proteins (for example, B2-KLH, A2-BSA, A2-KLH), or at specific conjugation ratios (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)), the blood group antigen trisaccharide analogues can be effectively developed on the T line to the extent that the blood group antigen trisaccharide analogues can be stably and effectively immobilized on NC membranes or other types of solid phase media (for example, ELISA plates, microspheres of various materials or magnetic beads), and can effectively and specifically capture the corresponding blood group antibodies, thus the blood group antigen trisaccharide analogues can be used for rapid and simple qualitative detection of blood group antibodies (for example, can be read by the naked eye), blood group antigen trisaccharide analogues can be stably immobilized on NC membranes or other types can be detected at home, and can be done in a small scale.
It will be apparent to those skilled in the art that the blood group antigen trisaccharide analog protein conjugates of the present invention can also be used in assay systems employing other labeling means (e.g., enzymatic, fluorescent, or other luminescent labels). In addition, besides labeling the antibody, the blood group antigen trisaccharide analogue protein conjugate can be labeled by different labeling methods, and can be used for various heterogeneous or homogeneous detection according to actual requirements.
Table 2a
Table 2b
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Table 2c
Table 2d
Example III
Quantitative validation of blood group antigen trisaccharide B analog protein conjugates
3.1 blood group antigen trisaccharide B coupled KLH protein experiment
1) Detection results of 100-fold dilution of Standard antibody
The main experimental reagents used in this experiment include: latex microspheres (125 μl latex microspheres, co-labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH 8.5).
The loading mode of this experiment is as follows: the A, B antibody standard (Millipore, batch No. JHE2103, cat# JH-1L-BK; millipore, batch No. JMC2103, cat# JM-1L-BK) was diluted 100-fold with R1 to obtain a diluted A, B antibody standard having a volume of 200. Mu.L, and then 200. Mu.L of a reagent R2 (i.e., the latex microsphere was diluted 100-fold with TBS) was added to obtain 400. Mu.L of a sample to be tested. In this experiment, the detection wavelength was 340nm.
The above experimental results are summarized in the following table:
TABLE 3a
| Coupling ratio | OD value of antibody A | OD value of antibody B | P/N |
| B1:KLH＝5:1 | 0.251 | 0.362 | 1.442 |
| B1:KLH＝10:1 | 0.239 | 0.338 | 1.416 |
| B1:KLH＝20:1 | 0.253 | 0.384 | 1.519 |
| B1:KLH＝40:1 | 0.257 | 0.417 | 1.622 |
| B2:KLH＝5:1 | 0.237 | 0.367 | 1.547 |
| B2:KLH＝10:1 | 0.236 | 0.405 | 1.715 |
| B2:KLH＝20:1 | 0.261 | 0.642 | 2.459 |
| B2:KLH＝40:1 | 0.227 | 0.424 | 1.869 |
| B3:KLH＝5:1 | 0.243 | 0.350 | 1.442 |
| B3:KLH＝10:1 | 0.229 | 0.343 | 1.498 |
| B3:KLH＝20:1 | 0.234 | 0.334 | 1.427 |
| B3:KLH＝40:1 | 0.251 | 0.405 | 1.614 |
| B4:KLH＝5:1 | 0.237 | 0.342 | 1.441 |
| B4:KLH＝10:1 | 0.242 | 0.362 | 1.497 |
| B4:KLH＝20:1 | 0.248 | 0.374 | 1.508 |
| B4:KLH＝40:1 | 0.252 | 0.410 | 1.627 |
The experimental results show that after the antigen B and the KLH are coupled, no matter which type of B1-B4 is adopted, and no matter which coupling ratio of the antigen B and the KLH is in the range of 5:1-40:1, the OD value (more than or equal to 0.334) of the antigen B combined with the B antibody is obviously higher than the OD value (the highest OD value can only reach 0.261) obtained when the A antibody is added, and the P/N value (positive serum OD value/negative serum OD value) can reach more than 1.4, namely, the antigen B adopted by the invention can well distinguish the A antibody from the B antibody in the detection of blood type antibodies, and has better specificity. Notably, for B1-KLH, B3-KLH and B4-KLH, the P/N value was highest at a coupling ratio of 40:1, i.e.the best discrimination between antibodies A and B. The P/N value of B2-KLH is higher in the coupling ratio range employed in the present invention (5:1-40:1), especially at coupling ratios of 20:1, with P/N values as high as 2.459 being optimal.
2) Detection results of dilution of Standard antibody by 200-fold
In order to further verify the detection effect of the blood group antigen trisaccharide B analogue protein conjugate (B-KLH) synthesized by the invention when the concentration of the antibody is low, the experiment dilutes the standard antibody by 200 times for detection. The main experimental reagents used in this experiment include: latex microspheres (125 μl latex microsphere labeled 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH 8.5). The loading mode of this experiment is as follows: the A, B antibody standard was diluted 200-fold with R1 (Millipore, lot number JHE2103, cat number JH-1L-BK; millipore, lot number JMC2103, cat number JM-1L-BK), to obtain a diluted antibody standard having a volume of 200. Mu.L, and then 200. Mu.L of a reagent R2 (the latex microsphere was diluted 200-fold with TBS) was added thereto to obtain 400. Mu.L of a sample to be tested. In this experiment, the detection wavelength was 340nm.
The results of the above experiments are summarized in the following table:
TABLE 3b
| Coupling ratio | OD value of antibody A | OD value of antibody B | 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 |
The above experimental results show that even though the standard antibody is diluted 200 times, the OD value (0.300 or more) of the antigen B combined with the B antibody is obviously higher than that obtained when the A antibody is added, the P/N value can almost reach 1.3 or more (except that only the coupling ratio of B1-KLH to B3-KLH is 5:1) no matter which type of B1-B4 is adopted or the coupling ratio of the antigen B to KLH is 5:1-40:1. In other words, the antigen B employed in the present invention can distinguish well between the a-antibody and the B-antibody in the detection of blood group antibodies even in the case of a low antibody concentration.
3.2 blood group antigen trisaccharide B binding BSA protein experiment
1) Detection results of 100-fold dilution of Standard antibody
The main experimental reagents used in this experiment include: latex microspheres (125 μl latex microspheres, co-labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH 8.5).
The loading mode of this experiment is as follows: the A, B antibody standard (Millipore, lot number JHE2103, cat number JH-1L-BK; millipore, lot number JMC2103, cat number JM-1L-BK) was diluted 100 times with R1 to obtain a diluted antibody standard having a volume of 200. Mu.L, and then 200. Mu.L of a reagent R2 (100 times the dilution of the latex microsphere with TBS) was added thereto to obtain 400. Mu.L of a sample to be tested. In this experiment, the detection wavelength was 340nm.
The above experimental results are summarized in the following table:
TABLE 3c
| Coupling ratio | OD value of antibody A | OD value of antibody B | 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 |
The above experimental results show that the OD value of the antigen B combined with the B antibody is different from the OD value obtained when the A antibody is added, but is not the same as the difference of the OD value when the antigen B is coupled with KLH when the A antibody and the B antibody are identified, no matter which type of B1-B4 is adopted or the coupling ratio of the antigen B and the BSA is adopted in the range of 5:1-40:1, and the P/N value of multiple groups of data is between 1.231 and 1.678. The above results demonstrate that the blood group antigen trisaccharide B (B1-B4) analog conjugated BSA protein also detects B antibodies, and that the a antibodies and B antibodies are distinguished in the above experiments, but their ability and effect are inferior to those of the blood group antigen trisaccharide B (B1-B4) analog conjugated KLH protein.
2) Detection results of dilution of Standard antibody by 200-fold
In order to further verify the detection effect of the blood group antigen trisaccharide B analogue protein conjugate (B-BSA) synthesized by the invention when the concentration of the antibody is low, the experiment is carried out by diluting the standard antibody by 200 times. The main experimental reagents used in this experiment include: latex microspheres (125 μl latex microsphere labeled 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH 8.5). The loading mode of this experiment is as follows: the A, B antibody standard was diluted 200-fold with R1 (Millipore, lot number JHE2103, cat number JH-1L-BK; millipore, lot number JMC2103, cat number JM-1L-BK), to obtain a diluted antibody standard having a volume of 200. Mu.L, and then a reagent R2 was added (the latex microsphere was diluted 200-fold with TBS) to obtain 400. Mu.L of the sample to be tested. In this experiment, the detection wavelength was 340nm.
The above experimental results are summarized in the following table:
TABLE 3d
| Coupling ratio | OD value of antibody A | OD value of antibody B | 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 |
The above experimental results were similar to the 100-fold dilution of standard antibody, and the OD value of antigen B binding to B antibody was different from that obtained when A antibody was added, regardless of the type of B1-B4 and the ratio of coupling to BSA in the range of 5:1-40:1, but clearly was not as different from that when antigen B was coupled to KLH, in recognizing the A antibody and the B antibody. Only B2: at bsa=20:1, the P/N value can reach 1.421;
B3: at bsa=20:1, the P/N value can reach 1.394. In conjunction with NC membrane colour experiments (table 2 a), only B2: at bsa=20:1, the chromogenic reaction can be seen with the naked eye on the test card. The above results demonstrate that the blood group antigen trisaccharide B (B1-B4) analogue conjugated BSA protein is also able to detect B antibodies to some extent, distinguishing a antibodies from B antibodies, but its ability is significantly inferior to the blood group antigen trisaccharide B (B1-B4) analogue conjugated KLH protein.
Example IV
Quantitative verification of blood group antigen trisaccharide A analogue protein conjugate (optical density experiment)
4.1 blood group antigen trisaccharide A binding BSA protein experiment
1) Detection results of 100-fold dilution of Standard antibody
The main experimental reagents used in this experiment include: latex microspheres (125 μl latex microspheres, co-labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH 8.5).
The loading mode of this experiment is as follows: the A, B antibody standard (Millipore, lot number JHE2103, cat number JH-1L-BK; millipore, lot number JMC2103, cat number JM-1L-BK) was diluted 100 times with R1 to obtain a diluted antibody standard having a volume of 200. Mu.L, and then 200. Mu.L of a reagent R2 (100 times the dilution of the latex microsphere with TBS) was added thereto to obtain 400. Mu.L of a sample to be tested. In this experiment, the detection wavelength was 340nm.
The above experimental results are summarized in the following table:
table 4a
| Coupling ratio | OD value of antibody A | OD value of antibody B | P/N |
| A1:BSA＝10:1 | 0.281 | 0.235 | 1.194 |
| A1:BSA＝20:1 | 0.284 | 0.244 | 1.163 |
| A1:BSA＝40:1 | 0.312 | 0.239 | 1.305 |
| A1:BSA＝60:1 | 0.307 | 0.251 | 1.223 |
| A1:BSA＝80:1 | 0.281 | 0.253 | 1.109 |
| A2:BSA＝10:1 | 0.420 | 0.228 | 1.844 |
| A2:BSA＝20:1 | 0.453 | 0.231 | 1.959 |
| A2:BSA＝40:1 | 0.619 | 0.224 | 2.764 |
| A2:BSA＝60:1 | 0.428 | 0.236 | 1.813 |
| A2:BSA＝80:1 | 0.407 | 0.225 | 1.809 |
| A3:BSA＝10:1 | 0.416 | 0.243 | 1.711 |
| A3:BSA＝20:1 | 0.290 | 0.239 | 1.212 |
| A3:BSA＝40:1 | 0.407 | 0.241 | 1.688 |
| A3:BSA＝60:1 | 0.410 | 0.242 | 1.696 |
| A3:BSA＝80:1 | 0.392 | 0.227 | 1.725 |
| A4:BSA＝10:1 | 0.415 | 0.238 | 1.744 |
| A4:BSA＝20:1 | 0.364 | 0.244 | 1.491 |
| A4:BSA＝40:1 | 0.408 | 0.237 | 1.723 |
| A4:BSA＝60:1 | 0.432 | 0.245 | 1.764 |
| A4:BSA＝80:1 | 0.404 | 0.237 | 1.705 |
The above experimental results show that when antigen a employed in the present invention is coupled to BSA, the vast majority of A2-A4 (except a3:bsa=20:1, a4:bsa=20:1) is higher than the OD of antibody a bound to a (0.4 or more) than the OD of antibody B bound (up to only 0.245) regardless of the coupling ratio with BSA in the range of 10:1-80:1. Experiments prove that the antigen A2-A4 adopted by the invention can well distinguish the A antibody from the B antibody in the detection of blood group antibodies, and particularly the distinguishing effect of A2: BSA=40:1 is the best. In contrast, A1-BSA coupling was less able to distinguish between the recognition of the A and B antibodies, with P/N values only slightly above 1.
2) Detection results of dilution of Standard antibody by 200-fold
In order to further verify the detection effect of the synthesized blood group antigen trisaccharide A analogue protein conjugate (A-BSA) when the concentration of the antibody is low, the experiment dilutes the standard antibody by 200 times for detection. The main experimental reagents used in this experiment include: latex microspheres (125 μl latex microsphere labeled 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH 8.5). The loading mode of this experiment is as follows: the A, B antibody standard was diluted 200-fold with R1 (Millipore, lot number JHE2103, cat number JH-1L-BK; millipore, lot number JMC2103, cat number JM-1L-BK), to obtain a diluted antibody standard having a volume of 200. Mu.L, and then 200. Mu.L of a reagent R2 (the latex microsphere was diluted 200-fold with TBS) was added thereto to obtain 400. Mu.L of a sample to be tested. In this experiment, the detection wavelength was 340nm.
The above experimental results are summarized in the following table:
table 4b
| Coupling ratio | OD value of antibody A | OD value of antibody B | P/N |
| A1:BSA＝10:1 | 0.273 | 0.235 | 1.161 |
| A1:BSA＝20:1 | 0.269 | 0.244 | 1.102 |
| A1:BSA＝40:1 | 0.304 | 0.239 | 1.271 |
| A1:BSA＝60:1 | 0.282 | 0.251 | 1.125 |
| A1:BSA＝80:1 | 0.277 | 0.253 | 1.093 |
| A2:BSA＝10:1 | 0.382 | 0.228 | 1.674 |
| A2:BSA＝20:1 | 0.396 | 0.231 | 1.715 |
| A2:BSA＝40:1 | 0.575 | 0.224 | 2.569 |
| A2:BSA＝60:1 | 0.400 | 0.236 | 1.694 |
| A2:BSA＝80:1 | 0.387 | 0.225 | 1.718 |
| A3:BSA＝10:1 | 0.394 | 0.243 | 1.621 |
| A3:BSA＝20:1 | 0.267 | 0.239 | 1.119 |
| A3:BSA＝40:1 | 0.368 | 0.241 | 1.527 |
| A3:BSA＝60:1 | 0.365 | 0.242 | 1.508 |
| A3:BSA＝80:1 | 0.368 | 0.227 | 1.622 |
| A4:BSA＝10:1 | 0.375 | 0.238 | 1.577 |
| A4:BSA＝20:1 | 0.316 | 0.244 | 1.296 |
| A4:BSA＝40:1 | 0.381 | 0.237 | 1.609 |
| A4:BSA＝60:1 | 0.397 | 0.245 | 1.622 |
| A4:BSA＝80:1 | 0.365 | 0.237 | 1.538 |
The above experimental results show that even when the standard sample is diluted 200 times and the concentration of the antibody is low, the antigen A2-A4 used in the invention can be used for coupling BSA, no matter which coupling ratio (A3: BSA=20:1, A4: BSA=20:1) is used for coupling BSA in the range of 10:1-80:1, the P/N value is relatively high, namely, the A antibody and the B antibody can still be well distinguished in the detection of blood group antibodies, and particularly, the distinction effect of A2: BSA=40:1 is the best. In contrast, A1-BSA coupling was less able to distinguish between the recognition of the A and B antibodies, with P/N values only slightly above 1.
4.2 blood group antigen trisaccharide A binding KLH protein experiment
1) Detection results of 100-fold dilution of Standard antibody
The main experimental reagents used in this experiment include: latex microspheres (125 μl latex microspheres, co-labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH 8.5).
The loading mode of this experiment is as follows: the A, B antibody standard (Millipore, lot number JHE2103, cat number JH-1L-BK; millipore, lot number JMC2103, cat number JM-1L-BK) was diluted 100 times with R1 to obtain a diluted antibody standard having a volume of 200. Mu.L, and then 200. Mu.L of a reagent R2 (100 times the dilution of the latex microsphere with TBS) was added thereto to obtain 400. Mu.L of a sample to be tested. In this experiment, the detection wavelength was 340nm.
The above experimental results are summarized in the following table:
table 4c
| Coupling ratio | OD value of antibody A | OD value of antibody B | 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 |
The above experimental results show that when the standard sample is diluted 100 times, the OD value (0.4 or more) of the antigen A binding A antibody is higher than the OD value (only up to 0.293) of the antigen B antibody, no matter which coupling ratio of the antigen A to the KLH is used in the range of 10:1-80:1, in the vast majority of the antigen A to the A2-A4 used in the invention is used for coupling the KLH. Experiments prove that the antigen A2-A4 adopted by the invention can well distinguish the A antibody from the B antibody in the detection of blood group antibodies, and particularly the distinguishing effect of A2:KLH=40:1 is the best. In contrast, A1-KLH coupling is relatively poor in distinguishing between the recognition of the A and B antibodies, with P/N values only slightly above 1.
2) Detection results of dilution of Standard antibody by 200-fold
In order to further verify the detection effect of the blood group antigen trisaccharide A analogue protein conjugate (A-KLH) synthesized by the invention when the concentration of the antibody is low, the experiment dilutes the standard antibody by 200 times for detection. The main experimental reagents used in this experiment include: latex microspheres (125 μl latex microsphere labeled 0.1mg antigen); reagent R1 (1g BSA,0.24gTris,0.1g PC300,5g PEG6000, 100mL water, pH 8.5). The loading mode of this experiment is as follows: the A, B antibody standard was diluted 200-fold with R1 (Millipore, lot number JHE2103, cat number JH-1L-BK; millipore, lot number JMC2103, cat number JM-1L-BK), to obtain a diluted antibody standard having a volume of 200. Mu.L, and then 200. Mu.L of a reagent R2 (the latex microsphere was diluted 200-fold with TBS) was added thereto to obtain 400. Mu.L of a sample to be tested. In this experiment, the detection wavelength was 340nm.
The above experimental results are summarized in the following table:
table 4d
| Coupling ratio | OD value of antibody A | OD value of antibody B | 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 |
| A2:KLH＝40:1 | 0.614 | 0.283 | 2.170 |
| A2:KLH＝60:1 | 0.447 | 0.264 | 1.694 |
| A2:KLH＝80:1 | 0.446 | 0.269 | 1.657 |
| A3:KLH＝10:1 | 0.443 | 0.277 | 1.601 |
| A3:KLH＝20:1 | 0.449 | 0.283 | 1.585 |
| A3:KLH＝40:1 | 0.435 | 0.274 | 1.588 |
| A3:KLH＝60:1 | 0.416 | 0.285 | 1.461 |
| A3:KLH＝80:1 | 0.439 | 0.293 | 1.498 |
| A4:KLH＝10:1 | 0.422 | 0.288 | 1.464 |
| A4:KLH＝20:1 | 0.407 | 0.271 | 1.503 |
| A4:KLH＝40:1 | 0.431 | 0.284 | 1.519 |
| A4:KLH＝60:1 | 0.420 | 0.275 | 1.527 |
| A4:KLH＝80:1 | 0.414 | 0.272 | 1.523 |
The above experimental results show that even when the standard sample is diluted 200 times, the antigen A2-A4 used in the invention is coupled with KLH, no matter which coupling ratio of KLH is used in the range of 10:1-80:1, the P/N value is relatively high, namely the A antibody and the B antibody can still be well distinguished in the detection of blood group antibodies, and particularly, the distinguishing effect of A2:KLH=40:1 is the best. In contrast, A1-KLH coupling is relatively poor in distinguishing between the recognition of the A and B antibodies, with P/N values only slightly above 1.
Example five
Blood group antigen trisaccharide analogue protein conjugate column agglutination neutralization experiment
In this example, a neutralization test is performed using a human ABO blood typing detection card (column agglutination method), i.e., a certain amount of antibodies are first neutralized with artificial antigen, and then indicator cells are added to check whether antibodies that have not been neutralized with artificial antigen are still present. If the specificity of the artificial antigen is strong and the binding efficiency with the antibody is high, the antibody can be sufficiently neutralized by the artificial antigen, then the indicator cell (corresponding to the antibody) is added with an antibody which can not react with the surface antigen of the indicator cell, so that the experimental result shows negative (no agglutination phenomenon); in contrast, if the specificity of the artificial antigen is weak and the binding efficiency with the antibody is low, the antibody is not sufficiently neutralized by the artificial antigen, then when the indicator cell (corresponding to the antibody) is added, the antibody capable of reacting with the surface antigen of the indicator cell remains, and the experimental result shows positive (there is an agglutination phenomenon). Through the experimental design, the reactivity of the artificially synthesized antigen (namely, the blood group antigen trisaccharide B analogue protein conjugate and the blood group antigen trisaccharide A analogue protein conjugate) and the antibody can be effectively verified, namely, whether the artificially synthesized antigen has good specificity in recognizing the corresponding antibody.
Information on a antibody standard used in this experiment: the brand Millipore, lot number JHE2103, cat# JH-1L-BK. Information on B antibody standard used in this experiment: brand Millipore, lot number JMC2103, cat# JM-1L-BK.
5.1B-BSA antigen detection antibodies
1) Firstly diluting the B antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with normal saline; then mixing diluted antibody (using normal saline as negative control) with B antigen (0.01 mg/mL) with different configurations and different BSA coupling ratios in equal volume, and incubating for about 15min at room temperature; then taking 40 mu L of the incubated mixture as an object to be detected and adding the object to be detected into the ABO blood type inverse typing detection card; finally, 10. Mu.L of indicator cell B (with blood group antigen B) was added, and the result was read after centrifugation. The results are shown in the following table:
table 5a
2) Firstly diluting the A antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with normal saline; then mixing diluted antibody (using normal saline as negative control) with B antigen (0.01 mg/mL) with different configurations and different BSA coupling ratios in equal volume, and incubating for about 15min at room temperature; taking 40 mu L of the incubated mixture as an object to be detected, adding the object to be detected into the ABO blood group reverse typing detection card, adding 10 mu L of indicator cell B (with blood group antigen B), centrifuging and reading the result. The results are shown in the following table:
Table 5b
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Experimental results: table 5a shows that the neutralizing capacity of B antigen (B1-B4) conjugated BSA for B antibody is overall weaker, compared to B-BSA internally, that the neutralizing capacity is relatively stronger with B2-BSA (negative on all conjugation ratios when B antibody is diluted 10000 times), especially with B2:BSA=20:1 (sufficient neutralization when B antibody is diluted 1000 times). Under the condition of diluting the B antibody by 1000 times, the B1-BSA and the B3-BSA show better neutralizing capacity of the B antibody relative to other coupling ratios of the isotype antigens when the coupling ratio is 20:1; the B4-BSA exhibits better neutralizing capacity for B antibodies than other conjugation ratios for isotype antigens at a conjugation ratio of 40:1. Table 5B shows that the control experiment using the A antibody and the B antigen (B1-B4) coupled BSA protein should not undergo neutralization reaction and also should not react with the indicator cell B, so that the experimental results are negative, and the experimental system is proved to run normally.
5.2B-KLH antigen detection antibodies
1) Firstly diluting the B antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with normal saline; then mixing diluted antibody (using normal saline as negative control) with B antigen (0.01 mg/mL) with different configurations and different KLH coupling ratios in equal volume, and incubating for about 15min at room temperature; then taking 40 mu L of the incubated mixture as an object to be detected and adding the object to be detected into the ABO blood type inverse typing detection card; finally, 10. Mu.L of indicator cell B was added, and the result was read after centrifugation. The results are shown in the following table:
Table 5c
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2) Firstly diluting the A antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with normal saline; then mixing diluted antibody (using normal saline as negative control) with B antigen (0.01 mg/mL) with different configurations and different KLH coupling ratios in equal volume, and incubating for about 15min at room temperature; then taking 40 mu L of the incubated mixture as an object to be detected and adding the object to be detected into the ABO blood type inverse typing detection card; finally, 10. Mu.L of indicator cell B was added, and the result was read after centrifugation. The results were as follows:
table 5d
Experimental results: table 5c shows that the neutralizing ability of B antigen (B1-B4) conjugated KLH to B antibody is relatively good with B2-KLH, especially with B2: KLH=20:1, which is the strongest (B2: KLH=20:1 can still sufficiently neutralize B antibody even in a high concentration environment where B antibody is diluted only 10-fold); b2: klh=40:1 also shows better specificity, and B antibody can be sufficiently neutralized in a higher concentration environment where B antibody is diluted 100 times. The B1-KLH, the B3-KLH and the B4-KLH show better neutralizing capacity of the B antibody relative to other coupling ratios of the isotype antigen when the coupling ratio is 40:1, and the B antibody can be fully neutralized in an environment of diluting the B antibody 1000 times. Table 5d shows that the control experiment using the A antibody and the conjugated KLH of the B antigen (B1-B4) should not react with the indicator cell B and thus the experimental results are negative, which proves that the experimental system operates normally.
5.3A-BSA antigen detection antibodies
1) Firstly diluting the A antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with normal saline; then mixing the diluted antibody (physiological saline is used as a negative control) with antigen A (0.01 mg/mL) with different configurations and different BSA coupling ratios in an equal volume, and incubating for about 15min in a room temperature environment; then taking 40 mu L of the incubated mixture as an object to be detected and adding the object to be detected into the ABO blood type inverse typing detection card; finally, 10. Mu.L of indicator cell A was added and the result read after centrifugation. The results were as follows:
table 6a
2) Firstly diluting the B antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with normal saline; then mixing the diluted antibody (physiological saline is used as a negative control) with antigen A (0.01 mg/mL) with different configurations and different BSA coupling ratios in an equal volume, and incubating for about 15min in a room temperature environment; then taking 40 mu L of the incubated mixture as an object to be detected and adding the object to be detected into the ABO blood type inverse typing detection card; finally, 10. Mu.L of indicator cell A was added and the result read after centrifugation. The results were as follows:
table 6b
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Experimental results:
table 6a shows that the neutralizing ability of the A antigen (A1-A4) coupled BSA to the A antibody is relatively good, and the neutralizing ability is strongest with A2-BSA, especially with A2: BSA=40:1 (A2: BSA=40:1 can still sufficiently neutralize the A antibody even in a high concentration environment where the A antibody is diluted only 10 times); A2-BSA also exhibits better specificity at other coupling ratios (10:1-20:1, 60:1-80:1), and can fully neutralize the A antibody in a higher concentration environment where the A antibody is diluted 100 times. A3-BSA and A4-BSA are coupled in a ratio of 40:1-80:1 exhibit a better neutralizing ability of the a antibody than other conjugation ratios to isotype antigen, and can be sufficiently neutralized in an environment where the a antibody is diluted 1000-fold. Table 6B shows that the control experiment using antibody B and the conjugated KLH of antigen A (A1-A4) should not undergo neutralization reaction and also should not react with indicator cell A, so that the experimental results are negative, and the experimental system is proved to run normally.
5.4A-KLH antigen detection antibodies
1) Firstly diluting the A antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with normal saline; then mixing the diluted antibody (physiological saline is used as a negative control) with the antigen A (0.01 mg/mL) with different configurations and different KLH coupling ratios in an equal volume, and incubating for about 15min in a room temperature environment; then taking 40 mu L of the incubated mixture as an object to be detected and adding the object to be detected into the ABO blood type inverse typing detection card; finally, 10. Mu.L of indicator cell A was added and the result read after centrifugation. The results were as follows:
table 6c
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2) diluting the B antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with normal saline; then mixing the diluted antibody (physiological saline is used as a negative control) with the antigen A (0.01 mg/mL) with different configurations and different KLH coupling ratios in an equal volume, and incubating for about 15min in a room temperature environment; then taking 40 mu L of the incubated mixture as an object to be detected and adding the object to be detected into the ABO blood type inverse typing detection card; finally, 10. Mu.L of indicator cell A was added and the result read after centrifugation. The results were as follows:
TABLE 6d
Experimental results: table 6c shows that the neutralizing ability of the A antigen (A1-A4) coupled KLH to the A antibody is relatively good also with A2-KLH, especially with A2: KLH=40:1, which is the strongest (A2: KLH=40:1 can still sufficiently neutralize the A antibody even in a high concentration environment where the A antibody is diluted only 10-fold); A2-KLH also showed better specificity at other coupling ratios (10:1-20:1, 60:1-80:1), and all could still fully neutralize the A antibody in the higher concentration environment where the A antibody was diluted 100-fold. A3-KLH and A4-KLH also exhibit a certain neutralizing capacity of the A antibody at the respective coupling ratios (10:1 to 80:1), and the A antibody can be sufficiently neutralized in an environment where the A antibody is diluted 1000-fold. Table 6d shows that the control experiment using antibody B and the conjugated KLH of antigen A (A1-A4) should not undergo neutralization reaction and also should not react with indicator cell A, so that the experimental results are negative, and the experimental system is proved to run normally.
In the above experiments, the blood group antigen trisaccharide a analogue protein conjugates with obvious or more obvious experimental effects are summarized in table 7 a; the blood group antigen trisaccharide a analog protein conjugates with obvious or more obvious experimental effects are summarized in table 7 b.
Table 7a
Table 7b
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The embodiments of the present invention have been described above with reference to the accompanying drawings, but the present invention is not limited to the above-described embodiments, which are merely illustrative and not restrictive, and many forms may be made by those having ordinary skill in the art without departing from the spirit of the present invention and the scope of the claims, which are to be protected by the present invention.
Claims (12)
1. The application of the blood group antigen trisaccharide conjugate in preparing blood group antibody detection reagent is characterized in that the blood group antigen trisaccharide conjugate is blood group antigen trisaccharide analogue protein conjugate; the blood group antigen trisaccharide analogues comprise blood group antigen trisaccharide B analogues; the blood group antigen trisaccharide B analogues comprise B1 type, B2 type, B3 type and B4 type; the blood group antigen trisaccharide B analogue is coupled with hemocyanin or bovine serum albumin to form a blood group antigen trisaccharide B analogue protein conjugate;
When the blood group antigen trisaccharide B analogue is B2 type, the coupling ratio of the blood group antigen trisaccharide B analogue to the hemocyanin is 5:1-40:1, and the coupling ratio of the blood group antigen trisaccharide B analogue to the bovine serum albumin is 20:1;
when the blood group antigen trisaccharide B analogue is B1 type, B3 type or B4 type, the coupling ratio of the blood group antigen trisaccharide B analogue to the hemocyanin is 40:1; the chemical formulas of the B1 type, the B2 type, the B3 type and the B4 type are as follows:
2. the use of claim 1, wherein said blood group antigen trisaccharide analog further comprises a blood group antigen trisaccharide a analog; the blood group antigen trisaccharide A analogue is coupled with hemocyanin or bovine serum albumin to form a blood group antigen trisaccharide A analogue protein conjugate; the blood group antigen trisaccharide A analogues comprise A1 type, A2 type, A3 type and A4 type, and the chemical formula is as follows:
3. the use according to claim 2, wherein when said blood group antigen trisaccharide a analogue is
In the A2 type, the coupling ratio of the blood group antigen trisaccharide A analogue to the hemocyanin is 10:1-80:1; when the blood group antigen trisaccharide A analogue is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analogue to the hemocyanin is 10:1-80:1.
4. The use according to claim 2, wherein when said blood group antigen trisaccharide a analogue is
In the A2 type, the coupling ratio of the blood group antigen trisaccharide A analogue to the bovine serum albumin is 10:1-80:1; when the blood group antigen trisaccharide A analogue is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analogue to bovine serum albumin is 10:1 or 40:1-80:1.
5. The use according to claim 1, wherein the blood group antibody detection reagent is used for immunochromatographic detection, densitometric detection and column agglutination detection.
6. The use of claim 3, wherein the blood group antibody detection reagent is used for immunochromatographic detection, and when the blood group antigen trisaccharide A analogue is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analogue to the hemocyanin is 10:1 or 40:1-80:1.
7. The immunochromatography detection kit is characterized by comprising a detection card, wherein the detection card adopts a nitrocellulose membrane; the conjugate of the blood group antigen trisaccharide B analogue and hemocyanin or bovine serum albumin is coated on the detection card and is used for detecting blood group antibodies in a serum sample; the blood group antigen trisaccharide B analogues comprise B1 type, B2 type, B3 type and B4 type; when the blood group antigen trisaccharide B analogue is B2 type, the coupling ratio of the blood group antigen trisaccharide B analogue to the hemocyanin is 5:1-40:1, and the coupling ratio of the blood group antigen trisaccharide B analogue to the bovine serum albumin is 20:1; when the blood group antigen trisaccharide B analogue is B1 type, B3 type or B4 type, the coupling ratio of the blood group antigen trisaccharide B analogue to hemocyanin is 40:1; the chemical formulas of the B1 type, the B2 type, the B3 type and the B4 type are as follows:
8. The kit of claim 7, wherein the test card is further coated with a conjugate of a blood group antigen trisaccharide a analog with hemocyanin or a conjugate with bovine serum albumin; the blood group antigen trisaccharide A analogues comprise A1 type, A2 type, A3 type and A4 type, and the chemical formula is as follows:
9. the kit of claim 8, wherein when said blood group antigen trisaccharide a analog is of type A2, said blood group antigen trisaccharide a analog is coupled to hemocyanin in a ratio of 10:1 to 80:1; when the blood group antigen trisaccharide A analogue is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analogue to hemocyanin is 10:1 or 40:1-80:1.
10. The kit of claim 8, wherein when the blood group antigen trisaccharide a analog is of type A2, the coupling ratio of the blood group antigen trisaccharide a analog to bovine serum albumin is from 10:1 to 80:1; when the blood group antigen trisaccharide A analogue is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analogue to bovine serum albumin is 10:1 or 40:1-80:1.
11. A column agglutination kit comprising an ABO blood group reverse typing detection card, a blood group antigen trisaccharide analogue protein conjugate; the blood group antigen trisaccharide analogue protein conjugate comprises coupling of blood group antigen trisaccharide B analogues with hemocyanin or bovine serum albumin; the blood group antigen trisaccharide B analogues comprise B1 type, B2 type, B3 type and B4 type; when the blood group antigen trisaccharide B analogue is B2 type, the coupling ratio of the blood group antigen trisaccharide B analogue to hemocyanin is 10:1-40:1, and the coupling ratio of the blood group antigen trisaccharide B analogue to bovine serum albumin is 20:1; when the blood group antigen trisaccharide B analogue is B1 type, B3 type or B4 type, the coupling ratio of the blood group antigen trisaccharide B analogue to hemocyanin coupling is 40:1; the chemical formulas of the B1 type, the B2 type, the B3 type and the B4 type are as follows:
12. The column agglutination kit of claim 11, wherein said blood group antigen trisaccharide analog protein conjugate further comprises a blood group antigen trisaccharide a analog conjugated with a hemocyanin or bovine serum albumin; the blood group antigen trisaccharide A analogues comprise A2 type, A3 type and A4 type; when the blood group antigen trisaccharide A analogue is A2 type, the coupling ratio of the blood group antigen trisaccharide A analogue to hemocyanin or bovine serum albumin is 10:1-80:1; when the blood group antigen trisaccharide A analogue is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analogue to hemocyanin is 10:1-80:1, and the coupling ratio of the blood group antigen trisaccharide A analogue to bovine serum albumin is 10:1 or 40:1-80:1; the chemical formulas of the A2 type, the A3 type and the A4 type are as follows:
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| CN202311224022.1A CN117269517B (en) | 2022-10-18 | 2022-12-05 | Application of blood group antigen trisaccharide B analogue protein conjugate in blood group antibody detection |
| CN202311223836.3A CN117269516A (en) | 2022-10-18 | 2022-12-05 | Application of blood group antigen trisaccharide A analogue protein conjugate in blood group antibody detection |
| CN202311223686.6A CN117269515A (en) | 2022-10-18 | 2022-12-05 | Blood group antigen trisaccharide B analogue protein conjugate and application thereof |
| CN202311223493.0A CN117269514A (en) | 2022-10-18 | 2022-12-05 | Blood group antigen trisaccharide A analogue protein conjugate and application thereof |
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| CN202311223493.0A Division CN117269514A (en) | 2022-10-18 | 2022-12-05 | Blood group antigen trisaccharide A analogue protein conjugate and application thereof |
| CN202311224022.1A Division CN117269517B (en) | 2022-10-18 | 2022-12-05 | Application of blood group antigen trisaccharide B analogue protein conjugate in blood group antibody detection |
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| CN117269517A (en) * | 2022-10-18 | 2023-12-22 | 天津德祥生物技术股份有限公司 | Application of blood group antigen trisaccharide B analogue protein conjugate in blood group antibody detection |
| CN117402833A (en) * | 2023-12-14 | 2024-01-16 | 天津德祥生物技术股份有限公司 | Hybridoma cell strain secreting specific antibody, erythrocyte A antibody and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117269517A (en) * | 2022-10-18 | 2023-12-22 | 天津德祥生物技术股份有限公司 | Application of blood group antigen trisaccharide B analogue protein conjugate in blood group antibody detection |
| CN117269517B (en) * | 2022-10-18 | 2024-04-16 | 天津德祥生物技术股份有限公司 | Application of blood group antigen trisaccharide B analogue protein conjugate in blood group antibody detection |
| CN117402833A (en) * | 2023-12-14 | 2024-01-16 | 天津德祥生物技术股份有限公司 | Hybridoma cell strain secreting specific antibody, erythrocyte A antibody and application |
| CN117402833B (en) * | 2023-12-14 | 2024-03-12 | 天津德祥生物技术股份有限公司 | Hybridoma cell strain secreting specific antibody, erythrocyte A antibody and application |
Also Published As
| Publication number | Publication date |
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| CN117269514A (en) | 2023-12-22 |
| CN117269517B (en) | 2024-04-16 |
| CN117269515A (en) | 2023-12-22 |
| CN116868059B (en) | 2024-04-05 |
| CN117269517A (en) | 2023-12-22 |
| CN117269516A (en) | 2023-12-22 |
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