CN115343485A - 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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Abstract
The invention belongs to the field of blood group antibody detection, and relates to application of blood group antigen-trisaccharide conjugate in blood group antibody detection. The blood group antigen trisaccharide conjugate is a blood group antigen trisaccharide analogue protein conjugate; including blood group antigen trisaccharide B analogs; coupling with hemocyanin or bovine serum albumin to form 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 5-40, and the coupling ratio of the blood group antigen trisaccharide B analogue to the bovine serum albumin is 20; 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. The blood group antigen trisaccharide analogue protein conjugate has good specificity, can identify corresponding blood group antibodies, has high detection accuracy, and can be applied to detection of various blood group antibodies.
Description
Technical Field
The invention belongs to the field of blood type antibody detection, relates to application of blood type antigen trisaccharide conjugates in blood type antibody detection, and particularly relates to application of blood type antigen trisaccharide A analogue protein conjugates and blood type antigen trisaccharide B analogue protein conjugates in blood type antibody detection.
Background
Blood grouping is the primary task before clinical transfusion, because blood of different blood types is subjected to hemolysis due to the coagulation of antigen and antibody when transfused with each other, and further the life safety of people can be endangered, so correct blood grouping is a prerequisite for ensuring transfusion safety. Current methods of blood grouping include orthotyping and retrotyping. The positive typing method is used for detecting erythrocyte antigens, and the negative typing method is used for detecting antibodies in serum, wherein the detection of human blood group antigens/antibodies of A, B and O is the most important. It can be classified into different blood types according to the difference of erythrocyte membrane surface antigen in human blood. The red blood cells of the type A blood are provided with blood group antigen A (hereinafter referred to as "A antigen"), and blood group antibody B (hereinafter referred to as "B antibody") is contained in the blood serum; the red blood cells of the B type blood are provided with blood group antigen B (hereinafter referred to as "B antigen"), blood group antibody A (hereinafter referred to as "A antibody") is contained in the serum, the red blood cells of the AB type blood are provided with the A antigen and the B antigen, and the serum is not provided with the A antibody and the B antibody; both A and B antigens are absent from erythrocytes in blood type O, and both A and B antibodies are present in the serum.
Currently, the normal and reverse typing of the common blood types generally adopts an agglutination method, and the principle is to judge the result by utilizing the macroscopic erythrocyte agglutination caused by the reaction of blood type antigens and antibodies. Detecting red blood cell antigens of the sample using IgM anti-A or anti-B reagents when positive; conversely, the IgM blood group antibodies in the serum of a sample are determined using red blood cell reagents of known types A or B. However, because fresh red blood cells are not easy to store (the antigenicity of blood group antigens on the surface of the red blood cells is gradually reduced along with the prolonging of the storage time), the red blood cell reagent of type A or B for the reverse titration detection has higher requirements on the storage condition and the transportation condition, the cost of the reverse titration detection is increased, and the application of the reverse titration detection is limited. Some technical schemes in the prior art try to extract natural blood group antigens (for example, natural A antigens and natural B antigens are extracted through erythrocyte membranes) to replace complete erythrocytes in the inverse quantitative detection, but the related extraction and preparation processes are complex and difficult to produce in mass, and the extracted natural blood group antigens still have high requirements on storage conditions. The artificial synthetic blood group antigen is one of the research directions for solving the technical problems, but the existing artificial synthetic blood group antigen has the technical problems in the aspects of specificity, affinity, stability and the like, and cannot be effectively applied to inverse quantitative detection.
Disclosure of Invention
In view of the above, the present invention provides an application of blood group antigen-trisaccharide conjugate in blood group antibody detection. The blood group antigen trisaccharide conjugate is a blood group antigen trisaccharide analogue protein conjugate, and is collectively called blood group antigen trisaccharide analogue in the following before unconjugated protein for convenient distinction.
The application of blood group antigen trisaccharide analogue in preparing blood group antibody detection reagent is characterized in that the blood group antigen trisaccharide analogue comprises blood group antigen trisaccharide B analogue; the blood group antigen trisaccharide B analogue is coupled with Hemocyanin (Keyhole Lipmet Hemocyanin, KLH) or Bovine Serum Albumin (Bovine Serum Albumin, BSA) 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 5-40; 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.
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 is 20.
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; 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 10-80.
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.
In some embodiments, when the blood group antigen trisaccharide a analog is type A2, the coupling ratio of the blood group antigen trisaccharide a analog to bovine serum albumin is 10; 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 or 1-80.
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.
In some embodiments, the blood group antibody detection reagent is used for immunochromatography, densitometry, or column agglutination.
In some embodiments, the blood group antibody detection reagent is used in an immunochromatographic assay, 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 or 1 to 40.
The invention also provides an immunochromatography detection kit which is characterized by comprising a detection card, wherein the detection card adopts a nitrocellulose membrane; the detection card is coated with a conjugate of blood group antigen trisaccharide B analogue and hemocyanin or bovine serum albumin 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 hemocyanin is 5-40; when the blood group antigen trisaccharide B analogue is B1 type, B3 type or B4 type, the coupling ratio of the trisaccharide B analogue to hemocyanin is 40.
In some embodiments, the test card is further coated with a conjugate of blood group antigen trisaccharide a analog and hemocyanin or a conjugate of bovine serum albumin.
In some embodiments, when the blood group antigen trisaccharide a analogue is of type A2, the coupling ratio of the blood group antigen trisaccharide a analogue to hemocyanin is 10-1; 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 or 1-80.
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 10-80; 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 or 1-80.
The invention also provides a column agglutination kit, which is characterized by comprising an ABO blood type reverse typing detection card and blood type antigen trisaccharide analogue protein conjugates; the blood group antigen trisaccharide analogue protein conjugate comprises blood group antigen trisaccharide B analogue and 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 10-40; 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.
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 10-80; 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 hemocyanin is 10 to 1, and the coupling ratio of the blood group antigen trisaccharide a analog to bovine serum albumin is 10 or 1 to 40.
Has the 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) (see tables 7a and 7B) adopted by the technical scheme of the invention have good specificity, can be used as artificially synthesized blood group antigens, can distinguish and identify corresponding blood group antibodies, have high detection accuracy, can be used for detecting various types of blood group antibodies, and have huge potential clinical application value. Especially, when the conjugate is applied to immunochromatography detection, part of blood group antigen trisaccharide analogue protein conjugates adopted by the technical scheme of the invention can be stably fixed on detection test paper, and can efficiently capture corresponding blood group antibodies, and the chromogenic effect (for example, chromogenic through a colloidal gold label) is easy to identify and visible to naked eyes, so that the conjugate has the potential of being applied to scenes such as rapid blood group screening, POCT detection, general human blood group self-check and the like.
2) The blood group antigen trisaccharide analogue protein conjugate is synthesized by a chemical process, industrial-grade mass production is easy to realize, and the conjugate can be used as a blood group antigen raw material in various experimental systems, so that a red blood cell reagent or a natural blood group antigen can be replaced in inverse definite detection, the complicated process of extraction and preparation of the natural blood group antigen is avoided, and the cost of blood group detection is reduced.
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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 is obvious that the drawings in the following description are some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive exercise.
FIG. 1 is a synthesis scheme of blood group antigen trisaccharide B analogue type I of the present invention.
FIG. 2 is a synthetic route diagram of blood group antigen trisaccharide B analogue type II of the present invention.
FIG. 3 is a synthetic route diagram of trisaccharide B analogue type III of the blood group antigen of the present invention.
FIG. 4 is a synthetic route diagram of blood group antigen trisaccharide B analogue 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 clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
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.
In this specification, certain embodiments may be disclosed in a range of formats. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, the description of range 1-6 should be taken 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, e.g., 1,2,3,4,5 and 6. The above rules apply regardless of the breadth of the range.
The "blood group antigen trisaccharide A analogue" described in the present invention refers to four compounds represented by the basic chemical formulas A1 to A4 and derivatives thereof, i.e., a type I A antigen compound (chemical formula A1, hereinafter referred to as "A1" or "A1"), a type II A antigen compound (chemical formula A2, hereinafter referred to as "A2" or "A2"), a type III A antigen compound (chemical formula A3, hereinafter referred to as "A3" or "A3"), and a type IV A4 antigen compound (chemical formula A4, hereinafter referred to as "A4" or "A4"), respectively.
The "blood group antigen trisaccharide B analogue" according to the present invention refers to four compounds represented by the basic chemical formulas B1 to B4 and derivatives thereof, i.e., 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"), respectively.
The blood group antigen trisaccharide analogue protein conjugate refers to a protein conjugate formed by coupling the blood group antigen trisaccharide A analogue with BSA or KLH protein (hereinafter referred to as "A-BSA" or "A-KLH"), and a protein conjugate formed by coupling the blood group antigen trisaccharide B analogue with BSA or KLH protein (hereinafter referred to as "B-BSA" or "B-KLH").
EXAMPLE I preparation of blood group antigen trisaccharide analogue protein conjugates
1.1 Preparing blood group antigen trisaccharide A analogue protein conjugate.
In some embodiments, two target sugar blocks may be synthesized first, followed by glycosylation to synthesize a target sugar chain, according to the modular assembly strategy of the Bovin topic group (Drouillar S, et al, large-scale synthesis of H-antisense oligonucleotides by expressing a helicobacter pylori 1,2-fucosyltransferase in metabolic engineered Escherichia coli cells [ J ]. Angew Chem, 2006, 118 (11): 1810-1812), the main reaction steps being illustrated below.
Then, the end position of the target sugar chain generated by the method and butyramide connected with carboxyl undergo addition reaction to generate the antigen trisaccharide A analogue of the invention, namely a total tetrasaccharide (A1-A4), and the chemical structural formula is shown in 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 a lysine residue of BSA protein or KLH protein (for example, a section of an alkane chain connected with azide groups is pre-arranged on an initial substrate and is used as a connecting arm during sugar chain synthesis, and then the coupling of the blood group antigen trisaccharide analogue and a carrier protein is realized through a double-activated ester reagent), so that the blood group antigen trisaccharide A analogue protein conjugates (A1-BSA, A2-BSA, A3-BSA, A4-BSA; A1-KLH, A2-KLH, A3-KLH and A4-KLH) are synthesized, and the chemical formula of the blood group antigen trisaccharide A analogue protein conjugates 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 blood group antigen trisaccharide a analogue to protein (BSA or KLH) coupling ranges from 10 to 1, see table 1 for details.
1.2 Preparing blood group antigen trisaccharide B analogue protein conjugate.
Firstly synthesizing 4 disaccharide precursors by introducing a azide group-containing linking arm into the reducing end of a starting monosaccharide (GlcNAc, glaNAc) by a chemical method, and then chemically synthesizing GlcNAc beta ProN 3 ，GalNAcαProN 3 ，GalNAcβProN 3 Monosaccharide is used as a receptor, and the synthesis of 4 types of precursors is completed by a one-pot multi-enzyme method, and the chemical formula is as follows.
Blood group antigen trisaccharide B analogue disaccharide precursor type I
Blood group antigen trisaccharide B analogue disaccharide precursor II
Blood group antigen trisaccharide B analogue disaccharide precursor type III
Blood group antigen trisaccharide B analogue disaccharide precursor tetratype
Then, an alpha 1-3 galactosyltransferase 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 structures are shown in chemical formulas B1-B4), and the specific synthetic route is shown in figures 1-4.
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 on a lysine residue of BSA protein or KLH protein (for example, a section of an alkane chain connected with azide group is pre-arranged on an initial substrate as a connecting arm during sugar chain synthesis, and then the coupling of the blood group antigen trisaccharide analogue and carrier protein is realized through a double-activated ester reagent), and the antigen trisaccharide B analogue protein conjugate (B1-BSA, B2-BSA, B3-BSA, B4-BSA; B1-KLH, B2-KLH, B3-KLH, B4-KLH) is synthesized, and 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 blood group antigen trisaccharide B analogue to KLH coupling ranges from 5 to 1, see table 1 for details.
TABLE 1 blood group antigens trisaccharide analogs and protein coupling ratios
Example two
Qualitative verification of blood group antigen trisaccharide analogue protein conjugate (antigen immobilization immunochromatography).
In order to research whether the trisaccharide analogue protein conjugate of the blood group antigen synthesized by the invention can be used for simple and rapid qualitative blood group detection, the invention adopts an antigen solidification immunochromatography technology for verification. In order to detect the antibody A and the antibody B in serum, a detection card assembled by a Satorius Sartorius Nitrocellulose (NC) membrane CN140 is adopted, and the NC membrane comprises a detection line (namely a T line) and a quality control line (namely a C line). 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 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). The blood group antibody A or antibody B contained in the sample can be combined with anti-human mu-chain antibody labeled by colloidal gold to form a compound and chromatographed on an NC membrane, when the compound chromatographs to a T line, the blood group antibody A or B is specifically combined with the 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 artificially synthesized antigen A, and the blood group antibody B is combined with the artificially synthesized antigen B). If there is no corresponding antibody A or antibody B in the sample, no complex is formed, and no color develops at the T-line. Based on the above, when the sample contains the antibody A and does not contain the antibody B, the sample is type A blood; the sample contains the antibody B, does not contain the antibody A and is B type blood; the sample does not contain the antibody A and the antibody B and is AB type blood; the sample contains both antibody A and antibody B, and is blood type O. The sample is firstly chromatographed to a T line, and then chromatographed to a C line, wherein the C line is developed when all samples are detected, otherwise, the test is invalid.
The detection card coated with blood group antigen trisaccharide A analogue protein conjugates (A-BSA, A-KLH) is called an A detection card in the invention; the test card coated with blood group antigen trisaccharide B analogue protein conjugates (B-BSA, B-KLH) is referred to herein as the B test card.
And respectively adding 1 mu L of standard blood type antibody A (brand Millipore, batch number JHE2103, cargo number JH-1L-BK) to each A detection card, adding 80 mu L of sample diluent (0.01 MPBS), standing for about 15-20 min, observing the color development conditions of the C line and the T line by naked eyes, and taking a picture for recording.
And respectively adding 1 mu L of standard blood type antibody B (brand Millipore, batch number JMC2103, cargo number JM-1L-BK) to each B detection card, adding 80 mu L of sample diluent (0.01 MPBS), standing for about 15-20 min, observing the color development conditions at the C line and the T line by naked eyes, and taking a picture for recording.
The above test results are summarized in tables 2a to 2d, and the coloration is indicated by "+" sign, and the non-visibility to the naked eye is indicated by "-" sign.
The detection result shows that: for blood group antigens trisaccharide B analogues, B2 coupled to BSA or KLH gave an effective coloration (visible to the naked eye) on the T-line. However, the color development effect of B2-BSA was only significant at a coupling ratio of 20. In addition, when the coupling ratio of B-KLH is 40, the color development effect of B1-KLH, B3-KLH and B4-KLH can be seen by naked eyes. For blood group antigen trisaccharide a analogs, A2-A4, whether coupled with BSA or KLH, formed blood group antigen trisaccharide analog protein conjugates, developed efficient color development on the T-line (coupling ratio: 10, 1, 40, 1, 60, 1, 80. In addition, neither A1, nor KLH, was effectively colored at the coupling ratios tested in the present invention.
The above experimental results show that when a specific blood group antigen trisaccharide analog is coupled with a specific protein (e.g., B2-KLH, A2-BSA, A2-KLH), or at a specific coupling ratio (20 (B2-BSA): 40.
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 using other labeling means (e.g., enzyme, fluorescent, or other luminescent labels). In addition, besides the labeled antibody, different labeling methods can be used for labeling the blood group antigen trisaccharide analogue protein conjugate, and the conjugate can be used for various heterogeneous or homogeneous detection according to actual requirements.
Table 2a
Table 2b
Table 2c
Table 2d
EXAMPLE III
And (3) quantitative verification of blood group antigen trisaccharide B analogue protein conjugate.
3.1 Blood group antigen trisaccharide B was coupled to KLH protein.
1) Test results obtained by diluting standard substance antibody 100 times
The main experimental reagents adopted in the experiment comprise: latex microspheres (125 μ L latex microspheres, total labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH8.5).
The loading mode for this experiment was as follows: the antibody A and B standards (brand Millipore, lot number JHE2103, product number JH-1L-BK; brand Millipore, lot number JMC2103, product number JM-1L-BK) were diluted 100-fold with R1 to give a diluted antibody A and B standard having a volume of 200. Mu.L, and then the reagent R2 (i.e., the above latex microspheres were diluted 100-fold with TBS) was added to give 200. Mu.L in total of 400. Mu.L of the sample to be assayed. In this experiment, the detection wavelength was 340nm.
The results of the above experiments are summarized in the following table:
table 3a
The above experimental results show that, after the antigen B adopted by the present invention is coupled with KLH, no matter which type of B1-B4 is adopted, and no matter which coupling ratio with KLH is in the range of 5-40. Notably, the highest P/N value was obtained for B1-KLH, B3-KLH and B4-KLH at a coupling ratio of 40. The P/N values of B2-KLH are higher in the range of coupling ratios used according to the invention (5.
2) Test results obtained by diluting the standard substance antibody by 200 times
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 antibody concentration is low, the experiment dilutes the standard antibody by 200 times for detection. The main experimental reagents adopted by the experiment comprise: latex microspheres (125 μ L latex microspheres labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH8.5). The loading mode of this experiment was as follows: the antibody standards A and B were diluted 200-fold with R1 (brand Millipore, lot JHE2103, cat JH-1L-BK; brand Millipore, lot JMC2103, cat JM-1L-BK) to give a volume of 200. Mu.L of the diluted antibody standard, and then 200. Mu.L of the reagent R2 (the latex microspheres were diluted 200-fold with TBS) was added to give 400. Mu.L of the sample to be assayed. In this experiment, the detection wavelength was 340nm.
The results of the above experiments are summarized in the following table:
table 3b
The above experimental results show that even when the standard antibody is diluted 200-fold, the OD value (0.300 or more) of the antigen B combined with the B antibody is significantly higher than that obtained when the A antibody is added (up to 0.261) regardless of the type of B1-B4 and the coupling ratio of KLH in 5. In other words, the antigen B used in the present invention can well distinguish between A antibody and B antibody in the detection of blood group antibodies even in the case where the antibody concentration is low.
3.2 Blood group antigen trisaccharide B binds to BSA protein assay.
1) And (5) diluting the standard antibody by 100 times to obtain a detection result.
The main experimental reagents adopted in the experiment comprise: latex microspheres (125 μ L latex microspheres, total labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH8.5).
The loading mode of this experiment was as follows: the antibody standards A and B (brand Millipore, lot number JHE2103, cat # JH-1L-BK; brand Millipore, lot number JMC2103, cat # JM-1L-BK) were diluted 100-fold with R1 to give a diluted antibody standard having a volume of 200. Mu.L, and then a reagent R2 (the above latex microspheres were diluted 100-fold with TBS) was added thereto at 200. Mu.L to give 400. Mu.L of a sample to be assayed. In this experiment, the detection wavelength was 340nm.
The results of the above experiments are summarized in the following table:
table 3c
The above experimental results show that, in the antigen B used in the present invention, regardless of the type B1 to B4 and the coupling ratio of BSA to BSA in the range of 5 to 1-40, the OD value of the antibody B bound to the antigen B is different from the OD value obtained when the antibody a is added, but the P/N value of the data sets is not as high as the difference between the OD values when the antibody a and the OD value when the antibody B are recognized when the antigen B is coupled to KLH, and is in the range of 1.231 to 1.678. The results demonstrate that blood group antigen trisaccharide B (B1-B4) analogue coupled BSA protein can also detect B antibody, distinguishing A antibody from B antibody in the above experiment, but the ability and effect are inferior to those of blood group antigen trisaccharide B (B1-B4) analogue coupled KLH protein.
2) And (5) diluting the standard substance antibody by 200 times to obtain the detection result.
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 antibody concentration is low, the experiment dilutes the standard antibody by 200 times for detection. The main experimental reagents adopted in the experiment comprise: latex microspheres (125 μ L latex microspheres labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH8.5). The loading mode of this experiment was as follows: the antibody standards A and B were diluted 200-fold with R1 (brand Millipore, lot number JHE2103, cat # JH-1L-BK; brand Millipore, lot number JMC2103, cat # JM-1L-BK) to give a volume of 200. Mu.L of the diluted antibody standard, and then the reagent R2 (the above latex microspheres were diluted 200-fold with TBS) was added to give 400. Mu.L of the sample to be assayed. In this experiment, the detection wavelength was 340nm.
The results of the above experiments are summarized in the following table:
table 3d
The above experimental results are similar to those of the test results obtained by diluting the standard antibody 100-fold, and the OD values obtained when antigen B was bound to B antibody were different from those obtained when A antibody was added, but were significantly inferior to those obtained when antigen B was conjugated to KLH, regardless of the types of B1 to B4 and regardless of the coupling ratios to BSA in 5 to 40. Only B2: when BSA = 20; b3: when BSA =20, the P/N value can reach 1.394. In combination with NC membrane development experiments (table 2 a), only B2: BSA =20, a color reaction can be seen with naked eyes on the detection card. The above results demonstrate that blood group antigen trisaccharide B (B1-B4) analogue coupled BSA protein can also detect antibody B to some extent, distinguishing antibody A from antibody B, but its ability is significantly inferior to blood group antigen trisaccharide B (B1-B4) analogue coupled KLH protein.
Example four
Quantitative validation (densitometric experiments) of blood group antigen trisaccharide a analogue protein conjugates.
4.1 Blood group antigen trisaccharide a binds to BSA protein assay.
1) And (5) diluting the standard substance antibody by 100 times to obtain a detection result.
The main experimental reagents adopted in the experiment comprise: latex microspheres (125 μ L latex microspheres, total labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH8.5).
The loading mode of this experiment was as follows: the antibody standards A and B (brand Millipore, lot number JHE2103, cat # JH-1L-BK; brand Millipore, lot number JMC2103, cat # JM-1L-BK) were diluted 100-fold with R1 to give a diluted antibody standard having a volume of 200. Mu.L, and then a reagent R2 (the above latex microspheres were diluted 100-fold with TBS) was added thereto at 200. Mu.L to give 400. Mu.L of a sample to be assayed. In this experiment, the detection wavelength was 340nm.
The results of the above experiments are summarized in the following table:
table 4a
The above experimental results show that when antigen a used in the present invention is coupled to BSA, the OD value of antigen a bound to a antibody (0.4 or more) is higher than the OD value of bound B antibody (up to only 0.245) regardless of which coupling ratio with BSA in 10-80. Experiments prove that the antigen A2-A4 adopted by the invention can well distinguish the antibody A from the antibody B in the detection of blood group antibodies, and particularly, the distinguishing effect is best when A2: BSA = 40. 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 higher than 1.
2) And (5) diluting the standard antibody by 200 times to obtain a detection result.
In order to further verify the detection effect of the blood group antigen trisaccharide A analogue protein conjugate (A-BSA) synthesized by the invention when the antibody concentration is low, the experiment dilutes the standard antibody by 200 times for detection. The main experimental reagents adopted by the experiment comprise: latex microspheres (125 μ L latex microspheres labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH8.5). The loading mode of this experiment was as follows: the antibody standards A and B were diluted 200-fold with R1 (brand Millipore, lot JHE2103, cat JH-1L-BK; brand Millipore, lot JMC2103, cat JM-1L-BK) to give a volume of 200. Mu.L of the diluted antibody standard, and then 200. Mu.L of the reagent R2 (the latex microspheres were diluted 200-fold with TBS) was added to give 400. Mu.L of the sample to be assayed. In this experiment, the detection wavelength was 340nm.
The results of the above experiments are summarized in the following table:
table 4b
The above experimental results show that even in the case where the antigen A2-A4 coupled to BSA used in the present invention is low after the standard sample is diluted 200-fold, the P/N value is relatively high regardless of which coupling ratio (except A3: BSA =20, A4: BSA = 20). 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 higher than 1.
4.2 Blood group antigen trisaccharide a was tested in combination with KLH protein.
1) And (5) diluting the standard antibody by 100 times to obtain a detection result.
The main experimental reagents adopted by the experiment comprise: latex microspheres (125 μ L latex microspheres, total labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH8.5).
The loading mode for this experiment was as follows: the antibody standards A and B (brand Millipore, lot number JHE2103, cat # JH-1L-BK; brand Millipore, lot number JMC2103, cat # JM-1L-BK) were diluted 100-fold with R1 to give a diluted antibody standard having a volume of 200. Mu.L, and then a reagent R2 (the above latex microspheres were diluted 100-fold with TBS) was added thereto at 200. Mu.L to give 400. Mu.L of a sample to be assayed. In this experiment, the detection wavelength was 340nm.
The results of the above experiments are summarized in the following table:
table 4c
The results of the above experiments show that when antigen a used in the present invention is coupled to KLH after 100-fold dilution of the standard sample, the OD value of antigen a-binding a antibody (0.4 or more) is higher than the OD value of binding B antibody (up to only 0.293), regardless of which coupling ratio with KLH is in the range of 10. Experiments prove that the antigen A2-A4 adopted by the invention can well distinguish the antibody A from the antibody B in the detection of blood group antibodies, and particularly, the distinguishing effect is best when A2: KLH = 40. In contrast, the A1-KLH conjugate was relatively poor at discriminating between the recognition A and B antibodies, with P/N values only slightly above 1.
2) And (5) diluting the standard substance antibody by 200 times to obtain the detection result.
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 antibody concentration is low, the experiment dilutes the standard antibody by 200 times for detection. The main experimental reagents adopted by the experiment comprise: latex microspheres (125 μ L latex microspheres labeled with 0.1mg antigen); reagent R1 (1g BSA,0.24g Tris,0.1g PC300,5g PEG6000, 100mL water, pH8.5). The loading mode for this experiment was as follows: the antibody standards A and B were diluted 200-fold with R1 (brand Millipore, lot number JHE2103, cat # JH-1L-BK; brand Millipore, lot number JMC2103, cat # JM-1L-BK) to give a volume of 200. Mu.L of the diluted antibody standard, and then 200. Mu.L of the reagent R2 (the above latex microspheres were diluted 200-fold with TBS) was added thereto to give 400. Mu.L of the sample to be assayed. In this experiment, the detection wavelength was 340nm.
The results of the above experiments are summarized in the following table:
table 4d
The above experimental results show that even after 200-fold dilution of the standard sample, when the antigen A2-A4 used in the present invention is coupled to KLH, the P/N value is relatively high regardless of which coupling ratio of KLH to KLH is in the range of 10 to 80. In contrast, the A1-KLH conjugate was relatively poor at discriminating between the recognition A and B antibodies, with P/N values only slightly above 1.
EXAMPLE five
Blood group antigen trisaccharide analogue protein conjugate column agglutination neutralization assay.
In this embodiment, a neutralization test is performed by using a human ABO blood type reverse typing test card (column agglutination method), i.e., a certain amount of antibody is neutralized by using an artificial antigen, and then indicator cells are added to check whether the antibody which is not neutralized by the artificial antigen still exists. If the artificial antigen has strong specificity and high binding efficiency with the antibody, the antibody can be fully neutralized by the artificial antigen, and no antibody capable of reacting with the surface antigen of the indicator cell is added (corresponding to the antibody), so that the experimental result shows negative (no agglutination phenomenon); on the contrary, if the specificity of the artificial antigen is weak, the binding efficiency with the antibody is low, and the antibody cannot be sufficiently neutralized by the artificial antigen, when the indicator cell (corresponding to the antibody) is added, the antibody capable of reacting with the indicator cell surface antigen still exists, and thus the experimental result shows positive (there is an agglutination phenomenon). The experimental design can effectively verify 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, namely whether the artificially synthesized antigen has good specificity when identifying the corresponding antibody.
Information on antibody a standards used in this experiment: the brand Millipore, batch number JHE2103, cat number JH-1L-BK. Information on antibody B standards used in this experiment: the brand Millipore, batch number JMC2103, cat # JM-1L-BK.
5.1 B-BSA antigen detection antibody.
1) Diluting the standard substance of the antibody B by 10 times, 100 times, 1000 times, 10000 times and 100000 times with normal saline; then mixing the diluted antibody (using physiological saline as a 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 a substance to be detected and adding the substance into the ABO blood type reverse typing detection card; finally, 10. Mu.L of indicator cell B (with blood group antigen B) is added, and the result is read after centrifugation. The results are given in the following table:
table 5a
2) Diluting the A antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with physiological saline; then mixing the diluted antibody (using physiological saline as a 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; and taking 40 mu L of the incubated mixture as a substance to be detected, adding the substance to the ABO blood type reverse typing detection card, adding 10 mu L of indicator cell B (blood group-bearing antigen B), and centrifuging to read the result. The results are given in the following table:
table 5b
The experimental results are as follows: table 5a shows that the neutralizing capacity of B antigen (B1-B4) coupled BSA on B antibody is overall weaker, and that B2-BSA is relatively better (the experimental results are negative at all coupling ratios when B antibody is diluted 10000 times), especially the neutralizing capacity is relatively stronger when B2: BSA = 20. In the case of diluting the B antibody 1000 times, the B1-BSA and the B3-BSA show better B antibody neutralizing capacity relative to other coupling ratios of the same type antigen when the coupling ratio is 20; B4-BSA exhibited better B antibody neutralizing ability at a coupling ratio of 40. And in the table 5B, the control experiment adopting the antibody A, the BSA protein coupled with the B antigen (B1-B4) does not generate a neutralization reaction and also does not generate a reaction with the indicator cell B, so the experimental results are negative, and the experiment system is proved to run normally.
5.2 B-KLH antigen detection antibody.
1) 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 (using physiological saline as a 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 a substance to be detected and adding the substance into the ABO blood type reverse typing detection card; finally, 10. Mu.L of indicator cell B was added, and the results were read after centrifugation. The results are given in the following table:
table 5c
2) Diluting the A antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with physiological saline; then mixing the diluted antibody (using physiological saline as a 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 a substance to be detected and adding the substance into the ABO blood type reverse typing detection card; finally, 10. Mu.L of indicator cell B was added, and the results were read after centrifugation. The results are as follows:
table 5d
The experimental results are as follows: table 5c shows that the B antigen (B1-B4) coupled to KLH has a relatively good neutralizing effect on B antibody, and the neutralizing effect is strongest when B2-KLH is used, particularly when B2: KLH =20 is used as the neutralizing agent (even in a high concentration environment where B antibody is diluted 10 times, B2: KLH = 20; b2: KLH =40, 1, and the B antibody can be sufficiently neutralized even in a high concentration environment in which the B antibody is diluted 100-fold. B1-KLH, B3-KLH and B4-KLH show better B antibody neutralizing capacity relative to other conjugation ratios of the same type antigen when the conjugation ratio is 40. Table 5d shows the control experiment using antibody A, and the coupling of B antigen (B1-B4) to KLH should not produce neutralization reaction, and also should not produce reaction with indicator cell B, so the experimental results are all negative, which proves that the experimental system operates normally.
5.3 A-BSA antigen detection antibody.
1) Diluting the A antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with physiological saline; then mixing the diluted antibody (physiological saline is used as a negative control) with the A 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 a substance to be detected and adding the substance into the ABO blood type reverse typing detection card; finally, 10. Mu.L of indicator cell A was added, and the results were read after centrifugation. The results are as follows:
table 6a
2) Diluting the standard substance of the antibody B 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 A 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 a substance to be detected and adding the substance into the ABO blood type reverse typing detection card; finally, 10. Mu.L of indicator cell A was added, and the results were read after centrifugation. The results are as follows:
table 6b
The experimental results are as follows: table 6a shows that the neutralizing ability of the a antigen (A1-A4) coupled to BSA to a antibody is relatively good with A2-BSA, and especially the most potent with A2: BSA = 40; A2-BSA also exhibits better specificity in other coupling ratios (10-20, 1, 60. A3-BSA and A4-BSA were mixed at a coupling ratio of 40:1, exhibits better neutralizing ability of the A antibody compared with other coupling ratio of the same type antigen, and can sufficiently neutralize the A antibody under the environment of diluting the A antibody by 1000 times. Table 6B shows the control experiment using antibody B, and the A antigen (A1-A4) coupled with KLH should not react with neutralization reaction and indicator cell A, so the experimental results are all negative, thus proving that the experimental system runs normally.
5.4 A-KLH antigen detection antibody.
1) Diluting the A antibody standard substance by 10 times, 100 times, 1000 times, 10000 times and 100000 times with physiological saline; then mixing the diluted antibody (physiological saline as a negative control) with A 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 a substance to be detected and adding the substance into the ABO blood type reverse typing detection card; finally, 10. Mu.L of indicator cell A was added, and the results were read after centrifugation. The results were as follows:
table 6c
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 as a negative control) with A 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 a substance to be detected and adding the substance into the ABO blood type reverse typing detection card; finally, 10. Mu.L of indicator cell A was added, and the result was read after centrifugation. The results are as follows:
table 6d
The experimental results are as follows: table 6c shows that the neutralizing ability of the a antigen (A1-A4) coupled to KLH on the a antibody is relatively good with A2-KLH, and especially the neutralizing ability is strongest with A2: KLH = 40; A2-KLH also exhibits better specificity at other coupling ratios (10. A3-KLH and A4-KLH also exhibited a certain neutralizing ability of the A antibody at each coupling ratio (10. Table 6d shows the control experiment using antibody B, and the coupling of A antigen (A1-A4) to KLH should not produce neutralization reaction, nor reaction with indicator cell A, so the experimental results are all negative, thus proving that the experimental system operates normally.
In the above experiments, the blood group antigen trisaccharide a analogue protein conjugates with significant or more significant experimental effects are summarized in table 7 a; the blood group antigen trisaccharide a analogue protein conjugates with significant or more significant experimental effects are summarized in table 7 b.
Table 7a
Table 7b
While the present invention has been described with reference to the particular illustrative embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but is intended to cover various modifications, equivalent arrangements, and equivalents thereof, which may be made by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (12)
1. The application of the blood group antigen tri-saccharide conjugate in preparing a blood group antibody detection reagent is characterized in that the blood group antigen tri-saccharide conjugate is a blood group antigen tri-saccharide analogue protein conjugate; including blood group antigen trisaccharide B analogs; 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 analog is B2 type, the coupling ratio of the blood group antigen trisaccharide B analog to the hemocyanin is 5-40, and the coupling ratio of the blood group antigen trisaccharide B analog to the bovine serum albumin is 20;
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.
2. The use of claim 1 wherein 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.
3. The use according to claim 2, wherein when the blood group antigen trisaccharide a analogue is of type A2, the ratio of coupling of the blood group antigen trisaccharide a analogue to the hemocyanin is 10; 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.
4. The use of claim 2, wherein when the blood group antigen trisaccharide a analog is of type A2, the blood group antigen trisaccharide a analog has a coupling ratio to bovine serum albumin of 10 to 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 or 1-80.
5. The use of claim 1, wherein the blood group antibody detection reagent is used for immunochromatography, densitometry, or column agglutination.
6. The use according to claim 3, wherein the blood group antibody detection reagent is used for immunochromatography detection, and when the blood group antigen trisaccharide A analog is A3 type or A4 type, the coupling ratio of the blood group antigen trisaccharide A analog to the hemocyanin is 10.
7. The immunochromatography detection kit is characterized by comprising a detection card, wherein the detection card adopts a nitrocellulose membrane; the detection card is coated with a conjugate of blood group antigen trisaccharide B analogue and hemocyanin or bovine serum albumin and is used for detecting blood group antibodies in a serum sample; when the blood group antigen trisaccharide B analogue is B2, the coupling ratio of the blood group antigen trisaccharide B analogue to hemocyanin is 5-40; when the blood group antigen trisaccharide B analogue is B1 type, B3 type or B4 type, the coupling ratio to hemocyanin is 40.
8. The kit of claim 7, wherein the test card is further coated with a conjugate of a blood group antigen trisaccharide A analog and hemocyanin or a conjugate of bovine serum albumin.
9. The kit of claim 8, wherein when the blood group antigen trisaccharide a analog is of type A2, the blood group antigen trisaccharide a analog has a coupling ratio to hemocyanin of 10 to 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 or 1-80.
10. The kit of claim 8, wherein when the blood group antigen trisaccharide a analog is type A2, the coupling ratio of the blood group antigen trisaccharide a analog to bovine serum albumin is 10 to 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 or 40.
11. A column agglutination kit comprising an ABO blood group reverse typing test card, a blood group antigen trisaccharide analogue protein conjugate; the blood group antigen trisaccharide analogue protein conjugate comprises blood group antigen trisaccharide B analogue and hemocyanin or bovine serum albumin coupled; when the blood group antigen trisaccharide B analogue is of B2 type, the coupling ratio of the blood group antigen trisaccharide B analogue to the hemocyanin is 10-40; 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.
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 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 10-80; 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 hemocyanin is 10 to 1, and the coupling ratio of the blood group antigen trisaccharide a analog to bovine serum albumin is 10 or 1 to 40.
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