CN114397464A - Coupling compound of erythrocyte membrane fragments and carrier, coupling method and application thereof - Google Patents
Coupling compound of erythrocyte membrane fragments and carrier, coupling method and application thereof Download PDFInfo
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- CN114397464A CN114397464A CN202210294520.2A CN202210294520A CN114397464A CN 114397464 A CN114397464 A CN 114397464A CN 202210294520 A CN202210294520 A CN 202210294520A CN 114397464 A CN114397464 A CN 114397464A
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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
- G01N33/80—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
Abstract
The invention belongs to the field of blood type antibody detection, and particularly relates to a coupling compound of an erythrocyte membrane fragment and a carrier, a coupling method and application thereof. The coupling compound is formed by coupling erythrocyte membrane fragments and a carrier, wherein the carrier is a latex microsphere. The preparation method comprises the following steps: ultrasonication, carrier activation, coupling, dialysis sealing, centrifugal collection and storage. The invention carries out ultrasonic crushing treatment on the conjugate membrane fragments to make the conjugate membrane fragments become small fragments with uniform surface structures, and removes impurities generated by ultrasonic crushing and electrostatic attraction by dialysis treatment to make the membrane fragments in a stretched state as much as possible. And each part of the membrane fragment can be fully contacted and connected with the carrier microsphere, so that the coupling efficiency can be obviously improved. The process of sealing after coupling is closed liquid system dialysis, so that sealing liquid and the like can be fully contacted with the coupling compound, and a better liquid sealing effect is achieved.
Description
Technical Field
The invention belongs to the field of blood type antibody detection, and particularly relates to a coupling compound of an erythrocyte membrane fragment and a carrier, a coupling method and application thereof.
Background
The research and detection of human cell membrane antigen and its specific antibody are one of the most basic contents of biological science, immunological experiment research and clinical detection. Human A, B, O and Rh blood group antigen/antibody testing are routine clinical testing items. Blood grouping is based on the agglutination of cells by antigen-antibody reactions. The use of red blood cell surface antigens to detect blood group antibodies in serum is called retrotyping. In clinical blood transfusion, the safety of blood transfusion can be ensured only by ensuring the consistency of the identification results of the positive and negative shaping methods.
For blood group antibody detection, the traditional technology is a test tube method, a paper sheet method or a column agglutination method, the methods all need a fresh red blood cell indicator, the red blood cell indicator is a red blood cell suspension, and the survival cycle of the red blood cells can only be stored for 3-6 months and cold chain transportation is needed. Red blood cells present certain risks for use as a blood product.
The erythrocyte membrane can better protect the activity of the carried substance and has longer and more controllable life span. These properties make the "carrier red blood cells" a very valuable transport carrier.
CN101387648A discloses a magnetic bead kit of erythrocyte membrane antigen and its application in blood group antibody detection, which describes that magnetic beads coated with blood group antigen on the surface are used to replace erythrocytes for blood group reverse typing detection. However, it is known that blood group antigens are membrane proteins, which have difficulty in maintaining the three-dimensional structure in an in vivo environment in vitro, and gradually lose antigenicity (ability to react with corresponding blood group antibodies). Therefore, the magnetic beads coated with blood group antigens cannot solve the problem that red blood cells are difficult to store for a long time.
Common chromogenic labels are enzymes, colloidal gold, and latex microspheres. Although the technology of using the enzyme as a marking material is mature, the enzyme is greatly influenced by temperature, the stability is poor, and the effective period of the product is short; colloidal gold is the most used marking material in the current commercial test strip, but the signal method has high cost and cannot be used for accurate detection; the latex microspheres can form stable alternation in liquid, compared with colloidal gold, the microspheres have larger particles, can save the using amount of antibodies and have higher sensitivity; meanwhile, large-scale production can be carried out, and the cost is lower; the stability and the sensitivity are better. Therefore, the covalent bonding between the latex microspheres with different groups on the surface and the antibody to form a complex with a stable structure is a hot spot of current research in the fields of disease diagnosis, biological medicine and the like.
However, in the process of labeling the antigen by the existing carrier, most of the antigen is natural antigen or synthetic antigen with small molecular weight. The coupling of the erythrocyte membrane fragments and the carrier is not suitable for the traditional marking method, and the adoption of the existing marking method can cause the coupling efficiency and the coupling strength of the carrier microspheres and the erythrocyte membrane fragments to be lower, thereby causing the antigenicity to be lower.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a coupling compound of erythrocyte membrane fragments and a carrier, a coupling method and application thereof, and aims to solve the problem that the antigenicity is lower due to the fact that the coupling efficiency and coupling strength of carrier microspheres and erythrocyte membrane fragments are lower because a labeling method is not appropriate in the prior art.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the coupling compound of the red cell membrane fragments and the carrier is formed by coupling the red cell membrane fragments and the carrier, and the carrier is a latex microsphere.
The coupling compound of the erythrocyte membrane fragments and the carrier has higher antigenicity, can be used for human blood group antibody detection, can accurately reflect the types of blood group antibodies, achieves the aim of replacing fresh erythrocytes, provides convenience for the detection of the blood group antibodies, and breaks the limit that the detection of the blood group antibodies depends on the erythrocytes.
Further, the concentration ratio of the red cell membrane fragments to the carrier is 35-45: 1, preferably 40: 1.
in the invention, the latex microspheres are carboxyl latex microspheres.
The invention also provides a coupling method of the erythrocyte membrane fragments and the carrier, wherein the coupling method comprises the following steps:
s1, ultrasonic crushing: under the ice bath condition, carrying out ultrasonic crushing, resuspension and dialysis on the erythrocyte membrane fragments to obtain the erythrocyte membrane fragments subjected to ultrasonic crushing;
s2, activating the carrier: ultrasonically activating the latex microspheres, centrifuging and cleaning an activation buffer solution, adding an MES solution of EDC, immediately mixing uniformly, centrifuging to obtain activated latex microspheres, and keeping the activated latex microspheres in the dark at normal temperature for later use;
s3, coupling: uniformly mixing the red cell membrane fragments subjected to ultrasonic crushing with the standby activated latex microspheres, centrifuging to obtain a coupling compound, and keeping the coupling compound away from light at normal temperature for standby;
s4, dialysis sealing: redissolving the coupling compound for later use by using a confining liquid, uniformly mixing, and dialyzing to obtain the coupling compound after dialysis;
s5, centrifugal collection: centrifuging the dialyzed coupling compound, and discarding the supernatant;
s6, storage: adding storage buffer, resuspending the conjugate complex, and cold storing.
The invention couples the red cell membrane fragment and the carrier prepared by extraction by an immune labeling technology, and displays the form and the content of the antigen in a reaction system by the enhanced amplification effect of a label. Such a carrier coupled to erythrocytes is not limited to colloidal gold, magnetic beads, latex, fluorescein, fluorescent microspheres, enzymes, or radionuclides, etc.
The invention carries out ultrasonic crushing treatment on the conjugate membrane fragments to make the conjugate membrane fragments become small fragments with uniform surface structures, and removes impurities generated by ultrasonic crushing and electrostatic attraction by dialysis treatment to make the membrane fragments in a stretched state as much as possible; each part of the membrane fragment can be fully contacted and connected with the carrier microsphere, so that the coupling efficiency can be obviously improved, and the detection result can be improved; the process of sealing after coupling is closed liquid system dialysis, so that sealing liquid and the like can be fully contacted with the coupling compound, and a better liquid sealing effect is achieved.
Further, in step S2, the ultrasonic activation is ultrasonic activation at a frequency of 40kHz for 30 min.
In the invention, the influence of the ultrasonic activation time in the carrier activation process on the dilution sensitivity of the prepared coupling compound for detecting A, B antibody is examined. The result shows that compared with 20min or 40min ultrasonic activation, the coupling compound obtained by 30min ultrasonic activation has higher antigenicity, can better improve the coupling efficiency of the carrier latex microspheres and the membrane antigen, and improves the detection result. Therefore, the ultrasonic activation of the invention selects ultrasonic activation for 30min at the frequency of 40 kHz.
Further, in step S1, the ultrasonication is performed at a power of 200W, and the ultrasonication is stopped for 5S every 5S, and the total ultrasonication time is 30 min.
Further, in step S3, the concentration ratio of the erythrocyte membrane fragments to the latex microspheres is 35-45: 1, preferably 40: 1.
further, the air conditioner is provided with a fan,
in step S1, the resuspension is performed using 0.1M, pH =8.0 Tris-cl to a volume of 10 ml; the dialysis is carried out by adopting a dialysis bag with the molecular weight cutoff of 10KD and normal saline as dialysis buffer solution, and the dialysis time is 24 h;
in step S4, the dialysis is performed by using a dialysis bag with a cut-off molecular weight of 100KD and a confining liquid as a dialysis buffer, and the dialysis time is 8 h.
Further, the air conditioner is provided with a fan,
in step S2, the centrifugation is performed for 30min at 1000 rpm;
in step S3, the centrifugation is performed for 2.5h at 1000 rpm;
in step S5, the centrifugation is performed at 18000rpm for 15 min.
Further, the air conditioner is provided with a fan,
preparing EDC by using 0.05M MES, wherein the concentration of the prepared solution is 10 mg/mL;
the activating buffer is 0.05M, pH =6.1 MES buffer;
the confining liquid is Gly with mass fraction of 7.5%, BSA solution with mass fraction of 1%, and NaN with mass fraction of 0.05%3(ii) a Wherein Gly is glycine and accounts for 7.5 percent of the whole confining liquid by mass; the BSA solution with the mass fraction of 1% means that the BSA solution accounts for 1% of the mass fraction of the whole blocking solution; NaN with mass fraction of 0.05%3Refers to NaN3The mass fraction of the sealing liquid is 0.05 percent;
the storage buffer solution comprises 0.05M, pH =8.1 of Tris buffer solution, 1% of BSA solution by mass fraction and 0.05% of NaN by mass fraction3(ii) a Wherein, a Tris buffer of 0.05M, pH =8.1 means that the concentration of Tris buffer at pH 8.1 is 0.05M of the entire storage buffer; the BSA solution with the mass fraction of 1% means that the BSA solution accounts for 1% of the mass fraction of the whole storage buffer solution; NaN with mass fraction of 0.05%3Refers to NaN3The mass fraction of the buffer solution in the whole storage is 0.05%.
The invention also provides application of the coupling compound in blood group antibody detection or in preparation of a blood group antibody detection kit.
Compared with the prior art, the invention has the following advantages:
(1) the coupling compound of the erythrocyte membrane fragments and the carrier has higher antigenicity, can be used for human blood group antibody detection, can accurately reflect the types of blood group antibodies, achieves the aim of replacing fresh erythrocytes, provides convenience for the detection of the blood group antibodies, and breaks the limit that the detection of the blood group antibodies depends on the erythrocytes;
(2) the invention carries out ultrasonic crushing treatment on the conjugate membrane fragments to make the conjugate membrane fragments become small fragments with uniform surface structures, and removes impurities generated by ultrasonic crushing and electrostatic attraction by dialysis treatment to make the membrane fragments in a stretched state as much as possible; each part of the membrane fragment can be fully contacted and connected with the carrier microsphere, so that the coupling efficiency can be obviously improved, and the detection result can be improved; the process of sealing after coupling is closed liquid system dialysis, so that sealing liquid and the like can be fully contacted with the coupling compound, and a better liquid sealing effect is achieved.
Drawings
FIG. 1 is a graph showing the sensitivity of detection of A, B antigen in a conjugated complex according to the present invention;
FIG. 2 is a 2-fold sensitivity of detection of A, B antibody dilution by conjugate complexes of the invention;
FIG. 3 is a 4-fold dilution sensitivity of the conjugate complex detection A, B antibody of the present invention;
FIG. 4 shows the 8-fold sensitivity of detection of A, B antibody dilution by conjugate complexes of the invention;
FIG. 5 is a 16-fold dilution sensitivity of the conjugate complex detection A, B antibody of the present invention;
FIG. 6 shows the 32-fold sensitivity of detection of A, B antibody dilution of the conjugate complexes of the present invention;
FIG. 7 shows the sensitivity of detection of A, B antibody dilutions 64 times for the conjugate complexes of the invention;
FIG. 8 shows the sensitivity of detection of A, B antigen in the conjugate complex of the comparative example;
FIG. 9 shows the sensitivity of detecting A, B antibody dilution 2-fold for the conjugate complex of the comparative example;
FIG. 10 shows the sensitivity of detection of A, B antibody diluted 4-fold for the conjugate complex of the comparative example;
FIG. 11 is an 8-fold dilution sensitivity of the conjugate complex detection A, B antibody of the comparative example;
FIG. 12 is a 16-fold sensitivity of detection of A, B antibody dilution with conjugate complexes of the comparative example;
FIG. 13 is a 32-fold dilution sensitivity of the conjugate complex detection A, B antibody of the comparative example;
FIG. 14 shows the sensitivity of detection of A, B antibody diluted 64 times for the conjugate complex of the comparative example;
FIG. 15 shows the sensitivity of detecting A, B antigen in situ for 20min of conjugate complex by ultrasonic activation;
FIG. 16 shows the 2-fold sensitivity of detection of A, B antibody dilution for 20min of conjugate complexes by ultrasonic activation;
FIG. 17 shows the sensitivity of A, B antibody dilution 4-fold for 20min ultrasonic activation of conjugate complex detection;
FIG. 18 shows the 8-fold sensitivity of detection of A, B antibody dilution for 20min of conjugate complexes by ultrasonic activation;
FIG. 19 shows the 16-fold sensitivity of detection of A, B antibody dilution for 20min ultrasonically activated conjugate complexes;
FIG. 20 shows the sensitivity of A, B antibody dilution 32 times for 20min of conjugate complex detection by ultrasonic activation;
FIG. 21 shows the sensitivity of A, B detection of primary antibody for 40min of conjugation complex by ultrasonic activation;
FIG. 22 shows the 2-fold sensitivity of detection of A, B antibody dilution for conjugate complexes with ultrasonic activation for 40 min;
FIG. 23 shows the sensitivity of A, B antibody dilution 4-fold for conjugate complex detection with ultrasonic activation for 40 min;
FIG. 24 shows 8-fold sensitivity of detection of A, B antibody dilution for conjugate complexes with ultrasonic activation for 40 min;
FIG. 25 shows the 16-fold sensitivity of detection of A, B antibody dilution for 40min of conjugate complexes by ultrasonic activation;
FIG. 26 shows the sensitivity of A, B antibody dilution 32 fold for 40min of conjugate complex detection by ultrasonic activation.
Detailed Description
The following are specific embodiments of the present invention, which are intended to further illustrate the invention and not to limit it.
In the following examples, the red blood cell fragments used were prepared as follows:
1) taking 3mL of anticoagulated mixed whole blood (taken from 6-10 blood samples of A type or other blood types), adding the anticoagulated mixed whole blood into a 15m L centrifuge tube filled with 10mL of 0.01mol/L PBS (pH 7.2), centrifuging for 5min at 3000r/min, and discarding the supernatant and white blood cells and platelet layers under the supernatant to obtain about 1.5mL of packed red blood cells;
2) washing with 4 deg.C pre-cooled 0.01mol/L PBS (pH7.2) with volume 3 times of packed red blood cells for 2 times, and centrifuging at 4 deg.C for 15min at 5000 r/min;
3) with V: V = 40: 1, adding 0.01mol/L PBS (pH7.2) precooled at 4 ℃ to the precipitate, standing at 4 ℃ for 2 hours, centrifuging at 9000r/min for 20min, and removing the supernatant;
4) repeating the step 3) for 4 times until no red blood cells are visible to the naked eye, and obtaining about 800 microliter of precipitate which is the red blood cell membrane fragment sample carrying the membrane antigen.
Multiple preparations were carried out in this way, and the red cell membrane fragments obtained were either used immediately (for membrane antigen detection) or were dispensed directly and stored at 4 ℃ or-20 ℃ respectively.
In the following examples, EDC was prepared using 0.05M MES as an MES solution, and the concentration of the prepared solution was 10 mg/mL;
the activating buffer solution is as follows: MES buffer 0.05M, pH = 6.1;
the confining liquid is as follows: 7.5% of Gly, 1% of BSA solution and 0.05% of NaN3(ii) a Wherein Gly is glycine and accounts for 7.5 percent of the whole confining liquid by mass; the BSA solution with the mass fraction of 1% means that the BSA solution accounts for 1% of the mass fraction of the whole blocking solution; NaN with mass fraction of 0.05%3Refers to NaN3The mass fraction of the sealing liquid is 0.05 percent;
the storage buffer solution is as follows: tris buffer 0.05M, pH =8.1, BSA solution at 1% mass fraction, and NaN at 0.05% mass fraction3(ii) a Wherein, a Tris buffer of 0.05M, pH =8.1 means that the concentration of Tris buffer at pH 8.1 is 0.05M of the entire storage buffer; the BSA solution with the mass fraction of 1% means that the BSA solution accounts for 1% of the mass fraction of the whole storage buffer solution; NaN with mass fraction of 0.05%3Refers to NaN3The mass fraction of the buffer solution in the whole storage is 0.05%.
Example 1
S1, ultrasonic crushing in ice bath: 1 beaker is taken and put into ice blocks, and a penicillin bottle filled with 5mLA type erythrocyte membrane fragments (5 mg/ml) is fixedly arranged at the center of the beaker. The beaker was placed on an ultrasonic platform. Turning on the cell crusher, setting power 200W, total working time 30M, ultrasonic on time 5S and ultrasonic off time 5S. Resuspend to volume 10ml with 0.1M, pH =8.0 Tris-cl. Putting the A-type erythrocyte membrane fragments subjected to ultrasonic treatment into a dialysis bag with the molecular weight cutoff of 10KD, and dialyzing for 24 hours by using physiological saline as dialysis buffer. Eliminating static electricity and balancing the buffer system.
S2, activating the carrier: ultrasonically activating JSR114nm latex microspheres for 30min at the frequency of 40kHz, centrifugally cleaning the activated buffer solution for 2 times, preparing EDC10mg/mL by using 0.05M MES, immediately and uniformly mixing the mixture in a vortex manner (the mixture is ready for use), fixing a centrifugal tube on a shaking bed, centrifuging the mixture for 30min at 1000rpm, and keeping the mixture away from light at normal temperature.
S3, coupling: the treated fragment of the type A erythrocyte membrane and JSR114nm latex microspheres are mixed according to the weight ratio of 40: 1, and immediately mixing by swirling. The tube was fixed on a shaker and centrifuged at 1000rpm for 2.5h, protected from light at room temperature.
S4, dialysis sealing: and (3) redissolving the coupled compound by using 3000mL of confining liquid, and shaking and uniformly mixing. Adding dialysis bag with cut-off molecular weight of 100KD, dialyzing with dialysis buffer solution as confining liquid for 8 hr.
S5, centrifugal collection: the dialyzed conjugate complex was centrifuged at 18000rpm for 15min and the supernatant was discarded.
S6, storage: add 800uL of storage buffer, resuspend the conjugate complex, and store at 4 ℃.
Example 2
Referring to example 1, the difference from example 1 is that the red blood cell membrane fragments used are B-type red blood cell membrane fragments.
Example 3
Referring to example 1, the difference from example 1 is that the red blood cell membrane fragments used are O-type red blood cell membrane fragments.
Example 4
Referring to example 1, the difference from example 1 is that the red cell membrane fragments used are AB type red cell membrane fragments.
Example 5
Referring to example 1, the difference from example 1 is that the a-type erythrocyte membrane fragments treated in the coupling process of step S3 were mixed with JSR114nm latex microspheres according to a 35: 1 concentration ratio.
Example 6
Referring to example 1, the difference from example 1 is that the a-type erythrocyte membrane fragments treated in the coupling process of step S3 were mixed with JSR114nm latex microspheres according to a ratio of 45: 1 concentration ratio.
Comparative example
1) Taking a penicillin bottle filled with 5mLA type erythrocyte membrane fragments (5 mg/ml), and placing at room temperature;
2) activating a carrier: ultrasonically activating JSR114nm latex microspheres for 30min at the frequency of 40kHz, centrifugally cleaning the activated buffer solution for 2 times, preparing EDC10mg/mL by using 0.05M MES, immediately and uniformly mixing the mixture in a vortex manner (the mixture is ready for use), fixing a centrifugal tube on a shaking bed, centrifuging the mixture for 30min at 1000rpm, and keeping the mixture away from light at normal temperature;
3) coupling: the treated fragment of the type A erythrocyte membrane and JSR114nm latex microspheres are mixed according to the weight ratio of 40: 1, and immediately mixing by swirling. Fixing the centrifugal tube on a shaker, centrifuging at 1000rpm for 2.5h, and keeping out of the sun at normal temperature;
4) and (3) centrifugal collection: centrifuging the centrifuged coupling compound at 18000rpm for 15min, and removing the supernatant;
5) and (3) storage: add 800uL of storage buffer, resuspend the conjugate complex, and store at 4 ℃.
Test example 1
This test example examined the dilution sensitivity of A, B antibody for detection of the conjugate complexes prepared in the present invention.
Experimental materials: the conjugated complex (denoted as No. 2A membrane ball), Millipore-A antibody, Millipore-B antibody, normal saline, 5% PEG6000 normal saline solution and conjugated microsphere dilution buffer (namely the storage buffer) after the JSR114nm latex microsphere is conjugated with the type A erythrocyte membrane fragments prepared in the example 1.
The experimental method comprises the following steps: the coupled compound (namely No. 2A membrane ball) prepared in example 1 and obtained by coupling the A-type erythrocyte membrane fragments with JSR114nm latex microspheres is diluted by 40 times by using a coupled microsphere dilution buffer solution, and is respectively mixed with A, B antibody or a sample for reaction, and an OD value change curve in the reaction time is detected at the wavelength of 340 nm.
Detection A, B antibody dilution sensitivity results:
the A antigen coupled microspheres (i.e. No. 2A membrane spheres) are respectively mixed with A, B antibody and A, B antibody diluted by physiological saline for reaction, and the result of detecting OD value is as follows (the detection antibody-dotted line is negative, and the solid line is positive).
The results are shown in FIGS. 1 to 7.
Test example 2
This test example examined the dilution sensitivity of A, B antibody for the conjugate complexes prepared in the comparative examples.
Experimental materials: the conjugated complex (marked as No. 1A membrane ball), Millipore-A antibody, Millipore-B antibody, physiological saline, 5% PEG6000 physiological saline solution and conjugated microsphere dilution buffer (namely the storage buffer) after the JSR114nm latex microsphere is conjugated by the A type erythrocyte membrane fragments prepared by the comparative example.
The experimental method comprises the following steps: and (3) diluting the coupled compound (namely the No. 1A membrane ball) prepared by the comparative example and obtained by coupling the A-type erythrocyte membrane fragments with JSR114nm latex microspheres by 40 times by using a coupled microsphere dilution buffer solution, mixing and reacting with A, B antibody or a sample respectively, and detecting the change curve of the OD value within the reaction time at the wavelength of 340 nm.
Detection A, B antibody dilution sensitivity results:
the A antigen coupled microspheres (i.e. No. 1A membrane spheres) are respectively mixed with A, B antibody and A, B antibody diluted by physiological saline for reaction, and the result of detecting OD value is as follows (the detection antibody-dotted line is negative, and the solid line is positive).
The results are shown in FIGS. 8 to 14.
From the results of test example 1 and test example 2, it can be seen that the conjugate prepared by the coupling method of the example has higher antigenicity, and can better improve the coupling efficiency of the carrier latex microspheres and the membrane antigen and improve the detection result, compared with the coupling method of the comparative example.
Test example 3
The test example examines the influence of the ultrasonic activation time in the carrier activation process on the dilution sensitivity of the prepared coupling compound for detecting A, B antibody.
The test method comprises the following steps: referring to example 1, except that the ultrasonic activation time was 20min and 40min, respectively, wherein the coupled complex obtained at 20min was designated as membrane sphere No. 3 a, and the coupled complex obtained at 40min was designated as membrane sphere No. 4 a. The membrane beads A No. 3 and the membrane beads A No. 4 were diluted 40-fold with a coupled microsphere dilution buffer, and the dilution sensitivity results of the A, B antibody were measured according to the method of test example 1.
Wherein the detection result of ultrasonic activation for 20min is shown in FIGS. 15 to 20, and the detection result of ultrasonic activation for 40min is shown in FIGS. 21 to 26 (detection antibody-dotted line is negative, solid line is positive).
From the test results and the results of the test example 1, it can be seen that, compared with the ultrasonic activation for 20min or 40min, the coupling compound obtained by the ultrasonic activation for 30min has higher antigenicity, and can better improve the coupling efficiency of the carrier latex microspheres and the membrane antigen and improve the detection results.
Claims (10)
1. A method for coupling erythrocyte membrane fragments with carriers is characterized by comprising the following steps:
s1, ultrasonic crushing: under the ice bath condition, carrying out ultrasonic crushing, resuspension and dialysis on the erythrocyte membrane fragments to obtain the erythrocyte membrane fragments subjected to ultrasonic crushing;
s2, activating the carrier: ultrasonically activating the latex microspheres, centrifugally cleaning the activated buffer solution, adding an MES solution of EDC, immediately mixing uniformly, centrifuging to obtain activated latex microspheres, and keeping the activated latex microspheres away from light at normal temperature for later use;
s3, coupling: uniformly mixing the red cell membrane fragments subjected to ultrasonic crushing with the standby activated latex microspheres, centrifuging to obtain a coupling compound, and keeping the coupling compound away from light at normal temperature for standby;
s4, dialysis sealing: redissolving the coupling compound for later use by using a confining liquid, uniformly mixing, and dialyzing to obtain the coupling compound after dialysis;
s5, centrifugal collection: centrifuging the dialyzed coupling compound, and discarding the supernatant;
s6, storage: adding storage buffer, resuspending the conjugate complex, and cold storing.
2. The coupling process according to claim 1, wherein in step S2, the ultrasonic activation is ultrasonic activation at a frequency of 40kHz for 30 min.
3. The coupling process according to claim 1, wherein in step S1, the ultrasonication is performed at a power of 200W, and the total ultrasonication time is 30min for every 5S sonications.
4. The coupling method according to claim 1, wherein in step S3, the concentration ratio of the erythrocyte membrane fragments to the latex microspheres is 35-45: 1.
5. the coupling method according to claim 4, wherein the concentration ratio of the red cell membrane fragments to the latex microspheres is 40: 1.
6. the coupling method according to claim 1, wherein in step S1, the resuspension is performed by resuspending to a volume of 10ml with 0.1M, pH =8.0 Tris-cl; the dialysis is carried out by adopting a dialysis bag with the molecular weight cutoff of 10KD and normal saline as dialysis buffer solution, and the dialysis time is 24 h;
in step S4, the dialysis is performed by using a dialysis bag with a cut-off molecular weight of 100KD and a confining liquid as a dialysis buffer, and the dialysis time is 8 h.
7. The coupling method according to claim 1,
in step S2, the centrifugation is performed for 30min at 1000 rpm;
in step S3, the centrifugation is performed for 2.5h at 1000 rpm;
in step S5, the centrifugation is performed at 18000rpm for 15 min.
8. The coupling method according to any one of claims 1 to 7,
preparing EDC by using 0.05M MES, wherein the concentration of the prepared solution is 10 mg/mL;
the activating buffer is 0.05M, pH =6.1 MES buffer;
the confining liquid is Gly with mass fraction of 7.5%, BSA solution with mass fraction of 1%, andNaN with mass fraction of 0.05%3；
The storage buffer solution comprises 0.05M, pH =8.1 of Tris buffer solution, 1% of BSA solution by mass fraction and 0.05% of NaN by mass fraction3。
9. Use of a conjugate complex obtained by the conjugation method according to any one of claims 1-8 for blood group antibody detection.
10. The use according to claim 9, characterized in that the use in the detection of blood group antibodies is the preparation of a kit for the detection of blood group antibodies.
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| CN117471110A (en) * | 2023-12-27 | 2024-01-30 | 天津德祥生物技术股份有限公司 | Blocking liquid and blocking method for blocking antigen-microsphere indication system |
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