CN117517645A - Method for separating serum by anti-erythrocyte antibody magnetic particles and application - Google Patents
Method for separating serum by anti-erythrocyte antibody magnetic particles and application Download PDFInfo
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- CN117517645A CN117517645A CN202311492237.1A CN202311492237A CN117517645A CN 117517645 A CN117517645 A CN 117517645A CN 202311492237 A CN202311492237 A CN 202311492237A CN 117517645 A CN117517645 A CN 117517645A
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- 239000006249 magnetic particle Substances 0.000 title claims abstract description 75
- 210000002966 serum Anatomy 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 26
- 210000004369 blood Anatomy 0.000 claims abstract description 49
- 239000008280 blood Substances 0.000 claims abstract description 49
- 210000003743 erythrocyte Anatomy 0.000 claims abstract description 37
- 238000001514 detection method Methods 0.000 claims abstract description 26
- 239000000427 antigen Substances 0.000 claims abstract description 16
- 102000036639 antigens Human genes 0.000 claims abstract description 16
- 108091007433 antigens Proteins 0.000 claims abstract description 16
- 239000011248 coating agent Substances 0.000 claims abstract description 7
- 238000000576 coating method Methods 0.000 claims abstract description 7
- 238000000926 separation method Methods 0.000 claims abstract description 7
- 238000007885 magnetic separation Methods 0.000 claims abstract description 6
- 238000012545 processing Methods 0.000 claims abstract description 4
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 12
- 241000710799 Rubella virus Species 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 9
- 239000003085 diluting agent Substances 0.000 claims description 7
- 238000002965 ELISA Methods 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000011161 development Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 238000011895 specific detection Methods 0.000 claims description 3
- 239000012089 stop solution Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000011160 research Methods 0.000 abstract description 7
- 238000011282 treatment Methods 0.000 abstract description 3
- 239000011324 bead Substances 0.000 description 13
- 239000000523 sample Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 6
- 238000003759 clinical diagnosis Methods 0.000 description 5
- 210000000601 blood cell Anatomy 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 206010018910 Haemolysis Diseases 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000010241 blood sampling Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 206010042772 syncope Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000001112 coagulating effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
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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
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
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- 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
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/19—Rubella virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
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Abstract
A method for separating serum by anti-erythrocyte antibody magnetic particles and application thereof relate to the field of medical examination and detection, in particular to an improvement of a serum separation method and application thereof. The specific method comprises the following steps: preparing anti-RBC antibody magnetic particles: coating the anti-RBC antibody on the surface of the magnetic particles to form anti-RBC antibody magnetic particles; 2. adding the whole blood sample into prepared anti-RBC antibody magnetic particles in proportion, and allowing the magnetic particles to adsorb red blood cells in blood; 3. through magnetic separation technology, the anti-RBC antibody magnetic particles are combined with specific antigens on the surface of the red blood cells to separate out the fixed red blood cells, and the plasma can be left in the tube; 4. the separated serum sample is collected in a clean environment for further detection or processing. It provides a method for coating magnetic particles with anti-RBC antibody (anti-erythrocyte antibody) so as to effectively remove erythrocyte in whole blood and obtain high-quality serum sample. The method can also be used in the fields of biological research, detection, treatment and the like.
Description
Technical Field
The invention relates to the field of medical examination and detection, in particular to a serum separation method and an application improvement.
Background
In the medical test, clinical diagnosis requires collecting a whole blood sample and separating serum or plasma, and two methods currently in common use are precipitation and centrifugation. However, both methods require time, which affects the real-time and sensitivity of the detection.
A new method has been developed to remove red blood cells from whole blood using magnetic particles. This method is fast, simple, sensitive and requires no additional equipment. However, this method also has its drawbacks. The effect is poor for special samples, such as hemolyzed samples, red blood cell clot samples, etc., so that the sample needs to be collected with the addition of an anticoagulant to prevent the red blood cells from coagulating. This increases the cost and affects the detection result.
Disclosure of Invention
The invention aims at overcoming the defects and shortcomings of the prior art, and provides a method for separating serum by using anti-red blood cell antibody magnetic particles and application thereof, and provides a method for efficiently removing red blood cells in whole blood and obtaining a high-quality serum sample by coating anti-RBC antibodies (anti-red blood cell antibodies) on the magnetic particles. The method can also be used in the fields of biological research, detection, treatment and the like.
In order to achieve the above object, the method for separating serum by using anti-erythrocyte antibody magnetic particles in the invention comprises the following steps: 1. preparing anti-RBC antibody magnetic particles: coating the anti-RBC antibody on the surface of the magnetic particles to form anti-RBC antibody magnetic particles; 2. adding the whole blood sample into prepared anti-RBC antibody magnetic particles in proportion, and allowing the magnetic particles to adsorb red blood cells in blood; 3. through magnetic separation technology, the anti-RBC antibody magnetic particles are combined with specific antigens on the surface of the red blood cells to separate out the fixed red blood cells, and the plasma can be left in the tube; 4. the separated serum sample is collected in a clean environment for further detection or processing.
The application of the anti-erythrocyte antibody magnetic particle separation serum in detecting the serum rubella virus IgG antibody is as follows: detection is carried out by using enzyme-linked immunosorbent assay (ELISA) of anti-RBC antibody magnetic particles, and the specific detection steps are as follows:
1. preparing anti-RBC antibody magnetic particles: mixing anti-RBC antibody magnetic particles uniformly, adding 600ug to an enzyme-labeled hole respectively, performing magnetic adsorption, and removing supernatant diluent;
2. adding 100ul whole blood into anti-RBC antibody magnetic particles, mixing uniformly, and standing at 37 ℃ for incubation for 10min to allow the magnetic particles to adsorb red blood cells in the blood;
3. adsorbing the magnetic particles and the red blood cell-combined compound on the bottom of the enzyme-labeled hole through a magnetic device, so that red blood cells in whole blood are separated from serum;
4. transferring 50ul serum into an enzyme-labeled plate hole coated with rubella virus antigen, incubating at 37 ℃ for 30min, and washing the hole after the binding with the antigen, so that the part which is not bound with the antigen is washed off;
5. adding 100ul of labeled enzyme-labeled secondary antibody, and incubating at 37 ℃ for 30min to combine with the IgG antibody combined with the antigen to form a specific complex;
6. washing the wells again, removing unbound secondary antibodies, adding 50ul of color development liquid A, B each, and incubating at 37 ℃ for 15min;
7. adding 50ul of stop solution;
8. OD values were read at a wavelength of 450 nm.
Detection result: 1) Positive coincidence rate: the test results of 96 rubella virus patient samples show that the rubella virus IgG antibodies are positive in 96 cases, the positive coincidence rate (+/+) is 96/96, and no missed test occurs; 2) Negative coincidence rate: the detection result of 96 healthy human samples is 88 cases of rubella virus IgG antibody negative, the negative coincidence rate (-/-) is 88/96, 8 cases of false positive appear, and through investigation, 6 cases of the cases appear a small amount of red blood cells to be broken, so that the false positive is caused; 3) Repeatability: the same positive sample is used for repeated detection for 10 times, the detection results are positive for the rubella virus IgG antibody, and the variation coefficient of the absorbance value is not more than 15.0%.
The invention provides a means for efficiently and quickly removing red blood cells in whole blood, and avoids other complicated separation steps. The magnetic particles of the anti-RBC antibody are utilized to separate the red blood cells, so that the selectivity and the efficiency are high, the high quality of serum is ensured, and the pollution to serum components is avoided. The magnetic separation technology is adopted, so that devices such as centrifugation, a suction pipe and the like are not needed in the operation process, and the automatic operation can be realized. The method can be applied to the fields of biology and medical research, including but not limited to the fields of hematology, clinical diagnosis, microbiology and the like, so that the application range of the method in scientific research and clinical application is widened. The application and clinical diagnosis can reduce the multitube blood sampling of patients and avoid syncope caused by excessive blood loss. In clinical assay detection, the anti-RBC antibody magnetic particles improve the detection sensitivity and specificity, reduce non-specific background signals and improve the detection accuracy.
After the technical scheme is adopted, the invention provides a method for efficiently removing red blood cells in whole blood and obtaining a high-quality serum sample by coating the anti-RBC antibody (anti-red blood cell antibody) on the magnetic particles, so that the subsequent detection and detection are more convenient and accurate; the method can also be used in the fields of biological research, detection, treatment and the like.
Detailed Description
The technical scheme adopted by the specific embodiment is as follows:
the method for separating serum from the medium anti-erythrocyte antibody magnetic particles comprises the following steps: 1. preparing anti-RBC antibody magnetic particles: coating the anti-RBC antibody on the surface of the magnetic particles to form anti-RBC antibody magnetic particles; 2. adding the whole blood sample into prepared anti-RBC antibody magnetic particles in proportion, and allowing the magnetic particles to adsorb red blood cells in blood; 3. through magnetic separation technology, the anti-RBC antibody magnetic particles are combined with specific antigens on the surface of the red blood cells to separate out the fixed red blood cells, and the plasma can be left in the tube; 4. the separated serum sample is collected in a clean environment for further detection or processing.
The application of the anti-erythrocyte antibody magnetic particle separation serum in detecting the serum rubella virus IgG antibody is as follows: detection is carried out by using enzyme-linked immunosorbent assay (ELISA) of anti-RBC antibody magnetic particles, and the specific detection steps are as follows:
1. preparing anti-RBC antibody magnetic particles: mixing anti-RBC antibody magnetic particles uniformly, adding 600ug to an enzyme-labeled hole respectively, performing magnetic adsorption, and removing supernatant diluent;
2. adding 100ul whole blood into anti-RBC antibody magnetic particles, mixing uniformly, and standing at 37 ℃ for incubation for 10min to allow the magnetic particles to adsorb red blood cells in the blood;
3. adsorbing the magnetic particles and the red blood cell-combined compound on the bottom of the enzyme-labeled hole through a magnetic device, so that red blood cells in whole blood are separated from serum;
4. transferring 50ul serum into an enzyme-labeled plate hole coated with rubella virus antigen, incubating at 37 ℃ for 30min, and washing the hole after the binding with the antigen, so that the part which is not bound with the antigen is washed off;
5. adding 100ul of labeled enzyme-labeled secondary antibody, and incubating at 37 ℃ for 30min to combine with the IgG antibody combined with the antigen to form a specific complex;
6. washing the wells again, removing unbound secondary antibodies, adding 50ul of color development liquid A, B each, and incubating at 37 ℃ for 15min;
7. adding 50ul of stop solution;
8. OD values were read at a wavelength of 450 nm.
The ratio test for optimizing whole blood and anti-RBC magnetic particles in this embodiment is as follows:
(1) taking 5 EP tubes, respectively adding 50ul of whole blood and 100ul of anti-RBC antibody magnetic particles, uniformly mixing, adding 100ul of magnetic particle diluent into one of the EP tubes as a control, and placing the EP tubes in a 37 ℃ incubator for incubation.
1:50ul whole blood +250ug anti-RBC magnetic beads, incubated at 37℃for 10min
2:50ul whole blood+500 ug anti-RBC magnetic beads, incubated at 37℃for 10min
3:50ul whole blood +250ug anti-RBC magnetic beads, incubated at 37℃for 20min
4:50ul whole blood+500 ug anti-RBC magnetic beads, incubated at 37℃for 20min
5: blank, 50ul whole blood+100 ul dilution, incubation at 37℃for 30min
Conclusion: the blank control showed hemolysis, which may be caused by red blood cell rupture due to excessive mixing speed or excessive concentration of diluent ions, thus failing the experiment.
(2) 6 EP tubes were taken, and different volumes of whole blood and 100ul of anti-RBC antibody magnetic particles were added, mixed well, one of them was added with 100ul of magnetic particle dilution as a control, and incubated in a 37℃incubator.
1:5ul whole blood +25ug anti-RBC magnetic beads, incubated at 37℃for 10min
2:10ul whole blood +25ug anti-RBC magnetic beads, incubated at 37℃for 10min
3:30ul whole blood +25ug anti-RBC magnetic beads, incubated at 37℃for 10min
4:50ul whole blood +25ug anti-RBC magnetic beads, incubated at 37℃for 10min
5:50ul whole blood+50 ug anti-RBC magnetic beads, incubated at 37℃for 10min
6: blank, 50ul whole blood, incubation at 37℃for 10min
Conclusion: the control did not show hemolysis and this experiment was significant. Condition 1 had the best effect of removing erythrocytes, but still had residual erythrocytes that were not removed. As whole blood volume increases, the more residual red blood cells are exposed at the same time, and after serum is diluted, the test requirements for different project tests are not necessarily met, and the serum content is too small for project tests, so that the ratio 1 is as follows: and 5, continuously increasing the proportion of the RBC-resistant magnetic particles, removing the diluent from the magnetic particles, and optimizing the proportion of the whole blood to the RBC-resistant magnetic particles.
(3) Taking 4 EP tubes, respectively adding magnetic particles with different volumes, removing diluent after adsorbing the magnetic particles by a magnetic rack, adding 50ul whole blood, uniformly mixing, and placing in a 37 ℃ incubator for 10min.
1:100ul whole blood +200ug anti-RBC magnetic beads incubated at 37℃for 10min
2:100ul whole blood +400ug anti-RBC magnetic beads, incubated at 37℃for 10min
3:100ul whole blood +600ug anti-RBC magnetic beads incubated at 37 ℃ for 10min
4:100ul whole blood +800ug anti-RBC magnetic beads incubated at 37℃for 10min
Conclusion: as anti-RBC magnetic particles increase, the fewer residual red blood cells are, greater than 1:6, the proportion of residual erythrocytes is very small, and the feasibility of further evaluating serum isolated against erythrocyte antibody magnetic particles is required.
It avoids other complicated separation steps. The magnetic particles of the anti-RBC antibody are utilized to separate the red blood cells, so that the selectivity and the efficiency are high, the high quality of serum is ensured, and the pollution to serum components is avoided. The magnetic separation technology is adopted, so that devices such as centrifugation, a suction pipe and the like are not needed in the operation process, and the automatic operation can be realized. The method can be applied to the fields of biology and medical research, including but not limited to the fields of hematology, clinical diagnosis, microbiology and the like, so that the application range of the method in scientific research and clinical application is widened. The application and clinical diagnosis can reduce the multitube blood sampling of patients and avoid syncope caused by excessive blood loss. In clinical assay detection, the anti-RBC antibody magnetic particles improve the detection sensitivity and specificity, reduce non-specific background signals and improve the detection accuracy.
The foregoing is merely illustrative of the present invention and not restrictive, and other modifications and equivalents thereof may occur to those skilled in the art without departing from the spirit and scope of the present invention.
Claims (8)
1. A method for separating serum by anti-erythrocyte antibody magnetic particles, which is characterized by comprising the following steps: the method for separating serum by anti-erythrocyte antibody magnetic particles comprises the following steps: 1. preparing anti-RBC antibody magnetic particles: coating the anti-RBC antibody on the surface of the magnetic particles to form anti-RBC antibody magnetic particles; 2. adding the whole blood sample into prepared anti-RBC antibody magnetic particles in proportion, and allowing the magnetic particles to adsorb red blood cells in blood; 3. through magnetic separation technology, the anti-RBC antibody magnetic particles are combined with specific antigens on the surface of the red blood cells to separate out the fixed red blood cells, and the plasma can be left in the tube; 4. the separated serum sample is collected in a clean environment for further detection or processing.
2. An application of anti-erythrocyte antibody magnetic particles in separating serum is characterized in that: the application of anti-erythrocyte antibody magnetic particle separation serum in detecting the serum rubella virus IgG antibody.
3. The use of anti-erythrocyte antibody magnetic particles for separating serum according to claim 2, characterized in that it comprises the following specific applications: detection is carried out by using enzyme-linked immunosorbent assay (ELISA) of anti-RBC antibody magnetic particles, and the specific detection steps are as follows:
1. preparing anti-RBC antibody magnetic particles: mixing anti-RBC antibody magnetic particles uniformly, adding 600ug to an enzyme-labeled hole respectively, performing magnetic adsorption, and removing supernatant diluent;
2. adding 100ul whole blood into anti-RBC antibody magnetic particles, mixing uniformly, and standing at 37 ℃ for incubation for 10min to allow the magnetic particles to adsorb red blood cells in the blood;
3. adsorbing the magnetic particles and the red blood cell-combined compound on the bottom of the enzyme-labeled hole through a magnetic device, so that red blood cells in whole blood are separated from serum;
4. transferring 50ul serum into an enzyme-labeled plate hole coated with rubella virus antigen, incubating at 37 ℃ for 30min, and washing the hole after the binding with the antigen, so that the part which is not bound with the antigen is washed off;
5. adding 100ul of labeled enzyme-labeled secondary antibody, and incubating at 37 ℃ for 30min to combine with the IgG antibody combined with the antigen to form a specific complex;
6. washing the wells again, removing unbound secondary antibodies, adding 50ul of color development liquid A, B each, and incubating at 37 ℃ for 15min;
7. adding 50ul of stop solution;
8. OD values were read at a wavelength of 450 nm.
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| CN202311492237.1A CN117517645A (en) | 2023-11-10 | 2023-11-10 | Method for separating serum by anti-erythrocyte antibody magnetic particles and application |
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