CN116626307A - IgA antibody detection kit and detection method - Google Patents
IgA antibody detection kit and detection method Download PDFInfo
- Publication number
- CN116626307A CN116626307A CN202310584352.5A CN202310584352A CN116626307A CN 116626307 A CN116626307 A CN 116626307A CN 202310584352 A CN202310584352 A CN 202310584352A CN 116626307 A CN116626307 A CN 116626307A
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- iga
- antibody
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- microsphere
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- 238000001514 detection method Methods 0.000 title claims abstract description 47
- 239000000126 substance Substances 0.000 claims abstract description 35
- 239000004005 microsphere Substances 0.000 claims abstract description 24
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 12
- 238000011534 incubation Methods 0.000 claims abstract description 6
- 230000008878 coupling Effects 0.000 claims abstract description 5
- 238000010168 coupling process Methods 0.000 claims abstract description 5
- 238000005859 coupling reaction Methods 0.000 claims abstract description 5
- 230000003248 secreting effect Effects 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 3
- 241001494479 Pecora Species 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 210000004251 human milk Anatomy 0.000 claims description 3
- 235000020256 human milk Nutrition 0.000 claims description 3
- 241001529936 Murinae Species 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 7
- 239000000427 antigen Substances 0.000 abstract description 4
- 102000036639 antigens Human genes 0.000 abstract description 4
- 108091007433 antigens Proteins 0.000 abstract description 4
- 229940099472 immunoglobulin a Drugs 0.000 description 49
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 5
- 208000029138 selective IgA deficiency disease Diseases 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 206010020751 Hypersensitivity Diseases 0.000 description 3
- 208000007924 IgA Deficiency Diseases 0.000 description 3
- 206010039915 Selective IgA immunodeficiency Diseases 0.000 description 3
- 208000003441 Transfusion reaction Diseases 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 231100000517 death Toxicity 0.000 description 3
- 230000036039 immunity Effects 0.000 description 3
- 201000007156 immunoglobulin alpha deficiency Diseases 0.000 description 3
- 229940027941 immunoglobulin g Drugs 0.000 description 3
- 206010066173 Allergic transfusion reaction Diseases 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical group O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004400 mucous membrane Anatomy 0.000 description 2
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- 208000035143 Bacterial infection Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 239000007987 MES buffer Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 238000007818 agglutination assay Methods 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000012503 blood component Substances 0.000 description 1
- 201000001383 blood group incompatibility Diseases 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
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- 210000002826 placenta Anatomy 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
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Classifications
-
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- 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
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an IgA antibody detection kit and a detection method, wherein the IgA antibody detection kit comprises a reagent and a kit body, and the reagent is arranged in the kit body; the reagent comprises a specific fluorescence coding microsphere, a standard substance and a fluorescein labeled secondary antibody, wherein the specific fluorescence coding microsphere is formed by coupling an anti-IgA polyclonal antibody to the surface of the microsphere with fluorescence intensity. The IgA antibody detection method is to incubate the specific fluorescent coding microsphere with the sample to be detected and the standard substance respectively, add the fluorescein labeled secondary antibody to continue the incubation reaction, and analyze after the reaction is finished to obtain the detection result. The invention can realize the rapid completion of IgA antigen/antibody specificity quantitative detection by one-time reaction, and the detection process is simple, convenient and accurate.
Description
Technical Field
The invention relates to the field of immunoassay detection, in particular to an IgA antibody detection kit and a detection method.
Background
Immunoglobulin a (IgA) is not only an important antibody in mucosal immunity, but also a 2 nd main antibody family next to immunoglobulin G (IgG) in blood, and plays a defensive function against bacterial and viral infections. IgA is classified into two types: i.e., serotype IgA and secretory IgA. Serotype IgA is present in serum, which has the immune function of certain IgG and IgM, and specific IgA can neutralize antigens in blood, while alternative complement immune systems can also be present. Secretory IgA is present in secretions, for example: saliva, tears and milk all contain IgA antibodies. Secretory IgA is the main antibody of local mucous membrane anti-infection immunity of organism, so it is also called mucous membrane immunity antibody. IgA cannot be produced by placenta, and there is no IgA antibody in the serum of newborn animals, but secretory IgA can be obtained from breast milk.
IgA normal content values vary according to hospitals using different detection techniques, and also vary among adults and children. In general, the normal range for humoral immune detection of immunoglobulins in adults is 80-350mg/dL, and IgA deficiency is usually defined in laboratory studies as a serum content of less than 5mg/dL, and when the content is less than 0.05mg/dL, igA is called complete deficiency. Epidemiological investigation of six ethnic selective IgA deficiency diseases in different regions of our country showed a prevalence of 1:4100, and the prevalence of different ethnic groups also varies, the han nationality, which occupies a large population, is 1:2600.
the IgA-induced transfusion immune response is divided into two types depending on the antibody: one is that when IgA-containing plasma, whole blood or IVIG (containing a trace amount of IgA) is administered to a patient completely deficient in IgA, the administration of the IgA-containing blood or plasma product again results in severe allergic reactions and even death. The other is blood group incompatibility transfusion between different subclasses of IgA and allotypes, and immune transfusion reaction can also be generated.
anti-IgA may cause transfusion reactions in selective IgA deficiency (sIgAD) recipients, and if recipients are transfused with IgA-containing blood components, allergic reactions may result, and severe cases may even be shock, death. IgA transfusion allergic response rate is about 1:20000-47000, accounting for 3.1% of transfusion-related deaths. In recent years, igA transfusion reactions have been reported, but IgA allergic transfusion reactions have not been evaluated reliably. Because the diagnosis is not standardized, most patients with severe allergic transfusion reactions lack laboratory examination and follow-up. Our country lacks detection of IgA deficient patients and further lacks blood to provide IgA (-). So that the transfusion of IgA deficiency patients in China is at great risk at present.
In order to avoid the risk of transfusion allergic reactions, rapid and effective detection of IgA content and IgA antibodies is required. Many methods for IgA diagnosis and IgA antibody measurement have been reported, such as radioimmunoassay, passive agglutination assay, passive agglutination inhibition assay, ELISA, solid phase erythrocyte adhesion assay, particle size immunoassay, etc. However, some of the above methods have high laboratory requirements, have radiation pollution, some have complicated operation and long experiment time, are not suitable for screening work for blood donors, and are not beneficial to popularization and application.
Disclosure of Invention
The invention mainly solves the technical problem of providing the IgA antibody detection kit and the IgA antibody detection method, which are simple and convenient to apply, high-efficiency and accurate and reliable in result.
In order to solve the technical problems, the invention adopts a technical scheme that: the IgA antibody detection kit comprises a reagent and a kit body, wherein the reagent is arranged in the kit body; the reagent comprises a specific fluorescence coding microsphere, a standard substance and a fluorescein labeled secondary antibody, wherein the specific fluorescence coding microsphere is formed by coupling an anti-IgA polyclonal antibody to the surface of the microsphere with fluorescence intensity.
In a preferred embodiment of the invention, the anti-IgA polyclonal antibody is a murine anti-IgA monoclonal antibody, a sheep anti-IgA polyclonal antibody or a rabbit anti-IgA polyclonal antibody.
In a preferred embodiment of the present invention, the standard substance is secretory IgA extracted from breast milk.
In a preferred embodiment of the present invention, the standard substances are five groups.
In a preferred embodiment of the invention, the concentration of reactive protein of five groups of the standard substances is 2.0. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0. Mu.g/mL, respectively.
In a preferred embodiment of the present invention, the fluorescein in the fluorescein-labeled secondary antibody is FITC-series fluorescent substance, PE-series fluorescent substance, APC-series fluorescent substance or CY-series fluorescent substance.
The IgA antibody detection method adopts the IgA antibody detection kit to detect, and comprises the following steps: (1) Incubating the specific fluorescent coding microsphere with a sample to be detected and a standard substance respectively; (2) adding a fluorescein-labeled secondary antibody to continue the incubation reaction; and (3) after the reaction is finished, analyzing to obtain a detection result.
In a preferred embodiment of the present invention, the sample to be tested in step (1) is serum or plasma.
In a preferred embodiment of the invention, the analysis in step (3) is performed using a flow cytometer.
The beneficial effects of the invention are as follows: the IgA antibody detection kit and the detection method can realize the rapid completion of IgA antigen/antibody specificity quantitative detection by one-time reaction, and the detection process is simple, convenient and accurate.
Drawings
For a clearer description of the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly introduced below, it being obvious that the drawings in the description below are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art, wherein:
FIG. 1 is a schematic flow chart of an embodiment of the IgA antibody detection method of the present invention;
FIG. 2 is a graph showing comparison of detection results of the IgA antibody detection method of the present invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Embodiment one:
the IgA antibody detection kit comprises a reagent and a kit body, wherein the reagent is arranged in the kit body; the reagent comprises a specific fluorescence coding microsphere, a standard substance and a fluorescein labeled secondary antibody, wherein the specific fluorescence coding microsphere is formed by coupling an anti-IgA polyclonal antibody to the surface of the microsphere with fluorescence intensity.
The anti-IgA polyclonal antibody is a mouse anti-IgA monoclonal antibody, a sheep anti-IgA polyclonal antibody or a rabbit anti-IgA polyclonal antibody, and in the embodiment, the anti-IgA polyclonal antibody is a mouse anti-IgA monoclonal antibody, and the subsequent method also adopts the antibody for detection. The standard substance is a human IgA antigen standard substance. In this embodiment, the standard substances are five groups, and the concentration of IgA reaction protein of the sample to be detected can be quantified by the concentrations of the reaction proteins of the five standard substances, wherein the concentrations of the reaction proteins of the five groups of standard substances are respectively 2.0 mug/mL, 1 mug/mL, 0.5 mug/mL, 0.25 mug/mL and 0 mug/mL. The fluorescein in the second antibody is FITC series fluorescent substance, PE series fluorescent substance, APC series fluorescent substance or CY series fluorescent substance, in this embodiment, the fluorescein in the second antibody is FITC series fluorescent substance.
Referring to fig. 1 and 2, an IgA antibody detection method is provided, and the detection method using the IgA antibody detection kit includes the steps of:
(1) For the specific fluorescent coding microsphere in the kit, coupling an anti-IgA polyclonal antibody to the surface of the corresponding microsphere with fluorescence intensity to prepare the specific fluorescent coding microsphere; the specific process comprises the following steps: firstly, activating the fluorescent microspheres to obtain activated fluorescent microspheres; adding MES buffer solution containing glycerol into the activated fluorescent microspheres, carrying out ultrasonic mixing, then adding IgA antibody solution, carrying out ultrasonic reaction, and carrying out light-shielding rotary mixing; after the reaction is finished, adding a sealing liquid into the precipitate, carrying out ultrasonic mixing, carrying out light-shielding rotary mixing, centrifuging, removing supernatant, and then continuing to add the sealing liquid, carrying out ultrasonic mixing and centrifuging; collecting the centrifuged precipitate, placing in glycine solution, and dispersing with ultrasound to obtain fluorescent microsphere and antibody complex
(2) Incubating and reacting the specific fluorescent coding microspheres with a sample to be detected and a standard substance respectively, wherein the sample to be detected is serum or plasma, and five groups of the standard substances are incubated and reacted with the specific fluorescent coding microspheres to serve as a control group; the incubation was performed at 37℃for 20 minutes, or at ambient temperature for 45 minutes.
(3) Adding a fluorescein-labeled secondary antibody to continue the incubation reaction; the incubation was performed at 37℃for 20 minutes, or at ambient temperature for 45 minutes.
(4) And after the reaction is finished, adopting a flow cytometer for analysis to obtain a detection result. Calculating the concentration of the IgA standard substance and the corresponding fluorescence signal value, and fitting a standard curve to obtain a curve equation, wherein the fluorescence value of the zero value standard substance is taken as a local deduction. Substituting the fluorescent signal value of the sample into a curve equation, and multiplying the fluorescent signal value by the corresponding dilution times to calculate the concentration of IgA in the sample.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.
Claims (9)
1. The IgA antibody detection kit is characterized by comprising a reagent and a kit body, wherein the reagent is arranged in the kit body; the reagent comprises a specific fluorescence coding microsphere, a standard substance and a fluorescein labeled secondary antibody, wherein the specific fluorescence coding microsphere is formed by coupling an anti-IgA polyclonal antibody to the surface of the microsphere with fluorescence intensity.
2. The IgA antibody detection kit of claim 1 wherein the anti-IgA polyclonal antibody is a murine anti-IgA monoclonal antibody, a sheep anti-IgA polyclonal antibody or a rabbit anti-IgA polyclonal antibody.
3. The IgA antibody detection kit according to claim 1, wherein the standard substance is secretory IgA extracted from breast milk.
4. The IgA antibody detection kit of claim 1 wherein the standard substances are in five groups.
5. The IgA antibody test kit of claim 4 wherein the five groups of standard substances have reactive protein concentrations of 2.0 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.25 μg/mL, 0 μg/mL, respectively.
6. The IgA antibody detection kit according to claim 1, wherein the fluorescein in the fluorescein-labeled secondary antibody is FITC-series fluorescent substance, PE-series fluorescent substance, APC-series fluorescent substance or CY-series fluorescent substance.
7. An IgA antibody detection method, characterized in that it adopts the IgA antibody detection kit of any one of claims 1-6, comprising the steps of: (1) Incubating the specific fluorescent coding microsphere with a sample to be detected and a standard substance respectively; (2) adding a fluorescein-labeled secondary antibody to continue the incubation reaction; and (3) after the reaction is finished, analyzing to obtain a detection result.
8. The IgA antibody detection method of claim 7 wherein the sample to be tested in step (1) is serum or plasma.
9. The IgA antibody detection method of claim 7 wherein the analysis in step (3) is performed using a flow cytometer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310584352.5A CN116626307A (en) | 2023-05-23 | 2023-05-23 | IgA antibody detection kit and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310584352.5A CN116626307A (en) | 2023-05-23 | 2023-05-23 | IgA antibody detection kit and detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116626307A true CN116626307A (en) | 2023-08-22 |
Family
ID=87591434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310584352.5A Pending CN116626307A (en) | 2023-05-23 | 2023-05-23 | IgA antibody detection kit and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116626307A (en) |
-
2023
- 2023-05-23 CN CN202310584352.5A patent/CN116626307A/en active Pending
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