CN103954766A - Transferring receptor detection kit and preparation method thereof - Google Patents
Transferring receptor detection kit and preparation method thereof Download PDFInfo
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- CN103954766A CN103954766A CN201410035490.9A CN201410035490A CN103954766A CN 103954766 A CN103954766 A CN 103954766A CN 201410035490 A CN201410035490 A CN 201410035490A CN 103954766 A CN103954766 A CN 103954766A
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- 239000004816 latex Substances 0.000 claims abstract description 100
- 229920000126 latex Polymers 0.000 claims abstract description 100
- 239000004005 microsphere Substances 0.000 claims abstract description 84
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 71
- 238000000034 method Methods 0.000 claims abstract description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000004094 surface-active agent Substances 0.000 claims abstract description 16
- 239000000375 suspending agent Substances 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims description 103
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 51
- 239000008363 phosphate buffer Substances 0.000 claims description 51
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 claims description 45
- 238000006243 chemical reaction Methods 0.000 claims description 25
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical group OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 24
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 18
- 239000010452 phosphate Substances 0.000 claims description 18
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- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical group [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 10
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- -1 antiseptic Substances 0.000 claims description 6
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- 239000006172 buffering agent Substances 0.000 claims description 4
- 230000009514 concussion Effects 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 2
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
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- 230000035945 sensitivity Effects 0.000 abstract description 7
- 108010033576 Transferrin Receptors Proteins 0.000 abstract description 6
- 125000003277 amino group Chemical group 0.000 abstract description 4
- 102000005962 receptors Human genes 0.000 abstract description 4
- 108020003175 receptors Proteins 0.000 abstract description 4
- 230000001965 increasing effect Effects 0.000 abstract description 3
- 102000007238 Transferrin Receptors Human genes 0.000 abstract 3
- 239000012581 transferrin Substances 0.000 abstract 3
- 238000010382 chemical cross-linking Methods 0.000 abstract 2
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- 230000002335 preservative effect Effects 0.000 abstract 2
- 239000002738 chelating agent Substances 0.000 abstract 1
- 230000002035 prolonged effect Effects 0.000 abstract 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 30
- 229910052742 iron Inorganic materials 0.000 description 15
- 239000000523 sample Substances 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- 102000004190 Enzymes Human genes 0.000 description 10
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 7
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- 102100026745 Fatty acid-binding protein, liver Human genes 0.000 description 5
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- 102000008857 Ferritin Human genes 0.000 description 3
- 108050000784 Ferritin Proteins 0.000 description 3
- 238000008416 Ferritin Methods 0.000 description 3
- 102000002070 Transferrins Human genes 0.000 description 3
- 108010015865 Transferrins Proteins 0.000 description 3
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 1
- BHNQPLPANNDEGL-UHFFFAOYSA-N 2-(4-octylphenoxy)ethanol Chemical compound CCCCCCCCC1=CC=C(OCCO)C=C1 BHNQPLPANNDEGL-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 108010017480 Hemosiderin Proteins 0.000 description 1
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- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
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- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 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
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
-
- 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
-
- 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- 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
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/79—Transferrins, e.g. lactoferrins, ovotransferrins
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a transferrin receptor content detection kit, which comprises a reagent I and a reagent II, wherein the components of the reagent I comprise a buffer, a preservative, a chelating agent, a surfactant and water, the components of the reagent II comprise anti-transferrin receptor antibody coated latex microsphere particles, a buffer, a preservative, an auxiliary suspending agent and water, and the particle size of the anti-transferrin receptor antibody coated latex particles is 120-200 nm. The invention further relates to a preparation method for the transferrin receptor content detection kit, wherein the anti-transferrin receptor antibody coated latex microsphere particles are prepared by using a chemical cross-linking method. According to the present invention, the amino group latex microsphere particles with the appropriate particle size are selected and the chemical cross-linking method is adopted to prepare the latex microsphere particles, such that the sensitivity and the stability of the prepared transferrin receptor detection kit are increased, and the shelf life and the bottle opening stable phase are prolonged.
Description
Technical field
The present invention relates to TfR content detection field, relate in particular to a kind of TfR detection kit and preparation method.
Background technology
Iron is distributed in human body in three kinds of different modes: approximately 80% iron participates in the haemoglobin of transhipment oxygen and stores the myoglobins of oxygen, also has in several enzymes that are present in cellular oxidation and respiratory chain.Superfluous iron is combined with ferritin and hemosiderin in order to using, because free iron is virose to cell.0.1% the iron of only having an appointment betransported in blood circulation, they are combined with transferrins, from " place of production "---and the albumen of being combined with the iron with physiological function is (for example: synthetic haemoglobin marrow) (for example: the ferritin in liver) be transported to " storage place ".In healthy human body, absorb the iron of about 1mg every day from food, and have the iron of equivalent to lose.Red blood cell contains most iron, has in addition small part but the iron that is absolutely necessary offers muscle and enzyme, makes them carry out normal activity.Superfluous iron is combined with ferritin, is stored in different cells.
The total amount of the iron of iron/generation level of participation normal physiological activity can be reflected by the amount of soluble transferrin receptor (sTfR).The cell receptor of this solubility can mediate the transferrins that combines iron and enter erythroid cells precursor.The soluble recepter of circulation is proportional with the density with the cell of acceptor, along with the increase of the demand of iron and increasing of red cell volume are increased.
TfR is the glycoprotein of wearing film, and it tends in conjunction with combining ferrous transferrins, and enters cell by receptor mediated endocytosis.All body cells are all at its surface expression TfR, but 75～80% TfR is present in the precursor of erythroid cells of marrow.In the tissue of liver and placenta, the density of TfR is also very high.The outside lease making of TfR becomes soluble transferrin receptor after shearing, and it becomes certain ratio with the concentration of acceptor on cell.The fundamental purpose of measuring soluble transferrin receptor is whether to diagnose iron deficiency.Because it is a very sensitive mark, the anaemia that diagnostic result can cause with chronic disease distinguishes.Again because it is relevant with erythropoietic amount, so it can be used as the mark the earliest that monitoring red blood cell generates result for the treatment of.
Mostly its methodology of testing product of TfR is immunoturbidimetry and the euzymelinked immunosorbent assay (ELISA) based on common at present.Common immunoturbidimetry is to utilize the antigen-antibody reaction in reaction system to form immune complex, and turbidity appears in compound polymerization within a certain period of time.The immunochemistry quantivative approach that this detection method is more traditional is responsive, quick, easy, but quantity and the molecular weight of the immune complex forming while requiring reaction reach a certain height, otherwise is just difficult to measure.Euzymelinked immunosorbent assay (ELISA) is to make antigen or antibody and certain enzyme connect into enzyme-labelled antigen or antibody, and this enzyme-labelled antigen or antibody had both retained its immunocompetence, retained again the activity of enzyme.The substrate that finally adds enzyme reaction of measuring, substrate is become coloured product by enzymatic, the amount of coloured product is directly related with the amount of tested substance in sample, this detection method is because the catalytic efficiency of enzyme is very high, therefore iodine effect greatly, thereby make assay method reach very high susceptibility.But euzymelinked immunosorbent assay (ELISA) detects complex operation, automaticity is low, consuming time very long, is unfavorable for the fast detecting of clinical laboratory, simultaneously because enzyme linked immunological is applicable to batch detection, thereby be not suitable for the timely detection of single sample.Latex enhancing immune turbidimetry is a kind of body fluid albumen homogeneous phase immunoturbidimetry detection method occurring in recent years, it is by the surface-crosslinked antibody at polymer latex microballoon, after the crosslinked microballoon that has antibody is combined with antigen, can flock together rapidly at short notice, change astigmatic performance or the light transmission of reactant liquor.And, reactant liquor astigmatism performance or the change of light transmission (being absorbance) and the concentration of tested antigen have stronger correlativity, can reflect the concentration of tested antigen within the specific limits, this detection method has been save ELISA method and has repeatedly been hatched and washed the loaded down with trivial details operation stepss such as plate, a few minutes just can obtain result, time saving and energy saving.In addition, the interference of the extraneous factors such as many manual operation factors and reagent, environment has also correspondingly been avoided in the simplification of this method operation steps, can reflect more truly the content of measured matter.But latex enhancing immune turbidimetry detects, and TfR kit still exists that sensitivity and stability have much room for improvement, shelf life and uncork shorter problem stationary phase.
Summary of the invention
In order to solve above technical matters, the invention provides a kind of TfR detection kit and preparation method, by selecting the amino group latex microsphere particle of appropriate particle size and using chemical crosslink technique to prepare latex microsphere particle, make the TfR detection kit of preparation improve sensitivity and stability, extended shelf life and uncork stationary phase.
A TfR reagent box for detecting content, described detection kit is comprised of reagent I and reagent II, and the component of described reagent I comprises: buffering agent, antiseptic, sequestrant, surfactant and water; The component of described reagent II comprises: latex microsphere particle, buffering agent, antiseptic, suspending agent and the water of coated anti-TfR antibody; The particle diameter of the latex particle of described coated anti-TfR antibody is 120～200nm.
In technique scheme, described anti-TfR antibody is monoclonal antibody or polyclonal antibody.
In technique scheme, described damping fluid is phosphate buffer; Described antiseptic is Proclin 300; Described sequestrant is disodium ethylene diamine tetraacetate; Described surfactant is polysorbas20; Described suspending agent is ethylene glycol.
In technique scheme, in every 1L reagent I, the consumption of each component is antiseptic: 0.05-2mL, and sequestrant is 0.1-5g, surfactant 0.1-3mL, and surplus is phosphate buffered solution.
In technique scheme, in every 1L reagent I, the consumption of each component is antiseptic: 0.1-1mL, and sequestrant is 1-3g, surfactant 1-2mL, and surplus is phosphate buffered solution.
In technique scheme, in every 1L reagent II, the consumption of each component is: coated anti-TfR antibody latex microsphere particle 0.1-5g, antiseptic 0.05-2mL, suspending agent 5-50mL, surplus is phosphate buffered solution.
In technique scheme, in every 1L reagent II, the consumption of each component is: coated anti-TfR antibody latex microsphere particle 1-3g, antiseptic 0.1-1mL, suspending agent 10-30mL, and surplus is phosphate buffered solution;
In technique scheme, the pH value scope of described reagent I and reagent II is 6.5～8.5.
In technique scheme, comprise the following steps: (1) prepares reagent I: antiseptic, sequestrant, surfactant dissolves, in phosphate buffer, are fully dissolved, mix, and use phosphate buffer constant volume, obtain reagent I; (2) prepare reagent II: the latex microsphere particle solution of the coated anti-TfR antibody of preparation; Antiseptic, suspending agent are dissolved in phosphate buffer; Latex microsphere particle solution and mixed solution are mixed, and constant volume, obtains reagent II.
In technique scheme, the latex microsphere particle of described coated anti-TfR antibody adopts chemical crosslink technique to prepare, and the latex microsphere particle of the coated TfR antibody of described chemical crosslink technique preparation comprises the following steps:
(1) washing latex: get activation damping fluid dilution for appropriate amounts of ammonia base latex microsphere particle solution, concussion mixes, then adds surfactant, and concussion mixes, and the centrifugal supernatant of abandoning retains precipitation, and precipitation is resuspended with 6mmol/L phosphate buffer;
(2) preparation of antibody-solutions: get antibody-solutions, with the dilution of 6mmol/L phosphate buffer, adjust pH to 5.0;
(3) preparation of EDC solution: get EDC powder and be mixed with deionized water the EDC solution that concentration is 10mg/mL;
(4) preparation of antibody coupling latex microsphere particle: the resuspended liquid of step (1) gained latex microsphere particle is mixed with step (2) gained antibody-solutions, cultivate 15-30 minute under room temperature; Add again step (3) gained EDC solution; The pH to 6.0-7.0 that adjusts reaction system with 0.1M sodium hydroxide solution, is shaking bed reaction 2-3 hour under room temperature condition;
(5) in the middle system of having reacted of step (4), add BSA solution, reaction 10-15 minute, the centrifugal supernatant of abandoning, and with 6mmol/L phosphate buffer washing precipitation 2 times, after use the resuspended precipitation of 6mmol/L phosphate buffer, both must be coated with the latex microsphere particle solution of anti-TfR antibody.
Detection kit of the present invention is screened latex particle particle diameter and type by experiment, the amino group latex microsphere particle that preferably diameter is 120-200nm is as preparing one of raw material of detection kit of the present invention, and suitable particle diameter can effectively guarantee stability, sensitivity and the good detection range of linearity of prepared detection kit; The latex microsphere particle of the coated preferable particle size of antibody of anti-TfR adopts specific chemical crosslink technique, mode by the group on antibody and latex microsphere particle by chemical bond combines, the stability of chemical bond is stronger, can guarantee the stability of the latex microsphere particle of coated antibody in detection kit, and then guaranteed the stability of detection kit quality, extended shelf life and uncork stationary phase of kit.
Accompanying drawing explanation
Fig. 1 is TfR reagent box for detecting content standard working curve prepared by embodiment of the present invention 1-5;
Fig. 2 is the detection kit of the embodiment of the present invention 2 preparations and the correlation figure that enzyme-linked immunologic detecting kit is measured its result of TfR content in human serum;
Fig. 3 is the detection kit thermal stability assay figure of the embodiment of the present invention 4 preparations;
The coated latex microsphere particle schematic diagram of the antibody of anti-TfR in the detection kit preparation process that Fig. 4 provides for the embodiment of the present invention.
embodiment
Below in conjunction with drawings and Examples, technical scheme of the present invention is described in detail.
Embodiment 1
The preparation of the latex microsphere particle solution of coated anti-TfR antibody:
(1) amino latex microsphere particle (purchased from Poly Microspheres company) washing latex: getting 0.5mL particle diameter is that 120 nm concentration are 10%(w/v) is in 10 mL centrifuge tubes, in centrifuge tube, add phosphate (PBS) damping fluid that 4.5 mL 0.12 mol/LpH values are 7.4, the centrifugal latex microsphere particle that obtains, with twice latex microsphere particle of deionized water repeated washing, rear use 5 mL concentration are the resuspended latex microsphere particle of phosphate (PBS) damping fluid that 6 mmol/L pH values are 7.4;
(2) preparation of antibody-solutions: get a certain amount of antibody-solutions (purchased from Taiwan Abnova Corporation), with the dilution of 6mmol/L phosphate buffer, adjust pH to 5.0;
(3) preparation of EDC solution: get the 10 mL deionized water dissolvings for EDC powder of 10 mg, being mixed with concentration is the EDC solution of 10mg/mL;
(4) preparation of antibody coupling latex microsphere particle: get 5mL(1) the resuspended liquid of step gained latex microsphere particle and 5mL(2) step gained antibody-solutions mixes according to 1:1, cultivates 25 minutes under room temperature; (3) the step gained EDC solution that adds again 3mL; The pH to 6.5 that adjusts reaction system with 0.1M sodium hydroxide solution, is shaking bed reaction 2.5 hours under room temperature condition.
(5) in the system of having reacted to (4) step, add 2 mL 1%(w/v) the BSA solution of concentration, react 15 minutes, the centrifugal supernatant of abandoning, and with 6mmol/L phosphate buffer washing precipitation 2 times, after use the resuspended precipitation of 1mL6mmol/L phosphate buffer, both must be coated with the latex microsphere particle solution of anti-TfR antibody.
The preparation of detection kit:
1, the preparation of reagent I:
By described use, measure each component:
Procline 300 0.5mL
Disodium ethylene diamine tetraacetate 1.5g
Polysorbas20 1.2mL
By above-mentioned each components dissolved, in 900mL pH, be that 7.0 concentration are in 0.05mol/L phosphate buffer, with 1mol/L sodium hydroxide solution regulator solution pH to 7.0, rear is that 7.0 concentration are that 0.05mol/L phosphate buffer is settled to 1L with pH, seals 4 ℃ of preservations, standby.
2, the preparation of reagent II:
By described consumption, measure each component:
Procline300 0.5mL
Ethylene glycol 25mL
By above-mentioned each components dissolved, in 800mLpH, be that 7.0 concentration are in 0.05mol/L phosphate buffer, the latex particle solution that adds 35 parts of above-mentioned coated liver type fatty acid binding protein antibody that prepare after stirring, fully dissolving, after fully mixing, with 1mol/L sodium hydroxide solution regulator solution pH to 7.0, with pH, be that 7.0 concentration are that 0.05mol/L phosphate buffer is settled to 1L afterwards, seal 4 ℃ of preservations, standby.
Embodiment 2
The preparation of the latex microsphere particle solution of coated anti-TfR antibody:
(1) amino latex microsphere particle (purchased from Poly Microspheres company) washing latex: getting 0.6mL particle diameter is that 160 nm concentration are 10%(w/v) is in 10mL centrifuge tube, in centrifuge tube, add phosphate (PBS) damping fluid that 4.4mL 0.12mol/L pH value is 7.4, the centrifugal latex microsphere particle that obtains, with twice latex microsphere particle of deionized water repeated washing, rear is the resuspended latex microsphere particle of phosphate (PBS) damping fluid that 5mmol/L pH value is 7.4 by 5mL concentration;
(2) preparation of antibody-solutions: get a certain amount of antibody-solutions (purchased from Taiwan Abnova Corporation), with the dilution of 6mmol/L phosphate buffer, adjust pH to 5.0;
(3) preparation of EDC solution: get the EDC powder 10mL deionized water dissolving of 10mg, being mixed with concentration is the EDC solution of 10mg/mL;
(4) preparation of antibody coupling latex microsphere particle: get 5mL(1) the resuspended liquid of step gained latex microsphere particle and 5mL(2) step gained antibody-solutions mixes according to 1:1, cultivates 20 minutes under room temperature; (3) the step gained EDC solution that adds again 4mL; The pH to 6.5 that adjusts reaction system with 0.1M sodium hydroxide solution, is shaking bed reaction 2.5 hours under room temperature condition;
(5) in the system of having reacted to (4) step, add 3mL 1%(w/v) the BSA solution of concentration, react 20 minutes, the centrifugal supernatant of abandoning, and with 6mmol/L phosphate buffer washing precipitation 2 times, after use the resuspended precipitation of 1mL5mmol/L phosphate buffer, both must be coated with the latex microsphere particle solution of anti-TfR antibody.
The preparation of detection kit:
1, the preparation of reagent I:
By described use, measure each component:
Procline300 0.25mL
Disodium ethylene diamine tetraacetate 2g
Polysorbas20 1.5mL
By above-mentioned each components dissolved, in 900mL pH, be that 7.2 concentration are in 0.05mol/L phosphate buffer, with 1mol/L sodium hydroxide solution regulator solution pH to 7.2, rear is that 7.2 concentration are that 0.05mol/L phosphate buffer is settled to 1L with pH, seals 4 ℃ of preservations, standby.
2, the preparation of reagent II:
By described consumption, measure each component:
Procline300 0.25mL
Ethylene glycol 20mL
By above-mentioned each components dissolved, in 800mL pH, be that 7.2 concentration are in 0.05mol/L phosphate buffer, the latex particle solution that adds 30 parts of above-mentioned coated liver type fatty acid binding protein antibody that prepare after stirring, fully dissolving, after fully mixing, with 1mol/L sodium hydroxide solution regulator solution pH to 7.2, with pH, be that 7.2 concentration are that 0.05mol/L phosphate buffer is settled to 1L afterwards, seal 4 ℃ of preservations, standby.
Embodiment 3
The preparation of the latex microsphere particle solution of coated anti-TfR antibody:
(1) amino latex microsphere particle (purchased from Poly Microspheres company) washing latex: getting 0.8mL particle diameter is that 200 nm concentration are 10%(w/v) is in 10mL centrifuge tube, in centrifuge tube, add phosphate (PBS) damping fluid that 4.2mL0.12mol/L pH value is 7.4, the centrifugal latex microsphere particle that obtains, with twice latex microsphere particle of deionized water repeated washing, rear is the resuspended latex microsphere particle of phosphate (PBS) damping fluid that 5mmol/L pH value is 7.4 by 5mL concentration;
(2) preparation of antibody-solutions: get a certain amount of antibody-solutions (purchased from Taiwan Abnova Corporation), with the dilution of 6mmol/L phosphate buffer, adjust pH to 5.0;
(3) preparation of EDC solution: get the EDC powder 10mL deionized water dissolving of 10mg, being mixed with concentration is the EDC solution of 10mg/mL;
(4) preparation of antibody coupling latex microsphere particle: get 5mL(1) the resuspended liquid of step gained latex microsphere particle and 5mL(2) step gained antibody-solutions mixes according to 1:1, cultivates 20 minutes under room temperature; (3) the step gained EDC solution that adds again 4mL; The pH to 6.5 that adjusts reaction system with 0.1M sodium hydroxide solution, is shaking bed reaction 2.5 hours under room temperature condition.
(5) in the system of having reacted to (4) step, add 3mL 1%(w/v) the BSA solution of concentration, react 20 minutes, the centrifugal supernatant of abandoning, and with 6mmol/L phosphate buffer washing precipitation 2 times, after use the resuspended precipitation of 1mL5mmol/L phosphate buffer, both must be coated with the latex microsphere particle solution of anti-TfR antibody.
The preparation of detection kit:
1, the preparation of reagent I:
By described use, measure each component:
Procline300 0.6mL
Disodium ethylene diamine tetraacetate 1.8g
Polysorbas20 1.0mL
By above-mentioned each components dissolved, in 900mLpH, be that 7.2 concentration are in 0.05mol/L phosphate buffer, with 1mol/L sodium hydroxide solution regulator solution pH to 7.2, rear is that 7.2 concentration are that 0.05mol/L phosphate buffer is settled to 1L with pH, seals 4 ℃ of preservations, standby.
2, the preparation of reagent II:
By described consumption, measure each component:
Procline300 0.6mL
Ethylene glycol 15mL
By above-mentioned each components dissolved, in 800mLpH, be that 7.2 concentration are in 0.05mol/L phosphate buffer, the latex particle solution that adds 26 parts of above-mentioned coated liver type fatty acid binding protein antibody that prepare after stirring, fully dissolving, after fully mixing, with 1mol/L sodium hydroxide solution regulator solution pH to 7.2, with pH, be that 7.2 concentration are that 0.05mol/L phosphate buffer is settled to 1L afterwards, seal 4 ℃ of preservations, standby.
Embodiment 4
The preparation of the latex microsphere particle solution of coated anti-TfR antibody:
(1) amino latex microsphere particle (purchased from Poly Microspheres company) washing latex: getting 0.5mL particle diameter is that 140nm concentration is 10%(w/v) is in 10mL centrifuge tube, in centrifuge tube, add phosphate (PBS) damping fluid that 4.5mL 0.12mol/L pH value is 7.2, the centrifugal latex microsphere particle that obtains, with twice latex microsphere particle of deionized water repeated washing, rear is the resuspended latex microsphere particle of phosphate (PBS) damping fluid that 5mmol/L pH value is 7.2 by 5mL concentration;
(2) preparation of antibody-solutions: get a certain amount of antibody-solutions (purchased from Taiwan Abnova Corporation), with the dilution of 5mmol/L phosphate buffer, adjust pH to 5.0;
(3) preparation of EDC solution: get the EDC powder 10mL deionized water dissolving of 10mg, being mixed with concentration is the EDC solution of 10mg/mL;
(4) preparation of antibody coupling latex microsphere particle: get 5mL(1) the resuspended liquid of step gained latex microsphere particle and 5mL(2) step gained antibody-solutions mixes according to 1:1, cultivates 18 minutes under room temperature; (3) the step gained EDC solution that adds again 2mL; The pH to 6.5 that adjusts reaction system with 0.1M sodium hydroxide solution, is shaking bed reaction 2.5 hours under room temperature condition.
(5) in the system of having reacted to (4) step, add 4mL 1%(w/v) the BSA solution of concentration, react 20 minutes, the centrifugal supernatant of abandoning, and with 5mmol/L phosphate buffer washing precipitation 2 times, after use the resuspended precipitation of 1mL 5mmol/L phosphate buffer, both must be coated with the latex microsphere particle solution of anti-TfR antibody.
The preparation of detection kit:
1, the preparation of reagent I:
By described use, measure each component:
Procline300 1.2mL
Disodium ethylene diamine tetraacetate 3.5g
Polysorbas20 2.5mL
By above-mentioned each components dissolved, in 900mL pH, be that 7.2 concentration are in 0.05mol/L phosphate buffer, with 1mol/L sodium hydroxide solution regulator solution pH to 7.2, rear is that 7.2 concentration are that 0.05mol/L phosphate buffer is settled to 1L with pH, seals 4 ℃ of preservations, standby.
2, the preparation of reagent II:
By described consumption, measure each component:
Procline300 1.2mL
Ethylene glycol 40mL
By above-mentioned each components dissolved, in 800mL pH, be that 7.2 concentration are in 0.05mol/L phosphate buffer, the latex particle solution that adds 32 parts of above-mentioned coated liver type fatty acid binding protein antibody that prepare after stirring, fully dissolving, after fully mixing, with 1mol/L sodium hydroxide solution regulator solution pH to 7.2, with pH, be that 7.2 concentration are that 0.05mol/L phosphate buffer is settled to 1L afterwards, seal 4 ℃ of preservations, standby.
Embodiment 5
The preparation of the latex microsphere particle solution of coated anti-TfR antibody:
(1) amino latex microsphere particle (purchased from Poly Microspheres company) washing latex: getting 0.5mL particle diameter is that 180nm concentration is 10%(w/v) is in 10mL centrifuge tube, in centrifuge tube, add phosphate (PBS) damping fluid that 4.5mL 0.12mol/L pH value is 7.6, the centrifugal latex microsphere particle that obtains, with twice latex microsphere particle of deionized water repeated washing, rear is the resuspended latex microsphere particle of phosphate (PBS) damping fluid that 6mmol/L pH value is 7.6 by 5mL concentration;
(2) preparation of antibody-solutions: get a certain amount of antibody-solutions (purchased from Taiwan Abnova Corporation), with the dilution of 6mmol/L phosphate buffer, adjust pH to 5.0;
(3) preparation of EDC solution: get the EDC powder 10mL deionized water dissolving of 10mg, being mixed with concentration is the EDC solution of 10mg/mL;
(4) preparation of antibody coupling latex microsphere particle: get 5mL(1) the resuspended liquid of step gained latex microsphere particle and 5mL(2) step gained antibody-solutions mixes according to 1:1, cultivates 20 minutes under room temperature; (3) the step gained EDC solution that adds again 4mL; The pH to 6.5 that adjusts reaction system with 0.1M sodium hydroxide solution, is shaking bed reaction 2.5 hours under room temperature condition.
(5) in the system of having reacted to (4) step, add 5mL 1%(w/v) the BSA solution of concentration, react 20 minutes, the centrifugal supernatant of abandoning, and with 6mmol/L phosphate buffer washing precipitation 2 times, after use the resuspended precipitation of 1mL 6mmol/L phosphate buffer, both must be coated with the latex microsphere particle solution of anti-TfR antibody.
The preparation of detection kit:
1, the preparation of reagent I:
By described use, measure each component:
Procline300 2.5mL
Disodium ethylene diamine tetraacetate 3.0g
Polysorbas20 2.2mL
By above-mentioned each components dissolved, in 900mL pH, be that 7.2 concentration are in 0.05mol/L phosphate buffer, with 1mol/L sodium hydroxide solution regulator solution pH to 7.2, rear is that 7.2 concentration are that 0.05mol/L phosphate buffer is settled to 1L with pH, seals 4 ℃ of preservations, standby.
2, the preparation of reagent II:
By described consumption, measure each component:
Procline300 2.5mL
Ethylene glycol 35mL
By above-mentioned each components dissolved, in 800mL pH, be that 7.2 concentration are in 0.05mol/L phosphate buffer, the latex particle solution that adds 35 parts of above-mentioned coated liver type fatty acid binding protein antibody that prepare after stirring, fully dissolving, after fully mixing, with 1mol/L sodium hydroxide solution regulator solution pH to 7.2, with pH, be that 7.2 concentration are that 0.05mol/L phosphate buffer is settled to 1L afterwards, seal 4 ℃ of preservations, standby.
The assessment of detection kit analytical performance
1, the range of linearity
Compound concentration is 0nmol/mL, 10nmol/mL, 20nmol/mL, 40nmol/mL, the TfR standard solution of 80nmol/mL, the TfR reagent box for detecting content prepared with embodiment of the present invention 1-5 detects it, draws each detection kit standard working curve.
The flow process that detection kit of the present invention detects TfR content in sample is as follows: to 5uL sample to be detected, (calibration tube is made sample with calibration object, blank be take distilled water as sample, calibration object is purchased from Taiwan Abnova Corporation company) in add the reagent I of 150uL fully to mix, in 37 ℃ of constant temperature 5 minutes, in mixed system, add 50uL reagent II again, mix, 37 ℃ of constant temperature are after 1 minute, blank tube zeroing, wavelength 540nm, measure respectively to manage after absorbance A Isosorbide-5-Nitrae minute and measure and respectively manage absorbance A 2, calculate Δ A=A2-A1.According to multiple spot calibration object concentration and corresponding absorbance changing value Δ A, adopt multiple spot gamma correction mode decision working curve, sample absorbance changes corresponding concentration value on working curve and is mensuration concentration.
Measurement result is shown in Fig. 1.The prepared detection kit of embodiment of the present invention 1-5 has good linearity as can be seen from Figure 1, the kit (embodiment 2) that another the present invention adopts optimized condition to prepare, the kit linearity that its standard working curve is prepared compared with all the other optimum conditions is better.
2, lowest detectable limit
Get the detection kit of the embodiment of the present invention 2 preparations, take 5% human serum albumins as dummy, according to the experimental technique of said determination " range of linearity ", replication 20 times, result of calculation average is 0.0006, standard deviation S is 0.00012, it is 0.00084 that the blank average of take adds twice standard deviation method calculating absorbance variable quantity, by concentration after dilution, be respectively 0.25nmol/mL, the TfR calibration object measured in solution of 0.5nmol/mL and 1.0nmol/mL, absorbance changing value is respectively 0.0002, 0.0008 and 0.0013, wherein concentration is that 0.5nmol/mL TfR calibration object solution Δ A adds twice standard deviation method a little more than blank average and carrys out calculated value, therefore, the sensitivity that detection kit of the present invention detects TfR is 0.5nmol/mL.
3, the correlation test of kit of the present invention and enzyme-linked immunoassay kit measured value
3.1 for examination kit
The TfR reagent box for detecting content that the embodiment of the present invention 2 is prepared, enzyme linked immunological kit (purchased from U.S. R & D Systems company);
3.2 test methods and result
The measured value of measuring TfR content in urine specimen by the prepared TfR reagent box for detecting content of the embodiment of the present invention 2 and the enzyme linked immunological kit purchased from U.S. R & D company compares, test findings as shown in Figure 2, the line retrace analysis of going forward side by side.As can be seen from Figure 2 prepared TfR detection kit and the enzyme-linked immunologic detecting kit of the embodiment of the present invention 2 has good correlativity aspect TfR content in measuring sample.
4, the repeatability of kit and accuracy test
Using TfR solution dilution purchased from Taiwan Abnova company to become concentration is 10nmol/mL sample, by the embodiment of the present invention 1, embodiment 2, the TfR reagent box for detecting content of embodiment 3 preparations is measured diluted sample, detection kit replication prepared by each embodiment 10 times, calculate respectively and measure average (M) and standard deviation (S), with S/M * 100%, calculate the coefficient of variation and carry out repeatability investigation, with (1-M/ concentration of specimens) * 100%, calculate relative deviation and carry out accuracy investigation, experimental result sees the following form 1, the embodiment of the present invention 2 adopts optimum particle diameter to prepare as shown in Table 1 its accuracy of detection kit and precision are better than the detection kit that adopts other particle diameters to prepare.
The repeatability of table 1 embodiment detection kit and accuracy result thereof
| Test sequence number | Embodiment 1 | Embodiment 2 | Embodiment 3 |
| 1 | 9.9 | 9.9 | 9.5 |
| 2 | 9.6 | 9.8 | 9.6 |
| 3 | 10.4 | 9.9 | 10.5 |
| 4 | 10.2 | 10.0 | 10.5 |
| 5 | 9.7 | 10.1 | 9.3 |
| 6 | 10.3 | 10.2 | 9.9 |
| 7 | 9.5 | 10.1 | 9.7 |
| 8 | 10.8 | 10.2 | 10.2 |
| 9 | 10.7 | 9.9 | 10.1 |
| 10 | 10.0 | 10.1 | 10.5 |
| Mean value (M) | 10.11 | 10.02 | 9.98 |
| Standard deviation (S) | 0.45 | 0.14 | 0.45 |
| The coefficient of variation (CV%) | 4.45% | 1.40% | 4.51% |
| Relative deviation (Bias%) | 1.10% | 0.20% | 0.20% |
5, the heat stability test of detection kit of the present invention test
The detection kit of the embodiment of the present invention 4 preparations is placed in to 37 ℃ of difference thermal treatment 0 day, 3 days, 5 days and 7 days, after the different processing times, measures respectively TfR calibration object, record its Δ A value, and draw its change curve.Test findings is shown in Fig. 3.From Fig. 3, the known detection kit of the present invention of result has good thermal stability, has compared with long shelf life and uncork stationary phase.
Damping fluid in detection kit of the present invention is in order to make reaction system can maintain certain pH value, various all damping fluid uses of energy conduct preparation detection kit of the present invention of damping fluid that can make reaction system maintain certain pH value, reagent I of the present invention and reagent II preferably phosphoric acid damping fluid are as the damping fluid of reagent preparation.
Antiseptic in detection kit of the present invention is in order to prevent that various microbial growths from causing product rotten, thereby testing result is brought to harmful effect, therefore, can stop the material of microbial reproduction all to use as the antiseptic of kit of the present invention, as: one or more potpourris in Sodium Benzoate, potassium sorbate, sodium azide, Proclin 300 etc.; For the properties of guarantee reagent box is in optimum condition, the preferred Proclin 300 of reagent I of the present invention and reagent II is as antiseptic.
Sequestrant in detection kit reagent I of the present invention is to detect the metallic ion in sample for complexing, avoid metallic ion to cause harmful effect to testing result, the various sequestrants of energy complexation of metal ions all can be applicable to the present invention, for example: disodium ethylene diamine tetraacetate, aminotriacetic acid etc.The preferred disodium ethylene diamine tetraacetate of the present invention (EDTA-2Na) is as sequestrant.
Surfactant in detection kit reagent I of the present invention is conducive to each component in course of reaction and detects the dispersed of each material in sample, make reaction system in homogeneous state, reach and reduce the object of sample turbidity on the impact of testing result, the material with solubilization all can be used as surfactant of the present invention, as serial in: tween series, Qu Latong, Nonidet P40 etc.For the more excellent performance of guarantee reagent box, the preferred polysorbas20 of the present invention is as surfactant.
Suspending agent in detection kit reagent II of the present invention can help latex microsphere particle to maintain good disperse state, prevent that latex microsphere particle from sinking the reliability of the stability of detection kit and testing result is brought to harmful effect, the various materials that can increase reaction system viscosity all can be as suspending agent of the present invention, and the preferred ethylene glycol of the present invention is as suspending agent.
Detection kit of the present invention is screened latex particle particle diameter and type by experiment, the amino group latex microsphere particle that preferably diameter is 120-200nm, as preparing one of raw material of detection kit of the present invention, can effectively guarantee stability, sensitivity, stability and the good detection range of linearity of prepared detection kit; The too little detectability of latex particle particle diameter can be higher, causes kit sensitivity not high, and stability that particle diameter is large is bad, easily sedimentation.
In kit preparation process of the present invention, the latex microsphere particle of the coated preferable particle size of antibody of anti-TfR adopts specific chemical crosslink technique (principle is shown in Fig. 4), by the carbonyl reaction on the antibody of the amino on amino latex microsphere particle and anti-TfR, generate more stable carbonnitrogen bond, can guarantee the stability of the latex microsphere particle of coated antibody in detection kit, and then guarantee the stability of detection kit quality; The anti-TfR monoclonal antibody adopting in detection kit preparation process of the present invention and the specific identification of Anti-TNF-α physical efficiency in conjunction with TfR albumen, guaranteed the specificity of detection kit, thereby guaranteed the accuracy of testing result.Detection kit of the present invention is by preferred ingredient and optimize the content of each component, makes to adopt detection kit that the art of this patent prepares performance in every respect all in optimum condition, can better meet the demand of detection.
It should be noted last that, above embodiment is only in order to implementer's case of this material to be described and unrestricted, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.
Claims (10)
1. a TfR reagent box for detecting content, is characterized in that, described detection kit is comprised of reagent I and reagent II, and the component of described reagent I comprises: buffering agent, antiseptic, sequestrant, surfactant and water; The component of described reagent II comprises: latex microsphere particle, buffering agent, antiseptic, suspending agent and the water of coated anti-TfR antibody; The particle diameter of the latex particle of described coated anti-TfR antibody is 120～200nm.
2. TfR reagent box for detecting content as claimed in claim 1, is characterized in that: described anti-TfR antibody is monoclonal antibody or polyclonal antibody.
3. TfR reagent box for detecting content as claimed in claim 1, is characterized in that: described damping fluid is phosphate buffer; Described antiseptic is Proclin 300; Described sequestrant is disodium ethylene diamine tetraacetate; Described surfactant is polysorbas20; Described suspending agent is ethylene glycol.
4. TfR reagent box for detecting content as described in claim 1, is characterized in that: in every 1L reagent I, the consumption of each component is antiseptic: 0.05-2mL, and sequestrant is 0.1-5g, surfactant 0.1-3mL, and surplus is phosphate buffered solution.
5. TfR reagent box for detecting content as described in claim 1, is characterized in that: in every 1L reagent I, the consumption of each component is antiseptic: 0.1-1mL, and sequestrant is 1-3g, surfactant 1-2mL, and surplus is phosphate buffered solution.
6. TfR reagent box for detecting content according to claim 1, it is characterized in that: in every 1L reagent II, the consumption of each component is: coated anti-TfR antibody latex microsphere particle 0.1-5g, antiseptic 0.05-2mL, suspending agent 5-50mL, surplus is phosphate buffered solution.
7. TfR reagent box for detecting content according to claim 1, it is characterized in that: in every 1L reagent II, the consumption of each component is: coated anti-TfR antibody latex microsphere particle 1-3g, antiseptic 0.1-1mL, suspending agent 10-30mL, surplus is phosphate buffered solution.
8. TfR reagent box for detecting content according to claim 1, is characterized in that: the pH value scope of described reagent I and reagent II is 6.5～8.5.
9. the preparation method of the TfR reagent box for detecting content described in any one in a claim 1-8, it is characterized in that: comprise the following steps: (1) prepares reagent I: by antiseptic, sequestrant, surfactant dissolves in phosphate buffer, fully dissolve, mix, and use phosphate buffer constant volume, obtain reagent I; (2) prepare reagent II: the latex microsphere particle solution of the coated anti-TfR antibody of preparation; Antiseptic, suspending agent are dissolved in phosphate buffer; Latex microsphere particle solution and mixed solution are mixed, and constant volume, obtains reagent II.
10. the preparation method of TfR reagent box for detecting content as claimed in claim 9, it is characterized in that: the latex microsphere particle of described coated anti-TfR antibody adopts chemical crosslink technique to prepare, the latex microsphere particle of the coated TfR antibody of described chemical crosslink technique preparation comprises the following steps:
(1) washing latex: get activation damping fluid dilution for appropriate amounts of ammonia base latex microsphere particle solution, concussion mixes, then adds surfactant, and concussion mixes, and the centrifugal supernatant of abandoning retains precipitation, and precipitation is resuspended with 6mmol/L phosphate buffer;
(2) preparation of antibody-solutions: get antibody-solutions, with the dilution of 6mmol/L phosphate buffer, adjust pH to 5.0;
(3) preparation of EDC solution: get EDC powder and be mixed with deionized water the EDC solution that concentration is 10mg/mL;
(4) preparation of antibody coupling latex microsphere particle: the resuspended liquid of step (1) gained latex microsphere particle is mixed with step (2) gained antibody-solutions, cultivate 15-30 minute under room temperature; Add again step (3) gained EDC solution; The pH to 6.0-7.0 that adjusts reaction system with 0.1M sodium hydroxide solution, is shaking bed reaction 2-3 hour under room temperature condition;
(5) in the middle system of having reacted of step (4), add BSA solution, reaction 10-15 minute, the centrifugal supernatant of abandoning, and with 6mmol/L phosphate buffer washing precipitation 2 times, after use the resuspended precipitation of 6mmol/L phosphate buffer, both must be coated with the latex microsphere particle solution of anti-TfR antibody.
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| CN110736735A (en) * | 2018-07-18 | 2020-01-31 | 博阳生物科技（上海）有限公司 | homogeneous phase chemiluminescence detection kit and application thereof |
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| CN111308087A (en) * | 2020-02-21 | 2020-06-19 | 安徽大千生物工程有限公司 | Determination kit for detecting soluble transferrin receptor sTfR and preparation and use method thereof |
| CN112409487A (en) * | 2020-11-27 | 2021-02-26 | 宁波赛珀生物技术有限公司 | Anti-soluble transferrin receptor antibodies and uses thereof |
| CN113125711A (en) * | 2019-12-31 | 2021-07-16 | 博阳生物科技（上海）有限公司 | Receptor reagent and application thereof |
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