CN111175516A - Vascular endothelial growth factor detection kit and preparation method thereof - Google Patents
Vascular endothelial growth factor detection kit and preparation method thereof Download PDFInfo
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Classifications
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
- G01N33/543—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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- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- G—PHYSICS
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- 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
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Abstract
The invention relates to the field of immunodetection, in particular to a vascular endothelial growth factor detection kit, which comprises a magnetic microsphere suspension coated with VEGF antibodies, a VEGF reference substance, a VEGF quality control substance, a VEGF marker, a concentrated washing solution and a luminescent substrate solution; wherein the VEGF marker labels the engineered VEGF antibody by an acridinium ester modification. The detection kit has the advantages of accurate detection result, good stability, high sensitivity and wide linear range.
Description
Technical Field
The invention relates to the field of immunodetection, in particular to a vascular endothelial growth factor detection kit and a preparation method thereof.
Background
VEGF, a protein isolated by Ferrara et al (1989) from bovine pituitary follicular cells that specifically promotes vascular endothelial cell growth, acts as a specific mitogen for vascular endothelial cells, and specifically promotes vascular endothelial cell growth and induces angiogenesis both in vitro and in vivo. VEGF is a highly glycosylated basic protein with a molecular weight of 45KD, is a highly specific factor for promoting vascular endothelial cell division and inducing angiogenesis, and can specifically act on vascular endothelial cells, promote angiogenesis, maintain the normal state and integrity of blood vessels and improve vascular permeability. The vascular endothelial cell growth factor participates in the expression of a plurality of pathologies and physiology, plays an important role in the whole expression process of the fracture healing, can be expressed and enhanced in the morbidity process of diabetic nephropathy, and can be used as a gene therapy medicament in the expression of leukemia and critical limb ischemia. It has been found that VEGF is expressed in many pathophysiological processes, such as vascular endothelial cells and macrophages in wound repair, diabetic retinopathy cells, cardiovascular and cerebrovascular diseases, liver diseases, etc., and specifically acts on VEGF receptors on the vascular endothelial cell membrane in an autocrine, paracrine and intracellular manner to induce intracellular signal transduction. Among the numerous angiogenic factors, VEGF is recognized as the major angiogenic inducing substance, which strongly promotes vascular endothelial mitosis and neovascularization. Therefore, VEGF detection is currently used as a good diagnostic tool in the diagnosis of diseases.
Most of the existing VEGF diagnostic kits use the previous generation detection technology, the signal amplification base is not enough, the content of VEGF in human serum is very little, the content of VEGF in normal human serum is 10-80pg/mL, and even if the patient is in the middle stage of tumor, the content of VEGF in serum is only 400-1000 pg/mL. The existing kit has low sensitivity, and once the content of VEGF is detected in practical application, the patient is basically marked as a patient with a late tumor stage, so that the aim of early diagnosis cannot be fulfilled; in addition, the condition of missed detection exists in the detection of samples, and the stability of the existing VEGF reagent is poor. Therefore, it is necessary to develop a kit for detecting vascular endothelial growth factor.
Disclosure of Invention
The first object of the present invention is to: aiming at the defects in the prior art, the kit for detecting the vascular endothelial growth factor is provided, and has the advantages of accurate detection result, good stability, high sensitivity and wide linear range.
In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:
a vascular endothelial growth factor detection kit comprises a magnetic microsphere suspension coated with a VEGF antibody, a VEGF reference substance, a VEGF quality control substance, a VEGF marker, a concentrated washing solution and a luminescent substrate solution; wherein the VEGF label labels the engineered VEGF antibody by an acridinium ester modification.
As an improved technical scheme, the acridinium ester modifier is acridinium ester DEMS-NHS, and the modified VEGF antibody is prepared by reacting a VEGF antibody with a bifunctional chelating agent.
As an improved technical scheme, the concentration of the magnetic microsphere suspension coated with the VEGF antibody is prepared according to the proportion of 1:50-1: 100;
diluting the concentration of the VEGF marker according to the proportion of the VEGF marker to the marker diluent of 1:500-1: 10000;
the VEGF reference substance and the VEGF quality control substance are both prepared from a VEGF standard substance and a reference substance diluent, and the concentration range of the VEGF reference substance is 0-2500 pg/mL;
the concentration of the VEGF quality control product is 125pg/mL and 1000 pg/mL.
As a further preferable technical scheme, the concentration of the VEGF antibody coated magnetic microsphere suspension is prepared according to the ratio of 1:50, and the concentration of the VEGF marker is diluted according to the ratio of 1:2000 to the marker diluent.
As a preferred technical scheme, the reference dilution is HEPES buffer solution with pH7.5, and each 1L of the buffer solution also contains 18-22g of sodium caseinate, 14-15g of SDS, 3.8-4g of DTT and 0.3-0.5mL of PC 300.
The concentrated washing solution is prepared according to the conditions that each 1LpH7.5 Tris-HCl buffer solution contains 7-10g of NaCl, 0.3-0.6ml of Tween-80 and 6-8ml of Proclin-300.
As a preferable technical scheme, the luminescence substrate solution comprises a luminescence substrate solution A and a luminescence substrate solution B, the pH of the luminescence substrate solution A is 2.5-2.6, and the components of the luminescence substrate solution A are hydrogen peroxide and nitric acid;
the pH value of the luminescence substrate liquid B is 12.3-12.7, and the components of the luminescence substrate liquid B are potassium hydroxide and TritonX-100.
A second object of the present invention is to: provides a preparation method of a detection kit of vascular endothelial growth factor.
In order to achieve the purpose of the invention, the technical scheme of the invention is as follows:
a preparation method of a detection kit for vascular endothelial growth factor comprises the following steps:
(1) preparation of VEGF magnetic microsphere suspension
Adding a magnetic bead mother solution into a magnetic bead coating buffer solution I, mixing uniformly, sucking the liquid, adding the magnetic bead coating buffer solution I, mixing uniformly, adding a VEGF antibody, mixing uniformly, adding a magnetic bead coating buffer solution II, mixing uniformly, reacting for a period of time, sucking the liquid after the reaction is finished, adding a magnetic bead coating buffer solution III, mixing uniformly, sucking the liquid after the reaction is finished, adding a magnetic bead preservation solution, mixing uniformly, adding a magnetic bead coating buffer solution IV, and mixing uniformly to obtain a VEGF magnetic microsphere suspension;
(2) preparing VEGF reference substance and quality control product
Diluting the VEGF standard substance into a series of reference substances according to a certain proportion by using a reference substance diluent, wherein the concentration range of the reference substances is 0pg/mL-2000 pg/mL; preparing quality control products with the concentrations of 125pg/mL and 1000pg/mL according to the above operation, subpackaging and freeze-drying;
(3) preparation of VEGF marker
preparation of solution
Using 0.1M N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid solution with pH of 7.5 as a buffer system to prepare working solution of the antibody with the concentration of 1 mg/mLVEGF;
② protein crosslinking reaction
Adding 300 mu L of glutaraldehyde with the volume concentration of 25% into 2ml of 1mg/mLVEGF antibody working solution, reacting to obtain a protein compound VEGF-GA, and dialyzing the protein compound in HBS buffer solution for 8 hours for later use;
③ acridinium ester labeling
Taking 0.1mg/mL acridine ester DMAE-NHS solution, adding 1mg/mL dialyzed VEGF-GA solution according to the volume ratio of 1:1, stirring, mixing uniformly, reacting to obtain VEGF-GA-DMAE reaction marker, and dialyzing for later use;
VEGF-GA-DMAE marker configuration
Mixing the VEGF-GA-DMAE reaction marker after dialysis with a marker diluent according to the volume ratio of 1: the proportion of 1000-5000 is configured;
(4) preparing concentrated washing liquid
The preparation is carried out according to the conditions that each 1L and 0.1M HEPES buffer system contains 7-10g NaCl, 0.5-2g KCl, 0.3-0.6ml Triton X-100, 6-8ml Proclin-300 and 0.5-1g NaN 3;
(5) preparing luminescent substrate solution A and luminescent substrate solution B
Luminescent substrate solution a: taking 10-20L of pure water, and adding 200-278.4ml of HNO3，100-132.8ml H2O2Adding pure water to a constant volume of 30-40L, and adjusting the pH value to 2.55 +/-0.5;
luminescent substrate solution B: taking 10-20L of pure water, adding 300-400g of KOH and 600-800ml of TritonX-100, fixing the volume of the pure water to 30-40L, and adjusting the pH to 12.50 +/-0.2;
(6) and (3) respectively subpackaging the magnetic microsphere suspension prepared in the steps (1) to (5), the VEGF marker, the reference substance, the quality control substance, the concentrated washing solution, the luminescent substrate solution A and the luminescent substrate solution B.
As an improved technical scheme, the magnetic particles are magnetic microspheres with the particle size of 1-3 mu m and the surfaces of which are wrapped with carboxyl or sulfonyl active groups, and VEGF magnetic microsphere suspension is prepared by coating 1.0-2.5mg of VEGF antibody according to the dosage of each 50mg of magnetic microspheres.
As a preferable technical scheme, the marker diluent in the step (3) is PBS buffer solution with pH7.5, and each 1LPBS buffer solution also contains 5-6g of calcium chloride, 0.02-0.04% v/v of Proclin-300 and 0.3-1.0% v/v of AES.
By adopting the technical scheme, compared with the prior art, the invention has the following advantages:
the VEGF antibody raw material is mainly a recombinant antibody Fab fragment expressed by CHO cells in vitro, the molecular force is about 50kD, the carboxyl on the antibody fragment is less, the cross-linking with acridine ester with NHS modifying groups is difficult, and the labeling efficiency is extremely low. According to the invention, the VEGF antibody fragment is treated, so that the amino group of the VEGF antibody fragment is changed into carboxyl, and the carboxyl reaction sites on the surface of the antibody are increased, thus the binding rate with the acridinium ester modifier is improved, and the sensitivity of the kit is greatly improved. The formula of each component in the kit product is a formula component obtained through multiple experiments, and the components are matched to ensure the stability and the accuracy of the kit product, so that the kit has a good linear relationship between 0 and 2500pg/mL, the sensitivity can reach 1pg/mL, the detection efficiency is greatly improved, and the omission factor is reduced.
Detailed Description
The present invention will be described in further detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A vascular endothelial growth factor detection kit comprises a magnetic microsphere suspension coated with a VEGF antibody, a VEGF reference substance, a VEGF quality control substance, a VEGF marker, a concentrated washing solution and a luminescent substrate solution; wherein the VEGF label labels the engineered VEGF antibody by an acridinium ester modification.
The acridine ester modifier is acridine ester DEMS-NHS, and the modified VEGF antibody is prepared by reacting a VEGF antibody with a bifunctional chelating agent.
Wherein the concentration of the magnetic microsphere suspension coated with the VEGF antibody is prepared according to the proportion of 1: 50;
wherein the concentration of the VEGF marker is diluted according to the ratio of the VEGF marker to the marker diluent of 1: 2000;
the VEGF reference substance and the VEGF quality control substance are both prepared from a VEGF standard substance and a reference substance diluent, and the concentration range of the VEGF reference substance is 0-2500 pg/mL;
wherein the concentration of the VEGF quality control product is 125pg/mL and 1000 pg/mL.
Wherein the reference dilution is HEPES buffer solution with pH7.5, and each 1L of the buffer solution also contains 18-22g sodium caseinate, 14-15g SDS, 3.8-4g DTT and 0.3-0.5mL PC 300.
Wherein the concentrated washing solution is prepared according to the conditions that each 1LpH7.5 Tris-HCl buffer solution contains 7-10g of NaCl, 0.3-0.6ml of Tween-80 and 6-8ml of Proclin-300.
The luminous substrate liquid comprises a luminous substrate liquid A and a luminous substrate liquid B, wherein the pH of the luminous substrate liquid A is 2.5, and the luminous substrate liquid A comprises hydrogen peroxide and nitric acid;
wherein the pH of the luminescence substrate liquid B is 12.5, and the components of the luminescence substrate liquid B are potassium hydroxide and TritonX-100.
The preparation method comprises the following steps:
(1) preparation of VEGF magnetic microsphere suspension
Adding M270 carboxyl magnetic bead mother liquor into magnetic bead coating buffer solution I, mixing, sucking liquid, adding magnetic bead coating buffer solution I (including 0.97g Tris, 6.59g Tris-HCI, 9.00g NaCl, and adding ddH2O to constant volume of 1L, and adjusting pH to 7.0 + -0.1 with 1M NaOH/1MHCI, mixing, adding VEGF antibody, mixing, adding magnetic bead coating buffer solution II (comprising 9.54g borax, 6.18g boric acid, and ddH)2O to constant volume of 1L, adjusting pH to 9.5 +/-0.1 with 1M NaOH/1M HCI, mixing uniformly for 16-20h, sucking liquid after reaction, adding magnetic bead coating buffer solution III (BS3(bis (sulfovinylidyl) sulfate) Thermo Fisher1mg and TRIS buffer solution prepared into 100 mu L), mixing uniformly, sucking liquid after reaction for 3h, and adding magnetic bead preserving solution (including Tris 0.97g, Tris-HCI 6.59g, NaCl 9.00g and ddH)2O to volume of 1L, adjusting pH to 7.4 + -0.1 with 1M NaOH/1M HCI, mixing, adding magnetic bead coating buffer solution IV (containing 14.5g Na)2HPO4.12H20、1.48gNaH2PO4.2H20. 0.50g PC300, 5.00g BSA, 0.50g Tween-20, using ddH2O is metered to 1L, the pH value is adjusted to 9.5 +/-0.1 by using 1M NaOH/1MHCI, and VEGF magnetic microsphere suspension is obtained after uniform mixing;
(2) preparing VEGF reference substance and quality control product
Diluting the VEGF standard substance into a series of reference substances according to a certain proportion by using a reference substance diluent, wherein the concentration range of the reference substances is 0pg/mL-2500pg/mL (the concentration ranges of the reference substances are 0pg/mL, 50pg/mL, 200pg/mL, 500pg/mL, 1200pg/mL and 2500pg/mL respectively); preparing quality control products with the concentrations of 125pg/mL and 1000pg/mL according to the above operation, subpackaging and freeze-drying;
(3) preparation of VEGF marker
preparation of solution
Using 0.1M N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid solution with pH of 7.5 as a buffer system to prepare working solution of the antibody with the concentration of 1 mg/mLVEGF;
② protein crosslinking reaction
Taking 2ml of 1mg/mLVEGF antibody working solution, adding 300 mu L of glutaraldehyde with the volume concentration of 25%, reacting to obtain a protein compound VEGF-GA, and then putting the protein compound in HBS buffer solution for dialysis for 8 hours for later use;
③ acridinium ester labeling
Taking 0.1mg/mL acridine ester DMAE-NHS solution, adding 1mg/mL dialyzed VEGF-GA solution according to the volume ratio of 1:1, stirring, mixing uniformly, reacting to obtain VEGF-GA-DMAE reaction marker, and dialyzing for later use;
VEGF-GA-DMAE marker configuration
Mixing the VEGF-GA-DMAE reaction marker after dialysis with a marker diluent according to the volume ratio of 1: the proportion of 1000-5000 is configured;
(4) preparing concentrated washing liquid
The preparation is carried out according to the conditions that each 1L and 0.1M HEPES buffer system contains 7-10g NaCl, 0.5-2g KCl, 0.3-0.6ml Triton X-100, 6-8ml Proclin-300 and 0.5-1g NaN 3;
(5) preparing luminescent substrate solution A and luminescent substrate solution B
Luminescent substrate solution a: taking 10-20L of pure water, and adding 200-278.4ml of HNO3，100-132.8ml H2O2Adding pure water to a constant volume of 30-40L, and adjusting the pH value to 2.55 +/-0.5;
luminescent substrate solution B: taking 10-20L of pure water, adding 300-400g of KOH and 600-800ml of TritonX-100, fixing the volume of the pure water to 30-40L, and adjusting the pH to 12.50 +/-0.2;
(6) and (3) respectively subpackaging the magnetic microsphere suspension prepared in the steps (1) to (5), the VEGF marker, the reference substance, the quality control substance, the concentrated washing solution, the luminescent substrate solution A and the luminescent substrate solution B.
When the VEGF magnetic microsphere suspension is prepared, 2mg of VEGF antibody is coated according to the dosage of each 50mg of magnetic microspheres, and the concentration of the antibody is 40 ug/mg.
Wherein, the marker diluent in the step (3) is PBS buffer solution with pH7.5, and each 1LPBS buffer solution also contains 5-6g of calcium chloride, 0.02-0.04% v/v of Proclin-300 and 0.3-1.0% v/v of AES.
Example 2
A vascular endothelial growth factor detection kit comprises a magnetic microsphere suspension coated with a VEGF antibody, a VEGF reference substance, a VEGF quality control substance, a VEGF marker, a concentrated washing solution and a luminescent substrate solution; wherein the VEGF label labels the engineered VEGF antibody by an acridinium ester modification.
The acridine ester modifier is acridine ester DEMS-NHS, and the modified VEGF antibody is prepared by reacting a VEGF antibody with a bifunctional chelating agent.
Wherein the concentration of the magnetic microsphere suspension coated with the VEGF antibody is prepared according to the proportion of 1: 80;
wherein the concentration of the VEGF marker is diluted according to the ratio of the VEGF marker to the marker diluent of 1: 1000;
the VEGF reference substance and the VEGF quality control substance are both prepared from a VEGF standard substance and a reference substance diluent, and the concentration range of the VEGF reference substance is 0-2500pg/mL (the concentrations of the reference substances are 0pg/mL, 50pg/mL, 200pg/mL, 500pg/mL, 1200pg/mL and 2500pg/mL respectively);
wherein the concentration of the VEGF quality control product is 125pg/mL and 1000 pg/mL.
Wherein the reference dilution is HEPES buffer solution with pH7.5, and each 1L of the buffer solution also contains 18-22g sodium caseinate, 14-15g SDS, 3.8-4g DTT and 0.3-0.5mL PC 300.
Wherein the concentrated washing solution is prepared according to the conditions that each 1LpH7.5 Tris-HCl buffer solution contains 7-10g of NaCl, 0.3-0.6ml of Tween-80 and 6-8ml of Proclin-300.
The luminous substrate liquid comprises a luminous substrate liquid A and a luminous substrate liquid B, wherein the pH of the luminous substrate liquid A is 2.5, and the luminous substrate liquid A comprises hydrogen peroxide and nitric acid;
wherein the pH of the luminescence substrate liquid B is 12.3, and the components of the luminescence substrate liquid B are potassium hydroxide and TritonX-100.
The preparation method comprises the following steps:
(1) preparation of VEGF magnetic microsphere suspension
Adding M270 carboxyl magnetic bead mother liquor into magnetic bead coating buffer solution I, mixing, sucking liquid, adding magnetic bead coating buffer solution I (including 0.97g Tris, 6.59g Tris-HCI, 9.00g NaCl, and adding ddH2O to constant volume of 1L, and adjusting pH to 7.0 + -0.1 with 1M NaOH/1MHCI, mixing, adding VEGF antibody, mixing, adding magnetic bead coating buffer solution II (comprising 9.54g borax, 6.18g boric acid, and ddH)2O to constant volume of 1L, adjusting pH to 9.5 +/-0.1 with 1M NaOH/1M HCI, mixing uniformly for 16-20h, sucking liquid after reaction, adding magnetic bead coating buffer solution III (BS3(bis (sulfovinylidyl) sulfate) Thermo Fisher1mg and TRIS buffer solution prepared into 100 mu L), mixing uniformly, sucking liquid after reaction for 3h, and adding magnetic bead preserving solution (including Tris 0.97g, Tris-HCI 6.59g, NaCl 9.00g and ddH)2O to 1L, adjusting pH to 7.4 + -0.1 with 1M NaOH/1M HCI, mixing, adding magnetic bead coating buffer solution IV (containing 14.5g Na)2HPO4.12H20、1.48gNaH2PO4.2H20. 0.50g PC300, 5.00g BSA, 0.50g Tween-20, using ddH2O is metered to 1L, the pH value is adjusted to 9.5 +/-0.1 by using 1M NaOH/1M HCI, and VEGF magnetic microsphere suspension is obtained after uniform mixing;
(2) preparing VEGF reference substance and quality control product
Diluting the VEGF standard substance into a series of reference substances according to a certain proportion by using a reference substance diluent, wherein the reference substance concentration ranges from 0pg/mL to 2500pg/mL (the specific concentrations of the reference substances are respectively 0pg/mL, 50pg/mL, 200pg/mL, 500pg/mL, 1200pg/mL and 2500 pg/mL); preparing quality control products with the concentrations of 125pg/mL and 1000pg/mL according to the above operation, subpackaging and freeze-drying;
(3) preparation of VEGF marker
preparation of solution
Using 0.1M N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid solution with pH of 7.5 as a buffer system to prepare working solution of the antibody with the concentration of 1 mg/mLVEGF;
② protein crosslinking reaction
Taking 2ml of 1mg/mLVEGF antibody working solution, adding 300 mu L of glutaraldehyde with the volume concentration of 25%, reacting to obtain a protein compound VEGF-GA, and then putting the protein compound in HBS buffer solution for dialysis for 8 hours for later use;
③ acridinium ester labeling
Taking 0.1mg/mL acridine ester DMAE-NHS solution, adding 1mg/mL dialyzed VEGF-GA solution according to the volume ratio of 1:1, stirring, mixing uniformly, reacting to obtain VEGF-GA-DMAE reaction marker, and dialyzing for later use;
VEGF-GA-DMAE marker configuration
Mixing the VEGF-GA-DMAE reaction marker after dialysis with a marker diluent according to the volume ratio of 1: the proportion of 1000-5000 is configured;
(4) preparing concentrated washing liquid
The preparation is carried out according to the conditions that each 1L and 0.1M HEPES buffer system contains 7-10g NaCl, 0.5-2g KCl, 0.3-0.6ml Triton X-100, 6-8ml Proclin-300 and 0.5-1g NaN 3;
(5) preparing luminescent substrate solution A and luminescent substrate solution B
Luminescent substrate solution a: taking 10-20L of pure water, and adding 200-278.4ml of HNO3，100-132.8ml H2O2Adding pure water to a constant volume of 30-40L, and adjusting the pH value to 2.55 +/-0.5;
luminescent substrate solution B: taking 10-20L of pure water, adding 300-400g of KOH and 600-800ml of TritonX-100, fixing the volume of the pure water to 30-40L, and adjusting the pH to 12.50 +/-0.2;
(6) and (3) respectively subpackaging the magnetic microsphere suspension prepared in the steps (1) to (5), the VEGF marker, the reference substance, the quality control substance, the concentrated washing solution, the luminescent substrate solution A and the luminescent substrate solution B.
Wherein, when preparing the VEGF magnetic microsphere suspension, 1.0mg of VEGF antibody is coated according to the dosage of each 50mg of magnetic microspheres, and the concentration of the antibody is 20 ug/mg.
Wherein, the marker diluent in the step (3) is PBS buffer solution with pH7.5, and each 1LPBS buffer solution also contains 5-6g of calcium chloride, 0.02-0.04% v/v of Proclin-300 and 0.3-1.0% v/v of AES.
Example 3
A vascular endothelial growth factor detection kit comprises a magnetic microsphere suspension coated with a VEGF antibody, a VEGF reference substance, a VEGF quality control substance, a VEGF marker, a concentrated washing solution and a luminescent substrate solution; wherein the VEGF label labels the engineered VEGF antibody by an acridinium ester modification.
The acridine ester modifier is acridine ester DEMS-NHS, and the modified VEGF antibody is prepared by reacting a VEGF antibody with a bifunctional chelating agent.
Wherein the concentration of the magnetic microsphere suspension coated with the VEGF antibody is prepared according to the proportion of 1: 100;
wherein the concentration of the VEGF marker is diluted according to the ratio of the VEGF marker to the marker diluent of 1: 5000;
the VEGF reference product and the VEGF quality control product are both prepared from a VEGF standard product and a reference product diluent, and the concentration range of the VEGF reference product is 0-2500pg/mL (the concentration ranges of the reference products are 0pg/mL, 50pg/mL, 200pg/mL, 500pg/mL, 1200pg/mL and 2500pg/mL respectively);
wherein the concentration of the VEGF quality control product is 125pg/mL and 1000 pg/mL.
Wherein the reference dilution is HEPES buffer solution with pH7.5, and each 1L of the buffer solution also contains 18-22g sodium caseinate, 14-15g SDS, 3.8-4g DTT and 0.3-0.5mL PC 300.
Wherein the concentrated washing solution is prepared according to the conditions that each 1LpH7.5 Tris-HCl buffer solution contains 7-10g of NaCl, 0.3-0.6ml of Tween-80 and 6-8ml of Proclin-300.
The luminous substrate liquid comprises a luminous substrate liquid A and a luminous substrate liquid B, wherein the pH of the luminous substrate liquid A is 2.6, and the luminous substrate liquid A comprises hydrogen peroxide and nitric acid;
wherein the pH of the luminescence substrate liquid B is 12.7, and the components of the luminescence substrate liquid B are potassium hydroxide and TritonX-100.
The preparation method comprises the following steps:
(1) preparation of VEGF magnetic microsphere suspension
Adding M270 carboxyl magnetic bead mother liquor into magnetic bead coating buffer solution I, mixing, sucking liquid, adding magnetic bead coating buffer solution I (including 0.97g Tris, 6.59g Tris-HCI, 9.00g NaCl, and adding ddH2O to constant volume of 1L, and adjusting pH to 7.0 + -0.1 with 1M NaOH/1MHCI, mixing, adding VEGF antibody, mixing, adding magnetic bead coating buffer solution II (comprising 9.54g borax, 6.18g boric acid, and ddH)2O to constant volume of 1L, adjusting pH to 9.5 +/-0.1 with 1M NaOH/1M HCI, mixing uniformly for 16-20h, sucking liquid after reaction, adding magnetic bead coating buffer solution III (BS3(bis (sulfocyanidyl) sulfate), Thermo Fisher1mg and TRIS buffer solution for 100 mu L), mixing uniformly, sucking liquid after 3h reaction, and adding magnetic bead preservation solution (including Tris 0.97g, Tris-HCI 6.59g, NaCl 9.00g and ddH)2O to 1L, adjusting pH to 7.4 + -0.1 with 1M NaOH/1M HCI, mixing, adding magnetic bead coating buffer solution IV (containing 14.5g Na)2HPO4.12H20、1.48gNaH2PO4.2H20. 0.50g PC300, 5.00g BSA, 0.50g Tween-20, using ddH2O is metered to 1L, the pH value is adjusted to 9.5 +/-0.1 by using 1M NaOH/1M HCI, and VEGF magnetic microsphere suspension is obtained after uniform mixing;
(2) preparing VEGF reference substance and quality control product
Diluting the VEGF standard substance into a series of reference substances according to a certain proportion by using a reference substance diluent, wherein the concentration range of the reference substances is 0pg/mL-2500pg/mL (the concentration ranges of the reference substances are 0pg/mL, 50pg/mL, 200pg/mL, 500pg/mL, 1200pg/mL and 2500pg/mL respectively); preparing quality control products with the concentrations of 125pg/mL and 1000pg/mL according to the above operation, subpackaging and freeze-drying;
(3) preparation of VEGF marker
preparation of solution
Using 0.1M N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid solution with pH of 7.5 as a buffer system to prepare working solution of the antibody with the concentration of 1 mg/mLVEGF;
② protein crosslinking reaction
Taking 2ml of 1mg/mLVEGF antibody working solution, adding 300 mu L of glutaraldehyde with the volume concentration of 25%, reacting to obtain a protein compound VEGF-GA, and then putting the protein compound in HBS buffer solution for dialysis for 8 hours for later use;
③ acridinium ester labeling
Taking 0.1mg/mL acridine ester DMAE-NHS solution, adding 1mg/mL dialyzed VEGF-GA solution according to the volume ratio of 1:1, stirring, mixing uniformly, reacting to obtain VEGF-GA-DMAE reaction marker, and dialyzing for later use;
VEGF-GA-DMAE marker configuration
Mixing the VEGF-GA-DMAE reaction marker after dialysis with a marker diluent according to the volume ratio of 1: the proportion of 1000-5000 is configured;
(4) preparing concentrated washing liquid
The preparation is carried out according to the conditions that each 1L and 0.1M HEPES buffer system contains 7-10g NaCl, 0.5-2g KCl, 0.3-0.6ml Triton X-100, 6-8ml Proclin-300 and 0.5-1g NaN 3;
(5) preparing luminescent substrate solution A and luminescent substrate solution B
Luminescent substrate solution a: taking 10-20L of pure water, and adding 200-278.4ml of HNO3，100-132.8ml H2O2Adding pure water to a constant volume of 30-40L, and adjusting the pH value to 2.55 +/-0.5;
luminescent substrate solution B: taking 10-20L of pure water, adding 300-400g of KOH and 600-800ml of TritonX-100, fixing the volume of the pure water to 30-40L, and adjusting the pH to 12.50 +/-0.2;
(6) and (3) respectively subpackaging the magnetic microsphere suspension prepared in the steps (1) to (5), the VEGF marker, the reference substance, the quality control substance, the concentrated washing solution, the luminescent substrate solution A and the luminescent substrate solution B.
Wherein the magnetic bead is M270 carboxyl magnetic bead, 2.5mg of VEGF antibody is coated according to the dosage of each 50mg of magnetic microsphere during the preparation of the VEGF magnetic microsphere suspension, and the concentration of the antibody is 50 ug/mg.
Wherein, the marker diluent in the step (3) is PBS buffer solution with pH7.5, and each 1LPBS buffer solution also contains 5-6g of calcium chloride, 0.02-0.04% v/v of Proclin-300 and 0.3-1.0% v/v of AES.
Example 4
The main product performance indexes of the vascular endothelial growth factor detection kit are as follows:
(1) minimum detection limit (sensitivity) of kit
And (3) parallelly measuring the sample diluent by 10 holes, calculating the average (M) and Standard Deviation (SD) of the measurement result to obtain a luminous value corresponding to M +2SD, substituting the luminous value into a dose-response curve, and calculating a corresponding concentration value, wherein the concentration value is the lowest detection limit of the kit. The detection result of the lowest detection limit index of the kit is shown in table 1.
TABLE 1 determination of the lowest detection Limit of the kit
And (4) conclusion: the average luminescence value M of VEGF sample dilution measured was 3025.1, SD was 238.6, and the value of (M +2SD) was added to the reaction curve, and the sensitivity was obtained as follows: 1pg/mL (<5pg/mL), satisfactory.
(2) Linearity of dose-response curves of kits
The reference samples of the double-well assay kit (the reference samples with the concentrations of 0pg/mL, 50pg/mL, 200pg/mL, 500pg/mL, 1200pg/mL and 2500pg/mL in sequence are subjected to two-well assay), are fitted by four parameters, and the linear detection result of the dose-reaction curve of the kit is shown in Table 2.
TABLE 2 kit dose-response Curve Linearity
And (4) conclusion: the linear correlation coefficient (r) of the VEGF dose-response curve determined was: 0.997(>0.9900), meets the requirements.
(3) Accuracy of the kit
The detection concentrations of the reference substances are 125pg/mL and 1000pg/mL, each concentration reference substance is detected for 3 times, the mean value M of the detection result is calculated, and the accuracy detection result of the kit is shown in Table 3.
TABLE 3 accuracy of the kit
| Numbering | 125pg/mL | 1000pg/mL |
| 1 | 126.52 | 962.25 |
| 2 | 126.28 | 998.12 |
| 3 | 127.03 | 1008.89 |
| Mean value of | 126.61 | 989.75 |
| Bias％ | 1.28％ | 1.02％ |
And (4) conclusion: the relative deviations (Bias%) of the 125pg/mL and 1000pg/mL accuracy references determined were: 1.29% and 1.02%, and the relative deviation (Bias%) of the measured value and the target value is less than 15.0%, which meets the requirement.
(4) Stability of the kit
The kit is placed in a 37 ℃ incubator for accelerated destructive experiments for 1 day, 3 days, 5 days and 7 days respectively, and the degree of decrease of the signal value of the kit is observed. According to the Arrhenius reaction equation, the condition that the acceleration of 37 ℃ for 7 days is equivalent to the condition that the storage is carried out at 4 ℃ for one year, the signal value reduction amplitude is less than 20 percent, the stability of the reagent is considered to be extremely excellent, the signal value reduction is less than 50 percent, the stability of the reagent is considered to be qualified, and the specific results are shown in tables 4 and 5.
TABLE 4 accelerated stability test
TABLE 5 accelerated stability test
The data in tables 4 and 5 show that the signal value reduction is less than 20%, and the kit has good stability.
From the measured data, the vascular endothelial growth factor detection kit designed by the invention has leading position in all quality parameters such as sensitivity, accuracy, stability and the like.
Example 5
An example of clinical detection of the vascular endothelial growth factor assay kit is as follows:
the kit in the embodiment 2 of the invention is adopted to carry out VEGF detection on 50 patients with initial tumors, the contrast reagent is the existing VEGF detection reagent in the market, and the data is shown in the following table;
as can be seen from the data table, the kit of the invention can detect VEGF abnormality of sample numbers 13, 17, 19, 20, 23, 32 and 33, and the contrast reagent is not detected, so that the kit of the invention has better sensitivity and accuracy.
Except for tumor samples which are specially detected due to the improvement of sensitivity, the kit has good consistency with the measured value of the existing products in the market, wherein Y is 0.9767x +0.518 (R)20.9869) with a correlation coefficient R greater than 0.95.
The patent is not limited to the above-mentioned embodiments, and those skilled in the art can make various changes without creative efforts from the above-mentioned conception, and fall within the protection scope of the patent.
Claims (9)
1. A vascular endothelial growth factor detection kit is characterized in that: the detection kit comprises a magnetic microsphere suspension coated with a VEGF antibody, a VEGF reference substance, a VEGF quality control substance, a VEGF marker, a concentrated washing solution and a luminescent substrate solution; wherein the VEGF label labels the engineered VEGF antibody by an acridinium ester modification.
2. The VEGF detection kit of claim 1, wherein: the acridine ester modifier is acridine ester DEMS-NHS, and the modified VEGF antibody is prepared by reacting a VEGF antibody with a bifunctional chelating agent.
3. The VEGF detection kit of claim 1, wherein: the concentration of the magnetic microsphere suspension coated with the VEGF antibody is prepared according to the proportion of 1:50-1: 100;
diluting the concentration of the VEGF marker according to the proportion of the VEGF marker to the marker diluent of 1:500-1: 10000;
the VEGF reference substance and the VEGF quality control substance are both prepared from a VEGF standard substance and a reference substance diluent, and the concentration range of the VEGF reference substance is 0-2500 pg/mL;
the concentration of the VEGF quality control product is 125pg/mL and 1000 pg/mL.
4. The VEGF detection kit of claim 1, wherein: the concentration of the VEGF antibody coated magnetic microsphere suspension is prepared according to the proportion of 1:50, and the concentration of the VEGF marker is diluted according to the proportion of 1:2000 to the marker diluent.
5. The VEGF detection kit of claim 1, wherein: the reference dilution is HEPES buffer solution with pH7.5, and each 1L of the buffer solution also contains 18-22g of sodium caseinate, 14-15g of SDS, 3.8-4g of DTT and 0.3-0.5mL of PC 300.
The concentrated washing solution is prepared according to the conditions that each 1LpH7.5 Tris-HCl buffer solution contains 7-10g of NaCl, 0.3-0.6ml of Tween-80 and 6-8ml of Proclin-300.
6. The VEGF detection kit of claim 1, wherein: the luminous substrate liquid comprises a luminous substrate liquid A and a luminous substrate liquid B, the pH of the luminous substrate liquid A is 2.5-2.6, and the luminous substrate liquid A comprises hydrogen peroxide and nitric acid;
the pH value of the luminescence substrate liquid B is 12.3-12.7, and the components of the luminescence substrate liquid B are potassium hydroxide and TritonX-100.
7. The method for preparing the vascular endothelial growth factor detection kit according to claim 1, wherein the preparation method comprises the following steps:
(1) preparation of VEGF magnetic microsphere suspension
Adding a magnetic bead mother solution into a magnetic bead coating buffer solution I, mixing uniformly, sucking the liquid, adding the magnetic bead coating buffer solution I, mixing uniformly, adding a VEGF antibody, mixing uniformly, adding a magnetic bead coating buffer solution II, mixing uniformly, reacting for a period of time, sucking the liquid after the reaction is finished, adding a magnetic bead coating buffer solution III, mixing uniformly, sucking the liquid after the reaction is finished, adding a magnetic bead preservation solution, mixing uniformly, adding a magnetic bead coating buffer solution IV, and mixing uniformly to obtain a VEGF magnetic microsphere suspension;
(2) preparing VEGF reference substance and quality control product
Diluting the VEGF standard substance into a series of reference substances according to a certain proportion by using a reference substance diluent, wherein the concentration range of the reference substances is 0pg/mL-2500 pg/mL; preparing quality control products with the concentrations of 125pg/mL and 1000pg/mL according to the above operation, subpackaging and freeze-drying;
(3) preparation of VEGF marker
preparation of solution
Using 0.1M N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid solution with pH of 7.5 as a buffer system to prepare working solution of the antibody with the concentration of 1 mg/mLVEGF;
② protein crosslinking reaction
Adding 300 mu L of glutaraldehyde with the volume concentration of 25% into 2ml of 1mg/mLVEGF antibody solution, reacting to obtain a protein compound VEGF-GA, and dialyzing the protein compound in HBS buffer solution for 8 hours for later use;
③ acridinium ester labeling
Taking 0.1mg/mL acridine ester DMAE-NHS solution, adding 1mg/mL dialyzed VEGF-GA solution according to the volume ratio of 1:1, stirring, mixing uniformly, reacting to obtain VEGF-GA-DMAE reaction marker, and dialyzing for later use;
VEGF-GA-DMAE marker configuration
Mixing the VEGF-GA-DMAE reaction marker after dialysis with a marker diluent according to the volume ratio of 1: the proportion of 1000-5000 is configured;
(4) preparing concentrated washing liquid
The preparation is carried out according to the condition that each 1L and 0.1M HEPES buffer system contains 7-10g NaCl, 0.5-2g KCl, 0.3-0.6ml TritonX-100, 6-8ml Proclin-300 and 0.5-1g NaN 3;
(5) preparing luminescent substrate solution A and luminescent substrate solution B
Luminescent substrate solution a: taking 10-20L of pure water, and adding 200-278.4ml of HNO3，100-132.8mlH2O2Adding pure water to a constant volume of 30-40L, and adjusting the pH value to 2.55 +/-0.5;
luminescent substrate solution B: taking 10-20L of pure water, adding 300-400g of KOH and 600-800ml of TritonX-100, fixing the volume of the pure water to 30-40L, and adjusting the pH to 12.50 +/-0.2;
(6) and (3) respectively subpackaging the magnetic microsphere suspension prepared in the steps (1) to (5), the VEGF marker, the reference substance, the quality control substance, the concentrated washing solution, the luminescent substrate solution A and the luminescent substrate solution B.
8. The method for preparing a vascular endothelial growth factor assay kit according to claim 7, wherein the method comprises the following steps: the pH value of the coating buffer solution I in the step (1) is 7.0 +/-0.1, and each 1L of the coating buffer solution I contains 0.97g of Tris-HCI, 6.59g of Tris-HCI and 9.00g of NaCl; the pH value of the coating buffer solution II is 9.5 +/-0.1, and each 1L of the coating buffer solution II contains 9.54g of borax and 6.18g of boric acid; the coating buffer solution III is prepared into 100 mu L by BS31mg and TRIS buffer solution, the pH of the magnetic bead preservation solution is 7.4 +/-0.1, and each 1L of the magnetic bead preservation solution contains 0.97g of Tris, 6.59g of Tris-HCI and 9.00g of NaCl; the pH of the coating buffer IV is 9.5 + -0.1, and each 1L of the coating buffer IV contains 14.5g of Na2HPO4.12H20、1.48gNaH2PO4.2H20. 0.50g PC300, 5.00g BSA and 0.50g Tween-20.
9. The method for preparing a vascular endothelial growth factor assay kit according to claim 7, wherein the method comprises the following steps: in the step (3), the marker diluent is PBS buffer solution with pH7.5, and each 1LPBS buffer solution also contains 5-6g of calcium chloride, 0.02-0.04% v/v of Proclin-300 and 0.3-1.0% v/v of AES.
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| US20030008410A1 (en) * | 1995-03-13 | 2003-01-09 | Hechinger Mark K. | Immunoassay apparatus, kit and methods |
| CN101377503A (en) * | 2008-03-25 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Hepatitis b virus preS2 antigen chemiluminescence immune analysis determination reagent kit and preparing method thereof |
| CN107561287A (en) * | 2017-08-30 | 2018-01-09 | 潍坊市康华生物技术有限公司 | A kind of vascular endothelial growth factor detection kit and its preparation and application |
| CN107807241A (en) * | 2017-11-03 | 2018-03-16 | 太原瑞盛生物科技有限公司 | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of intravascular ErbB1 |
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| CN114019174A (en) * | 2021-06-29 | 2022-02-08 | 北京健平金星生物科技有限公司 | Kit and application thereof in vascular endothelial growth factor detection |
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