CN113702642A - Raw material preparation and detection method of vascular endothelial growth factor detection kit - Google Patents
Raw material preparation and detection method of vascular endothelial growth factor detection kit Download PDFInfo
- Publication number
- CN113702642A CN113702642A CN202110722649.4A CN202110722649A CN113702642A CN 113702642 A CN113702642 A CN 113702642A CN 202110722649 A CN202110722649 A CN 202110722649A CN 113702642 A CN113702642 A CN 113702642A
- Authority
- CN
- China
- Prior art keywords
- vegf
- antibody
- detection
- magnetic bead
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N21/76—Chemiluminescence; Bioluminescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57411—Specifically defined cancers of cervix
-
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
-
- 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
- G01N33/574—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
-
- 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
- G01N2333/475—Assays involving growth factors
Abstract
The invention relates to the technical field of vascular endothelial growth factor detection, and discloses a raw material preparation and detection method of a vascular endothelial growth factor detection kit, which comprises the steps of preparing raw materials, wherein the raw material preparation comprises an antibody-coated magnetic bead working solution, a VEGF-labeled antibody, a VEGF calibrator, a VEGF quality control product and a sample diluent; the antibody-coated magnetic bead working solution is a magnetic microsphere suspension coated with a humanized VEGF antibody Fab segment, and the buffer system is TBS-T (20-200mM Tris, 0-1M NaCl, 0.0-1% BSA, 0.1-0.5% Tween-20, 0.1-1% Proclin-300, pH 6.5-9.0). The luminescent marker of choice in the present invention is the acridinium salt NSP-SA-NHS. So that luminescence detection is performed in a homogeneous liquid and the reaction time is shortened. Meanwhile, the bond level of C-N is larger than that of C-O, and the stability of the conjugate of the acridinium salt NSP-SA-NHS and the protein is higher. Based on the use of NSP-SA-NHS, the sensitivity, detection range and storage time of the detection system are improved, and the reaction time is shortened.
Description
Technical Field
The invention belongs to the technical field of vascular endothelial growth factor detection, and particularly relates to a raw material preparation method and a detection method of a vascular endothelial growth factor detection kit.
Background
Vascular Endothelial Growth Factor (VEGF) is a key regulator of physiological angiogenesis during embryogenesis, skeletal growth, and reproductive function. VEGF has been found to be expressed under a variety of pathological and physiological conditions and can therefore be used as a marker for in vitro diagnostics. At present, the VEGF in-vitro detection kit on the market mainly adopts a quantum dot immunofluorescence method, an enzyme linked method, a plate-type chemiluminescence method and a magnetic particle chemiluminescence method.
The existing kit has the following problems:
1. the sensitivity and the linear range need to be further improved. It was reported that the VEGF content of healthy volunteers was 16.27 + -8.54 pg/ml, while that of gastric cancer patients was 223.61 + -58.67 pg/ml, and that in the serum of the liver cancer group, the VEGF content was only 549.7 + -150.1 pg/ml. If the sensitivity of the kit is not high, the kit cannot play a good role in early screening. FIG. 1 shows the sensitivity comparison of related products which have been investigated so far. The comparison result shows that the linear range and the minimum detection quantity of the magnetic particle luminescence platform are relatively higher compared with quantum dot immunofluorescence and enzyme-linked immunosorbent assay platforms. The existing products are optimized on the platform, so that the raw material preparation and detection method of the vascular endothelial growth factor detection kit is designed to solve the problem. The highest sensitivity product in the existing products is 0.1pg/ml, but the upper limit of the linear range is only 800 pg/ml. This would result in having to dilute the high VEGF concentration samples at the time of actual detection, and the added extra step would reduce the reproducibility and authenticity of the data. The sensitivity of products with a relatively large detection linear range is insufficient (generally more than 1pg/ml), which may also cause the accuracy of detection data to be reduced, thereby affecting clinical judgment.
2. The process is complicated. At present, two main process routes of the VEGF magnetic particle chemiluminescence detection kit are provided. One technique is to introduce a pair of linkers between the antibody and the magnetic beads to perform the signal amplification (fig. 2 left); the other way is that the antibody is directly coated on the magnetic beads (fig. 2 right), and both the processes need to amplify the signal, which is complicated and not beneficial to the mass production of the kit.
3. The detection time is longer. The main detection modes of the VEGF kit on the market at present are a one-step method and a two-step method. The two-step method is to combine the antigen to be detected with the coating antibody, wash and then add the labeled antibody for incubation, and wash the unbound labeled antibody thoroughly to develop color (fig. 3A). The one-step method refers to incubating the antigen to be detected, the coated antibody and the labeled antibody together, washing thoroughly, and then developing (fig. 3B). Compared with the two-step method, the total incubation time required by the one-step method is shorter, generally about 25 minutes, while the total incubation time required by the current one-step method is still longer, so that a disease control scheme cannot be provided more quickly in clinic.
Disclosure of Invention
The invention aims to: in order to solve the problems in the background art, the invention provides a raw material preparation method and a detection method of a vascular endothelial growth factor detection kit.
In order to achieve the purpose, the invention provides the following technical scheme: a raw material preparation method of a vascular endothelial growth factor detection kit comprises the following steps: the kit comprises an antibody coated magnetic bead working solution, a VEGF labeled antibody, a VEGF calibrator, a VEGF quality control product and a sample diluent; the preparation method comprises the steps of taking 200 mu l of magnetic bead mother liquor, adding 0.1M borate buffer solution and 200 mu g of recombinant VEGF antibody Fab segment after resuspension, oscillating and mixing uniformly, and taking the magnetic bead as a tosyl activated magnetic bead, wherein the working solution of the antibody coated magnetic bead is a magnetic microsphere suspension for coating a humanized VEGF antibody Fab segment, and the buffer system is TBS-T (20-200mM Tris, 0-1M NaCl, 0.0-1% BSA, 0.1-0.5% Tween-20, 0.1-1% Proclin-300, and pH is 6.5-9.0); the VEGF labeled antibody is characterized in that an Fab section of a humanized VEGF antibody is labeled through an acridine ester modifier, the label is acridine salt NSP-SA-NHS or other acridine ester derivatives, the preparation method comprises the steps of taking 1mg of the Fab section of the recombinant VEGF antibody, diluting the Fab section to 2mg/ml through CBS buffer solution, adding 5mM NSP-SA-NHS solution, stirring uniformly, reacting to obtain the label, adding 200 ul of 5% DL-lysine, desalting through Sephadex G-25, and using an elution buffer system TBS-T (20-200mM Tris, 0-1M NaCl, 0.0-1% BSA, 0.1-0.5% Tween-20, 0.1-1% Proclin-300 and pH 6.5-9.0); the VEGF calibrator and the VEGF quality control product are both prepared from sample diluents, the concentration range of the VEGF calibrator is 0.5-50000pg/mL, and the concentrations of the VEGF quality control product are 200pg/mL, 400pg/mL and 4000 pg/mL; the sample diluent is PBS buffer solution with pH 6.5-9.0, and each liter of the sample diluent also contains 5-20g BSA and 1-10ml Proclin-300;
a detection method of a vascular endothelial growth factor detection kit comprises the following steps: a full-automatic chemiluminescence immunoassay analyzer is used as a detection tool, a sample, an antibody coated magnetic bead working solution and a VEGF labeled antibody are sequentially added into a reaction cup, magnetic separation is carried out after incubation for 15 minutes at 33-40 ℃, diluted concentrated washing liquid is used for cleaning for a plurality of times, the reaction cup is sent into a dark room, a luminescence excitation liquid A and a luminescence excitation liquid B are added for luminescence reaction, and a luminescence value is recorded.
Compared with the prior art, the invention has the beneficial effects that:
1. the luminescent marker of choice in the present invention is the acridinium salt NSP-SA-NHS. Compared with acridinium ester AE-NHS, DMAE-NHS and ME-DMAE-NHS, the acridinium ester derivative introduces propanesulfonic acid modification, increases hydrophilicity, is favorable for enhancing the water solubility of an antibody, so that luminescence detection is carried out in homogeneous liquid and reaction time is shortened. Meanwhile, the bond level of C-N is larger than that of C-O, and the stability of the conjugate of the acridinium salt NSP-SA-NHS and the protein is higher. Based on the use of NSP-SA-NHS, the sensitivity, detection range and storage time of the detection system are improved, and the reaction time is shortened.
2. The present invention selects tosyl activated magnetic beads that can react with both thiol and amino groups of the ligand without the need for a condensing agent. The magnetic beads have higher protein binding efficiency, and the binding capacity of the antibodies and the magnetic beads is improved without crosslinking the antibodies. Due to the improvement of the basic signal value, the signal does not need to be amplified by virtue of fluorescein isothiocyanate + corresponding antibody or avidin + biotin conjugate. Based on the two points, the production process is successfully simplified, and the production efficiency is improved.
3. The invention selects humanized recombinant anti-VEGF antibody Fab segment. Compared with the traditional mouse monoclonal antibody, the recombinant antibody is suitable for mass production, and the affinity of the recombinant antibody with the antigen is enhanced through modification. In addition, the use of Fab fragments of recombinant antibodies can effectively avoid non-specific binding of Fc fragments. The optimization helps to enhance the sensitivity of the product, reduce the reaction time and ensure the stability of the product.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a diagram illustrating sensitivity comparison of related products;
FIG. 2 is a schematic diagram of the main process route of the present VEGF kit;
FIG. 3 is a schematic diagram of the main detection mode of the present VEGF kit;
FIG. 4 is a schematic view of a preparation process of the present invention;
FIG. 5 kit regression analysis.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Referring to fig. 4, the raw material preparation and detection method of the vascular endothelial growth factor detection kit according to the present embodiment includes:
(1) preparing raw materials, wherein the raw material preparation comprises an antibody coated magnetic bead working solution, a VEGF labeled antibody, a VEGF calibrator, a VEGF quality control product and a sample diluent;
the preparation method comprises the steps of taking 200 mu l of mother solution of the magnetic beads, adding 0.1M borate buffer solution and 200 mu g of recombinant VEGF antibody Fab segment after resuspension, oscillating and mixing uniformly, wherein the magnetic beads are tosyl activated magnetic beads which can react with sulfydryl and amino of a ligand simultaneously and do not need a condensing agent. The magnetic beads have higher protein binding efficiency, and the binding capacity of the antibodies and the magnetic beads is improved without crosslinking the antibodies. Due to the improvement of the basic signal value, the signal does not need to be amplified by virtue of fluorescein isothiocyanate + corresponding antibody or avidin + biotin conjugate. Based on the two points, the production process is successfully simplified, and the production efficiency is improved;
the VEGF labeled antibody is characterized in that an Fab segment of a humanized VEGF antibody is labeled through an acridinium ester modifier, the marker is acridinium salt NSP-SA-NHS or other acridinium ester derivatives, and compared with acridinium ester AE-NHS, DMAE-NHS and ME-DMAE-NHS, the acridinium ester derivatives introduce propanesulfonic acid modification, so that the hydrophilicity is increased, the water solubility of the antibody is enhanced, the luminescence detection is carried out in homogeneous liquid, and the reaction time is shortened. Meanwhile, the bond level of C-N is larger than that of C-O, and the stability of the conjugate of the acridinium salt NSP-SA-NHS and the protein is higher. Based on the use of NSP-SA-NHS, the sensitivity, detection range and storage time of a detection system are improved, the reaction time is shortened, and meanwhile, compared with the traditional mouse monoclonal antibody, the humanized recombinant anti-VEGF antibody Fab segment is suitable for mass production, and the affinity of the humanized recombinant anti-VEGF antibody Fab segment with the antigen is enhanced through modification. In addition, the use of Fab fragments of recombinant antibodies can effectively avoid non-specific binding of Fc fragments. The optimization helps to enhance the sensitivity of the product, reduce the reaction time and ensure the stability of the product. The preparation method comprises the steps of taking 1mg of recombinant VEGF antibody Fab segment, diluting the recombinant VEGF antibody Fab segment to 2mg/ml by using CBS buffer solution, adding 5mM NSP-SA-NHS solution, stirring uniformly, and reacting to obtain the marker. Desalting with Sephadex G-25 after adding 200 μ l of 5% DL-lysine, and eluting with TBS-T (20-200mM Tris, 0-1M NaCl, 0.0% -1% BSA, 0.1% -0.5% Tween-20, 0.1% -1% Proclin-300, pH 6.5-9.0);
the VEGF calibrator and the VEGF quality control product are both prepared by sample diluent, and the concentration range of the VEGF calibrator is 0.5-50000 pg/mL; the concentration of the VEGF quality control product is 200pg/ml, 400pg/ml and 4000pg/ml, and specifically, the vascular endothelial growth factor antigen is configured into corresponding concentration by TBS-T buffer solution (20-200mM Tris, 0-1M NaCl, 0.0-1% BSA, 0.1-0.5% Tween-20, 0.1-1% Proclin-300, pH 6.5-9.0);
the sample diluent is PBS buffer solution with pH 6.5-9.0, and each liter of the sample diluent also contains 5-20g BSA and 1-10ml Proclin-300;
and then, respectively subpackaging the antibody-coated magnetic bead working solution, the VEGF-labeled antibody, the VEGF calibrator, the VEGF quality control product and the sample diluent.
(2) And the detection method comprises the following steps:
a full-automatic chemiluminescence immunoassay analyzer is used as a detection tool, and a sample, an antibody coated magnetic bead working solution and a VEGF labeled antibody are sequentially added into a reaction cup. Incubating at 33-40 deg.C for 15 min, magnetically separating, washing with diluted concentrated washing solution for several times, placing the reaction cup in dark room, adding luminescence excitation liquid A and B for luminescence reaction, and recording luminescence value.
Example 2
The main product performance indexes of the vascular endothelial growth factor detection kit are as follows:
(1) accuracy test of kit
And respectively preparing the VEGF international standard into 200pg/mL solutions, repeatedly detecting for 3 times, and calculating the relative deviation. Through verification, the detection result of the accuracy index of the kit is shown in table 1:
TABLE 1 determination of accuracy of kit
And (4) conclusion: the relative deviation of the measurement results is within the range of +/-10%, and the results of 3 times meet the requirements, and the accuracy of the above 3 batches of kits is qualified.
(2) Precision test of the kit
The method is carried out according to the scheme of American clinical laboratory standards Committee (NCCLS/CLSI) document EP5-A2, the batch precision and the laboratory precision are tested by adopting a multi-factor integrated nested design, different operators, different equipment and different places are tested once every day in the morning and afternoon, each sample is tested for 2 times repeatedly and continuously for 20 days, each equipment collects 80 data results, and the batch precision and the laboratory precision are calculated. Through verification, the detection result of the precision index of the kit is shown in table 2:
TABLE 2 determination of precision of kit
And (4) conclusion: different operators, different equipment and different places repeatedly detect the low-concentration sample and the high-concentration sample by the kit, the precision CV between batches and the precision CV between laboratories are less than 15 percent, and the kit meets the precision performance evaluation requirement.
(3) Linear interval of the kit
1 sample of blood vessel endothelium growth factor high concentration serum is selected, diluted by a diluent to 13 concentration levels, each diluted sample is tested repeatedly for 3 times, and the average value of the samples is calculated. And calculating a corresponding linear relation according to the concentration points which are gradually reduced according to the result, and determining the widest linear range of the kit. Through verification, the detection results of the precision indexes of the kit are shown in table 3 and figure 1:
TABLE 3 kit Linear Interval assay
And (4) conclusion: evaluating a linear interval by using the kit, wherein the correlation coefficient r is not lower than 0.9900 and has no outlier in the range of [ 0.5-50000 ] pg/mL; polynomial regression analysis is adopted for the linear interval, the Sig. in b2 and b3 in a quadratic equation and a cubic equation is more than 0.05, and the linear interval has no significant difference from zero, so that the data set has linearity. Therefore, the linear range of the kit is [ 0.5-50000 ] pg/mL.
(4) Sensitivity test of kit
And (5) detecting the blank sample by using the kit for 20 times, and determining the sensitivity. The detection results of the sensitivity indexes of the reagent are shown in the table 4 through verification:
and (4) conclusion: the average luminescence value of the blank sample is determined to be 364.70
The values were brought into the reaction curve to obtain a sensitivity of 0.5pg/mL, which met the requirements.
(5) Comparison of methodologies
40 samples assigned by a Jianping Venus vascular endothelial growth factor assay kit (enzyme-linked immunosorbent assay) are detected. Regression analysis data shows that y is 0.9945x +0.0050, r is more than 0.975, and p is less than 0.05, which proves that the product has good consistency with the same products on the market.
(6) Clinical diagnostic assay
The kit is adopted to detect clinical detection samples so as to judge whether the kit can be used for clinical auxiliary diagnosis.
a) Analysis of cervical cancer, gynecological benign tumor, cervicitis and normal female serum VEGF level
The serum of cervical cancer, gynecological benign tumor, cervicitis and normal female is selected for VEGF level analysis. The test results are shown in table 5:
TABLE 5 determination of cervical carcinoma, gynecological benign tumors, cervicitis and Normal female serum VEGF levels
And (4) conclusion: the positive rate of VEGF in the serum of a cervical cancer patient is obviously higher than that of benign tumor, cervicitis and normal female serum of gynecology, and the product can be used for clinical auxiliary diagnosis of cancer.
b) Analysis of serum VEGF levels in Breast cancer, benign lesions, Normal women
The serum of breast cancer, benign lesions, normal women was selected for VEGF level analysis. The test results are shown in table 6:
TABLE 6 Breast cancer, benign lesions, Normal female serum VEGF level determination
And (4) conclusion: the positive rate of the serum VEGF of the breast cancer patient is obviously higher than that of benign lesion and normal female serum, and the product can be used for clinical auxiliary diagnosis of cancer.
Meanwhile, in addition to the above embodiments, the following schemes may be adopted to achieve the effects of the present invention:
1) changing the luminous marker into acridinium ester and other acridinium ester derivatives;
2) changing the magnetic beads into magnetic beads or microbeads with other sizes or carrying other functional groups;
3) other recombinant anti-VEGF Fab fragments (either of prokaryotic or eukaryotic origin) were used to achieve similar results;
4) modifications to the above reagent concentrations and pH and other components;
5) modifying the concentration gradient of the quality control substance;
6) changing the light-emitting system into other light-emitting systems (such as alkaline phosphatase or horseradish peroxidase);
7) changing a tubular chemiluminescence platform into an electrochemical luminescence platform, a plate-type chemiluminescence platform, an enzyme-linked immunosorbent assay platform or a radionuclide-labeled immunization platform;
8) a full-automatic chemical luminescence platform is changed into a semi-automatic or manual chemical luminescence platform;
9) changing the one-step method into a two-step method for detection;
it is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (2)
1. A raw material preparation method of a vascular endothelial growth factor detection kit is characterized by comprising the following steps: the kit comprises an antibody coated magnetic bead working solution, a VEGF labeled antibody, a VEGF calibrator, a VEGF quality control product and a sample diluent;
the preparation method comprises the steps of taking 200 mu l of magnetic bead mother liquor, adding 0.1M borate buffer solution and 200 mu g of recombinant VEGF antibody Fab segment after resuspension, oscillating and mixing uniformly, and taking the magnetic bead as a tosyl activated magnetic bead, wherein the working solution of the antibody coated magnetic bead is a magnetic microsphere suspension for coating a humanized VEGF antibody Fab segment, and the buffer system is TBS-T (20-200mM Tris, 0-1M NaCl, 0.0-1% BSA, 0.1-0.5% Tween-20, 0.1-1% Proclin-300, and pH is 6.5-9.0);
the VEGF labeled antibody is characterized in that an Fab section of a humanized VEGF antibody is labeled through an acridine ester modifier, the label is acridine salt NSP-SA-NHS or other acridine ester derivatives, the preparation method comprises the steps of taking 1mg of the Fab section of the recombinant VEGF antibody, diluting the Fab section to 2mg/ml through CBS buffer solution, adding 5mM NSP-SA-NHS solution, stirring uniformly, reacting to obtain the label, adding 200 ul of 5% DL-lysine, desalting through Sephadex G-25, and using an elution buffer system TBS-T (20-200mM Tris, 0-1M NaCl, 0.0-1% BSA, 0.1-0.5% Tween-20, 0.1-1% Proclin-300 and pH 6.5-9.0);
the VEGF calibrator and the VEGF quality control product are both prepared from sample diluents, the concentration range of the VEGF calibrator is 0.5-50000pg/mL, and the concentrations of the VEGF quality control product are 200pg/mL, 400pg/mL and 4000 pg/mL;
the sample diluent is PBS buffer solution with pH of 6.5-9.0, and each liter of the sample diluent also contains 5-20g BSA and 1-10ml Proclin-300.
2. The method for detecting the VEGF-EGF detection kit of claim 1, wherein the method comprises: a full-automatic chemiluminescence immunoassay analyzer is used as a detection tool, a sample, an antibody coated magnetic bead working solution and a VEGF labeled antibody are sequentially added into a reaction cup, magnetic separation is carried out after incubation for 15 minutes at 33-40 ℃, diluted concentrated washing liquid is used for cleaning for a plurality of times, the reaction cup is sent into a dark room, a luminescence excitation liquid A and a luminescence excitation liquid B are added for luminescence reaction, and a luminescence value is recorded.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110722649.4A CN113702642A (en) | 2021-06-29 | 2021-06-29 | Raw material preparation and detection method of vascular endothelial growth factor detection kit |
| CN202111534659.1A CN114019174B (en) | 2021-06-29 | 2021-12-15 | Kit and application thereof in vascular endothelial growth factor detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110722649.4A CN113702642A (en) | 2021-06-29 | 2021-06-29 | Raw material preparation and detection method of vascular endothelial growth factor detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113702642A true CN113702642A (en) | 2021-11-26 |
Family
ID=78648251
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110722649.4A Pending CN113702642A (en) | 2021-06-29 | 2021-06-29 | Raw material preparation and detection method of vascular endothelial growth factor detection kit |
| CN202111534659.1A Active CN114019174B (en) | 2021-06-29 | 2021-12-15 | Kit and application thereof in vascular endothelial growth factor detection |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111534659.1A Active CN114019174B (en) | 2021-06-29 | 2021-12-15 | Kit and application thereof in vascular endothelial growth factor detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN113702642A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN116773813A (en) * | 2023-06-20 | 2023-09-19 | 北京健平金星医疗器械有限公司 | Preparation and application of VEGFR1 detection kit |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014083666A1 (en) * | 2012-11-29 | 2014-06-05 | ミライアル株式会社 | Method for measuring antigen-antibody reaction by sandwiching, and micro-channel chip |
| JP2019523404A (en) * | 2016-07-15 | 2019-08-22 | エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト | Method and means for detecting the level of total VEGF-A |
| CN106483293A (en) * | 2016-10-13 | 2017-03-08 | 广州华弘生物科技有限公司 | He4a clinical immunization detection kit and preparation method thereof |
| US20180149653A1 (en) * | 2016-11-29 | 2018-05-31 | Trustees Of Tufts College | Compositions and methods for diagnosing breast cancer |
| 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 |
| CN108037287A (en) * | 2017-12-22 | 2018-05-15 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of neutrophil elastase and preparation method thereof |
| CN112014566A (en) * | 2019-05-28 | 2020-12-01 | 上海奥普生物医药股份有限公司 | Amino-terminal brain natriuretic peptide precursor detection kit, preparation method and application |
| CN110568176A (en) * | 2019-09-09 | 2019-12-13 | 潍坊市康华生物技术有限公司 | method for directional coupling of antibody and magnetic bead |
| CN111044718B (en) * | 2019-12-20 | 2022-05-17 | 北京指真生物科技有限公司 | Diluent for whole blood immune luminescence analysis and analysis method thereof |
| CN111175516A (en) * | 2020-01-06 | 2020-05-19 | 潍坊市康华生物技术有限公司 | Vascular endothelial growth factor detection kit and preparation method thereof |
| CN113702642A (en) * | 2021-06-29 | 2021-11-26 | 北京健平金星生物科技有限公司 | Raw material preparation and detection method of vascular endothelial growth factor detection kit |
-
2021
- 2021-06-29 CN CN202110722649.4A patent/CN113702642A/en active Pending
- 2021-12-15 CN CN202111534659.1A patent/CN114019174B/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN116773813A (en) * | 2023-06-20 | 2023-09-19 | 北京健平金星医疗器械有限公司 | Preparation and application of VEGFR1 detection kit |
| CN116773813B (en) * | 2023-06-20 | 2024-01-30 | 北京健平金星生物医药有限公司 | Preparation and application of VEGFR1 detection kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114019174A (en) | 2022-02-08 |
| CN114019174B (en) | 2022-05-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DK160108C (en) | Method and equipment for direct or indirect detection of reaction between a specific binding agent and the corresponding acceptor substance | |
| CN114019174B (en) | Kit and application thereof in vascular endothelial growth factor detection | |
| US10830771B2 (en) | PIVKA-II assay method and method for manufacturing reagent or kit for PIVKA-II immunoassay | |
| CN107543932A (en) | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of calcitonin | |
| CN111781385A (en) | NT-proBNP detection kit and preparation method thereof | |
| CN112014577B (en) | Kit for improving GPC3 detection sensitivity and preparation method thereof | |
| CN111175494A (en) | Thyroglobulin antibody detection kit and use method thereof | |
| CN111289758B (en) | Kit for quantitative detection of H-FABP and method for quantitative detection of H-FABP | |
| CN113433318A (en) | Kit for detecting alpha-fetoprotein heteroplasmon AFP-L3 content and detection method and application thereof | |
| CN110988348B (en) | Free prostate specific antigen detection kit and preparation method thereof | |
| CN111007265A (en) | Simoa kit of preeclampsia risk prediction marker PIGF and application thereof | |
| CN111579781A (en) | Hepatitis C virus antibody detection kit, preparation method and detection method | |
| CN112166323A (en) | Direct immunoassay measurement of autoantibodies | |
| CN113933521A (en) | Magnetic particle chemiluminescence detection kit and preparation method and application thereof | |
| CN113917142A (en) | Kit for chemiluminescence immunoassay of tyrosine phosphatase autoantibody magnetic particles, preparation method and detection method | |
| JP6578119B2 (en) | Prostate-specific antigen measurement method and measurement kit | |
| US5436132A (en) | Quantitative determination of tenascin as glioma marker | |
| CN112834739A (en) | Kit for determining amino-terminal brain natriuretic peptide precursor in human blood by magnetic particle chemiluminescence method | |
| CN116718777B (en) | Preparation and application of PLGF detection kit | |
| JP3361323B2 (en) | Stabilization of calibrator containing cytokeratin | |
| WO2023163176A1 (en) | Reagent for use in detection or measurement of serine protease | |
| WO2023013725A1 (en) | Immunoassay for thryoglobulin and kit therefor | |
| CN115902206A (en) | Composition for alpha fetoprotein heterogenous body determination and application thereof | |
| CN113985036A (en) | Angiotensin I detection kit and preparation method thereof | |
| CN116125060A (en) | Chemiluminescence immune quantitative detection kit for colloid fiber acidic protein magnetic particles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20211126 |