CN116773813A - Preparation and application of VEGFR1 detection kit - Google Patents
Preparation and application of VEGFR1 detection kit Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—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
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
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Abstract
The invention discloses preparation and application of a VEGFR1 detection kit, and particularly relates to the field of biological detection. The kit comprises antibody coated magnetic bead working solution, VEGFR1 labeled antibody, VEGFR1 calibrator, VEGFR1 quality control product and sample diluent. The kit selects the acridine salt NSP-SA-NHS modifier to be coupled with the humanized anti-VEGFR 1 antibody Fab segment, so that the stability is high, the sensitivity, the detection range and the preservation time of a detection system are improved, and the reaction time is shortened; the linear range of the kit reaches 2.75-90000 pg/mL.
Description
Technical Field
The invention relates to the field of biological detection, in particular to preparation and application of a VEGFR1 detection kit.
Background
VEGFR-1 is a transmembrane protein, and is found by Shibiuya et al in 1990, VEGFR-1 is also called FLT, and VEGFR-1 is composed of 1338 amino acids and is divided into an extracellular region, a transmembrane region and an intracellular region. Wherein the extracellular domain comprises a 7 immunoglobulin-like domain, the transmembrane domain consists of 22 amino acids and the intracellular domain consists of 558 amino acids, wherein the intracellular domain comprises a tyrosine kinase domain bearing a tyrosine autophosphorylation site, which upon binding of VEGFR-1 to its ligand induces dimerization of the receptor, autophosphorylation of the intracellular phosphorylation site, resulting in a downstream series of cascade reactions.
sFLT-1 (soluble vascular endothelial growth factor receptor 1) is a splice of mRNAVEGFR-1, only 6 extracellular immunoglobulin-like domains and 31 amino acid residues at the tail end, and no transmembrane domain or intracellular domain, sFLT-1 (soluble vascular endothelial growth factor receptor 1) can be detected in peripheral blood and thus can be applied to hematological studies.
VEGFR-1 as a cell surface receptor for VEGFA, VEGFB and VEGFR1 plays a vital role in embryonic vasculature development, angiogenesis regulation, cell survival, cell migration, macrophage function and cancer cell invasion. VEGFR-1 has very high affinity with VEGFA and relatively low protein kinase activity; can act as a negative regulator of VEGFA signaling by limiting the amount of free VEGFA and preventing it from binding to VEGFR (KDR). VEGFR-1 is capable of interacting with its ligand, activating receptor tyrosine kinase activity, and thus, downstream signaling pathways. These signaling pathways include primarily the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI 3K) pathway. Therefore, the development of the kit for detecting the VEGFR-1 in human blood is important, which is beneficial to assisting in diagnosing the growth condition of early malignant tumors, realizing early detection and early treatment and improving the survival rate of patients. Therefore, the preparation of a reliable kit for detecting VEGFR-1 in human blood is not slow.
Disclosure of Invention
Therefore, the invention provides preparation and application of a VEGFR1 detection kit, which are used for solving the problems of complex detection means, long detection time and the like of the existing detection of malignant tumors.
In order to achieve the above object, the present invention provides the following technical solutions:
according to the VEGFR1 detection kit provided by the aspect of the invention, the kit comprises an antibody coated magnetic bead working solution, a VEGFR1 labeled antibody, a VEGFR1 calibrator, a VEGFR1 quality control product and a sample diluent.
Further, the coated magnetic bead working solution is a magnetic bead suspension coated with the antibody A;
the VEGFR1 labeled antibody is obtained by labeling antibody B by NSP-SA-NHS solution;
the antibody A and the antibody B are VEGFR1-Fab1 and VEGFR1-Fab2 respectively, and are composed of a light chain and a heavy chain; the light chain of VEGFR1-Fab1 is shown as SEQ ID NO.1, and the heavy chain is shown as SEQ ID NO. 2; the light chain of VEGFR1-Fab2 is shown as SEQ ID NO.3, and the heavy chain is shown as SEQ ID NO. 4.
Further, the mass ratio of the magnetic beads to the antibody A is 50:1-150:1; the magnetic beads in the antibody coated magnetic bead working solution are magnetic beads activated by tosyl; the amount of the substances of the antibody B and NSP-SA-NHS is 1:5-1:20.
Further, the method for coating the magnetic bead suspension of the antibody A comprises the steps of taking a magnetic bead mother solution, adding borate buffer solution and the antibody A after re-suspension, and shaking and mixing uniformly; then adding buffer solution TBS-T for dilution.
Further, the preparation method of the VEGFR1 labeled antibody comprises the steps of diluting an antibody B with a CBS buffer solution, adding an NSP-SA-NHS solution, uniformly stirring, reacting to obtain a label, adding DL-lysine, desalting with a Sephadex G-25 buffer solution, and eluting with a buffer system of TBS-T.
Further, the VEGFR1 calibrator and the VEGFR1 quality control product are prepared from sample diluent, and the concentration range of the VEGFR1 calibrator is 1.22-10000pg/mL; the concentration of VEGFR1 quality control was 20pg/mL and 2500pg/mL.
Further, the sample diluent is PBS buffer with pH of 6.5-9.0.
Further, the sample diluent also contains BSA and a preservative.
According to another aspect of the invention, the application of the VEGFR1 detection kit in preparing auxiliary diagnosis early tumor products is provided.
The invention has the following advantages:
1. the kit selects the acridine salt NSP-SA-NHS modifier to be coupled with the humanized anti-VEGFR 1 antibody Fab segment, so that the stability is high, the sensitivity, the detection range and the preservation time of a detection system are improved, and the reaction time is shortened; the linear range of the kit reaches 2.75-90000 pg/mL.
2. The preferred tosyl activated magnetic beads of the kit of the invention can react with both the thiol and amino groups of the ligand without the need for condensing agents. The magnetic beads have higher binding efficiency to proteins, and the antibodies do not need to be crosslinked to improve the binding capacity of the antibodies to the magnetic beads. Because of the improvement of the basic signal value, the signal is not amplified by the aid of fluorescein isothiocyanate plus corresponding antibody or avidin plus biotin linker. Based on the two points, the production process is successfully simplified, and the production efficiency is improved.
3. The invention establishes a system for detecting VEGFR-1 in human blood by screening antibody pairs with clinical application value and strong reactivity and performing series optimization on a detected reaction system such as coating concentration and the like, and prepares a commercially available detection kit. Compared with the existing kit, the detection kit has the advantages of short time consumption, high sensitivity, wide linear range, good precision, strong anti-interference performance and the like, can accurately reflect the content of VEGFR-1 in a sample, and has great clinical application prospects in the aspects of auxiliary diagnosis of early malignant tumor growth and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the ambit of the technical disclosure.
FIG. 1 is a graph showing the result of gel electrophoresis identification according to example 1 of the present invention;
wherein M-maeker;1-pET-28a (+) empty vector; 2-pET-28a (+) -VEGFR-1;
3-plasmid restriction map;
FIG. 2 is a graph showing the results of the electrophoresis analysis of the 37℃inducer protein polyacrylamide gel provided in example 1 of the present invention; wherein, M-marker;1-LB did not induce whole bacteria; 2-SB did not induce whole bacteria; 3-LB disruption supernatant; 4-LB crushing and sinking; crushing the supernatant by 5-SB; and 6-SB is crushed and precipitated.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Recombinant antigen and monoclonal antibody preparation
Vegfr1 prokaryotic expression:
the sequence of VEGFR-1 (P17948) in UniPort is referred to, and the signal peptide, extracellular region, transmembrane region and intracellular region of the target gene are predicted by using prediction software to know the binding region of the target gene and the ligand, so that the fragment expressed recombinantly is determined to be VEGFR-1D1-3. The target sequence is subjected to codon optimization by online codon optimization software Neopr organism (NovoPro), the obtained gene sequence is transformed into codons favored by escherichia coli to obtain a new gene for encoding vascular endothelial growth factor receptor-1 (VEGFR-1), restriction endonucleases Ndel and Xhol are respectively added into an upstream primer and a downstream primer, the N end is fused with a thrombin site and a 6xHis tag on a carrier for expression, and the C end contains a stop codon. And (3) designing a target gene and then carrying out total gene synthesis. And (3) constructing a vector and verifying: hpal and Xhol are added into the constructed plasmid to carry out double enzyme digestion, 1.5% agarose gel electrophoresis is used for identifying the result, the result is shown in figure 1, the band can be seen at about 500bp, and the successful verification is consistent with the expected size, which indicates that the target gene is successfully inserted into the constructed plasmid. The recombinant plasmid was transformed into DE3 E.coli expression strain and the strain was preserved by glycerol after culturing at 37℃for 12 hours.
2. Preparation of E.coli expressed VEGFR1 antigen
VEGFR1 induction expression:
the expression strain containing the recombinant plasmid is inoculated into LB culture medium to be cultured at different temperatures, the bacteria mud is collected to be ultrasonically crushed after the induction of different time by using IPTG to obtain a crushed supernatant, 15 percent of 10 percent polyacrylamide gel electrophoresis analysis results are shown in figure 2, and the induction and expression results of VEGFR1 are carried out at 37 ℃,4 ℃ and 16 ℃ to find that the target protein is in inclusion bodies but the target protein expression amount at 16 ℃ is more, so that the induction condition is finally determined to be 16 ℃ and the induction time is 48 hours.
3. Preparation of antibodies: mice were immunized with recombinant VEGFR1 antigen to obtain a pair of antibodies.
The method comprises the following steps: taking 50ug of recombinant antigen and an equal volume of Freund's complete adjuvant, fully emulsifying, and performing multipoint subcutaneous injection on a mice of proper age; taking 30 mug of antigen and an equal volume of Freund's incomplete adjuvant, fully mixing and emulsifying the antigen and the Freund's incomplete adjuvant, and performing subcutaneous multipoint injection on the mice after 2 weeks after the first immunization; taking 30 mug of antigen and an equal volume of Freund's incomplete adjuvant to fully emulsify after secondary immunization for 1 week, performing subcutaneous multipoint injection on the mice, and measuring the titer of blood sampling of tail veins of the mice after 7 days of injection; for mice with three immunizations titers combined, 30 μg of antigen was injected intraperitoneally as boost.
Mice meeting the requirements are sacrificed for lymphocytes. 50% PEG 4000 is used as a fusion agent, the fusion ratio of two cells of myeloma cells and lymphocytes is 1:3-1:5 under the water bath condition of 37 ℃, 50% PEG is added into spleen cells and myeloma cell clusters which are uniformly mixed and the supernatant is discarded in 1min, the mixture is kept still for 2min after shaking for 1min, and then 10ml of serum-free 1640 culture medium is added. The supernatant was centrifuged off and the cells were resuspended in 1640 medium supplemented with HAT. After fusion, the cells were placed into CO 2 Culturing for 7-9 days at 37 degrees in an incubator, and observing the fusion state. Screening was performed using an indirect ELISA plate beginning 10 days. The cell lines with strong positivity and high rarefaction number are selected from the initially screened positive clones, and 4 times of subcloning are carried out by using a limiting dilution method. Through the upper partThe screening process is to obtain two cell strains with high affinity and stable secretion of antibodies, and the corresponding antibody strains are respectively encoded into VEGFR1-Fab1 and VEGFR1-Fab 2.
Selecting BALB/c mice with normal growth and development for 6-8 weeks, wherein each mouse is firstly injected with 0.5ml of Freund incomplete adjuvant in an intraperitoneal mode; intraperitoneal injection was performed 2X 10 after 10 days 6 And a hybridoma cell. Ascites can be produced after the mice are inoculated with the hybridoma cells for 7-9 days, and the health condition and the ascites production of the mice are observed; mice were sacrificed when the ascites production was greater and the mice were less healthy, and the corresponding ascites was collected.
The collected mouse ascites was diluted with 3 volumes of 50mM acetic acid buffer (ph=4.0). Adding 3% n-octanoic acid to the mixed solution to precipitate ascites impurity. The mixture was centrifuged at 14500rpm at 4℃for 30min, and the supernatant was collected. The pH was adjusted to 7.4 with 1M sodium hydroxide solution. The solution was passed through an equilibrated protein a/G column, washed with phosphate solution, and eluted with 0.1M Gly-Hcl (ph=2.8), and the eluted product was pH-adjusted to 7.5 with 1M tris-Hcl (ph=9.0).
4. Humanized antibody preparation
This example designed two humanized VEGFR1 antibodies Fab, VEGFR1-Fab1 and VEGFR1-Fab2, respectively (present
In the examples, antibody A is VEGFR1-Fab1 and antibody B is VEGFR1-Fab2
Comprising the following steps:
(1) Immunizing mice with VEGFR1 recombinant antigen to obtain mouse monoclonal antibody with strong affinity to the antigen
Antibody variable region sequence sequencing was performed after cell line.
(2) Based on the obtained variable region sequence, an immune gene database IMGT (www.imgt.org) analysis was performed, and chimeric antibody design was performed with reference to a humanized antibody template. The optimized sequences were constructed to pcDNA3.1 (+) vector with HindIII and XhoI cleavage sites and transfected into HEK293T cell line, followed by chimeric antibody expression.
The VEGFR1-Fab1 light chain amino acid sequence is shown as a sequence 1 in a sequence table;
EIVLTQSPDFLSVTPKEKVTITCRASQSIGTTIHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSVEAEDAATYYCQQSNSWPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC；
the heavy chain amino acid sequence of the VEGFR1-Fab1 is shown as a sequence 2 in a sequence table;
QVQLQQSGGGLVKPSQSLSLTCTVSGFSLTSYGVHWVRQPPGKGLEWIGLIWSGGGTDYNPSLKSRLTISRDTSKNQVSFKISSLTAADTAVYYCARQLGLRAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGK；
the VEGFR1-Fab2 light chain amino acid sequence is shown in a sequence 3 in a sequence table:
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT；
the heavy chain amino acid sequence of the VEGFR1-Fab2 is shown as a sequence 4 in a sequence table:
QVQLQQSGGGLVKPSQSLSLTCTVSGFSLTSYGVHWVRQPPGKGLEWIGLIWSGGGTDYNPSLKSRLTISRDTSKNQVSFKISSLTAADTAVYYCARQLGLRAMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTI。
(3) Antibody purification was performed with Protein G packing. Protein concentration was measured according to BCA method. The concentration statistics are shown in Table 1.
TABLE 1
5. Antibody titer detection
The ELISA plates were coated with 100ng VEGFR1 recombinant antigen per well and incubated with a double dilution of VEGFR1Ab at 37℃for 1 hour. After the incubation, adding goat anti-human secondary antibody marked by HRP, finally adding TMB for color development, adding 2M sulfuric acid for stopping reaction, and placing the wavelength of an enzyme label at 450nM for detection. The detection results are shown in Table 2.
TABLE 2
| Valency of | 01 batch | 02 batch | Batch 03 |
| VEGFR1-1 | 400000 | 4000000 | 4000000 |
| VEGFR1-2 | 2000000 | 2000000 | 2000000 |
6. Preparation of magnetic particle detection reagent
The preparation method of the antibody coated magnetic bead working solution in the embodiment comprises the following steps: 200. Mu.l of magnetic bead mother liquor (Thermo Dynabeads TM M-280 Tosylated, product number 30110D, original concentration 100 mg/mL) was resuspended (gently blown with a pipette to suspend it sufficiently) and then mixed with 165. Mu.l of 0.1M borate buffer (BBS, pH 9.5) and 200. Mu.g of antibody A by shaking; buffer TBS-T (0.06M Tris-HCl buffer, 0.6M NaCl,0.05% BSA,0.5% Tween-20, 0.1% Proclin300, pH 7.5) was then added. Magnetic beads: the mass ratio of the antibody A is 100:1. The magnetic beads are tosyl activated magnetic beads.
The preparation method of the VEGFR1 labeled antibody in the embodiment comprises the following steps: 1mg of antibody B was diluted to 2mg/ml with CBS buffer, 16.5. Mu.L of 4mM NSP-SA-NHS solution was added, and after stirring well, the mixture was reacted to give a label, 200. Mu.L of 5% DL-lysine was added, and then desalted with Sephadex G-25, and the elution buffer system was TBS-T (0.06M Tris-HCl buffer, 0.6M NaCl,0.05% BSA,0.5% Tween-20,0.1%Proclin300,pH 7.5). The amount of antibody B and NSP-SA-NHS material in this example was 1:10.
The VEGFR1 calibrator and the VEGFR1 quality control product are prepared from sample diluent, and the concentration range of the VEGF calibrator is 1.22-10000pg/mL; the concentration of VEGFR1 quality control product is 20pg/ml, and 2500pg/ml, specifically, the vascular endothelial growth factor antigen is configured to the corresponding concentration by using a sample diluent buffer;
the sample diluent is PBS buffer with pH of 6.5-9.0, and each liter of the sample diluent also contains 5-20g BSA and 1-10ml Proclin-300.
Then, the antibody-coated magnetic bead working solution, the VEGFR1 labeled antibody, the VEGFR1 calibrator, the VEGFR1 quality control product and the sample diluent in the above steps are respectively packaged.
Example 2
The present embodiment is an application in VEGFR1 detection.
1. The specific detection method comprises the following steps:
1) The full-automatic chemiluminescence immunoassay analyzer is used as a detection tool. All samples, calibrators and quality controls were equilibrated to room temperature (18 ℃ -26 ℃) prior to testing.
2) And scanning a two-dimensional code of the reagent pack to automatically acquire parameters required by testing, and placing the reagent pack in a reagent position corresponding to the project after scanning the code.
3) And (3) performing instrument calibration by using a VEGFR1 calibrator, performing quality control by using a VEGFR1 quality control product, and performing target sample detection after determining that the measured concentration of the quality control product is in a quality control range.
4) The machine automatically and sequentially adds 20 mu L of sample dilution liquid with the volume of 4 times of sample to be detected into the reaction cup, fully mixes the sample dilution liquid, sequentially adds 100 mu L of magnetic bead working liquid and 100 mu LVEGFR1 labeled antibody, fully mixes the sample dilution liquid, and incubates the sample dilution liquid at 37 ℃ for 15min, and then carries out magnetic separation.
5) After magnetic separation, the supernatant was discarded, and 300. Mu.L of the diluted concentrated washing solution was added, and washing was repeated 3 times.
6) The reaction cup was sent to a darkroom, 100. Mu.L of luminescence excitation liquid A and 100. Mu.L of luminescence excitation liquid B were added to each to perform luminescence reaction, and luminescence values were recorded.
7) And automatically calculating the luminescence value measured according to the two-dimension code of the reagent pack to obtain the concentration value of the sample to be measured.
The main product performance indexes of the kit are as follows:
the Roche diagnostic test is assigned serum and repeated three times for detection, and the relative deviation is calculated. The verification shows that the detection result of the accuracy index of the kit is shown in Table 3.
TABLE 3 Table 3
| Detection result 1 | Detection result 2 | Detection result 3 | Mean value of | Reference concentration | Deviation of |
| 15.80 | 15.79 | 15.80 | 15.80 | 15.8 | -0.02％ |
| 53.17 | 53.17 | 53.17 | 53.17 | 53.2 | -0.05％ |
| 614.03 | 614.08 | 614.24 | 614.12 | 614 | 0.02％ |
| 38866.05 | 38880.35 | 38890.00 | 38878.80 | 38858 | 0.05％ |
| 70588.57 | 70490.05 | 70541.14 | 70539.92 | 70552 | -0.02％ |
Conclusion: the relative deviation of the measurement results is within +/-10%, and all the 3 results meet the requirements, and the accuracy of the above 3 batches of kits is equivalent to that of Roche products.
2. Precision test of kit
The experiment was performed according to the American clinical laboratory standards Committee (NCCLS/CLSI) document EP5-A2 protocol using a multi-factor integrated nested design for intra-batch precision and inter-laboratory precision, with different operators, different equipment, different sites, each test being repeated 2 times per sample in the morning and afternoon, and each equipment collecting 80 data results for 20 consecutive days, and the intra-batch precision and inter-laboratory precision being calculated. Proved by verification, the detection result of the precision index of the kit is shown as 4.
TABLE 4 Table 4
Conclusion: the kit repeatedly detects low-concentration samples and high-concentration samples by different operators, different equipment and different sites, and the precision between batches and the precision CV between laboratories are less than 15%, which indicates that the kit meets the precision performance evaluation requirement.
3. Linear interval of kit
1 VEGFR1 high concentration sample (basal serum supplemented with VEGFR1 recombinant antigen) was selected, 13 concentration levels were diluted with diluent, each diluted sample was tested 3 times repeatedly, and the mean value was calculated. And calculating a corresponding linear relation according to the concentration points of which the results gradually decrease, and determining the widest linear range of the kit. The test results of the precision index of the kit are shown in Table 5.
TABLE 5
Conclusion: evaluating the linear interval by the kit, wherein the correlation coefficient r is not lower than 0.9900 and has no outlier in the range of [ 2.75-90000 ] pg/mL; and (3) adopting polynomial regression analysis for the linear interval, wherein Sig in b2 and b3 in the quadratic equation and the cubic equation is more than 0.05, and no significant difference exists between the quadratic equation and zero, so that the data set has linearity. Therefore, the linear range of the kit is [ 2.75-90000 ] pg/mL.
4. Sensitivity test of kit
The kit is used for detecting blank samples, the detection is repeated for 20 times, and the sensitivity is determined. The test results of the sensitivity index of the reagent are shown in Table 6.
TABLE 6
Conclusion: the average luminescence value of the measured blank sample is 791.5, and the value is brought into a reaction curve, so that the sensitivity is 0.62pg/mL, and the sample meets the requirements.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
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Claims (9)
1. The kit is characterized by comprising antibody coated magnetic bead working solution, a VEGFR1 labeled antibody, a VEGFR1 calibrator, a VEGFR1 quality control product and a sample diluent.
2. The VEGFR1 detection kit according to claim 1, wherein the coated magnetic bead working fluid is a magnetic bead suspension coated with antibody a;
the VEGFR1 labeled antibody is obtained by labeling antibody B by NSP-SA-NHS solution;
the antibody A and the antibody B are VEGFR1-Fab1 and VEGFR1-Fab2 respectively, and are composed of a light chain and a heavy chain; the light chain of VEGFR1-Fab1 is shown as SEQ ID NO.1, and the heavy chain is shown as SEQ ID NO. 2; the light chain of VEGFR1-Fab2 is shown as SEQ ID NO.3, and the heavy chain is shown as SEQ ID NO. 4.
3. The VEGFR1 detection kit according to claim 2, wherein the mass ratio of magnetic beads to antibody a is 50:1-150:1; the magnetic beads in the antibody coated magnetic bead working solution are magnetic beads activated by tosyl; the amount of the substances of the antibody B and NSP-SA-NHS is 1:5-1:20.
4. The VEGFR1 detection kit as claimed in claim 2, wherein the method for coating the magnetic bead suspension of the antibody a is to take magnetic bead mother liquor, add borate buffer solution after re-suspension and mix the magnetic bead mother liquor with the antibody a by shaking; then adding buffer solution TBS-T for dilution.
5. The kit for detecting VEGFR1 according to claim 2, wherein the preparation method of the VEGFR1 labeled antibody is characterized in that after antibody B is diluted by CBS buffer solution, NSP-SA-NHS solution is added, after uniform stirring, the label is obtained through reaction, after DL-lysine is added, sephadexG-25 is used for desalting, and an elution buffer system is TBS-T buffer solution.
6. The VEGFR1 detection kit of claim 1, wherein the VEGFR1 calibrator and the VEGFR1 quality control are both formulated from a sample diluent, and the concentration range of the VEGFR1 calibrator is 1.22-10000pg/mL; the concentration of VEGFR1 quality control was 20pg/mL and 2500pg/mL.
7. The VEGFR1 detection kit as claimed in claim 1, wherein the sample diluent is PBS buffer at ph 6.5-9.0.
8. The VEGFR1 detection kit as claimed in claim 7, wherein the sample diluent further comprises BSA and a preservative.
9. Application of VEGFR1 detection kit in preparing auxiliary diagnosis early tumor products.
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Address after: Room 201-1, floor 2, building 2, yard 5, Yongfeng Road, Haidian District, Beijing 100094 Applicant after: Beijing Jianping Jinxing Biopharmaceutical Co.,Ltd. Address before: Room 201-1, floor 2, building 2, yard 5, Yongfeng Road, Haidian District, Beijing 100094 Applicant before: Beijing Jianping Jinxing Medical Instrument Co.,Ltd. |
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