CN111122848A - ELISA kit for screening HGFR (human chorionic gonadotropin receptor) targeted antibody and application - Google Patents
ELISA kit for screening HGFR (human chorionic gonadotropin receptor) targeted antibody and application Download PDFInfo
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Abstract
The invention discloses an ELISA kit for screening HGFR targeted antibodies, which is prepared by coating an ELISA plate with a recombinant human HGFR-ECD-Fc as an antigen. The ELISA kit takes recombinant human HGFR ectodomain Fc tag fusion protein (HGFR-ECD-Fc) as an antigen solid phase to be adsorbed on an ELISA plate; capturing an antibody capable of binding to the antigen through immobilized HGFR-ECD-Fc; after washing off the free components, the amount of the antibody was determined by an enzyme-labeled secondary antibody that specifically recognizes the captured antibody. The ELISA kit can be used for detecting HGFR targeted antibodies, has the advantages of strong specificity, high sensitivity and the like, can detect the antibody with the dilution degree of more than 700000 times, and has good application prospect.
Description
(I) technical field
The invention belongs to the field of detection, and mainly relates to an enzyme linked immunosorbent assay kit for screening an antibody of a targeted liver proliferation factor receptor, and a preparation method and application thereof.
(II) background of the invention
Hepatocyte Growth Factor Receptor (HGFR), a cell surface-localized single-transmembrane Receptor tyrosine kinase encoded by the proto-oncogene c-met. The only ligand for HGFR known so far is the liver proliferation Factor (HGF), which activates this signaling pathway by binding and inducing HGFR dimerization, thereby performing its function. Activation of the HGFR signaling pathway promotes cell proliferation, enhances cell migration ability, induces morphogenesis in epidermal cells, and inhibits apoptosis, and thus the signaling pathway is thought to be involved in tissue repair and regeneration. However, over-activation of the HGFR signaling pathway will stimulate cell overgrowth, thereby inducing the development of cancer; the enhancement of the cell migration ability will lead to metastatic spread of cancer cells; the activity of inhibiting apoptosis is closely related to cancer resistance.
Multiple studies have shown that over-activation of HGFR is closely linked to the development and progression of various cancers. The results of a study conducted on 38028 cancer patients showed that in 221 of these patients, HGFR was overactivated. In vitro studies also showed that cancer cells with overactivated HGFR proliferate faster and are more metastatic. Studies by scholars have found that HGFR overactivation is one of the major causes of glioblastoma. Epidermal growth factor receptor inhibitors are currently widely used in the treatment of various cancers caused by their over-activation, however, most patients develop drug resistance as their use lasts longer. The results of several studies on patients resistant to epidermal growth factor inhibitors indicate that compensatory cell proliferation signal activation by over-activation of HGFR is the main cause of drug resistance of patients to epidermal growth factor receptor inhibitors. Indirect clinical evidence suggests that inhibition of HGFR may have a therapeutic effect on cancer, such as Cabozantinib from BMS/Exelixis, which inhibits both VEGFR2 and HGFR, and was approved by the FDA for the treatment of thyroid cancer in 2012; pfeiffer's Crizotinib was approved by the FDA in 2011 for the treatment of non-small cell lung cancer, and this inhibitor inhibits both ALK and HGFR, with the anticancer effect believed to be associated with the inhibition of HGFR as well. In addition, excessive activation of HGFR has also been found in many patients resistant to Tyrosine Kinase Inhibitors (TKI), and it has been found that the combined use of HGFR inhibitors can eliminate the drug resistance effect. These evidences all indicate that inhibition of over-activation of the HGFR signaling pathway may have an effect in the treatment of cancer, and thus HGF and HGFR are candidate targets for anti-cancer drug development.
The development of selective inhibitors against HGF and HGFR is ongoing, but mainly based on small molecule compounds inhibiting tyrosine kinase activity that competitively bind to the ATP site. However, more inhibitors, such as PHA665752, SU11274, etc., are stopped before entering clinical trials due to strong cytotoxicity. A few inhibitors are currently in clinical trials, such as Capmatinib (INCB28060), SGX523 and the like, but currently there are no selective HGFR small molecule inhibitors available for clinical treatment worldwide. The emergence of monoclonal antibodies led to a second wave of biopharmaceutical products. Among antibody drugs, the anti-tumor monoclonal antibody drug is the heterophly protuberance and monopolizes half of the market. The monoclonal antibody medicine plays an important role in treating tumors immeasurably due to the advantages of high specificity, strong targeting property and low toxic and side effect. By the end of 2017, up to 77 FDA-approved monoclonal antibody drugs, such as Trastuzumab targeted to Her2 developed by Roche, are widely used for clinical treatment of breast cancer, gastric cancer, and esophageal cancer.
Because no anti-tumor monoclonal antibody medicine for targeting HGFR in clinic exists in the market at present, the anti-tumor monoclonal antibody for screening the antibody targeting HGFR and selecting the anti-tumor monoclonal antibody capable of selectively inhibiting HGF/HGFR signal channel has a great application prospect, and the ELISA kit for screening the HGFR targeting antibody provides a simple, convenient and efficient detection means for antibody preparation.
Disclosure of the invention
The ELISA kit prepared by the method is high in efficiency, simple and convenient, high in sensitivity and strong in specificity, and is suitable for detection of a large number of samples.
The technical scheme adopted by the invention is as follows:
the invention provides an ELISA kit for screening HGFR targeted antibodies, which is prepared by coating an ELISA plate with a recombinant human HGFR-ECD-Fc as an antigen.
Further, the kit is prepared according to the following method: (1) diluting the recombinant human HGFR-ECD-Fc into 0.1 mu g/mL antigen working solution by using coating buffer solution(ii) a Adding 100 mu L of antigen working solution into each hole of an enzyme label plate, incubating for 1 hour at 37 ℃, transferring to 4 ℃ for incubation for 24 hours, discarding the antigen working solution, adding 200 mu L of PBS into each hole, washing for 3 times, and then patting dry; the coating buffer consists of: 15mM Na2CO3，35mM NaHCO3The solvent is water, and the pH value is 9.6; the enzyme label plate is preferably a high adsorption enzyme label plate; (2) and (3) using PBS containing 1% Bovine Serum Albumin (BSA) in volume concentration as a blocking solution, adding 200 mu L of the blocking solution into each hole of the ELISA plate dried in the step (2), incubating for 2 hours at 37 ℃, discarding the blocking solution, adding 200 mu L of PBS into each hole, washing for 3 times, and drying to obtain the ELISA kit.
Further, the recombinant human HGFR-ECD-Fc is prepared by the following method: transfecting an expression plasmid pCAGGS-HGFR-ECD-Fc-His containing a recombinant human HGFR-ECD-Fc gene to CHO cells, and collecting a supernatant culture medium after one week; adding 2mL of nickel sepharose gel into each liter of culture medium, oscillating and adsorbing target protein, washing the nickel sepharose gel for 3 times by PBS, and dissolving out the protein by eluent to obtain recombinant human HGFR-ECD-Fc; the eluent composition is as follows: 50mM Tris, 0.5M NaCl, 0.01% Tween80, 100mM imidazole, solvent water, pH 8.0.
The invention also provides an application of the ELISA kit in detection of the HGFR targeted antibody, and the application method comprises the following steps:
(1) diluting a sample to be detected in PBS to prepare a sample diluent to be detected, adding the sample diluent into the ELISA kit according to the amount of 100 mu L per hole, incubating for 1 hour at 37 ℃, discarding liquid in the hole, adding 200 mu L PBS per hole, washing for 3 times and then patting dry;
(2) diluting goat anti-mouse IgG secondary antibody marked by HRP (horse radish peroxidase) in PBS (phosphate buffer solution) according to the volume ratio of 1:10000, adding 100 mu L of the secondary antibody into each hole in the step (1), incubating for 1 hour at 37 ℃, removing liquid in each hole, adding 200 mu L of PBS into each hole, washing for 3 times, and then drying by beating;
(3) adding 100 μ L chromogenic substrate working solution to step (2) per well, incubating at 37 deg.C for 10 min, and adding 50 μ L2 MH2SO4Stopping the reaction by using the aqueous solution, detecting the light absorption value of OD450, and determining the sample to be positive if the light absorption value is more than 2 times higher than that of a blank control hole; the chromogenic substrate working solution is prepared by mixing chromogenic substrates according to the volume ratio of 1:100Diluting the concentrated solution in a substrate buffer solution to prepare the compound; the chromogenic substrate concentrated solution is a dimethyl sulfoxide (DMSO) solution of 10mg/mL of 3,3',5,5' -Tetramethylbenzidine (TMB); the substrate buffer composition: 25mM citric acid, 50mM Na2HPO4，0.03％H2O2The solvent is water.
Compared with the prior art, the invention has the following beneficial effects: the invention provides an ELISA kit for screening HGFR targeted antibodies, which takes recombinant human HGFR ectodomain Fc tag fusion protein (HGFR-ECD-Fc) as an antigen solid phase to be adsorbed on an ELISA plate; capturing an antibody capable of binding to the antigen through immobilized HGFR-ECD-Fc; after washing off the free components, the amount of the antibody was determined by an enzyme-labeled secondary antibody that specifically recognizes the captured antibody.
The ELISA kit can be used for detecting HGFR targeted antibodies, has the advantages of strong specificity, high sensitivity and the like, can detect the antibody with the dilution degree of more than 700000 times, and has good application prospect.
(IV) description of the drawings
FIG. 1 is a bar graph of absorbance values of different concentrations of HGFR targeting antibody in diluted and PBS.
FIG. 2 is a bar graph of absorbance values of different concentrations of HGFR targeting antibody diluted in serum-containing medium.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1: preparation of antigen immobilized enzyme label
1) Recombinant human HGFR-ECD-Fc
The expression plasmid pCAGGS-HGFR-ECD-Fc-His (Huada gene) was transfected into CHO cells (institute of Chinese academy) using Lipofectamine 3000(ThermoFisher), and the supernatant medium was collected one week later. Adding 2mL of nickel sepharose (Biyunyan) into 1L of culture medium, oscillating and adsorbing the target protein, washing the nickel sepharose for 3 times by 10mL of PBS, and dissolving the protein by using 5mL of eluent (50mM Tris, 0.5M NaCl, 0.01% Tween80, 100mM imidazole, pH 8.0) to obtain the recombinant human HGFR-ECD-Fc for later use.
2) Coating antigen: recombinant human HGFR-ECD-Fc to coating buffer (15mM Na)2CO3，35mM NaHCO3pH9.6) was added to the reaction solution, and the reaction solution was diluted to 0.1. mu.g/mL of the antigen working solution. mu.L of antigen working solution was added to each well of a high adsorption microplate (Jet Biofil, FEP100008), incubated at 37 ℃ for 1 hour, and then incubated at 4 ℃ for 24 hours. Antigen working solution was discarded, 200. mu.L of PBS was added to each well, washed 3 times and then patted dry.
3) Antigen immobilized enzyme standard plate: PBS containing Bovine Serum Albumin (BSA) at a volume concentration of 1% was used as a blocking solution, and 200. mu.L of the blocking solution was added to each well of the plate blotted in step 2), followed by incubation at 37 ℃ for 2 hours. And (3) discarding the confining liquid, adding 200 mu LPBS into each hole, washing for 3 times, and then patting to dry to obtain the antigen immobilized enzyme target plate, thus obtaining the ELISA kit for later use.
Example 2 detection of different concentrations of HGFR targeting antibody dilutions
1) HGFR targeting antibody standard (Cell Signaling Technology) is diluted in PBS according to the volume ratio of 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:243000 and 1:729000 to prepare HGFR targeting antibody diluent. To each well of the antigen-immobilized enzyme standard prepared in example 1, 100. mu.L of the above dilution of HGFR targeting antibody at different concentrations was added, and incubated at 37 ℃ for 1 hour. The wells were discarded, 200. mu.L of PBS was added to each well, washed 3 times and then patted dry.
2) HRP-labeled goat anti-mouse IgG secondary antibody was diluted in PBS at a volume ratio of 1:10000, and 100. mu.L of the solution was added to each well and incubated at 37 ℃ for 1 hour. The wells were discarded, 200. mu.L of PBS was added to each well, washed 3 times and then patted dry.
3) The chromogenic substrate concentrate (10mg/mL TMB, solvent DMSO) was diluted in substrate buffer (25mM citric acid, 50mM Na) at a volume ratio of 1:1002HPO4，0.03％H2O2) As a chromogenic substrate working solution. mu.L of chromogenic substrate working solution was added to each well, incubated at 37 ℃ for 10 minutes, and 50. mu.L of stop solution (2M H) was added2SO4) And detecting the OD450 light absorption value.
3. Conclusion
As can be seen from the results in FIG. 1, the ELISA kit prepared by the method can be used for detecting HGFR targeting antibody diluted in PBS, and has strong sensitivity, and can detect antibody with dilution degree greater than 700000 times.
Example 3: detection of different concentrations of HGFR targeting antibodies diluted in serum-containing Medium
1) HGFR targeting antibody standard (Cell Signaling Technology) was diluted in DMEM medium containing 10% FBS at a volume ratio of 1:1000, 1:3000, 1:9000, 1:27000, 1:81000, 1:243000, 1:729000 as HGFR targeting antibody dilution. To each well of the antigen-immobilized enzyme standard prepared in example 1, 100. mu.L of the above dilution of HGFR targeting antibody at different concentrations was added, and incubated at 37 ℃ for 1 hour. The wells were discarded, 200. mu.L of PBS was added to each well, washed 3 times and then patted dry.
Step 2), 3) the same as in example 2.
3. Conclusion
Since hybridoma technology is generally used in actual screening of antibodies, the produced antibodies are mostly present in a serum-containing medium. The experiment simulates the complex environment in actual screening and detects the sensitivity of the kit. As can be seen from the results of fig. 2, the present ELISA kit can detect the HGFR targeting antibody diluted in DMEM medium containing 10% FBS, and the sensitivity is similar to the results shown in fig. 1, and thus, the present kit can be used for the detection of the HGFR targeting antibody in complex components.
Claims (6)
1. An ELISA kit for screening HGFR targeted antibodies is characterized in that the kit is prepared by coating an ELISA plate by taking recombinant human HGFR-ECD-Fc as an antigen.
2. The ELISA kit of claim 1, characterized in that the kit is prepared as follows: (1) diluting the recombinant human HGFR-ECD-Fc into 0.1 mu g/mL antigen working solution by using a coating buffer solution; adding 100 mu L of antigen working solution into each hole of an enzyme label plate, incubating for 1 hour at 37 ℃, transferring to 4 ℃ for incubation for 24 hours, discarding the antigen working solution, adding 200 mu LPBS into each hole, washing for 3 times, and patting dry; the coating buffer consists of: 15mM Na2CO3，35mM NaHCO3The solvent is water, and the pH value is 9.6; (2) PBS containing bovine serum albumin at a volume concentration of 1% was used as a blockingAnd (3) adding 200 mu L of confining liquid into each hole of the ELISA plate dried in the step (2), incubating for 2 hours at 37 ℃, removing the confining liquid, adding 200 mu L of PBS into each hole, washing for 3 times, and drying to obtain the ELISA kit.
3. The ELISA kit of claim 2 wherein the ELISA plate is a high adsorption ELISA plate.
4. The ELISA kit of claim 2, wherein the recombinant human HGFR-ECD-Fc is prepared as follows: transfecting an expression plasmid pCAGGS-HGFR-ECD-Fc-His containing a recombinant human HGFR-ECD-Fc gene to CHO cells, and collecting a supernatant culture medium after one week; adding 2mL of nickel sepharose gel into each liter of culture medium, oscillating and adsorbing target protein, washing the nickel sepharose gel for 3 times by PBS, and dissolving out the protein by eluent to obtain recombinant human HGFR-ECD-Fc; the eluent composition is as follows: 50mM Tris, 0.5M NaCl, 0.01% Tween80, 100mM imidazole, solvent water, pH 8.0.
5. Use of the ELISA kit of claim 1 to detect HGFR-targeting antibodies.
6. The application according to claim 5, characterized in that the method of application is:
(1) diluting a sample to be detected in PBS to prepare a sample diluent to be detected, adding the sample diluent into the ELISA kit according to the amount of 100 mu L per hole, incubating for 1 hour at 37 ℃, discarding liquid in the hole, adding 200 mu L PBS per hole, washing for 3 times and then patting dry;
(2) diluting goat anti-mouse IgG secondary antibody marked by HRP (horse radish peroxidase) in PBS (phosphate buffer solution) according to the volume ratio of 1:10000, adding 100 mu L of the secondary antibody into each hole in the step (1), incubating for 1 hour at 37 ℃, removing liquid in each hole, adding 200 mu L of PBS into each hole, washing for 3 times, and then drying by beating;
(3) adding 100 μ L chromogenic substrate working solution to step (2) per well, incubating at 37 deg.C for 10 min, and adding 50 μ L2 MH2SO4Stopping the reaction by using the aqueous solution, detecting the light absorption value of OD450, and determining the sample to be positive if the light absorption value is more than 2 times higher than that of a blank control hole; what is needed isThe chromogenic substrate working solution is prepared by diluting chromogenic substrate concentrated solution into substrate buffer solution according to the volume ratio of 1: 100; the chromogenic substrate concentrated solution is 10mg/mL dimethyl sulfoxide solution of 3,3',5,5' -tetramethyl benzidine; the substrate buffer composition: 25mM citric acid, 50mM Na2HPO4，0.03％H2O2The solvent is water.
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