Anti-epidermal growth factor antibody ELISA kit

Abstract

The present invention provides an enzyme-linked immunosorbent assay (ELISA) kit for anti-epidermal growth factor (EGF) antibody. Specifically, the present invention provides a detection kit, which is characterized by comprising: (a) an enzyme-labeled plate, which is an enzyme-labeled plate coated with EGF antigen; and (b) liquid reagents, which include: (b1) a standard for anti-human EGF antibody; (b2) a sample diluent; (b3) a washing solution; (b3) a working solution of HRP-labeled anti-human IgG antibody; (b4) a substrate, which is a substrate for HRP-labeled anti-human IgG antibody; and (b5) a termination solution. The kit of the present invention uses serum with a positive anti-human EGF antibody concentration as a standard, and can quantitatively detect the anti-EGF antibody concentration in the serum of clinical patients through a standard curve fitted by the detection results of the standard. It has the advantages of simple operation, high sensitivity, high precision, high accuracy, and can be used for rapid quantitative detection of a large number of samples. It can be used as an accompanying detection kit to meet the clinical use requirements of related vaccines with EGF as an antigen.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an anti-epidermal growth factor (EGF) antibody enzyme-linked immunosorbent assay kit. Background Art

[0002] Human epidermal growth factor (EGF) is a single-chain low molecular weight polypeptide composed of 53 amino acids. It is mainly distributed in the extracellular fluid of various tissues of the human body. It was first isolated from the mouse submandibular gland by American scientist Cohen in 1962. EGF acts on target cells through autocrine or paracrine action, which can promote cell mitosis, stimulate embryonic growth and development, increase the transport and metabolism of substances, etc., and has a wide range of physiological effects. EGF mainly exerts its function through EGFR. After EGF binds to EGFR, EGFR is activated under the induction of EGF, and the three-dimensional conformation of EGFR changes, causing EGFR to form homodimers or heterodimers. EGF / EGFR can activate multiple signaling pathways including Ras-Raf-MAPK, JAK-STAT, PI(3)K-Akt, etc. Studies in recent years have shown that EGF and EGFR are overexpressed in many solid tumors such as glioma, breast cancer, lung cancer, ovarian cancer, head and neck cancer, cervical cancer, esophageal cancer, prostate cancer, liver cancer, colon cancer, gastric cancer, etc. Many tumors produce a large amount of EGF through autocrine and paracrine methods, thereby overactivating the EGFR pathway. Overactivated EGFR enhances transcription, post-transcriptional activity or gene expression through multiple signaling pathways, causing cell phenotype transformation, leading to the occurrence and development of tumors. Therefore, reducing the content of EGF in tumor cells plays a vital role in inhibiting the proliferation of tumor cells, signal transduction between tumor cells, and thus inhibiting the development of tumors.

[0003] EGF-based tumor therapeutic vaccine targets human EGF. By stimulating the body to produce high-titer anti-EGF antibodies, it neutralizes EGF in the body, blocks the EGF-EGFR pathway, and then inhibits the growth of tumor cells, thus achieving the effect of treating cancer. CIMAvax-EGF developed by the Cuban Institute of Molecular Biology was approved by the Cuban Drug Administration in 2008 for the switch maintenance treatment of patients with stage IIIB / IV non-small cell lung cancer (NSCLC) after first-line chemotherapy. More than 5,000 advanced cancer patients have been vaccinated with this vaccine in different clinical trials. The clinical results of the CIMAvax-EGF vaccine show that the vaccine can induce the production of high levels of anti-EGF antibodies, keeping the EGF concentration in the serum at an undetectable low level. Patients with high anti-EGF antibody titers have a significantly longer survival period than the control group. The anti-EGF antibody level after EGF vaccine immunization is an important immunological index that needs to be monitored in the clinical trials and applications of EGF-based vaccines, and it has guiding significance for the prognosis and later medication guidance of tumor patients.

[0004] In the prior art, conventional anti-human EGF antibody titer detection uses the indirect ELISA method. The maximum serum dilution multiple when the OD value of the sample well / OD value of the negative control product ≥ 2.1 is the antibody titer of the test sample (see patent: ZL201720210309.2 for details). There is no standard curve calibration in the EGF antibody titer detection method, the inter-plate precision is poor, and the detection result error is generally between 50% and 200%. At the same time, a 96-well enzyme-linked immunosorbent assay (ELISA) plate can detect at most 6 serum samples, with a large workload. The conventional anti-EGF antibody titer detection method cannot meet the detection requirements of rapidity, precise quantification, and high throughput.

[0005] In summary, there is an urgent need in the art to develop a detection kit that can rapidly, highly precisely, and with high throughput detect the content of anti-EGF antibodies in serum. Summary of the Invention

[0006] The object of the present invention is to provide a detection kit for rapidly, highly precisely, highly accurately, and with high throughput quantitatively detecting anti-EGF antibodies in human serum.

[0007] In the first aspect of the present invention, a detection kit is provided, comprising:

[0008] (a) An enzyme-linked immunosorbent assay (ELISA) plate, which is an ELISA plate coated with EGF (epidermal growth factor) antigen; and

[0009] (b) A liquid reagent, which comprises:

[0010] (b1) A standard solution of anti-human EGF antibody;

[0011] (b2) A sample diluent;

[0012] (b3) Washing solution;

[0013] (b4) Working solution of HRP-labeled anti-human IgG antibody;

[0014] (b5) Substrate, where the substrate is the substrate of HRP-labeled anti-human IgG antibody; and

[0015] (b6) Stop solution.

[0016] In another preferred example, the EGF antigen is a recombinant human EGF antigen.

[0017] In another preferred example, the enzyme-linked immunosorbent assay (ELISA) plate is coated with 0.1 - 1 μg / well of EGF antigen.

[0018] In another preferred example, the ELISA plate is blocked with a blocking solution.

[0019] In another preferred example, the blocking solution is a PBS buffer containing 1 - 3% (w / v) BSA (optimally 2% (w / v)), 2 - 4% (w / v) protein protectant HT-HY (optimally 2% (w / v)), and 0.1 - 0.5% (v / v) Tween-20 (optimally 0.5% (w / v)).

[0020] In another preferred example, the ELISA plate is a 96-well ELISA plate.

[0021] In another preferred example, the standard solution of anti-human EGF antibody is prepared from the serum of subjects immunized with a recombinant human EGF-based vaccine, which induces the production of serum containing a high concentration of anti-EGF antibody.

[0022] In another preferred example, the kit further includes: a quality control product.

[0023] In another preferred example, the quality control product includes: a positive control product and a negative control product.

[0024] In another preferred example, the positive control product is a positive control product prepared by the following method: Take multiple portions of patient sera with a positive anti-EGF antibody titer, mix and centrifuge, take the supernatant, and dilute the supernatant with a sample diluent at an appropriate ratio to obtain a serum dilution sample.

[0025] In another preferred example, the OD value detected for the positive control product is 0.5 - 1.0. In another preferred example, the negative control product is a negative control product prepared by the following method: Take multiple portions of normal human sera, mix them in equal proportions and centrifuge, dilute the supernatant with a sample diluent at an appropriate ratio, mix and centrifuge again, take the supernatant, and dilute the supernatant with a sample diluent at an appropriate ratio to obtain a serum dilution sample.

[0026] In another preferred example, the OD value of the negative control product detection is ≤ 0.3.

[0027] In another preferred example, the washing solution is a concentrated washing solution.

[0028] In another preferred example, the concentrated washing solution is a 10×PBS buffer solution (i.e., 10-fold concentrated PBS buffer solution) containing 0.1 - 0.5% (v / v) Tween-20 (optimally 0.5%) and 0.8 - 1.5 g / L gentamicin sulfate (optimally 1%).

[0029] In another preferred example, the dosage ratio of Tween-20 to gentamicin sulfate is Tween-20 (ml) : gentamicin sulfate (g) = 1 - 5 : 0.8 - 1.5 (optimally, = 5 : 1).

[0030] In another preferred example, the pH value of the washing solution is 7.4 ± 0.2.

[0031] In another preferred example, each 1 L of the concentrated washing solution includes 1 - 5 ml of Tween-20.

[0032] In another preferred example, each 1 L of the concentrated washing solution includes 0.8 - 1.5 g (optimally, 1 g) of gentamicin sulfate.

[0033] In another preferred example, each 1 L of the concentrated washing solution includes the following components:

[0034] Tween-20 1～5ml Gentamicin Sulfate 0.8 - 1.5 g (optimally, 1 g) 10-fold concentrated PBS buffer the balance

[0035] And the pH value of the concentrated washing solution is 7.4 ± 0.2.

[0036] In another preferred example, the pH value of the sample diluent is 7.4 ± 0.2.

[0037] In another preferred example, each 1 L of the sample diluent includes 10 - 15 g (preferably, 10 - 12 g) of HT-AEP protein protectant.

[0038] In another preferred example, each 1 L of the sample diluent includes the following components:

[0039] HT-AEP protein protectant 10 - 15 g (preferably, 10 - 12 g) PBS buffer the balance

[0040] And the pH value of the sample diluent is 7.4 ± 0.2.

[0041] In another preferred example, the sample diluent is a PBS buffer solution containing 1 - 1.5% (w / v) HT-AEP protein protectant (optimally 1 - 1.2% (w / v)), and the pH value of the sample diluent is 7.4 ± 0.2.

[0042] In another preferred example, the working solution of HRP-labeled anti-human IgG antibody comprises Tween-20, HT-AES protectant, HRP-labeled anti-human IgG antibody and PBS buffer solution.

[0043] In another preferred example, the anti-human IgG antibody is donkey anti-human IgG antibody

[0044] In another preferred example, the working solution of HRP-labeled anti-human IgG antibody is prepared by diluting HRP-labeled anti-human IgG antibody 10,000 - 50,000 times with PBS buffer solution containing 1 - 1.5%

[0045] (w / v) HT-AES protectant (optimally 1%), 0.01 - 0.05% (v / v) Tween-20 (optimally 0.05%).

[0046] In another preferred example, the pH value of the PBS buffer solution is 7.4 ± 0.2.

[0047] In another preferred example, the PBS buffer solution comprises: NaCl, KCl, Na2HPO4 and KH2PO4.

[0048] In another preferred example, in the PBS buffer solution, the mass ratio of NaCl, KCl, Na2HPO4 and KH2PO4 is 80 ± 8:2 ± 0.2:14.4 ± 1.44:2.4 ± 0.24; preferably, it is 80 ± 4:2 ± 0.1:14.4 ± 0.72:2.4 ± 0.12.

[0049] In another preferred example, the PBS buffer solution contains 80 ± 8 g / L of NaCl, 2 ± 0.2 g / L of KCl, 14.4 ± 1.44 g / L of Na2HPO4 and 2.4 ± 0.24 g / L of KH2PO4.

[0050] In another preferred example, the PBS buffer solution contains 80 ± 4 g / L of NaCl, 2 ± 0.1 g / L of KCl, 14.4 ± 0.72 g / L of Na2HPO4 and 2.4 ± 0.12 g / L of KH2PO4.

[0051] In another preferred example, the substrate is TMB chromogenic solution.

[0052] In another preferred example, the terminating solution is sulfuric acid solution.

[0053] In another preferred example, in the terminating solution, the volume ratio of 98% concentrated sulfuric acid to water is 50 - 60:440 - 450.

[0054] In another preferred embodiment, the kit further comprises: (c) an instruction manual.

[0055] In another preferred embodiment, the instruction manual describes the method of using the kit. In another preferred embodiment, the instruction manual further optionally describes a standard curve for detection, and the standard curve is as shown in Formula I:

[0056]

[0057] where A = -0.023, B = 0.892, C = 147.9, D = 1.955; x represents the concentration of anti-EGF antibody (RU / ml), and y represents the average OD value.

[0058] In another preferred embodiment, the standard solution of anti-human EGF antibody is used to obtain the standard curve.

[0059] In another preferred embodiment, the standard solution of anti-human EGF antibody is prepared from a serum sample whose OD value is still ≥1 after being diluted at least 1000 - 3000 times (such as 2000 times).

[0060] In another preferred embodiment, the standard solution of anti-human EGF antibody comprises one or more (such as 1 to X - 1 kinds) of standards with known anti-EGF antibody concentrations, and the concentration of anti-EGF antibody in at least one standard is Cx RU / mL, where C X ≥800, X is 3, 4, 5, 6, 7, 8, 9 or 10, preferably, X is 5, 6, 7 or 8; and when the concentration is C X , the OD value of this standard is ≥1.

[0061] In another preferred embodiment, the kit comprises standards for obtaining some or all of the concentrations required for the standard curve. When the kit only provides standards for some concentrations, those skilled in the art can prepare other required concentrations.

[0062] In another preferred embodiment, the standard solution of anti-human EGF antibody comprises X - 1 kinds of standards with anti-EGF antibody concentrations of Cx, C X-1 , …, C2 and C1 RU / mL respectively.

[0063] In another preferred embodiment, Cx > C X-1 > … > C2 > C1.

[0064] In another preferred embodiment, Cx, C X-1 , …, C2 and C1 are in an arithmetic or geometric progression, preferably in a geometric progression. More preferably, Cx = 2 * C X-1 , …, C3 = 2 * C2 and C2 = 2 * C1.

[0065] In another preferred example, the kit further includes: a box body, and an enzyme-linked immunosorbent assay (ELISA) plate and containers for storing various liquid reagents are provided inside the box body.

[0066] In the second aspect of the present invention, a preparation method of a detection kit as described in the first aspect is provided, which is characterized by including the following steps:

[0067] (S1) Coating the ELISA plate with EGF antigen; and

[0068] (S2) Preparing various liquid reagents.

[0069] In another preferred example, the preparation method further includes the step: (S3) Preparing one or more standard products.

[0070] In another preferred example, the preparation method further includes the step: (S4) Preparing a quality control product.

[0071] In another preferred example, step (S1) includes the steps:

[0072] (S1.1) Providing a coating solution and a blocking solution containing EGF antigen;

[0073] (S1.2) Adding the coating solution to some or all of the wells of the ELISA plate for antigen coating, and removing the coating solution to obtain an ELISA plate coated with antigen; and

[0074] (S1.3) Adding the blocking solution to some or all of the wells of the ELISA plate coated with antigen for blocking, and removing the blocking solution to obtain an ELISA plate coated with EGF antigen.

[0075] In another preferred example, the concentration of EGF antigen in the coating solution is 1 - 10 μg / ml.

[0076] In another preferred example, the coating solution is obtained by diluting EGF antigen with a coating dilution solution to the required concentration.

[0077] In another preferred example, the coating dilution solution includes Na2CO3 and NaHCO3.

[0078] In another preferred example, in the coating dilution solution, the mass ratio of Na2CO3 to NaHCO3 is 1.59 ± 0.16: 2.93 ± 0.29.

[0079] In another preferred example, each 1 L of the coating dilution solution includes 1.59 ± 0.16 g of Na2CO3.

[0080] In another preferred example, each 1 L of the coating dilution solution includes 2.93 ± 0.29 g of NaHCO3.

[0081] In another preferred example, each 1 L of the coating diluent comprises the following components:

[0082] <![CDATA[Na2CO3]]> 1.59±0.16g <![CDATA[NaHCO3.]]> 2.93±0.29g

[0083] In another preferred example, the sample diluent further comprises the balance of water.

[0084] In another preferred example, in step (S1.2), the dosage of the coating solution is 100 ± 10 μl / well (preferably 100 ± 5 μl / well).

[0085] In another preferred example, in step (S1.2), the coating of the antigen is carried out at 2 - 8°C.

[0086] In another preferred example, in step (S1.2), the coating time of the antigen is 12 - 18 h.

[0087] In another preferred example, in step (S1.2), after removing the coating solution, a step of washing the plate is further included.

[0088] In another preferred example, in step (S1.3), the dosage of the blocking solution is 200 ± 20 μl / well (preferably 200 ± 10 μl / well).

[0089] In another preferred example, in step (S1.3), the blocking is carried out at 37 ± 2°C.

[0090] In another preferred example, in step (S1.3), the blocking time is 1.5 - 3 h.

[0091] In another preferred example, in step (S1.3), after removing the blocking solution, a step of vacuum drying and / or vacuum placing is further included.

[0092] In another preferred example, the standard solution of the anti - human EGF antibody uses relative units (RU / ml) as the concentration unit, and a standard curve is drawn using the standard product; to draw the standard curve, multiple measurement points need to be set up. According to the number of measurement points, the corresponding standard products are prepared. Generally, 4 - 12 measurement points are set, and preferably 5 - 8 measurement points.

[0093] In another preferred example, the method for setting the concentration unit of the standard solution of the anti - human EGF antibody is as follows: the upper and lower limits of the concentration unit are determined according to the upper and lower limits of OD450 respectively. For the middle part, the OD450 values are set with the corresponding concentration units according to conventional methods such as geometric progression, arithmetic progression or proportion. The upper limit of the concentration unit is generally set to 100 RU / ml - 1000 RU / ml (such as 800 RU / mL), the lower limit of the concentration unit is set to 0 RU / ml, and generally 3 - 10 concentration setting points are set in the middle of the concentration unit, and preferably 5 - 8 concentration setting points.

[0094] In another preferred example, the upper limit of the detection OD value of the concentration unit of the anti-human EGF antibody standard solution is generally greater than 1.0. The lower limit of the detection OD value of the concentration unit of the anti-human EGF antibody standard solution is generally less than 0.3.

[0095] In another preferred example, in step (S2), the anti-human EGF antibody standard solution is prepared by the following method, which includes the steps:

[0096] i. Select a serum sample for preparing the standard, dilute the serum sample, and the OD value of the diluted serum sample after dilution is ≥ 1; and stipulate that the concentration of the diluted serum sample is C X , so as to obtain a standard product (upper concentration limit) with an anti-EGF antibody concentration of C X RU / mL; where C X ≥ 800, X is 3, 4, 5, 6, 7, 8, 9 or 10 (preferably, X is 5, 6, 7 or 8); and

[0097] Optionally, ii. Dilute (such as dilute with a sample diluent) the standard product with an anti-EGF antibody concentration of C X RU / mL obtained in step i to obtain standard products with anti-EGF antibody concentrations of C X-1 ,..., C2 and C1 RU / mL respectively.

[0098] In the third aspect of the present invention, there is provided a use of the detection kit as described in the first aspect, which is used to detect anti-epidermal growth factor (EGF) antibodies in a sample.

[0099] In another preferred example, the sample is a serum sample.

[0100] In another preferred example, the detection is a quantitative detection.

[0101] In the fourth aspect of the present invention, there is provided a quality inspection standard finished product verification standard, mainly including the following indicators:

[0102] (1) Precision determination: Take three different concentrations of quality control serum within the linear range and repeat the determination 20 times, calculate the within-batch coefficient of variation, CV should be ≤ 10%, and the between-batch relative range, R should be ≤ 20%.

[0103] (2) Accuracy detection: Detect two internal reference products with determined values of the enterprise, the low-value deviation does not exceed ±20%, and the high-value deviation does not exceed ±25%.

[0104] (3) Sensitivity: Take the sample diluent, repeat the determination 20 times, calculate the average value and the standard deviation SD, +3SD is the sensitivity of the kit, and the sensitivity should be ≤ 5 RU / ml.

[0105] (4) Linearity: After fitting the readings of the standard products with a four-parameter logistic curve or other appropriate mathematical models, the correlation coefficient should be not less than 0.990.

[0106] (5) Stability: After accelerating the kit at 37 °C for 7 days, the physical inspection, within-batch precision determination, accuracy determination, sensitivity determination, and linearity determination all meet the above requirements.

[0107] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0108] Figure 1 Shows a typical standard curve example diagram of Example 5 of the present invention. Detailed Embodiments

[0109] Through long-term and in-depth research, the inventors first provided a detection kit for detecting and further improving each reagent (such as enzyme-linked immunosorbent assay (ELISA) plates, washing solutions, diluents, etc.) provided in the detection kit, so as to provide a kit with good stability that can still meet various quality inspection / release standards under accelerated tests. Furthermore, a detection kit for rapidly, highly precisely, highly accurately, and high-throughput quantitatively detecting anti-EGF antibodies in human serum is provided, and the reagent can meet the detection requirements of anti-EGF antibodies accompanying the clinical research and application of EGF vaccines prepared based on EGF. Based on this, the inventors completed the present invention.

[0110] Terms

[0111] Unless otherwise specified, each abbreviation in this article has the conventional meaning understood by those skilled in the art. For example, EGF refers to epidermal growth factor, and BSA refers to bovine serum albumin.

[0112] The following elaborates in detail the specific operation method for preparing an enzyme-linked immunosorbent assay (ELISA) detection kit for anti-EGF antibodies.

[0113] Kit and Its Quality Control Method

[0114] The first object of the present invention is to provide a detection kit for rapidly, highly precisely, highly accurately, and high-throughput quantitatively detecting anti-EGF antibodies in human serum, and the reagent can meet the detection requirements of anti-EGF antibodies accompanying the clinical research and application of EGF vaccines prepared based on EGF.

[0115] In a specific embodiment, the present invention provides a detection kit, including:

[0116] (a) an ELISA plate, wherein the ELISA plate is an ELISA plate coated with EGF (epidermal growth factor) antigen; and

[0117] (b) a liquid reagent, the liquid reagent comprising:

[0118] (b1) anti-human EGF antibody standard solution;

[0119] (b2) sample diluent;

[0120] (b3) washing liquid;

[0121] (b4) HRP-labeled anti-human IgG antibody working solution;

[0122] (b5) a substrate, which is a substrate for an HRP-labeled anti-human IgG antibody; and

[0123] (b6) Stop solution.

[0124] Preferably, the EGF antigen is a recombinant human EGF antigen.

[0125] In another preferred embodiment, the ELISA plate is coated with human EGF antigen and then blocked with PBS buffer containing 1-3% BSA (optimally 2%), 2-4% protein protectant HT-HY (optimally 2%), and 0.1-0.5% Tween-20 (optimally 0.5%).

[0126] In another preferred embodiment, the ELISA plate is a 96-well ELISA plate.

[0127] In another preferred embodiment, the anti-human EGF antibody standard solution is prepared from serum containing high concentration of anti-EGF antibodies induced by subjects immunized with a recombinant human EGF-based vaccine.

[0128] In another preferred embodiment, the anti-human EGF antibody standard in the kit is derived from a subject immunized with an EGF vaccine, and the high-concentration anti-EGF antibody serum is induced and prepared.

[0129] In another preferred embodiment, the anti-human EGF antibody standard solution uses relative units (RU / ml) as concentration units, and uses the standard product to draw a standard curve; to draw the standard curve, multiple measurement points need to be set up. According to the number of measurement points, the corresponding standard product is prepared, generally 4-12 measurement points are set, preferably 5-8 measurement points.

[0130] In another preferred example, the method for setting the concentration unit of the anti-human EGF antibody standard solution is as follows: The upper and lower limits of the concentration unit are determined according to the upper and lower limits of OD450 respectively. For the middle part, the corresponding concentration units are set for the OD450 values according to conventional methods such as geometric progression, arithmetic progression or proportion. The upper limit of the concentration unit is generally set to 100 RU / ml or 1000 RU / ml, the lower limit of the concentration unit is set to 0 RU / ml, and generally 3 - 10 concentration setting points are set in the middle of the concentration unit, preferably 5 - 8 concentration setting points.

[0131] In another preferred example, the OD value detected for the upper limit of the concentration unit of the anti-human EGF antibody standard solution is generally greater than 1.0. The OD value detected for the lower limit of the concentration unit of the anti-human EGF antibody standard solution is generally less than 0.3.

[0132] In another preferred example, the kit further includes: a quality control product.

[0133] In another preferred example, the quality control product includes: a positive quality control product and a negative quality control product.

[0134] In another preferred example, the positive quality control product is prepared by taking the sera of multiple patients with positive anti-EGF antibody titers, mixing and centrifuging, taking the supernatant, and diluting the supernatant with the sample diluent at an appropriate ratio to obtain a serum-diluted sample.

[0135] In another preferred example, the OD value detected for the positive quality control product is generally between 0.5 - 1.0.

[0136] In another preferred example, the negative quality control product is prepared by taking multiple normal human sera, mixing them in equal proportion and centrifuging, then diluting the supernatant with the sample diluent at an appropriate ratio, mixing and centrifuging again, and taking the supernatant, and diluting the supernatant with the sample diluent at an appropriate ratio to obtain a serum-diluted sample.

[0137] In another preferred example, the OD value detected for the negative quality control product is generally less than 0.3.

[0138] In another preferred example, the washing solution is a concentrated washing solution, which is 10×PBS buffer containing 1 - 5% Tween - 20 (optimally 5%) and 0.8 - 1.5 g / L gentamicin sulfate (optimally 1%), and the pH value is 7.4 ± 0.2.

[0139] In another preferred example, the sample diluent is PBS buffer containing 1 - 1.5% HT - AEP (optimally 1%), and the pH value of the sample diluent is 7.4 ± 0.2.

[0140] In another preferred example, the working solution of the HRP-labeled anti-human IgG antibody is the HRP-labeled anti-human IgG antibody diluted 10,000 - 50,000 times with PBS buffer containing 1 - 1.5% HT-AES protectant (optimally 1%) and 0.01 - 0.05% Tween-20 (optimally 0.5%).

[0141] In another preferred example, the PBS buffer comprises: NaCl, KCl, Na2HPO4, and KH2PO4.

[0142] In another preferred example, in the PBS buffer, the mass ratio of NaCl, KCl, Na2HPO4, and KH2PO4 is 80 ± 8:2 ± 0.2:14.4 ± 1.44:2.4 ± 0.24; preferably, it is 80 ± 4:2 ± 0.1:14.4 ± 0.72:2.4 ± 0.12.

[0143] In another preferred example, the substrate is TMB chromogenic solution.

[0144] In another preferred example, the termination solution is sulfuric acid solution.

[0145] In another preferred example, the kit further comprises: a box body, and an enzyme-linked immunosorbent assay (ELISA) plate and containers for storing each liquid reagent are provided in the box body.

[0146] The kit of the present invention uses the indirect ELISA method to detect anti-EGF antibodies in human serum, and its basic principle is as follows: The antigen coated on the ELISA plate is recombinant human EGF. After diluting the test serum and the standard product to be detected and adding them into the reaction wells, after incubation, the specific anti-EGF antibodies in the serum bind to the solid-phase human EGF antigen in the reaction wells, forming a solid-phase antigen-antibody complex; wash away the unbound antibody components, add the HRP-labeled anti-human IgG antibody for incubation, and the anti-EGF antibodies bound to the solid-phase human EGF antigen then bind to the HRP-labeled anti-human IgG antibody; wash away the unbound enzyme-labeled antibody components, add the substrate TMB, and finally add the termination solution to terminate the reaction. According to the standard product or the standard curve in the box body, the anti-EGF antibodies can be quantitatively detected.

[0147] In another specific embodiment, the present invention also provides the quality inspection standard and finished product verification standard of the present invention, mainly the following indicators:

[0148] (1) Precision determination: Take three different concentrations of quality control sera within the linear range and repeat the determination 20 times, calculate the within-batch coefficient of variation, CV should be ≤ 10%, and the relative range between batches, R should be ≤ 20%.

[0149] (2) Accuracy test: Two internal reference samples of the enterprise are tested. The deviation of the low value does not exceed ±20%, and the deviation of the high value does not exceed ±25%.

[0150] (3) Sensitivity: Take the sample diluent and measure it 20 times repeatedly. Calculate the average value and the standard deviation SD. +3SD is the sensitivity of the kit, and the sensitivity should be ≤5 RU / ml.

[0151] (4) Linearity: After fitting the readings of the standard products with a four-parameter logistic curve or other appropriate mathematical models, the correlation coefficient should be not less than 0.990.

[0152] (5) Stability: After accelerating the kit at 37°C for 7 days, the physical inspection, within-batch precision measurement, accuracy measurement, sensitivity measurement, and linearity measurement all meet the above requirements.

[0153] The main advantages of the present invention include:

[0154] 1. The within-batch coefficient of variation CV of the kit detected by the present invention is ≤10%, and the relative range between batches, R ≤20%.

[0155] 2. The deviation of the low value detected by the kit of the present invention does not exceed ±20%, and the deviation of the high value does not exceed ±25%.

[0156] 3. The sensitivity of the kit of the present invention: ≤5 RU / ml.

[0157] 4. The linearity of the kit of the present invention: In the range of 12.5 - 800 RU / ml, after fitting with a four-parameter logistic curve or other appropriate mathematical models, the correlation coefficient should be not less than 0.990.

[0158] 5. The kit of the present invention has good stability.

[0159] In addition, the kit of the present invention also has the advantages of fast detection speed, high precision, high accuracy, and high throughput.

[0160] In summary, the anti-epidermal growth factor (EGF) antibody enzyme-linked immunosorbent assay kit of the present invention is easy to operate, has high sensitivity and accuracy, and can be used for rapid quantitative detection of a large number of samples, which has great guiding significance for EGF vaccine clinical trials and applications based on EGF.

[0161] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually in accordance with conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0162] Unless otherwise specified, each reagent used in the examples of this application can be obtained from commercial sources.

[0163] Embodiment 1:

[0164] Preparation of enzyme label coated with EGF antigen:

[0165] (1) Preparation of coating diluent: Dissolve 1.59 g NaCO3 and 2.93 g NaHCO3 in 800 ml purified water. After fully dissolved, add water to make up to 1000 ml. Filter with a 0.22 μm filter membrane and store at 2-8°C.

[0166] (2) Preparation of blocking solution: Weigh 20 g (2% w / v) BSA and 30 g (3% w / v) protein protectant HT-TY and dissolve them in 800 ml 1× PBST solution. After fully dissolved, add water to make up to 1000 ml. Filter with a 0.22 μm filter membrane and store at 2-8°C.

[0167] (3) ELISA plate: purchased from CORNING, model: 9018, specification: 96 copies / set.

[0168] The recombinant EGF lyophilized powder made by our company was diluted to 1μg / ml with coating diluent, and the coating antigen concentration was 1μg / ml. Then the coating solution with antigen was added to each well of the ELISA plate, 100μl / well, and placed at 2-8℃ for 18h. The ELISA plate was taken out, the coating solution was shaken off, the plate was washed 3 times with PBST and patted dry, 200μl / well of blocking solution was added, and it was placed at 37℃ for 2h. After that, the ELISA plate was taken out, the blocking solution was discarded, and it was patted dry. The ELISA plate was placed in a vacuum drying oven for vacuum drying and stored in the vacuum oven for 1h; finally, the ELISA plate strips were put into aluminum foil bags; vacuum sealers were used to seal, labels were affixed, and batch numbers were stamped.

[0169] Embodiment 2:

[0170] Liquid reagent configuration:

[0171] (1) Preparation of concentrated detergent: Taking the preparation of 1000 ml of 10 times concentrated detergent as an example, the preparation ratio is as follows:

[0172]

[0173] Preparation method: Weigh the corresponding volume or mass of each component of the concentrate according to the above ratio, dissolve in 800ml of purified water, adjust the pH value to 7.4 with NaOH, and then make up to 1000ml with purified water.

[0174] After filtration with a 0.22μm filter membrane, dispense into 50ml / bottle and store at 2-8℃.

[0175] (2) Preparation of sample diluent: Taking the preparation of 1000 ml of sample diluent as an example, the proportion is as follows:

[0176]

[0177] Preparation method: Weigh the components of the concentrated solution with the corresponding volume or mass according to the above proportion, dissolve them in 800 ml of purified water, adjust the pH value to 7.4 with NaOH, and then make up the volume to 1000 ml with purified water.

[0178] After filtering by a 0.22 μm filter membrane, it is dispensed into 50 ml / vial and stored at 2 - 8 °C.

[0179] (3) Preparation of HRP-labeled anti-human IgG antibody: Purchase the HRP-labeled donkey anti-IgG antibody (product number 709 - 036 - 098) from Jackson Immunoresearch. After dissolving it in 0.5 ml of pure water, add 0.5 ml of glycerol to prepare the storage solution of HRP-labeled anti-human IgG antibody. Taking the preparation of 1000 ml of sample working solution as an example, the proportion is as follows:

[0180]

[0181] Preparation method: Weigh the components of the concentrated solution with the corresponding volume or mass according to the above proportion, dissolve them in 800 ml of purified water, adjust the pH value to 7.4 with NaOH, and then make up the volume to 1000 ml with purified water.

[0182] After filtering by a 0.22 μm filter membrane, it is dispensed into 20 ml / vial and stored at 2 - 8 °C.

[0183] (4) Preparation of TMB chromogenic solution: The TMB chromogenic solution is purchased from Yeasen Biotech Co., Ltd (product number 36602ES60), dispensed into brown vials of 20 ml / vial, and stored at 2 - 8 °C.

[0184] (5) Preparation of termination solution: Taking the preparation of 500 ml of termination solution as an example, the proportion is as follows:

[0185] 98% concentrated sulfuric acid (H2SO4): 55 ml

[0186] Purified water: 445 ml

[0187] Preparation method: Take 55 ml of 98% concentrated sulfuric acid and 445 ml of purified water respectively, then slowly add the concentrated sulfuric acid into the purified water, mix well, dispense into 10 ml / vial, and store at 2 - 8 °C.

[0188] Example 3:

[0189] (1) Preparation of standard product:

[0190] ① After immunization with the EGF-based vaccine, the positive serum of the subjects against EGF antibody was taken. The positive serum of the subjects against EGF antibody was diluted 2,000 times with the sample diluent to an appropriate concentration. The OD value of the diluted serum should be higher than 1.0. This serum diluent was assigned as the standard product with a concentration of 800 RU / ml.

[0191] ② The standard product assigned as 800 RU / ml was serially diluted to prepare standard products with concentrations of 400, 200, 100, 50, 25, and 12.5 RU / ml respectively. They were separately aliquoted into standard vials, 1 ml per vial, and stored at 2 - 8 °C. Using the sample diluent as the blank (OUR / ml), the OD value reading should be less than 0.3; OD(800 RU / ml) > OD(400 RU / ml) >

[0192] OD(200 RU / ml) > OD(100 RU / ml) > OD(50 RU / ml) > OD(25 RU / ml) > OD(12.5 RU / ml) >

[0193] OD(0 RU / ml).

[0194] (2) Preparation of positive control product:

[0195] Five serum samples from patients with positive anti-EGF antibody titers were taken, mixed and centrifuged, and the supernatant was taken. The supernatant was diluted with the sample diluent at an appropriate ratio to obtain the serum-diluted sample. The OD value of the positive control product for detection was generally between 0.5 and 1.0. After filtration and sterilization, it was aliquoted into standard vials, 1 ml per vial, and stored at 2 - 8 °C.

[0196] (3) Preparation of negative control product:

[0197] Ten serum samples from healthy individuals were taken, 10 μl from each serum sample. They were fully mixed in equal proportion and centrifuged. The supernatant was taken after centrifugation. The supernatant was diluted 2,000 times with the sample diluent, filtered and sterilized, and then aliquoted into standard vials, 1 ml per vial, and stored at 2 - 8 °C. The OD value of the negative control product for detection was generally lower than 0.3.

[0198] Example 4:

[0199] Detection steps for testing samples with the kit of the present invention:

[0200] (1) Preparation work

[0201] ① Thirty minutes before the experiment, the experimental reagents were taken out of the refrigerator and used after the temperature was balanced with the room temperature. The reagents were fully mixed before use.

[0202] ② Prepare the application washing solution: dilute the concentrated washing solution (10×) in the kit at a ratio of 1:10 (i.e., add 90 ml of purified water to 10 ml of concentrated washing solution). The diluted application washing solution can be stored at 2-8°C for one week.

[0203] ③ Dilute the serum to be tested: dilute the serum to be tested with sample diluent at a ratio of 1:2000, mix thoroughly, and repeat the test. The diluted serum sample should be used within 8 hours.

[0204] (2) Operation steps

[0205] ① Add samples: add the standard, serum to be tested, positive quality control and negative quality control to the ELISA plate, 100 μl per well, and incubate in a 37°C constant temperature incubator for 60 minutes (start counting after adding the last sample).

[0206] ② Wash the plate: Pour out the liquid in the reaction wells. Add 300μl / well of the application washing solution, then pour out the liquid in the reaction wells, turn it upside down on absorbent paper and pat it dry. There should be no visible residue or bubbles in the reaction wells. Repeat 3 times.

[0207] ③ Add HRP-labeled anti-human IgG antibody: 100 μl per well and incubate in a 37°C constant temperature incubator for 60 minutes.

[0208] ④Wash the plate, same as step ②.

[0209] ⑤ Color development: Add 100 μl of TMB color development solution to each well and incubate at room temperature in the dark for 20 minutes.

[0210] ⑥Terminate the reaction: Add 50 μl of stop solution to each well, gently shake to mix, and terminate the reaction.

[0211] ⑦ Determine the absorbance value (OD): After adding the stop solution, the absorbance value must be determined within 20 minutes, with a reading at 450nm and a reference wavelength of 540nm or 570nm.

[0212] ⑧Result calculation:

[0213] Since there is no international reference serum and standard substance for anti-EGF antibody, relative units (RU / ml) are used for quantitative determination. The absorbance value of the standard substance is the Y-axis, and the concentration of each standard substance (relative unit number) is the X-axis. A curve is drawn. It is recommended to use a four-parameter mathematical model fitting method to draw the curve, and the concentration of the antibody in the serum is calculated based on this standard curve.

[0214] Figure 1 is an example of a typical standard curve.

[0215] ⑨Result determination:

[0216] The sera of 120 healthy volunteers or patients not immunized with the EGF vaccine were detected using this system. The mean ± 2SD was set as the cut-off value for anti-EGF antibody positivity. The cut-off value of this kit was approximately 25 RU / ml.

[0217] If the absorbance value of the serum specimen exceeded 800 RU / ml, the test result could be reported as >800 RU / ml, and it was recommended to measure the specimen at a greater dilution.

[0218] ≤25 RU / ml Negative for anti-EGF antibody

[0219] ≥25 RU / ml Positive for anti-EGF antibody

[0220] Example 5:

[0221] Quality control of test samples using the kit of the present invention:

[0222] (1) Intra-assay precision determination: Take high, medium, and low three quality control products at 400, 100, and 25 RU / ml as test samples, and repeat the determination 20 times. After the determination is completed, calculate according to calculation formulas (1) and (2):

[0223]

[0224]

[0225] Where:

[0226] X - Series of measured values;

[0227] - Measured mean;

[0228] n - Number of determinations;

[0229] S - Standard deviation;

[0230] CV - Intra-assay coefficient of variation.

[0231] The results were as follows:

[0232] Sample number Number of replicates (n) Average concentration CV (%) 1 20 415 10 2 20 97 4 3 20 21 3

[0233] (2) Inter-assay relative range: Take the quality control product at 100 RU / ml as the test sample, and test this product of 3 different batches respectively. Each batch is tested 3 times. Calculate the mean of the 3 determinations for each batch respectively, and calculate its inter-assay relative range, and calculate according to formulas (4) and (5).

[0234]

[0235]

[0236] Wherein:

[0237] ―― The average value of three determinations per batch (i = 1, 2, 3);

[0238] ―― The maximum value in;

[0239] ―― The minimum value in;

[0240] R ―― The relative range between batches.

[0241] The results are as follows:

[0242]

[0243] (3) Accuracy detection: Take high, medium, and low three quality control products at 400, 100, and 25 RU / ml as the determination samples, operate according to the kit instruction manual, repeat the test 3 times, take the average value, and calculate the relative deviation, calculate according to formula (5),

[0244]

[0245] Wherein:

[0246] Bias ―― Relative deviation;

[0247] ―― The determination average value;

[0248] X ―― The assigned value of the calibrator, standard, or reference substance.

[0249] The results are as follows:

[0250] Labeled value 25 100 400 1 22 106 426 2 23 102 380 3 22 109 377 X mean 22 106 394 Relative deviation (%) 1 4 7

[0251] (4) Sensitivity detection: Take the sample diluent, repeat the determination 20 times, calculate the average value and the standard deviation SD, the average value + 3SD is the kit sensitivity, and the sensitivity should be ≤ 5 RU / ml.

[0252]

[0253]

[0254] Wherein:

[0255] X ―― The series of determination values;

[0256] ―― The determination average value;

[0257] n ―― The number of determinations;

[0258] S - Standard deviation;

[0259] LLD - Lowest limit of detection.

[0260] The results are as follows:

[0261] Result Sample diluent (RU / ml) 1 1.3 2 1.3 3 1.4 4 1.5 5 1.7 6 1.0 7 1.0 8 1.2 9 1.4 10 0.9 11 1.3 12 1.3 13 1.0 14 1.0 15 1.1 16 1.3 17 1.2 18 0.9 19 1.3 10 1.3 Average value 1.22 SD 0.22 LLD 1.87

[0262] (5) Linearity: In the range of 12.5 - 800 RU / ml, after fitting with a four - parameter logistic curve or other appropriate mathematical models, the correlation coefficient should be not less than 0.990.

[0263] The results are as Figure 1 shown.

[0264] Stability: After accelerating the kit at 37 degrees for 7 days, physical inspection, within - batch precision determination, accuracy determination, sensitivity determination, and linearity determination all meet the above requirements.

[0265] All documents mentioned in the present invention are cited herein by reference as if each individual document was specifically and individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A detection kit for anti-epidermal growth factor EGF antibodies in human serum, characterized in that, Comprising: (a) An enzyme-linked immunosorbent assay (ELISA) plate, which is an ELISA plate coated with EGF antigen. Among them, the ELISA plate is coated with a coating solution containing EGF antigen at a concentration of 1 - 10 μg / ml, and the coating solution is obtained by diluting the EGF antigen with a coating diluent to the required concentration. And each 1 L of the coating diluent contains 1.59 ± 0.16 g of Na2CO3 and 2.93 ± 0.29 g of NaHCO3. And the ELISA plate is blocked with a blocking solution, and the blocking solution is a PBS buffer containing 1 - 3% (w / v) BSA, 2 - 4% (w / v) protein protectant HT-HY, and 0.1 - 0.5% (v / v) Tween-20; and (b) Liquid reagents, which include: (b1) A standard solution of anti-human EGF antibody, which is used to obtain a standard curve, and the standard solution of anti-human EGF antibody is prepared from a serum sample with an OD value ≥ 1 after being diluted 1000 - 3000 times with a sample diluent. (b2) A sample diluent, where the sample diluent is a PBS buffer containing 1 - 1.5% (w / v) HT-AEP protein protectant, and the pH value of the sample diluent is 7.4 ± 0.

2. (b3) A washing solution, where the washing solution is a concentrated washing solution, and the concentrated washing solution is a 10-fold concentrated PBS buffer containing 0.1 - 0.5% (v / v) Tween-20 and 0.8 - 1.5 g / L gentamicin sulfate. (b4) A working solution of HRP-labeled anti-human IgG antibody, where the working solution of HRP-labeled anti-human IgG antibody is a PBS buffer containing 1 - 1.5% (w / v) HT-AES protectant and 0.01 - 0.05% (v / v) Tween-20, and the HRP-labeled anti-human IgG antibody is diluted 10000 - 50000 times to obtain the working solution. (b5) A substrate, which is a substrate for the HRP-labeled anti-human IgG antibody; and (b6) A termination solution.

2. The detection kit according to claim 1, characterized in that, The EGF antigen is a recombinant human EGF antigen.

3. The detection kit according to claim 1, wherein The detection kit meets the following indicators: (1) Precision determination: Take three different concentrations of quality control sera within the linear range and repeat the determination 20 times, calculate the within-batch coefficient of variation, CV should ≤ 10%, and the relative range between batches, R should ≤ 20%. (2) Accuracy detection: Detect two internal reference standards of the enterprise, the low-value deviation should not exceed ±20%, and the high-value deviation should not exceed ±25%. (3) Sensitivity: Take the sample diluent and repeat the determination 20 times, calculate the average value and the standard deviation SD, +3SD is the sensitivity of the kit, and the sensitivity should ≤ 5 RU / ml. (4) Linearity: After fitting the standard product readings with a four-parameter logistic curve or other appropriate mathematical models, the correlation coefficient should not be less than 0.990; and (5) Stability: After accelerating the kit at 37 degrees for 7 days, physical inspection, within-batch precision determination, accuracy determination, sensitivity determination, and linearity determination all meet the above requirements.

4. The detection kit according to claim 1, wherein The described kit can quantitatively detect the concentration of anti-EGF antibodies in clinical patients by fitting a standard curve.

5. The detection kit according to claim 1, characterized in that, The described kit further includes: (c) an instruction manual.

6. The detection kit according to claim 5, wherein, The instruction manual describes the usage method of the kit.

7. The detection kit according to claim 1, wherein, The described kit further includes: a kit body, and an enzyme-linked immunosorbent assay (ELISA) plate and containers for storing various liquid reagents are provided inside the kit body.

8. The detection kit according to claim 1, wherein The substrate is a TMB chromogenic solution.

9. The detection kit according to claim 1, characterized in that, The stop solution is a sulfuric acid solution.

10. The detection kit according to claim 1, characterized in that, The described kit further includes: a quality control product; and the quality control product includes: a positive control product and a negative control product.

11. The detection kit according to claim 10, wherein, The OD value detected by the positive control product is 0.5 - 1.0, and the OD value detected by the negative control product is ≤ 0.

3.

12. A method for preparing a detection kit as described in claim 1, characterized in that, It includes the following steps: (S1) Coating the ELISA plate with EGF antigen; and (S2) Preparing various liquid reagents.

13. The preparation method according to claim 12, characterized in that, The described preparation method further includes the step: (S3) Preparing one or more standard products.

14. The preparation method according to claim 13, wherein The described preparation method further includes the step: (S4) Preparing a quality control product.

15. The preparation method according to claim 12, characterized in that Step (S1) includes the steps: (S1.1) Providing a coating solution and a blocking solution containing EGF antigen; (S1.2) Adding the coating solution to some or all of the wells of the ELISA plate for antigen coating, and removing the coating solution to obtain an ELISA plate coated with antigen; and (S1.3) Adding the blocking solution to some or all of the wells of the ELISA plate coated with antigen for blocking, and removing the blocking solution to obtain an ELISA plate coated with EGF antigen.

16. Use of a detection kit as described in claim 1, characterized in that, It is used to detect anti-epidermal growth factor (EGF) antibodies in human serum samples.

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