CN111239410A - Kit for detecting liver cancer, hepatitis and/or liver cirrhosis and application of kit in AKR1B10 and AFP combined quantitative determination - Google Patents
Kit for detecting liver cancer, hepatitis and/or liver cirrhosis and application of kit in AKR1B10 and AFP combined quantitative determination Download PDFInfo
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Abstract
The invention belongs to the technical field of medical biology, and discloses a determination kit for simultaneously detecting the contents of aldo-keto reductase 1B10 and alpha fetoprotein by using a time-resolved fluorescence immunoassay method, which can be used for screening, diagnosing, judging curative effect, evaluating prognosis or monitoring relapse of diseases such as liver cancer, hepatitis, liver cirrhosis and the like. The kit comprises: an enzyme label plate, a calibrator, an AKR1B10 detection antibody marked by europium, an AFP detection antibody marked by terbium and an enhancement solution. AKR1B10 and AFP in serum to be detected are respectively combined with AKR1B10 and AFP coating antibodies of an enzyme label plate, two detection antibodies are added to form a double-antibody sandwich compound, and enhancement liquid is added to dissociate europium ions and terbium ions on the compound and respectively emit high-intensity fluorescence at the wavelengths of 340nm and 295 nm. The fluorescence intensity is directly proportional to the concentration of AKR1B10 and AFP in the sample. The kit can be used for simultaneously detecting the contents of two markers, and greatly improves the sensitivity and specificity of detection.
Description
Technical Field
The invention relates to the technical field of medical biology, in particular to a kit for jointly detecting the contents of aldehyde ketone reductase 1B10 (AKR 1B 10) and Alpha Fetoprotein (AFP) by using a time-resolved fluorescence immunoassay method, which can be used for screening, diagnosing, judging the curative effect, evaluating the prognosis or monitoring the recurrence of liver cancer, hepatitis, cirrhosis and other liver diseases.
Background
In adults, AFP is produced by liver cells, the content of AFP in serum is very small, but the function of producing AFP is recovered when the liver cells are cancerated, and the content of AFP in the serum is increased rapidly along with the deterioration of the condition of the liver.
The Aldo-keto Reductase 1B10 (Aldo-keto Reductase 1B10, AKR1B 10) gene is positioned on chromosome 7q33, encodes a protein consisting of 316 amino acids, namely AKR1B10 protein, the molecular weight is 36.02kDa, and the AKR1B10 protein can be secreted from tumor cells into blood circulation through a non-classical secretion pathway mediated by the amino acids, mainly has the function of protecting the cells from carboxyl toxicity damage, and stabilizes acetyl coenzyme A carboxylase α (ACC α) to prevent ACC α from degrading, thereby regulating fatty acid and lipid synthesis and promoting the growth, proliferation and migration of the tumor cells.
A recent article published in Hepatology (2019, 69(6):2489, 2501. doi: 10.1002/hep.30519.Epub 2019 Apr 6) reports that 1244 subjects including liver cancer patients (HCC), Healthy Controls (HC) and liver benign tumor patients (BLT), chronic hepatitis B patients (CHB) and liver cirrhosis patients (LC) were enrolled from three hospitals and divided into exploratory discovery cohorts (203), training cohorts (519) and validation cohorts (522). The detection of AKR1B10 and AFP proteins was analyzed from three cohorts and the results showed: the detection sensitivity of AKR1B10 is 72.7%, the sensitivity of AFP is 65.1%, and when the two indexes are detected together, the sensitivity can reach 87.5%. The validation queue of 522 participants confirmed this result. Therefore, the combination of AKR1B10 and AFP can improve the detection rate and accuracy of HCC, and has wider clinical diagnosis value.
Disclosure of Invention
In view of the above problems, the present inventors have developed a combined assay kit (time-resolved immunofluorescence assay) for detecting the tumor-specific markers aldo-ketoreductase 1B10 (AKR 1B 10) and alpha-fetoprotein (AFP). The kit uses europium ion (Eu)3+) And terbium ion (Tb)3+) Coupled to the AKR1B10 antibody and the AFP antibody, respectively, two different proteins, AKR1B10 and AFP, were detected simultaneously in the same reaction by using different excitation light wavelengths of the two ions. The kit can be used for detecting AKR1B10 and AFP proteins in serum, urine, milk, intestinal juice, stool or tissue samples of a subject, and the kit can be used for simultaneously detecting the contents of two markers, thereby greatly improving the sensitivity and specificity of detection.
Specifically, the present invention relates to the following:
the invention provides a kit for detecting liver cancer, hepatitis and/or cirrhosis, which is characterized by comprising an europium-labeled aldoketoreductase 1B10 detection antibody and a terbium-labeled alpha-fetoprotein detection antibody.
Further, the composition also comprises an enhancement solution which comprises β -NTA and TOPO, more preferably, the enhancement solution is 25 mu mol/L β -NTA, 50 mu mol/L TOPO, 0.1% Triton X-100, 0.5% anhydrous acetic acid, 0.1% potassium hydrogen phthalate and the pH value is 3.0-4.0.
Still further, the kit also comprises aldehyde ketoreductase 1B10 antigen and alpha fetoprotein antigen standard, preferably, the antigen is prepared by dissolving the antigen in a sample buffer solution at a certain concentration, specifically, the aldehyde ketoreductase antigen concentration is 0, 375, 750, 1500, 3000, 6000pg/mL respectively, the alpha fetoprotein antigen concentration is 0, 5, 10, 50, 200, 650ng/mL respectively, the buffer solution is 10mmol/L Tris.HCl (pH7.5 +/-0.1), 1% BSA, 0.05% Tween20, 0.05% Proclin 300.
Still further, a washing solution, such as Tris-HCl, sodium chloride, Tween20 and Proclin300 or a mixed solution thereof, is included in the kit in the form of a concentrated washing solution, and is used after being diluted appropriately at the time of detection. The preparation method of the preferred cleaning solution is as follows: 50mM Tris.HCl (pH 7.5 +/-0.1), 21% sodium chloride and 2.5% Tween20, fully mixing, storing at 2-8 ℃, and diluting by 25 times before use.
Preferably, the europium-labeled aldoketoreductase 1B10 detection antibody is a polyclonal antibody or a monoclonal antibody, preferably a murine anti-monoclonal antibody; the terbium-labeled alpha-fetoprotein detection antibody is a polyclonal antibody or a monoclonal antibody, and is preferably a mouse anti-monoclonal antibody.
In one embodiment, the rabbit anti-aldoketoreductase 1B10 polyclonal antibody and the murine anti-alphafetoprotein monoclonal antibody that recognizes another antigenic site are coated in a microplate.
In a preferred mode, the aldehyde ketone reductase 1B10 detection antibody is prepared by coupling a mouse anti-aldehyde ketone reductase 1B10 monoclonal antibody and europium ions; the alpha-fetoprotein detection antibody is prepared by coupling a mouse anti-alpha-fetoprotein monoclonal antibody and terbium ions.
Preferably, the kit further comprises: sample diluent: 10mmol/L Tris.HCl (pH7.5. + -. 0.1), 1% BSA, 0.05% Tween20, 0.05% Proclin 300. And 25-fold dilution of the following concentrated rinse solutions: 50mM Tris.HCl (pH7.5. + -. 0.1), 21% sodium chloride, 2.5% Tween 20; or the kit may contain the concentrated cleaning solution and may be further diluted by 25 times when used.
The subject to which the kit of the invention can be applied is selected from serum, urine, milk, intestinal fluid, stool or a tissue sample. The detection purpose is selected from screening, diagnosis, curative effect judgment, prognosis evaluation or relapse monitoring of liver cancer, hepatitis and cirrhosis.
The invention also provides application of the kit in vitro quantitative detection of the aldoketoreductase 1B10 and the alpha fetoprotein for non-diagnostic purposes, such as the condition that the detection result is not applicable to a subject from which a sample is derived, such as unknown blood and the like.
The reagent components (such as some necessary buffers) not described in detail in the kit of the present invention, the external package of the kit, and the independent packaging containers for each reagent component can be performed according to the conventional operations in the field, and the requirements are met.
The process steps not described in detail in the process of the invention can also be carried out with reference to conventional operations in the art. For example, before detection, each reagent component can be placed at room temperature (18-25 ℃) for balance, and fully and uniformly mixed before sample addition, and the like; the use of the instrument, such as a time-resolved immunofluorescence analyzer, was performed according to the instructions.
The AKR1B10 and AFP determination kit provided by the invention has the following advantages:
a) the invention uses AKR1B10 antibody and AFP antibody to coat the enzyme label plate, and uses europium ion (Eu)3+) Labelling of the AKR1B10 detection antibody, terbium ion (Tb)3+) An AFP detection antibody is marked, and specific immunological combination is generated with two antigens of AKR1B10 and AFP;
b) the invention can simultaneously determine the contents of AKR1B10 and AFP, greatly improves the sensitivity and specificity of clinical diagnosis, can also greatly reduce the detection time and cost, and has no similar products clinically at present;
c) the invention adopts a time-resolved immunofluorescence analysis method, reduces the background value to a negligible degree through time delay and wavelength resolution, and has the characteristics of higher signal-to-noise ratio, strong fluorescence signal, wide linear range, simple and convenient operation and the like;
d) the kit has good stability and the effective period can reach more than one year.
Drawings
FIG. 1 is a linear graph of the calibration curve of the kit of the present invention (the left graph is the AKR1B10 antigen standard curve; the right graph is the AFP antigen).
FIG. 2 shows that the kit of the invention can detect the content of AKR1B10 and AFP protein in the serum of normal human and liver cancer patients (the left figure is AKR1B 10; the right figure is AFP).
FIG. 3 shows that the kit of the present invention can detect the content of AKR1B10 and AFP protein in the serum of normal human and hepatitis patients (the left figure is AKR1B 10; the right figure is AFP).
FIG. 4 is a diagram showing the test kit of the present invention for measuring the content of AKR1B10 and AFP protein in the serum of normal persons and liver cirrhosis patients (AKR 1B10 on the left side; AFP on the right side).
Detailed description of the invention
The present invention provides an aldehyde ketone reductase 1B10 (AKR 1B 10) and Alpha Fetoprotein (AFP) assay kit (time-resolved immunofluorescence assay), the specific examples are provided only to more clearly illustrate the present invention, and the present invention is further described with reference to the following examples. The examples are for illustration only and do not limit the invention in any way. Some of the reagent materials used in the examples are commercially available (AKR 1B10 detection antibody is a patent product of this company, patent No. ZL 20141057977.0, and grant No. CN 104650234B), the equipment used is also the existing equipment in the field, and the experimental methods without specific conditions are all conventional methods and conditions well known in the field.
Example 1 preparation of AKR1B10 and AFP assay kit.
The preparation method of the coated enzyme label plate comprises the following steps:
preparing a mixed solution of 5ug/mL rabbit anti-AKR 1B10 polyclonal antibody and 10ug/mL mouse anti-AFP monoclonal antibody by using 10mmol/L Tris.HCl (pH7.5 +/-0.1) coating buffer solution, injecting 200uL of mixed solution into each pore of an enzyme label plate, placing the mixed solution at the temperature of 2-8 ℃ for coating reaction for 12-15 hours, and then discarding the coated mixed solution. Injecting 400uL of blocking buffer solution of 10mmol/L Tris.HCl (pH7.5 +/-0.1) and 1% BSA into the micropores of the ELISA plate, placing the plate at 37 ℃ for blocking reaction for 2 hours, then removing the blocking buffer solution, and naturally drying.
Secondly, the preparation method of the calibrator mixed solution is as follows:
the sample is prepared by dissolving AKR1B10 antigen and AFP antigen in a sample buffer solution at a certain concentration. The work concentrations of AKR1B10 are respectively 0, 375, 750, 1500, 3000 and 6000pg/mL, the work concentrations of AFP are respectively 0, 5, 10, 50, 200 and 650ng/mL, the buffer solution is 10mmol/L Tris.HCl (pH7.5 +/-0.1), 1% BSA, 0.05% Tween20 and 0.05% Proclin 300.
Thirdly, the preparation method of the AKR1B10 detection antibody is as follows:
mixing mouse anti-AKR 1B10 monoclonal antibody with europium ion (Eu)3+) Coupling to prepare the AKR1B10 detection antibody.
(1) The murine anti-AKR 1B10 monoclonal antibody to be labeled was replaced by desalting column into a solution of 10 mmol/LTris. HCl (pH 7.5. + -. 0.1).
(2) Mixing the antibody obtained in step (1) with 0.2mg of europium ion (Eu)3+) Mixing, and reacting for 12-15 hours at 2-8 ℃ by gentle oscillation.
(3) Then, the labeled antibody solution was replaced with a solution of 10mmol/L Tris.HCl (pH 7.5. + -. 0.1), 1% BSA, 0.05% Tween20, 0.05% Proclin300 in a desalting column to remove free europium ions (Eu)3+) And storing at-80 ℃ to prepare the required AKR1B10 detection antibody.
Fourthly, the preparation method of the AFP detection antibody comprises the following steps:
the murine anti-AFP monoclonal antibody was conjugated to terbium ion (Tb)3+) Coupling to obtain AFP detecting antibody.
(1) The mouse anti-AFP monoclonal antibody to be marked is replaced by a desalting column into a Tris.HCl (pH7.5 +/-0.1) solution with the buffer solution of 10 mmol/L.
(2) The antibody of step (1) was mixed with 0.2mg terbium ion (Tb)3+) Mixing, and reacting for 12-15 hours at 2-8 ℃ by gentle oscillation.
(3) Then the labeled antibody solution is replaced by a desalting column into a solution with the buffer of 10mmol/L Tris.HCl (pH7.5 +/-0.1), 1% BSA, 0.05% Tween20 and 0.05% Proclin300, and free terbium ions (Tb) are removed3+) And storing at-80 ℃ to prepare the required AFP detection antibody.
Fifthly, the preparation method of the enhancing liquid comprises the following steps:
the enhancing solution is prepared by dissolving 25 μmol/L β -NTA, 50 μmol/L TOPO, 0.1% Triton X-100, 0.5% anhydrous acetic acid, and 0.1% potassium hydrogen phthalate in purified water, and adjusting pH to 3.0-4.0.
The AKR1B10 and AFP determination kit also comprises a cleaning solution, wherein the cleaning solution is mainly used for cleaning after each reaction step in the detection process, and the specific components of the cleaning solution can be carried out by referring to the conventional operation in the field. Usually Tris-HCl, NaCl, Tween20 and Proclin300 or their mixture, and in the form of concentrated washing solution, and is diluted appropriately for detection. The preparation method of the preferred cleaning solution is as follows: 50mM Tris.HCl (pH 7.5 +/-0.1), 21% sodium chloride and 2.5% Tween20, fully mixing, storing at 2-8 ℃, and diluting by 25 times before use.
Example 2 use of AKR1B10 and AFP assay kit to establish a calibration curve
The calibration curve is established as follows:
(1) respectively adding 100ul of calibrator into the micropores of the ELISA plate, performing oscillation incubation for 1 hour at room temperature, adding 300ul of cleaning solution into each hole, and repeatedly cleaning for 3 times;
(2) simultaneously adding 100ul of AKR1B10 detection antibody and 100ul of AFP detection antibody into the micropores of the ELISA plate, oscillating and incubating for 1 hour at room temperature, adding 300ul of cleaning solution into each hole, and repeatedly cleaning for 3 times;
(3) adding 200ul of enhancing solution into each well, and incubating for 5 minutes at room temperature in a shaking way;
(4) respectively detecting the fluorescence value of europium ions in the wavelength of 340nm and the fluorescence value of terbium ions in the wavelength of 295nm by using a time-resolved immunofluorescence analyzer;
(5) and (3) data analysis:
the standard curve was obtained by four-parameter linear fitting of the concentration values and luminescence values of the calibrator (see fig. 1). Wherein the abscissa represents the concentration value of the calibrator and the ordinate represents the luminescence value of the calibrator, AKR1B10 calibrator (R)2= 0.9966); AFP calibrator (R)2=0.9983）。
Example 3 detection of the content of AKR1B10 and AFP proteins in serum samples of Normal human and liver cancer patients Using AKR1B10 and AFP assay kit
(1) Respectively adding 100ul of serum samples to be detected into micropores of an enzyme label plate, oscillating and incubating for 1 hour at room temperature, adding 300ul of cleaning solution into each hole, and repeatedly cleaning for 3 times;
(2) simultaneously adding 100ul of AKR1B10 detection antibody and 100ul of AFP detection antibody into the micropores of the ELISA plate, oscillating and incubating for 1 hour at room temperature, adding 300ul of cleaning solution into each hole, and repeatedly cleaning for 3 times;
(3) adding 200ul of enhancing solution into each well, and incubating for 5 minutes at room temperature in a shaking way;
(4) respectively detecting the fluorescence value of europium ions in the wavelength of 340nm and the fluorescence value of terbium ions in the wavelength of 295nm by using a time-resolved immunofluorescence analyzer;
(5) and (3) data analysis:
selecting serum samples of 30 healthy normal persons and 126 liver cancer patients for detection, obtaining a standard curve by linearly fitting concentration values and fluorescence values of a calibrator through four parameters, calculating the contents of AKR1B10 and AFP proteins in the serum samples of the subjects, and analyzing the results by Graphpad 4.0 statistical software. the results of the t-test showed that the mean levels of AKR1B10 and AFP protein were statistically significantly different (p < 0.0001) in both groups (AKR 1B10 expression levels were 68.68 + -12.16 pg/mL and 1130 + -127.3 pg/mL in healthy normal persons and liver cancer patients, respectively; AFP expression levels were 3.924 + -0.3208 ng/mL and 298 + -57.51 ng/mL in healthy normal persons and liver cancer patients, respectively; see FIG. 2).
Example 4 measurement of AKR1B10 and AFP assay kits for the determination of the levels of AKR1B10 and AFP proteins in serum samples from Normal and hepatitis patients
The serum samples were tested by the same method as in example 3, and serum samples of 35 healthy normal persons and 59 hepatitis patients were tested, and a standard curve was obtained by four-parameter linear fitting of the concentration and fluorescence values of the calibrator, and the levels of AKR1B10 and AFP protein in the serum samples of the subjects were calculated, and the results were analyzed by Graphpad 4.0 statistical software. the results of the t-test showed significant statistical differences in the mean levels of AKR1B10 and AFP protein in both groups (p < 0.0001) (AKR 1B10 expression levels in healthy normal and hepatitis patients of 46.98 + -7.403 pg/mL and 179.9 + -7.403 pg/mL, respectively; AFP expression levels in healthy normal and hepatitis patients of 3.525 + -0.305 ng/mL and 41.67 + -5.169 ng/mL, respectively; see FIG. 3).
Example 5 AKR1B10 and AFP assay kit for determining the levels of AKR1B10 and AFP proteins in serum samples from normal and cirrhosis patients
The serum samples were tested by the same method as in example 3, 46 healthy and 77 patients with liver cirrhosis were selected for testing, a standard curve was obtained by four-parameter linear fitting of the concentration and fluorescence values of the calibrator, the AKR1B10 and AFP protein content in the serum samples of each subject was calculated, and the results were analyzed by Graphpad 4.0 statistical software. the results of the t-test showed a significant statistical difference in the mean levels of AKR1B10 and AFP protein in both groups (p < 0.0001) (66.03. + -. 7.326pg/mL and 263.1. + -. 25.34pg/mL for AKR1B10 in healthy normal and cirrhosis patients, respectively; 5.639. + -. 1.04ng/mL and 72.46. + -. 7.59ng/mL for AFP in healthy normal and cirrhosis patients, respectively; see FIG. 4).
Claims (10)
1. A kit for detecting liver cancer, hepatitis and/or cirrhosis is characterized by comprising an europium-labeled aldoketoreductase 1B10 detection antibody and a terbium-labeled alpha-fetoprotein detection antibody.
2. The kit of claim 1, further comprising an enhancing solution comprising β -NTA and TOPO, more preferably 25 μmol/L β -NTA, 50 μmol/L TOPO, 0.1% Triton X-100, 0.5% anhydrous acetic acid, 0.1% potassium hydrogen phthalate, pH 3.0-4.0, and a sample diluent comprising 10mmol/L Tris.HCl, 1% BSA, 0.05% Tween20, 0.05% Proclin300, pH7.5 ± 0.1.
3. The kit of claim 1, further comprising standards for aldehyde ketoreductase 1B10 antigen and alpha fetoprotein antigen, preferably, the antigens are dissolved in a sample buffer at a concentration, specifically, the concentrations of aldehyde ketoreductase 1B10 antigen are 0, 375, 750, 1500, 3000, 6000pg/mL, the concentrations of alpha fetoprotein antigen are 0, 5, 10, 50, 200, 650ng/mL, the buffer is 10mmol/L tris.hcl (ph 7.5 ± 0.1), 1% BSA, 0.05% Tween20, 0.05% Proclin 300.
4. The kit of claim 1, further comprising a washing solution, such as Tris-HCl, sodium chloride, Tween20 and Proclin300 or a mixture thereof, in the form of a concentrated washing solution, which is diluted appropriately for use in the assay;
the preparation method of the preferred cleaning solution is as follows: 50mM Tris.HCl (pH7.5 +/-0.1), 21% sodium chloride and 2.5% Tween20, fully mixing, storing at 2-8 ℃, and diluting by 25 times before use.
5. The kit of claim 1, wherein: the europium-labeled aldoketoreductase 1B10 detection antibody is a polyclonal antibody or a monoclonal antibody, preferably a mouse-anti-monoclonal antibody; the terbium-labeled alpha-fetoprotein detection antibody is a polyclonal antibody or a monoclonal antibody, and is preferably a mouse anti-monoclonal antibody.
6. The kit of claim 1, wherein: the rabbit anti-aldo-keto reductase 1B10 polyclonal antibody and the mouse anti-alpha fetoprotein monoclonal antibody recognizing another antigenic site are coated in a microplate.
7. The kit according to claim 5, wherein: the aldehyde ketone reductase 1B10 detection antibody is prepared by coupling a mouse anti-aldehyde ketone reductase 1B10 monoclonal antibody and europium ions; the alpha-fetoprotein detection antibody is prepared by coupling a mouse anti-alpha-fetoprotein monoclonal antibody and terbium ions.
8. A kit according to any one of claims 1 to 7, wherein the subject is selected from serum, urine, milk, intestinal fluid, stool or a tissue sample.
9. The kit of any one of claims 1 to 7, wherein the use of detection is selected from screening, diagnosis, judgment of efficacy, prognosis evaluation or recurrence monitoring of liver cancer, hepatitis and cirrhosis.
10. A kit according to any one of claims 1 to 8 for use in the quantitative determination of the levels of aldoketoreductase 1B10 and alpha fetoprotein for non-diagnostic purposes in vitro.
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| CN202010062548.4A CN111239410A (en) | 2020-01-19 | 2020-01-19 | Kit for detecting liver cancer, hepatitis and/or liver cirrhosis and application of kit in AKR1B10 and AFP combined quantitative determination |
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