CN109884316B - Kit for detecting tumor M2 type pyruvate kinase and preparation method thereof - Google Patents
Kit for detecting tumor M2 type pyruvate kinase and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kit for detecting tumor M2 pyruvate kinase and a preparation method thereof, wherein the kit comprises a test paper card, the test paper card comprises a bottom plate, the bottom plate is provided with a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, the ends of the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially connected along the flow direction of a sample, the combination pad adsorbs a capture antibody marked by quantum dots, the nitrocellulose membrane is sequentially provided with a detection belt and a quality control belt along the flow direction of the sample, the detection belt is coated with a detection antibody, and the quality control belt is coated with IgG; the capture antibody and the detection antibody are anti-tumor M2 pyruvate kinase antibodies, and the capture antibody and the detection antibody are combined with different epitopes of tumor M2 pyruvate kinase; and modifying the capture antibody by adopting monophosphoryl sugar before labeling the quantum dots. The kit has high sensitivity and specificity, and is quick, simple and convenient to operate, accurate in result, economical and applicable.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnosis, and particularly relates to a kit for detecting tumor M2 pyruvate kinase and a preparation method thereof.
Background
Colorectal cancer is one of three common malignant tumors in the world, and the death rate of the colorectal cancer is the third place. In recent years, the incidence of diseases is on a rising trend with the improvement of living standard and the change of dietary structure. However, early diagnosis of colorectal cancer is still at a low level at home and abroad, and about half of patients enter colorectal cancer at the middle stage and the late stage when being diagnosed definitely. Therefore, improving the early diagnosis of colorectal cancer is a key problem which needs to be solved urgently at home and abroad at present. There are several common techniques for early diagnosis of colorectal cancer: (1) the fecal occult blood test is a common screening method for colorectal cancer, is simple and convenient to operate and noninvasive, and has the main defects of low sensitivity, poor specificity and high false positive result; (2) the electronic colonoscope (endoscope for short) is the most effective means for discovering and diagnosing the colorectal cancer, is also the main method for diagnosing the colorectal cancer at the early stage at present, and has important clinical value particularly in the inspection of high risk groups; since it is a traumatic examinationThe method has the advantages that dangerous side effects such as bleeding and intestinal perforation are accompanied, certain equipment and instruments are needed, the requirement on the professional level of operators is high, and therefore, the method has great limitation on the examination of a large range of people; (3) the electrochemical luminescence technology detects common tumor markers such as CEA, CA199, CA242 and CA50, and is not suitable for early diagnosis of colorectal cancer due to low positive rate and lack of specificity; (4) the PCR technology for detecting the DNA in the serum or the feces of the patient and the concentration of the microRNA have certain values for the treatment monitoring and the prognosis evaluation of the colorectal cancer, but the PCR technology and the microRNA have to be further researched in the aspect of being applied to the early diagnosis of the colorectal cancer. In recent years, researchers at home and abroad are dedicated to searching for a new sensitive, specific, reliable and effective colorectal cancer biomarker and establishing noninvasive detection and experimental methods to improve the diagnosis rate of early diagnosis of colorectal cancer. Foreign scholars believe tumor M2Pyruvate kinase type (M)2PK) has higher sensitivity and specificity in plasma and excrement detection, and is expected to become a novel tumor marker for colorectal cancer.
Pyruvate kinase has 4 different isozymes, L type, R type, M1 type and M2 type, and the distribution of the 4 isozymes has tissue specificity. M2-PK is mainly distributed in lung tissues, renal distal convoluted tubules, embryonic tissues and proliferative or undifferentiated tissues, and exists in a tetrameric form with high activity under a normal cell activity state. In tumor cells, it has been demonstrated that tetrameric cleavage, conversion to predominantly dimeric form, and high expression due to tyrosine receptor phosphorylation occurs in tumor cells, and thus, elevation of tomor M2-PK, tomor M2-PK, is seen in patients with most malignancies. Although tumors derived from different tissues and tumors at different stages show differences in the expression level of tomor M2-PK, tomor M2-PK is very useful as a tumor marker. Tumor M2-PK is a novel tumor marker which is researched more in recent years, and has a good application prospect in early diagnosis and prognosis judgment of malignant tumors.
Human colorectal epithelium is renewed very rapidly, approximately 1% per hour, with approximately 10 days per day10Normal epithelial cells are exfoliated. 1 piece contains 1091cm size tumor tissue of cells if renewed at a normal rate, at least 1% of the total colorectal epithelial cells shed into the intestinal tract are derived from the tumor tissue. The tumor cells are dropped into the intestinal cavity and are discharged out of the body along with the excrement, and a foundation is laid for the detection of the dropping cell mutant gene and the expression product thereof in the excrement. The intestinal cavity-oriented tumor has a great deal of decomposition of endocrine metabolites or substances peculiar to certain tumors and fallen tumor cells to release intracellular substances, and the feces prevent the tumor marker from being metabolized and decomposed by the liver, so that the detection of the tumor marker by the feces is feasible. Therefore, the diagnosis of colorectal cancer by detecting the level of tomor M2-PK in feces is an objective and reliable experimental method.
As early as 1993, tomor M2-PK was proposed as a novel tumor marker at the seventh tumor marker seminar in Germany. Later in Germany and other European countries, the tomorM 2-PK kit and fecal occult blood are applied every year as auxiliary diagnosis for early screening of colorectal cancer. In recent years, researchers at home and abroad research tunor M2-PK, and an imported German enzyme-linked immunosorbent assay (ELISA) shows that tunor M2-PK has higher sensitivity and specificity in colorectal cancer diagnosis, namely 73% and 78% respectively, and the tunor M2-PK in feces is considered as a very ideal colorectal tumor marker. At present, the foreign scholars believe that the sensitivity of tumor M2-PK in feces is higher than that in serum, probably because the tumor decomposes tumor cells with necrosis on the surface of the tumor under the premise that the tumor secretes Tumor M2-PK into the blood circulation and the intestinal cavity simultaneously to release Tumor M2-PK, and when the tumor does not grow to a certain extent, the amount of Tumor M2-PK secreted into the blood circulation by the tumor is smaller, while the amount of Tumor M2-PK in the intestinal cavity is more than that of Tumor M2-PK in the blood circulation, which results in the detection of the content of Tumor M2-PK in feces which is significantly higher than that in the blood circulation.
Chinese invention patent 200610061296.3 discloses human tumor M2 pyruvate kinase antigenic determinant polypeptide, antibody and application thereof in diagnostic kit, which prepares two different epitope tunor M2-PK polyclonal antibodies by using two different source antigenic polypeptides, wherein one of them is used as coating antibody; combining the Tumor M2-PK in the sample with Tumor M2-PK specific polyclonal antibody coated on an enzyme label plate, adding an enzyme labeled secondary antibody, incubating for 30 minutes, washing the plate, fixing a fluorescent signal on the enzyme label plate, and detecting the fluorescence intensity by a fluorescence enzyme labeling instrument to determine the concentration of the Tumor M2-PK. However, the method requires the coating and sealing of an enzyme label plate, the treatment of a standard substance or detected serum, and the detection of a polyclonal antibody, and has the disadvantages of long incubation time, multiple plate washing times, and the need of matched professional equipment and professionals in the detection process.
Chinese invention patent CN106405094A discloses a chemiluminescence immunoassay kit for detecting tumor type M2 pyruvate kinase, which comprises reagents of tumor M2-PK magnetic separation reagent, enzyme-labeled tumor M2-PK antibody solution, standard, washing solution and chemiluminescence substrate solution (divided into a luminescence substrate A solution and a luminescence substrate B solution), wherein the luminescence substrate A is Tris-HCl buffer solution with the value of 0.1M, pH of 8.5, the buffer solution contains luminol with the final concentration of 5.0mg/mL, the luminescence substrate B is 0.1M citric acid buffer solution with the pH value of 4.5, and the buffer solution contains hydrogen peroxide with the final concentration of 100mg/mL and horseradish catalase with the final concentration of 15 mg/mL; the method realizes quantitative detection of tomor M2-PK by using the principle of a double-antibody sandwich method. However, this method requires expensive chemiluminescent analyzers and specialized personnel to be operationally equipped, and is only capable of detecting serum or plasma samples, and is not capable of detecting secretions, including urine and feces.
Therefore, how to develop a Tumor M2-PK detection method and detection products with short detection time, convenient operation and accurate detection result becomes a problem to be solved urgently.
The currently developed tomor M2-PK kit at home and abroad adopts an enzyme immunization method, the method has multiple detection steps, more influencing factors are generated in the operation process, the detection result is easy to cause deviation, and the method is time-consuming and labor-consuming. Although quantum dot biological labeling has been reported, quantum dot coupled antibodies and purification have the disadvantages of complex operation, low recovery rate, difficulty in realizing batch detection and scale and the like. Therefore, how to apply the quantum dot technology to the tumor M2 pyruvate kinase and detect the tumor with high efficiency and simplicity is an urgent problem to be solved.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a kit for detecting tumor M2 pyruvate kinase and a preparation method thereof, the kit adopts monophosphoryl sugar allosterically activates the surface of a capture antibody, enhances the combination of the capture antibody and a quantum dot and couples the tumor M2 pyruvate kinase, the kit provided by the invention firstly applies the capture antibody marked by the quantum dot to the detection of the tumor M2 pyruvate kinase, and the kit has high sensitivity and specificity, is quick and simple to operate, has accurate result, and is economical and applicable.
In order to achieve the purpose, the invention adopts the technical scheme that: a test paper card for detecting tumor M2 pyruvate kinase comprises a bottom plate, wherein the bottom plate is provided with a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, the ends of the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially connected along the flow direction of a sample, the combination pad adsorbs a capture antibody marked by a quantum dot, the nitrocellulose membrane is sequentially provided with a detection band and a quality control band along the flow direction of the sample, the detection band is coated with a detection antibody, and the quality control band is coated with IgG (preferably goat anti mouse IgG);
the capture antibody is an anti-tumor M2 pyruvate kinase antibody, and the surface of the capture antibody is modified by monophosphoryl sugar before the capture antibody is labeled with quantum dots; the detection antibody is an anti-tumor M2 pyruvate kinase antibody, and the capture antibody and the detection antibody are combined with different epitopes of tumor M2 pyruvate kinase;
the monophosphoryl sugar modified capture antibody is prepared by the following method: adding a monophosphoryl sugar and bovine serum albumin buffer solution into the capture antibody, shaking uniformly, and standing at room temperature for 1-2 h; then adjusting the pH value of the solution to 8-11; preferably, 200-500 mg of monophosphoryl sugar and 200-800 mg/mL of bovine serum albumin buffer solution are added into 300-800 mg of capture antibody, the capture antibody is shaken uniformly and then placed at room temperature for 1-2 hours, and preparation is made for adding the activated quantum dots: after monophosphoryl is added into the capture antibody, glycosylation modification is carried out on the N-terminal of the capture antibody, namely glycosylation modification is carried out on an Fc region, so that on one hand, the activity of the N-terminal of the antibody is enhanced, and the N-terminal of the antibody is combined with the carboxyl of the quantum dot; on the other hand, one or more aldehyde functional groups are generated on the surface of the antibody, and the aldehyde group is coupled with the hydrazide reaction of the quantum dot, so that the stability is greatly improved.
Preferably, the capture antibody and the detection antibody are two hybridoma cell lines for secreting the antibodies obtained by cell cloning of recombinant human tumor M2-PK antigen, namely an M2 type pyruvate kinase 33.26 hybridoma cell line and an M2 type pyruvate kinase 31.3 hybridoma cell line; the gene recombinant tulor M2-PK protein is prepared by adopting an engineering strain of escherichia coli carrying a tulor M2-PK protein gene, and the preparation is carried out according to a conventional method.
In addition, the invention also provides a preparation method of the test paper card, which comprises the following steps: the combination pad is sprayed with 0.1-5 mg/mL quantum dot marked capture antibody solution according to 1-3 muL/cm, the detection band is sprayed with 0.1-5 mg/mL detection antibody solution according to 1-3 muL/cm, and the quality control band is sprayed with 0.1-5 mg/mL IgG solution according to 1-3 muL/cm; the solvents of the capture antibody solution, the detection antibody solution and the IgG solution marked by the quantum dots are coating buffer solutions, and the coating buffer solutions comprise 20-150 mM NaCl, 0.05-3% PEG, 0.2-1% trehalose, 2-10 mg/mL BSA, and 0.05% NaN310 to 100mM PBS buffer solution.
As an improvement of the above technical solution, the quantum dot-labeled capture antibody is prepared by the following steps:
s1) quantum dot activation: activating carboxyl on the surface of the CdSe/ZnS quantum dot by adopting N-hydroxyl thiosuccinimide (carboxyl water-soluble CdSe/ZnS quantum dot); in the next reaction, under the action of EDC, carboxyl of the activated quantum dots and original amino on the surface of the capture antibody form amido bond;
s2) modifying the surface of the capture antibody by adopting monophosphoryl sugar to obtain an activated capture antibody; before the quantum dots are successfully coupled with the capture antibody, the surface structure of the capture antibody is modified by applying the allosteric activation of monophosphoryl sugar, so that the activity of the capture antibody is improved, the function of the N-terminal structure of the capture antibody is enhanced, and the binding with the quantum dots and the coupling of tumor M2 pyruvate kinase are facilitated;
s3) placing the activated quantum dots to room temperature, adding an EDC solution, an N-hydroxy sulpho succinimide solution and a BSA solution into the activated quantum dots, shaking up, adding the activated capture antibody, shaking up, incubating, adding the EDC solution and the N-hydroxy sulpho succinimide solution during incubating, adding methanol after incubating, mixing, oscillating in a dark place, adding β -mercaptoethanol for termination, dialyzing by β -mercaptoethanol after termination of reaction, and centrifuging after dialysis to remove supernatant, thereby obtaining the capture antibody marked by the quantum dots.
As a further improvement of the above technical solution, in step S1, the concentration of the activated quantum dots is 1 to 10 μ M; in step S2, the concentration of the capture antibody is 10-100 mug/ml; in step S3, the BSA solution has a concentration of 20-200 mg/mL and the mass ratio of the activated quantum dots to the activated capture antibody is (1:2) - (1: 10).
As a further improvement of the above technical solution, in step S3, when the activated quantum dot is coupled with the activated capture antibody, the pH value of the solution is 9-11, the concentration of the EDC solution is 9.38% (w/v), the concentration of the N-hydroxy thiosuccinimide solution is 10% (w/v), the mass ratio of the activated quantum dot to the EDC added twice is 5-10, and the mass ratio of the activated quantum dot to the N-hydroxy thiosuccinimide added twice is 5-10.
As an improvement of the technical scheme, the combination pad is sprayed with 0.1-5 mg/mL quantum dot-labeled capture antibody solution according to 2 muL/cm, the detection band is sprayed with 0.1-5 mg/mL detection antibody solution according to 2 muL/cm, and the quality control band is sprayed with 0.1-5 mg/mL IgG solution according to 2 muL/cm.
Preferably, the interval between the detection belt and the control belt is 5-7 mm, and after spraying, the detection belt and the control belt are placed in a constant-temperature constant-humidity preservation box for standby after being dried for 24 hours under the conditions that the humidity is less than 30% and the temperature is 35-37 ℃.
As an improvement of the technical scheme, the sample pad is soaked by adopting pretreatment liquid and then dried; before spraying, the bonding pad is soaked in a pretreatment solution and then dried; the pretreatment solution is 0.01M PBS buffer solution containing 0.02-0.1% Tween-20 and having a pH of 7-7.3; the pretreatment solution has good adaptability to various antigens and antibodies.
Preferably, the step of pre-treating the sample pad and conjugate pad is: soaking the pretreatment liquid for 1-2 h, taking out, drying at 36-38 ℃ or vacuum freeze-drying at 37 ℃, and storing for later use. The absorbent pad is made of absorbent cotton; the bottom plate is a PVCJ rubber plate and is used for connecting the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad together.
In addition, the invention also provides a kit containing the test paper card, the kit also comprises a card shell, the card shell comprises a back card and an upper cover, the back card is provided with a clamping groove, the test paper card is embedded in the clamping groove, the upper cover is provided with a test window and a sample adding hole, the position of the test window is matched with the positions of the detection belt and the quality control belt, and the position of the sample adding hole is matched with the position of the sample pad.
As an improvement of the technical scheme, the kit further comprises a sample separation buffer solution, wherein the sample separation buffer solution is a PBS buffer solution containing a medium, 0.45-0.9% of sodium chloride, 0.02-0.1% of Tween-20 and 20-200 mg/mL of bovine serum albumin; when the medium is cesium chloride, the concentration of the cesium chloride in the sample separation buffer solution is 10% -30%; when the medium is sucrose, the concentration of the sucrose in the sample separation buffer solution is 0.1-0.5 mol/L; when the medium is polysucrose, the concentration of the polysucrose in the sample separation buffer solution is 10-30% (w/v).
In addition, the invention also provides a quantitative detection method of the tumor M2 pyruvate kinase: preparing a series of tumor M2 type pyruvate kinase standard solutions (with the concentration of more than 5) with different concentrations, respectively carrying out immunochromatography detection by using a quantum dot fluorescence quantitative analyzer, making a standard curve for the corresponding concentration of each peak area, carrying out the same treatment on an unknown sample to be detected to obtain the peak area, and obtaining the content of the tumor M2 type pyruvate kinase in the sample according to a standard curve formula.
The invention has the beneficial effects that: the invention provides a kit for detecting tumor M2 pyruvate kinase and a preparation method thereof, the invention detects tumor M2 pyruvate kinase in feces by quantum dot marking and combining with fluorescence immunochromatography for the first time, monophosphoryl sugar is adopted before a capture antibody is marked by the quantum dot to modify the surface structure of the capture antibody, which is beneficial to combining with the quantum dot and coupling with the tumor M2 pyruvate kinase; the invention adopts the quantum dot marking technology and has the following advantages:
1) the sensitivity and specificity of the fluorescent quantum dot are superior to those of other organic fluorescent markers, such as fluorescein or fluorescent microspheres and the like, and the fluorescent quantum dot is determined by unique excellent optical characteristics and has the characteristics of strong fluorescence intensity, long fluorescence life and the like; the quantum dot labeling technology is combined with the immunochromatography technology, the antigen content in a sample is reflected through the change of fluorescence intensity, and the quantitative detection of the sample is realized; the existing colloidal gold test strip can only carry out qualitative detection;
2) the detection result has high specificity and sensitivity: the specificity and the sensitivity of detection are greatly improved, the detection result is objective and sensitive, and the sensitivity can reach 100-1000 times of that of the traditional colloidal gold test strip;
the detection kit has wide application prospect in early auxiliary diagnosis of CRC, and is particularly suitable for use in primary hospitals and physical examination screening diagnosis of middle and high risk groups.
Drawings
FIG. 1 is an axial view of a test card according to the present invention; the kit comprises a base plate 1, a bottom plate 2, a sample pad 3, a combination pad 4, a detection zone 5, a quality control zone 6, a nitrocellulose membrane 7, a water absorption pad 8 and a chromatography direction;
fig. 2 is a diagram showing the formation process of a quantum dot-labeled capture antibody.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the following detailed description and accompanying drawings.
The invention provides a test paper card (shown in figure 1) for detecting tumor M2 pyruvate kinase, which comprises a bottom plate, wherein the bottom plate is provided with a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, the ends of the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially connected along the flow direction of a sample, the combination pad adsorbs capture antibodies marked by quantum dots, the nitrocellulose membrane is sequentially provided with a detection belt and a quality control belt along the flow direction of the sample, the detection belt is coated with detection antibodies, and the quality control belt is coated with IgG;
the capture antibody is an anti-tumor M2 pyruvate kinase antibody, and the surface of the capture antibody is modified by monophosphoryl sugar before the capture antibody is labeled with quantum dots; the detection antibody is an anti-tumor M2 pyruvate kinase antibody, and the capture antibody and the detection antibody are combined with different epitopes of tumor M2 pyruvate kinase.
Preferably, the reagent card is prepared by the following steps: the combination pad is sprayed with 0.1-5 mg/mL quantum dot marked capture antibody solution according to 1-3 muL/cm, the detection band is sprayed with 0.1-5 mg/mL detection antibody solution according to 1-3 muL/cm, and the quality control band is sprayed with 0.1-5 mg/mL IgG solution according to 1-3 muL/cm; the solvents of the capture antibody solution, the detection antibody solution and the IgG solution marked by the quantum dots are coating buffer solutions, and the coating buffer solutions comprise 20-150 mM NaCl, 0.05-3% PEG, 0.2-1% trehalose, 2-10 mg/mL BSA, and 0.05% NaN310 to 100mM PBS buffer.
Preferably, the quantum dot-labeled capture antibody is prepared by the following steps (as shown in fig. 2):
s1) quantum dot activation: activating carboxyl on the surface of the CdSe/ZnS quantum dot by adopting N-hydroxyl thiosuccinimide;
s2) modification of the capture antibody with a monophosphoryl sugar: adding a monophosphoryl sugar and bovine serum albumin buffer solution into the capture antibody, shaking uniformly, and standing at room temperature for 1-2 h; adjusting the pH value of the solution to 8-11 to obtain an activated capture antibody;
s3) placing the activated quantum dots to room temperature, adding an EDC solution, an N-hydroxy sulpho succinimide solution and a BSA solution into the activated quantum dots, shaking up, adding the activated capture antibody, shaking up, incubating, adding the EDC solution and the N-hydroxy sulpho succinimide solution during incubating, adding methanol after incubating, mixing, oscillating in a dark place, adding β -mercaptoethanol for termination, dialyzing by β -mercaptoethanol after termination of reaction, and centrifuging after dialysis to remove supernatant, thereby obtaining the capture antibody marked by the quantum dots.
Preferably, in step S1, the concentration of the activated quantum dots is 1-10 μ M; in step S2, the concentration of the capture antibody is 10-100 mug/ml; in step S3, the BSA solution has a concentration of 20-200 mg/mL and the mass ratio of the activated quantum dots to the activated capture antibody is (1:2) - (1: 10).
More preferably, in step S3, when the activated quantum dot is coupled to the activated capture antibody, the pH of the solution is 9 to 11, the concentration of the EDC solution is 9.38% (w/v), the concentration of the N-hydroxythiosuccinimide solution is 10% (w/v), the mass ratio of the activated quantum dot to the EDC added twice is 5 to 10, and the mass ratio of the activated quantum dot to the N-hydroxythiosuccinimide added twice is 5 to 10.
Preferably, the combination pad is sprayed with 0.1-5 mg/mL quantum dot-labeled capture antibody solution according to 2 muL/cm, the detection band is sprayed with 0.1-5 mg/mL detection antibody solution according to 2 muL/cm, and the quality control band is sprayed with 0.1-5 mg/mL IgG solution according to 1-2 muL/cm.
Preferably, the sample pad is soaked by adopting a pretreatment solution and then dried; before spraying, the bonding pad is soaked in a pretreatment solution and then dried; the pretreatment solution is 0.01M PBS buffer solution containing 0.02-0.1% Tween-20 and having a pH of 7-7.3.
In addition, the invention also provides a kit containing the test paper card, and the kit also comprises a card shell, wherein the card shell comprises a back card and an upper cover, the back card is provided with a clamping groove, the test paper card is embedded in the clamping groove, the upper cover is provided with a test window and a sample adding hole, the position of the test window is matched with the positions of the detection belt and the quality control belt, and the position of the sample adding hole is matched with the position of the sample pad.
Preferably, the kit further comprises a sample separation buffer solution, wherein the sample separation buffer solution is a PBS buffer solution containing a medium, 0.45-0.9% of sodium chloride, 0.02-0.1% of Tween-20 and 20-200 mg/mL of bovine serum albumin; when the medium is cesium chloride, the concentration of the cesium chloride in the sample separation buffer solution is 10% -30%; when the medium is sucrose, the concentration of the sucrose in the sample separation buffer solution is 0.1-0.5 mol/L; when the medium is polysucrose, the concentration of the polysucrose in the sample separation buffer solution is 10-30% (w/v).
Example 1
This example provides a method for preparing a quantum dot-labeled capture antibody, comprising the steps of:
s1) quantum dot activation: activating carboxyl on the surface of the CdSe/ZnS quantum dot by adopting N-hydroxyl thiosuccinimide;
s2) modifying the capture antibody by adopting monophosphoryl sugar to obtain an activated capture antibody;
s3) balancing the carboxyl water-soluble CdSe/ZnS (1-10 mu M) quantum dots to room temperature, adding EDC solution (9.38%), N-hydroxyl sulphosuccinimide solution (10%) and BSA solution (20-200 mg/mL) into the activated quantum dots, shaking uniformly, adding the activated capture antibody (10-100 mu g/mL), shaking uniformly and incubating for 30-45 min, adding EDC solution (9.38%) and N-hydroxyl sulphosuccinimide solution (10%) during incubation, adding methanol after incubation, mixing and shaking in a dark place for 1.5-2 h, adding β -mercaptoethanol for termination, adding β -mercaptoethanol for stabilizing the quantum dots after termination of reaction, dialyzing, centrifuging for 3min under the condition of 15000g after dialysis, removing supernatant, and re-suspending the precipitate (namely the capture antibody marked by the quantum dots) in PBS for storage at 4 ℃;
wherein, in step S3, when the activated quantum dot is coupled with the activated capture antibody, the pH of the solution is 9, the mass ratio of the activated quantum dot to the EDC added twice is 5, and the mass ratio of the activated quantum dot to the N-hydroxythiosuccinimide added twice is 5; the mass ratio of the activated quantum dots to the activated capture antibody was 1: 2.
Example 2
This example provides a method for preparing a quantum dot-labeled capture antibody, which is different from example 1 in that: in step S3, when the activated quantum dot is coupled to the activated capture antibody, the pH of the solution is 10, the mass ratio of the activated quantum dot to the twice-added EDC is 7, and the mass ratio of the activated quantum dot to the twice-added N-hydroxythiosuccinimide is 8; the mass ratio of the activated quantum dots to the activated capture antibody was 1: 7.
Example 3
This example provides a method for preparing a quantum dot-labeled capture antibody, which is different from example 1 in that: in step S3, when the activated quantum dot is coupled to the activated capture antibody, the pH of the solution is 11, the mass ratio of the activated quantum dot to the twice-added EDC is 10, and the mass ratio of the activated quantum dot to the twice-added N-hydroxythiosuccinimide is 10; the mass ratio of the activated quantum dots to the activated capture antibody was 1: 10.
Example 4
The embodiment provides a test paper card for detecting tumor M2 pyruvate kinase, which comprises a bottom plate (PVC rubber plate), wherein the bottom plate is provided with a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, the ends of the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially connected with each other along the flow direction of a sample, the combination pad adsorbs a capture antibody marked by a quantum dot, the nitrocellulose membrane is sequentially provided with a detection band and a quality control band along the flow direction of the sample, the detection band is coated with a detection antibody, and the quality control band is coated with goat anti-mouse IgG; the capture antibody is an anti-tumor M2-type pyruvate kinase antibody, and the surface of the capture antibody is modified by monophosphoryl sugar before the capture antibody is marked with quantum dots; the detection antibody is an anti-tumor M2 pyruvate kinase antibody, and the capture antibody and the detection antibody are combined with different epitopes of tumor M2 pyruvate kinase;
soaking the sample pad by using pretreatment solution, and drying; before spraying, the bonding pad is soaked in a pretreatment solution and then dried; the pretreatment solution is 0.01M PBS buffer solution containing 0.1% Tween-20 and having a pH of 7.3;
the combination pad is sprayed with 0.1mg/mL quantum dot-labeled capture antibody solution according to 2 muL/cm, the detection band is sprayed with 0.1mg/mL detection antibody solution according to 2 muL/cm, and the quality control band is sprayed with 0.1mg/mL IgG solution according to 2 muL/cm; the solvents of the capture antibody solution, the detection antibody solution and the goat anti-mouse IgG solution marked by the quantum dots are coating buffer solution which comprises 20mM NaCl, 0.05 percent PEG, 0.2 percent trehalose, 2mg/mLBSA and 0.05 percent NaN310mM PBS buffer.
Example 5
This example provides a test paper card for detecting tumor type M2 pyruvate kinase, which differs from example 4 in that: 1) the combination pad is sprayed with a capture antibody solution marked by 5mg/mL quantum dots according to 1 mu L/cm, the detection band is sprayed with a detection antibody solution of 5mg/mL according to 1 mu L/cm, and the quality control band is sprayed with a 5mg/mLIgG solution according to 1 mu L/cm; 2) the pretreatment solution is 0.01M PBS buffer solution containing 0.08% Tween-20 and having pH of 7; 3) the solvents of the capture antibody solution, the detection antibody solution and the goat anti-mouse IgG solution marked by the quantum dots are coating buffer solution, and the coating buffer solution comprises 130mM NaCl, 2% PEG, 0.8% trehalose, 7mg/mL BSA and 0.05% NaN380mM PBS buffer.
Example 6
This example provides a test paper card for detecting tumor type M2 pyruvate kinase, which differs from example 4 in that: 1) the combination pad is sprayed with 0.1mg/mL quantum dot-labeled capture antibody solution according to 3 muL/cm, the detection band is sprayed with 0.1mg/mL detection antibody solution according to 3 muL/cm, and the quality control band is sprayed with 0.1mg/mL IgG solution according to 3 muL/cm; 2) the pretreatment solution is 0.01M PBS buffer solution containing 0.02% Tween-20 and having a pH of 7.2; 3) the solvents of the capture antibody solution, the detection antibody solution and the goat anti-mouse IgG solution marked by the quantum dots are coating buffer solution, and the coating buffer solution comprises 150mM NaCl, 3% PEG, 1% trehalose, 10mg/mL BSA and 0.05% NaN3100mM PBS buffer.
Example 7
The embodiment provides a kit comprising a test paper card for detecting tumor M2 pyruvate kinase, and the kit further comprises a card shell, wherein the card shell comprises a back card and an upper cover, the back card is provided with a clamping groove, the test paper card is embedded in the clamping groove, the upper cover is provided with a test window and a sample adding hole, the position of the test window is matched with the positions of a detection zone and a quality control zone, and the position of the sample adding hole is matched with the position of a sample pad; the kit also comprises a sample separation buffer solution, and the sample separation buffer solution is a PBS buffer solution containing a medium, 0.45% of sodium chloride, 0.02% of Tween-20 and 20mg/mL of bovine serum albumin; the medium is cesium chloride and the concentration of cesium chloride in the sample separation buffer is 10%, 20% or 30%.
Example 8
This example provides a kit comprising a test card for detecting tumor pyruvate kinase M2, which differs from example 7 in that: the sample separation buffer solution is PBS buffer solution containing medium, 0.6% sodium chloride, 0.08% Tween-20 and 120mg/mL bovine serum albumin; when the medium is sucrose, the concentration of sucrose in the sample separation buffer is 0.1, 0.3 or 0.5 mol/L.
Example 9
This example provides a kit comprising a test card for detecting tumor pyruvate kinase M2, which differs from example 7 in that: the sample separation buffer solution is a PBS buffer solution containing a medium, 0.9 percent of sodium chloride, 0.1 percent of Tween-20 and 200mg/mL of bovine serum albumin; the medium is polysucrose, and the concentration of polysucrose in the sample separation buffer is 10%, 20% or 30% (w/v).
Example 10
This example provides a method for detecting tumor type M2 pyruvate kinase in stool, comprising the steps of:
1) mixing 10-15 ml of fecal cell separation buffer solution with 5-7 g of feces to prepare a mixed solution, centrifuging the mixed solution at 10000 rpm for 10 minutes, collecting supernatant, and removing a precipitate impurity part; collecting supernatant, and placing in another test tube;
2) and adding 100 mu L of supernatant into a sample adding hole on a test paper card, reacting for 5-10 min, respectively carrying out immunochromatography detection by using a quantum dot fluorescence quantitative analyzer, making a standard curve for the corresponding concentration of each peak area, carrying out the same treatment on an unknown sample to be detected to obtain the peak area, and obtaining the M2 type pyruvate kinase of the tumor in the sample according to a standard curve formula.
Kit performance determination
Detection principle and method of kit
The immunofluorescence chromatography reaction comprises two immunology methods, namely a double antibody sandwich method and an indirect method. The double antibody sandwich method is a reaction method of a detection belt, an immune complex is formed by an antigen to be detected and a capture antibody marked by a quantum dot at a binding pad (the quantum dot-tunor M2-PK monoclonal antibody + tunor M2-PK antigen) and moves on a nitrocellulose membrane, the immune complex is combined with a detection belt antibody (namely an anti-human tunor M2-PK monoclonal antibody with different epitopes) on the membrane, and a solidified immune complex is formed (namely the quantum dot-tunor M2-PK monoclonal antibody + tunor M2-PK antigen + an anti-human tunor M2-PK monoclonal antibody with different epitopes) to form the immune complex). The indirect method is a reaction method of a quality control band, free quantum dot-tumor M2-PK monoclonal antibody continues to move in a chromatography manner, and is captured and combined by a second antibody (goat anti-mouse IgG antibody) on the quality control band to form a solidified immune complex.
Sensitivity detection of kits
The fluorescence value mean and standard deviation were calculated by taking a zero concentration standard (i.e., a blank control, which does not contain protein) as a sample and measuring 20 times. The fluorescence value obtained by adding 2 times of standard deviation to the average of the measured values was substituted into the standard curve equation y of 2.8858x +271.42(R2 of 0.9985). The calculated concentration is the lowest detection quantity, and the result is substituted into the standard curve equation according to the method to calculate the lowest detection quantity to be 0.1ng/ml, which shows that the kit and the detection method have high sensitivity for detecting human tunor M2-PK.
In addition, as shown in table 1, compared with an enzyme-linked immunosorbent assay (ELISA), the kit of the invention has a result of 12.3-36.8 ng/ml (normal reference value of the immunochromatography is less than 10ng/ml) in stool detection in colorectal cancer groups, while the ELISA result is 3.2-13.6 ng/ml (normal reference value of the ELISA is less than 6ng/ml), and the difference between the two has statistical significance (p is less than 0.01). The clinical detection result of the immunochromatography shows that the kit has higher sensitivity and good accuracy, and has no false positive or false negative result. And ELISA sensitivity is low, 6 colorectal cancer patients in 20 colorectal cancer patients are normal results, and a higher false negative result obviously exists.
TABLE 1 detection results (ng/ml) of tumor M2-PK in feces
Specific detection of kits
The invention discloses a tomor M2-PK detection kit for respectively detecting tomor M2-PK protein in human excrement and tomor M2-PK protein in human serum. The detection kit of the invention is adopted to respectively detect the tulor M2-PK content (ng/ml) in 10 parts of human feces (5 parts of CRC patients, the other 5 parts of health physical examination people), the tulor M2-PK content in 10 parts of serum (5 parts of CRC patients, the other 5 parts of health physical examination people), the tulor M2-PK content in 10 parts of mouse feces (5 parts of CRC animal models, the other 5 parts of normal mice), and the tulor M2-PK content (ng/ml) in mouse serum (5 parts of CRC animal models, the other 5 parts of normal mice), and the detection results are shown in a table 2: in Table 2, the numbers 1 to 5 are human CRC patients or murine CRC patients, and the numbers 6 to 10 are healthy humans or normal mice.
As shown in Table 2, the detection results show that the concentration of the feces tunor M2-PK protein of the human CRC patient is 18.9-26.6 ng/M, and the concentration of the feces tunor M2-PK protein of the healthy population is 0.4-4.6 ng/ml; the concentration of serum tunor M2-PK protein of a human CRC patient is 10.4-13.6 ng/ml, and the concentration of serum tunor M2-PK protein of healthy people is 0.3-2.7 ng/ml; the concentration of the feces tunor M2-PK protein of the mouse CRC animal model is 4.3-6.8 ng/ml, and the concentration of the feces tunor M2-PK protein of a normal mouse is 0.2-2.1 ng/ml; the concentration of serum tunor M2-PK protein of the mouse CRC animal model is 3.7-5.3 ng/ml, and the concentration of serum tunor M2-PK protein of a normal mouse is 0.3-1.5 ng/ml. Therefore, the kit can specifically determine the tulor M2-PK content in human feces and serum.
TABLE 2 Tumor M2-PK content (ng/ml) in human, murine feces and serum
| Categories | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Excrement and urine (human) | 26.6 | 19.2 | 21.2 | 18.9 | 19.9 | 4.6 | 4.2 | 3.7 | 2.3 | 0.4 |
| Serum (human) | 13.6 | 10.9 | 11.2 | 11.8 | 10.4 | 1.9 | 2.7 | 1.8 | 0.3 | 1.6 |
| Excrement and urine (mouse) | 5.7 | 6.8 | 6.4 | 5.6 | 4.3 | 2.1 | 1.9 | 0.2 | 1.2 | 1.8 |
| Serum (mouse) | 4.3 | 5.3 | 5.1 | 4.4 | 3.7 | 1.5 | 1.8 | 0.9 | 1.1 | 0.3 |
Stability of the kit
Three batches of reagents of the self-made kit are respectively placed at 4 ℃ for half a year and one year, and after being placed at 25-30 ℃ for 7-10 days, the linear relation between the fluorescence intensity values of all points of the reference standard product before being placed and the fluorescence intensity value of the zero standard product are compared, the stability of each batch of reagents is detected, and the result shows that no obvious difference exists between the results and the results before being placed.
Clinical application of kit
Clinical stool samples were tested into four groups: 20 colorectal cancer (CRC) groups were pathologically diagnosed, 20 colorectal polyps were pathologically diagnosed, 20 colorectal adenomas were pathologically diagnosed, and 20 normal control groups were healthy subjects.
As shown in table 3, the detection results are: the content range of the tulor M2-P in the feces of the normal control group is 0.3-2.4 ng/ml; the range of the tulor M2-PK content in the feces of the colorectal polyp group is 0.4-3.1 ng/ml; the range of the tulor M2-PK content in the feces of the colorectal adenoma group is 0.5-4.7 ng/ml; the content range of the tulor M2-PK in the feces of the CRC group is 12.3-36.8 ng/ml; the comparison of differences was statistically significant (p < 0.01). Therefore, the clinical detection results show that the kit has higher sensitivity and good accuracy, and has no false positive or false negative results.
TABLE 3 humor M2-PK content (ng/ml) in feces of each group
| Serial number | Normal control group | Colorectal polyp group | Group of colorectal adenomas | Colorectal cancer group |
| 1 | 0.3 | 0.6 | 4.1 | 12.3 |
| 2 | 0.5 | 0.4 | 1.5 | 13.5 |
| 3 | 1.2 | 1.3 | 2.2 | 22.2 |
| 4 | 0.8 | 0.9 | 1.8 | 36.8 |
| 5 | 0.4 | 0.8 | 0.9 | 20.9 |
| 6 | 1.5 | 1.9 | 4.7 | 23.7 |
| 7 | 1.2 | 2.8 | 3.2 | 30.2 |
| 8 | 0.7 | 1.7 | 2.7 | 24.7 |
| 9 | 1.3 | 1.3 | 3.3 | 16.3 |
| 10 | 1.9 | 1.4 | 1.9 | 18.9 |
| 11 | 1.1 | 1.2 | 3.1 | 33.7 |
| 12 | 1.4 | 1.6 | 2.4 | 18.4 |
| 13 | 0.7 | 1.7 | 2.8 | 12.8 |
| 14 | 0.6 | 0.8 | 1.4 | 17.3 |
| 15 | 1.3 | 1.4 | 2.1 | 15.5 |
| 16 | 2.4 | 0.9 | 1.8 | 18.8 |
| 17 | 2.1 | 2.1 | 3.7 | 13.7 |
| 18 | 1.9 | 1.7 | 2.9 | 26.9 |
| 19 | 2.3 | 3.1 | 2.3 | 24.3 |
| 20 | 0.5 | 2.7 | 0.5 | 36.8 |
In addition, the capture antibodies of the invention quantum dot labels were prepared on the premise of many failures:
the efficient coupling of quantum dots to antibodies and the maintenance of their biological activity is a crucial step, and the biggest challenge in successful coupling of quantum dots to antibodies is the stability of the coupled product. In theory, quantum dot and antibody coupling is feasible, but in practice, quantum dot and antibody coupling is not easy, so that the method is very labor-intensive and fails for many times. The reasons for its failure are the following:
1) in step S3), when the pH value of the solution is between 5 and 7, the quantum dots are aggregated and cannot be effectively coupled with the antibody;
2) in step S3), if the following condition is not satisfied: when the mass ratio of the activated quantum dots to the EDC added twice is 5-10 and the mass ratio of the activated quantum dots to the N-hydroxy thiosuccinimide added twice is 5-10, the carboxyl groups of the quantum dots are slowly combined with the amino groups of the antibody, but the combination is not firm, and the separation phenomenon occurs;
3) except for the conditions of setting the proportion of quantum dots to a coupling agent, proper PH value, adding BSA (bovine serum albumin), methanol and the like, the inventor adopts monophosphoryl sugar to modify a capture antibody before the quantum dots are coupled with the antibody to obtain an activated capture antibody; the surface structure of the antibody is changed for the quantum dot to be successfully coupled with the antibody, which creates the optimal condition for the quantum dot to be successfully coupled with the capture antibody.
Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (8)
1. A test paper card for detecting tumor M2 pyruvate kinase is characterized by comprising a bottom plate, wherein the bottom plate is provided with a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad, the ends of the sample pad, the combination pad, the nitrocellulose membrane and the water absorption pad are sequentially connected along the sample flow direction, the combination pad adsorbs capture antibodies marked by quantum dots, the nitrocellulose membrane is sequentially provided with a detection band and a quality control band along the sample flow direction, the detection band is coated with detection antibodies, and the quality control band is coated with IgG;
the capture antibody is an anti-tumor M2 pyruvate kinase antibody, and the surface of the capture antibody is modified by monophosphoryl sugar before the capture antibody is labeled with quantum dots; the detection antibody is an anti-tumor M2 pyruvate kinase antibody, and the capture antibody and the detection antibody are combined with different epitopes of tumor M2 pyruvate kinase;
the monophosphoryl sugar modified capture antibody is prepared by the following method: adding a monophosphoryl sugar and bovine serum albumin buffer solution into the capture antibody, shaking uniformly, and standing at room temperature for 1-2 h; and adjusting the pH value of the solution to 8-11.
2. The method of making a test card of claim 1, comprising the steps of: the combination pad is sprayed with 0.1-5 mg/mL quantum dot marked capture antibody solution according to 1-3 muL/cm, the detection band is sprayed with 0.1-5 mg/mL detection antibody solution according to 1-3 muL/cm, and the quality control band is sprayed with 0.1-5 mg/mL IgG solution according to 1-3 muL/cm; the solvents of the capture antibody solution, the detection antibody solution and the goat anti-mouse IgG solution marked by the quantum dots are coating buffer solutions, and the coating buffer solutions comprise 20-150 mM NaCl, 0.05-3% PEG, 0.2-1% trehalose, 2-10 mg/mL BSA and 0.05% NaN310 to 100mM PBS buffer.
3. The method of preparing a test paper card according to claim 2, wherein the quantum dot-labeled capture antibody is prepared by the steps of:
s1) quantum dot activation: activating carboxyl on the surface of the CdSe/ZnS quantum dot by adopting N-hydroxyl thiosuccinimide;
s2) modifying the surface of the capture antibody by adopting monophosphoryl sugar to obtain an activated capture antibody;
s3) placing the activated quantum dots to room temperature, adding an EDC solution, an N-hydroxy sulpho succinimide solution and a BSA solution into the activated quantum dots, shaking up, adding the activated capture antibody, shaking up, incubating, adding the EDC solution and the N-hydroxy sulpho succinimide solution during incubation, adding methanol after incubation, mixing, oscillating in a dark place, adding β -mercaptoethanol for termination, dialyzing by β -mercaptoethanol after termination of reaction, and centrifuging after dialysis to remove supernatant, thereby obtaining the capture antibody marked by the quantum dots.
4. The method for preparing a test paper card according to claim 3, wherein in step S1, the concentration of the activated quantum dots is 1 to 10 μ M; in step S2, the concentration of the capture antibody is 10-100 mug/ml; in step S3, the BSA solution has a concentration of 20-200 mg/mL and the mass ratio of the activated quantum dots to the activated capture antibody is (1:2) - (1: 10).
5. The method of claim 2, wherein the binding pad is coated with 0.1-5 mg/mL quantum dot-labeled capture antibody solution at 2 μ L/cm, the detection zone is coated with 0.1-5 mg/mL detection antibody solution at 2 μ L/cm, and the quality control zone is coated with 0.1-5 mg/mL IgG solution at 2 μ L/cm.
6. The method of preparing a test card according to claim 2, wherein the sample pad is soaked with a pretreatment solution and dried; before spraying, the bonding pad is soaked in a pretreatment solution and then dried; the pretreatment solution is 0.01M PBS buffer solution containing 0.02-0.1% Tween-20 and having a pH of 7-7.3.
7. A kit comprising the test paper card of claim 1, said kit further comprising a card housing, said card housing comprising a back card and a top cover, said back card having a slot, said test paper card being embedded in said slot, said top cover having a test window and a sample application hole, said test window being positioned to match the position of said test strip and said quality control strip, said sample application hole being positioned to match the position of said sample pad.
8. The kit of claim 7, further comprising a sample isolation buffer, wherein the sample isolation buffer is a PBS buffer comprising media, 0.45% to 0.9% sodium chloride, 0.02% to 0.1% Tween-20, and 20 to 200mg/mL bovine serum albumin; when the medium is cesium chloride, the concentration of the cesium chloride in the sample separation buffer solution is 10% -30%; when the medium is sucrose, the concentration of the sucrose in the sample separation buffer solution is 0.1-0.5 mol/L; when the medium is polysucrose, the concentration of the polysucrose in the sample separation buffer solution is 10-30%.
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