CN110865192A - Kit for determining GP73 based on latex enhanced immunoturbidimetry and preparation and use methods thereof - Google Patents

Kit for determining GP73 based on latex enhanced immunoturbidimetry and preparation and use methods thereof Download PDF

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CN110865192A
CN110865192A CN201911145541.2A CN201911145541A CN110865192A CN 110865192 A CN110865192 A CN 110865192A CN 201911145541 A CN201911145541 A CN 201911145541A CN 110865192 A CN110865192 A CN 110865192A
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芮双印
高耀辉
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ANHUI DAQIAN BIO-ENGINEERING Ltd Co
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Abstract

The invention provides a kit for measuring GP73 based on a latex enhanced immunoturbidimetry, which comprises a reagent R1 and a reagent R2; the reagent R1 comprises PBS buffer solution, PEG6000, BSA, NaCl, Tween 20 and NaN3EDTA; the reagent R2 comprises PBS buffer solution, BSA, NaCl, latex microspheres of cross-linked anti-human rGP73 monoclonal antibody 1, latex microspheres of cross-linked anti-human rGP73 monoclonal antibody 2, NaN3EDTA. The invention also provides a preparation and use method of the kit for measuring GP73 based on the latex enhanced immunoturbidimetry. The invention can be used on a full-automatic biochemical analyzer, has simple operation, low cost and high automation and saves the detection time; and, in high stabilityCompared with other products, the invention has higher sensitivity and specificity under the conditions of high performance and high precision.

Description

Kit for determining GP73 based on latex enhanced immunoturbidimetry and preparation and use methods thereof
Technical Field
The invention relates to the field of genetic engineering and immunological determination and analysis, in particular to a kit for determining GP73 based on a latex enhanced immunoturbidimetry method and a preparation and use method thereof.
Background
Golgi glycoprotein-73, also known as type II Golgi transmembrane protein, was designated GP73 because it shows a relative molecular mass of 7.3X 104 in SDS-PAGE. Modern researches show that GP73 presents a high expression state in various tumor tissues such as liver cancer, cholangiocarcinoma, prostatic cancer, seminoma, lung adenocarcinoma and the like, is constantly expressed in bile duct epithelial cells of normal liver, and has little or no expression of liver parenchymal cells. Recent research shows that GP73 is abnormally expressed in various diseases, but GP73 is closely related to liver diseases, particularly liver cancer, is expected to be a serological marker for early diagnosis of liver cancer and has sensitivity superior to AFP. The serum GP73 level can be used as an index for the prognosis evaluation of liver diseases and can also diagnose significant liver injury and cirrhosis; GP73 is a complementary indicator of ALT diagnosis of liver damage.
The method for detecting GP73 is mainly based on antigen-antibody reactions. At present, enzyme-linked immunosorbent assay (ELISA) methods are mostly adopted for detecting GP73 in laboratories. However, the GP73 detection using elisa can accurately detect the GP73 concentration in a sample, but the operation is relatively complicated and time-consuming, and cannot meet the requirement of rapid detection in outpatient or emergency.
The latex boosting immune ratio method is a detection method for dynamically measuring antigen-antibody combination: in a specific dilution system, antigen and antibody are combined, and when the combination proportion is proper, particles are formed and are separated out from a liquid phase; before and after the antigen and antibody are combined, turbidity changes occur; the turbidity change is detected by a full-automatic biochemical analyzer, and a linear curve is drawn by using a standard substance, so that the content of the substance to be detected in the corresponding sample can be obtained. The method does not need special instruments, and is simple and convenient to operate. In addition, the latex enhanced immunoturbidimetry can enhance the absorbance of reaction by using a latex carrier, so that the sensitivity of detection is greatly improved, the detection is realized automatically by a full-automatic biochemical analyzer, the detection is more convenient and rapid, the time is saved, and the requirement of clinical large sample detection can be met.
Accordingly, there is an urgent need for a kit for measuring GP73 based on latex-enhanced immunoturbidimetry, and a preparation method and a use method thereof.
Disclosure of Invention
The invention aims to provide a kit for measuring GP73 based on a latex enhanced immunoturbidimetry, which is simple to operate, short in time consumption, strong in specificity and high in sensitivity and can be used for a full-automatic biochemical analyzer, and a preparation and use method thereof.
The invention adopts the following technical scheme to solve the technical problems:
a kit for measuring GP73 based on a latex enhanced immunoturbidimetry comprises two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in corresponding content:
reagent R1:
Figure BDA0002282071620000021
the solvent is purified water;
reagent R2:
Figure BDA0002282071620000031
the solvent is purified water.
As one of the preferable modes of the invention, the components and the corresponding contents are specifically as follows:
reagent R1:
Figure BDA0002282071620000032
the solvent is purified water;
reagent R2:
Figure BDA0002282071620000033
Figure BDA0002282071620000041
the solvent is purified water.
As one of the preferable modes of the invention, the GP73 calibrator is further included, and the components and the corresponding contents are as follows:
Figure BDA0002282071620000042
the solvent is purified water.
As one of the preferable modes of the invention, the GP73 calibrator comprises the following components in percentage by weight:
Figure BDA0002282071620000043
the solvent is purified water.
In a preferred mode of the invention, rGP73 in the GP73 calibrator is recombinant human GP73 protein, and is obtained by purchase.
In a preferred embodiment of the present invention, the anti-human rGP73 monoclonal antibody 1 in the latex microsphere crosslinked with anti-human rGP73 monoclonal antibody 1 and the anti-human rGP73 monoclonal antibody 2 in the latex microsphere crosslinked with anti-human rGP73 monoclonal antibody 2 are two anti-human rGP73 monoclonal antibodies each having a rGP73 epitope recognition function.
In a preferred embodiment of the present invention, the method for obtaining the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 1 comprises: crosslinking an anti-human rGP73 monoclonal antibody 1 to a polystyrene latex microsphere by using the polystyrene latex microsphere with the diameter of 80-120 nm by adopting a chemical crosslinking method;
the method for obtaining the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 2 comprises the following steps: and (2) crosslinking the anti-human rGP73 monoclonal antibody 2 to the polystyrene latex microspheres by using the polystyrene latex microspheres with the diameters of 80-120 nm and adopting a chemical crosslinking method.
In a preferred embodiment of the present invention, the specific preparation methods of the anti-human rGP73 monoclonal antibody 1 and the anti-human rGP73 monoclonal antibody 2 are as follows:
① immunization of mice:
2mL of rGP73 antigen of 1mg/mL is taken and fully emulsified with an equivalent amount of Freund's adjuvant to form stable emulsion which is marked as emulsified antigen; injecting 0.3mL of emulsified antigen into the abdominal cavity of each mouse, and carrying out primary immunization; after 14 days, booster immunization with emulsified antigen was performed once; on 28 days, the immunization was boosted once again; on day 42, booster immunization was performed again with neat antigen that was not emulsified; removing eyeball and blood, and collecting spleen at 45 days;
② preparation of fused cells:
recovering SP2/0 cells and screening by using 8-Ag selective medium; spleen was prepared as single suspension cells, washed twice with PBS and resuspended, counted separately, 1X 10 in volume8Spleen cells and 1X 107Adding the suspension of SP2/0 cells into the same 50mL centrifuge tube, and supplementing incomplete culture solution to 30 mL; after fully and uniformly mixing, centrifuging for 15min under the condition of 1500 r/min;
the supernatant was aspirated and the deposited fused cells were suspended in 100mL of HAT medium; then, cells were seeded into 5 96-well flat-bottom cell plates, 0.2mL per well; after inoculation, the cells were incubated at 37 ℃ with 5% CO2Culturing under the condition of saturated humidity; when the fused cells grow to half of the plate holes, amplifying the monoclonal cells to a 24-hole plate for continuous culture, replacing the monoclonal cells with a complete culture medium of 20% fetal calf serum for culture, and collecting the culture supernatant of the fused cells;
③ screening positive fusion cell strain by indirect ELISA:
coating a polystyrene micropore plate with 10 mu g/mL rGP73 antigen, wherein each hole is 100 mu L, and then sealing for 60min at the temperature of 37 ℃ by using a sealing solution containing 5% calf serum; then, washing with PBST washing solution containing 0.05% Toween for 3 times, 5 min/time;
adding 100 mu L of fusion cell culture supernatant prepared in the step ② into the coated plate, placing the coated plate in a wet box for incubation at 37 ℃ for 60min, washing the coated plate for 3 times and 5 min/time by PBST (basic fibroblast growth factor) solution, adding 100 mu L of goat anti-mouse HRP-IgG with the dilution multiple of 1:5000 into the coated plate, placing the coated plate in the wet box for incubation at 37 ℃ for 90min, washing the coated plate for 3 times and 5 min/time by PBST solution, adding 100 mu L/hole of OPD substrate solution, placing the coated plate at room temperature for 15min for observation, adding 50 mu L of stop solution into each hole after obvious color change appears in the holes, stopping reaction, measuring the OD value of each hole, selecting two fusion cells with the largest OD value, cloning and carrying out expanded culture by a limiting dilution method;
respectively taking the culture supernatant of the fusion cells as a primary antibody, using goat anti-mouse IgG marked by HRP as a secondary antibody, using verified rGP73 as an antigen, using the existing anti-human GP73 monoclonal antibody as a reference primary antibody, and verifying that the two fusion hybridoma cells are positive to anti-human GP73 through WB;
④ purification of monoclonal antibody:
collecting two fused cell culture supernatants, respectively slowly dropwise adding saturated ammonium sulfate with the same volume into the supernatants, and standing at 4 deg.C for more than 3 hr; centrifuging at 4 deg.C at 4200r/min for 30min, discarding supernatant, and dissolving precipitate with 200mL PBS; adding saturated ammonium sulfate with a total volume of 33%, standing at 4 deg.C for 3 hr, and centrifuging at 4 deg.C at 4200r/min for 30 min; the pellet after centrifugation was dissolved in 200mL PBS and further filled into a 10kD dialysis bag; finally, the membrane is placed in an environment of 4 ℃, PBS dialysate with 20 times volume is adopted for dialysis for 12h for desalting, and the antihuman rGP73 monoclonal antibodies are obtained and are respectively marked as antihuman rGP73 monoclonal antibody 1 and antihuman rGP73 monoclonal antibody 2;
⑤ recombinant anti-human rGP73 monoclonal antibody was demonstrated:
rGP73 protein is used as an antigen, the prepared monoclonal antibody is used as a primary antibody, HRP-labeled rabbit anti-mouse IgG is used as a secondary antibody for preparing WB, and the antigen has a positive band at 73 kD; the two monoclonal antibodies are used for verifying rGP73 that a positive band appears, namely, the two monoclonal antibodies are successfully prepared.
The preparation method of the kit for measuring GP73 based on the latex enhanced immunoturbidimetry comprises the following steps:
(1) preparation of anti-human rGP73 monoclonal antibody 1 and anti-human rGP73 monoclonal antibody 2:
① immunization of mice:
2mL of rGP73 antigen of 1mg/mL is taken and fully emulsified with an equivalent amount of Freund's adjuvant to form stable emulsion which is marked as emulsified antigen; injecting 0.3mL of emulsified antigen into the abdominal cavity of each mouse, and carrying out primary immunization; after 14 days, booster immunization with emulsified antigen was performed once; on 28 days, the immunization was boosted once again; on day 42, booster immunization was performed again with neat antigen that was not emulsified; removing eyeball and blood, and collecting spleen at 45 days;
② preparation of fused cells:
recovering SP2/0 cells and screening by using 8-Ag selective medium; spleen was prepared as single suspension cells, washed twice with PBS and resuspended, counted separately, 1X 10 in volume8Spleen cells and 1X 107Adding the suspension of SP2/0 cells into the same 50mL centrifuge tube, and supplementing incomplete culture solution to 30 mL; after fully and uniformly mixing, centrifuging for 15min under the condition of 1500 r/min;
the supernatant was aspirated and the deposited fused cells were suspended in 100mL of HAT medium; then, cells were seeded into 5 96-well flat-bottom cell plates, 0.2mL per well; after inoculation, the cells were incubated at 37 ℃ with 5% CO2Culturing under the condition of saturated humidity; when the fused cells grow to half of the plate holes, amplifying the monoclonal cells to a 24-hole plate for continuous culture, replacing the monoclonal cells with a complete culture medium of 20% fetal calf serum for culture, and collecting the culture supernatant of the fused cells;
③ screening positive fusion cell strain by indirect ELISA:
coating a polystyrene micropore plate with 10 mu g/mL rGP73 antigen, wherein each hole is 100 mu L, and then sealing for 60min at the temperature of 37 ℃ by using a sealing solution containing 5% calf serum; then, washing with PBST washing solution containing 0.05% Toween for 3 times, 5 min/time;
adding 100 mu L of fusion cell culture supernatant prepared in the step ② into the coated plate, placing the coated plate in a wet box for incubation at 37 ℃ for 60min, washing the coated plate for 3 times and 5 min/time by PBST (basic fibroblast growth factor) solution, adding 100 mu L of goat anti-mouse HRP-IgG with the dilution multiple of 1:5000 into the coated plate, placing the coated plate in the wet box for incubation at 37 ℃ for 90min, washing the coated plate for 3 times and 5 min/time by PBST solution, adding 100 mu L/hole of OPD substrate solution, placing the coated plate at room temperature for 15min for observation, adding 50 mu L of stop solution into each hole after obvious color change appears in the holes, stopping reaction, measuring the OD value of each hole, selecting two fusion cells with the largest OD value, cloning and carrying out expanded culture by a limiting dilution method;
respectively taking the culture supernatant of the fusion cells as a primary antibody, using goat anti-mouse IgG marked by HRP as a secondary antibody, using verified rGP73 as an antigen, using the existing anti-human GP73 monoclonal antibody as a reference primary antibody, and verifying that the two fusion hybridoma cells are positive to anti-human GP73 through WB;
④ purification of monoclonal antibody:
collecting two fused cell culture supernatants, respectively slowly dropwise adding saturated ammonium sulfate with the same volume into the supernatants, and standing at 4 deg.C for more than 3 hr; centrifuging at 4 deg.C at 4200r/min for 30min, discarding supernatant, and dissolving precipitate with 200mL PBS; adding saturated ammonium sulfate with a total volume of 33%, standing at 4 deg.C for 3 hr, and centrifuging at 4 deg.C at 4200r/min for 30 min; the pellet after centrifugation was dissolved in 200mL PBS and further filled into a 10kD dialysis bag; finally, the membrane is placed in an environment of 4 ℃, PBS dialysate with 20 times volume is adopted for dialysis for 12h for desalting, and the antihuman rGP73 monoclonal antibodies are obtained and are respectively marked as antihuman rGP73 monoclonal antibody 1 and antihuman rGP73 monoclonal antibody 2;
⑤ recombinant anti-human rGP73 monoclonal antibody was demonstrated:
rGP73 protein is used as an antigen, the prepared monoclonal antibody is used as a primary antibody, HRP-labeled rabbit anti-mouse IgG is used as a secondary antibody for preparing WB, and the antigen has a positive band at 73 kD; the two monoclonal antibodies are used for verifying rGP73 that a positive strip appears, namely the two monoclonal antibodies are successfully prepared;
(2) preparation of latex microspheres of cross-linked anti-human rGP73 monoclonal antibody 1 and latex microspheres of cross-linked anti-human rGP73 monoclonal antibody 2:
crosslinking the anti-human rGP73 monoclonal antibody 1 obtained in the step (1) on a polystyrene latex microsphere by using a polystyrene latex microsphere with the diameter of 80-120 nm and adopting a chemical crosslinking method to obtain a latex microsphere of a crosslinked anti-human rGP73 monoclonal antibody 1;
crosslinking the anti-human rGP73 monoclonal antibody 2 obtained in the step (1) on a polystyrene latex microsphere by using a polystyrene latex microsphere with the diameter of 80-120 nm and adopting a chemical crosslinking method to obtain a latex microsphere of a crosslinked anti-human rGP73 monoclonal antibody 2;
(3) preparation of GP73 latex enhanced immunoturbidimetry kit
① formulation reagent R1:
according to the component content of the reagent R1, mixing the components in the same container, and uniformly mixing to obtain a reagent R1;
② formulation reagent R2:
mixing the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 1, the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 2 and the rest of other component substances prepared in the step (2) in the same container according to the component content of the reagent R2, and uniformly mixing to prepare a reagent R2;
③ formulation of GP73 calibrator:
the GP73 calibrator contained the following components and corresponding contents:
Figure BDA0002282071620000101
the solvent is purified water;
the GP73 calibrator was prepared by mixing rGP73 and the remaining other ingredients in the same vessel according to the ingredient content of the GP73 calibrator.
The use method of the kit for measuring GP73 based on the latex enhanced immunoturbidimetry comprises the following specific steps:
(1) sucking 10 μ L of sample, adding 180 μ L of reagent R1, and incubating at 37 deg.C for 5 min;
(2) adding 60 μ L reagent R2, and incubating at 37 deg.C;
(3) after incubation for 1min, reading a light absorption value A1, after incubation for 3min, reading a light absorption value A2, and calculating delta A;
the calibration method is 6-point calibration, a full-automatic biochemical analyzer is adopted for detection, and the concentrations of calibrators are set as follows: 0. 18.7, 55.7, 166.7, 500 and 1500 ng/mL; and (4) according to the calibration value, calculating the GP73 content in the sample according to the Delta A.
The kit is a GP73 latex enhanced turbidimetric assay kit based on a plurality of monoclonal antibodies as common cross-linking substrates, and consists of a reagent R1 and a reagent R2; the reagent R1 contains buffer solution (PBS buffer solution), surfactant (PEG6000, Tween-20), stabilizer (BSA, NaCl, EDTA) and preservative (NaN)3) (ii) a The reagent R2 contains buffer solution (PBS buffer solution), stabilizer (BSA, NaCl, EDTA), preservative (NaN)3) And polystyrene latex microspheres (Cross-Linked)Latex microspheres of anti-human rGP73 monoclonal antibody 1 and latex microspheres of cross-linked anti-human rGP73 monoclonal antibody 2).
Compared with the prior art, the invention has the advantages that:
(1) compared with other methods, the kit provided by the invention utilizes a latex enhanced immunity transmission turbidimetry to measure GP73, the detection signal is amplified by multiple times, and the detection sensitivity is improved; moreover, the method can be used for a full-automatic biochemical analyzer, has low cost, simple operation, high automation degree and quick detection time, and is convenient for clinical application;
(2) compared with the similar latex enhanced immunotransmittance turbidimetric assay kit for GP73, the kit provided by the invention adopts a mode of combining two anti-human GP73 monoclonal antibodies, can respectively identify two antigen epitopes, has higher sensitivity compared with a single monoclonal antibody kit, and has better specificity compared with a plurality of monoclonal antibody kits;
(3) according to the invention, in the process of marking the antibody, the antibody is coupled with the latex microspheres with uniform sizes by adopting a special marking method, so that the stability of the latex antibody particles is improved, and the activity of the antibody is ensured not to be changed, thereby improving the stability and sensitivity of the detection kit.
Drawings
FIG. 1 shows the Western Blot identification of the anti-human rGP73 monoclonal antibody in example 4 (lane M: protein Marker 26616; lane 1: negative cell culture control; lane 2: sample after purification of anti-human rGP73 monoclonal antibody 1; lane 3: sample after purification of anti-human rGP73 monoclonal antibody 2; lane 4: GP73 monoclonal antibody control of abcam);
FIG. 2 is a graph of the linear relationship between the kit of the present invention and a commercial GP73 detection kit in example 6;
FIG. 3 is a linear range linear regression plot of the kit of the invention in example 6.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The kit for measuring GP73 based on the latex enhanced immunoturbidimetry comprises two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in corresponding content:
reagent R1:
Figure BDA0002282071620000121
the solvent is purified water.
Reagent R2:
Figure BDA0002282071620000122
the solvent is purified water.
In addition, the GP73 calibrator is also included, and the components and corresponding contents are as follows:
Figure BDA0002282071620000131
the solvent is purified water.
Further, rGP73 in the GP73 calibrator was recombinant human GP73 protein (obtained from the purchase source).
Further, the method for obtaining the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 1 in the reagent R2 comprises the following steps: polystyrene latex microspheres with a diameter of 80nm were used and anti-human rGP73 monoclonal antibody 1 was cross-linked to the polystyrene latex microspheres using a chemical cross-linking method. The method for obtaining the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 2 comprises the following steps: polystyrene latex microspheres with a diameter of 80nm were used and anti-human rGP73 monoclonal antibody 2 was cross-linked to the polystyrene latex microspheres using a chemical cross-linking method.
Further, the anti-human rGP73 monoclonal antibody 1 and the anti-human rGP73 monoclonal antibody 2 are specifically two anti-human rGP73 monoclonal antibodies respectively having rGP73 epitope recognition function, and the preparation methods of the two are detailed in example 4.
Example 2
The kit for measuring GP73 based on the latex enhanced immunoturbidimetry comprises two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in corresponding content:
reagent R1:
Figure BDA0002282071620000132
Figure BDA0002282071620000141
the solvent is purified water.
Reagent R2:
Figure BDA0002282071620000142
the solvent is purified water.
In addition, the GP73 calibrator is also included, and the components and corresponding contents are as follows:
Figure BDA0002282071620000143
the solvent is purified water.
Further, rGP73 in the GP73 calibrator was recombinant human GP73 protein (obtained from the purchase source).
Further, the method for obtaining the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 1 in the reagent R2 comprises the following steps: polystyrene latex microspheres 120nm in diameter were used and anti-human rGP73 monoclonal antibody 1 was cross-linked to the polystyrene latex microspheres using a chemical cross-linking method. The method for obtaining the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 2 comprises the following steps: polystyrene latex microspheres 120nm in diameter were used and chemically cross-linked to anti-human rGP73 monoclonal antibody 2.
Further, the anti-human rGP73 monoclonal antibody 1 and the anti-human rGP73 monoclonal antibody 2 are specifically two anti-human rGP73 monoclonal antibodies respectively having rGP73 epitope recognition function, and the preparation methods of the two are detailed in example 4.
Example 3
The kit for measuring GP73 based on the latex enhanced immunoturbidimetry comprises two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in corresponding content:
reagent R1:
Figure BDA0002282071620000151
the solvent is purified water.
Reagent R2:
Figure BDA0002282071620000161
the solvent is purified water.
In addition, the GP73 calibrator is also included, and the components and corresponding contents are as follows:
Figure BDA0002282071620000162
the solvent is purified water.
Further, rGP73 in the GP73 calibrator was recombinant human GP73 protein (obtained from the purchase source).
Further, the method for obtaining the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 1 in the reagent R2 comprises the following steps: polystyrene latex microspheres with a diameter of 100nm were used and anti-human rGP73 monoclonal antibody 1 was cross-linked to the polystyrene latex microspheres using a chemical cross-linking method. The method for obtaining the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 2 comprises the following steps: polystyrene latex microspheres with a diameter of 100nm were used and anti-human rGP73 monoclonal antibody 2 was cross-linked to the polystyrene latex microspheres using a chemical cross-linking method.
Further, the anti-human rGP73 monoclonal antibody 1 and the anti-human rGP73 monoclonal antibody 2 are specifically two anti-human rGP73 monoclonal antibodies respectively having rGP73 epitope recognition function, and the preparation methods of the two are detailed in example 4.
Example 4
This example is a method for preparing the kit for measuring GP73 based on latex enhanced immunoturbidimetry in examples 1-3 above, comprising the following steps:
(1) preparation of anti-human rGP73 monoclonal antibody 1 and anti-human rGP73 monoclonal antibody 2:
① immunization of mice:
2mL of rGP73 antigen of 1mg/mL is taken and fully emulsified with an equivalent amount of Freund's adjuvant to form stable emulsion which is marked as emulsified antigen; injecting 0.3mL of emulsified antigen into the abdominal cavity of each mouse, and carrying out primary immunization; after 14 days, booster immunization with emulsified antigen was performed once; on 28 days, the immunization was boosted once again; on day 42, booster immunization was performed again with neat antigen that was not emulsified; removing eyeball and blood, and collecting spleen at 45 days;
② preparation of fused cells:
recovering SP2/0 cells and screening by using 8-Ag selective medium; spleen was prepared as single suspension cells, washed twice with PBS and resuspended, counted separately, 1X 10 in volume8Spleen cells and 1X 107Adding the suspension of SP2/0 cells into the same 50mL centrifuge tube, and supplementing incomplete culture solution to 30 mL; after fully and uniformly mixing, centrifuging for 15min under the condition of 1500 r/min;
the supernatant was aspirated and the deposited fused cells were suspended in 100mL of HAT medium; then, cells were seeded into 5 96-well flat-bottom cell plates, 0.2mL per well; after inoculation, the cells were incubated at 37 ℃ with 5% CO2Culturing under the condition of saturated humidity; when the fused cells grow to half of the plate holes, amplifying the monoclonal cells to a 24-hole plate for continuous culture, replacing the monoclonal cells with a complete culture medium of 20% fetal calf serum for culture, and collecting the culture supernatant of the fused cells;
③ screening positive fusion cell strain by indirect ELISA:
coating a polystyrene micropore plate with 10 mu g/mL rGP73 antigen, wherein each hole is 100 mu L, and then sealing for 60min at the temperature of 37 ℃ by using a sealing solution containing 5% calf serum; then, washing with PBST washing solution containing 0.05% Toween for 3 times, 5 min/time;
adding 100 mu L of fusion cell culture supernatant prepared in the step ② into the coated plate, placing the coated plate in a wet box for incubation at 37 ℃ for 60min, washing the coated plate for 3 times and 5 min/time by PBST (peripheral blood pressure) solution, adding goat anti-mouse HRP-IgG (diluted by 1:5000 times), placing each hole with 100 mu L of wet box for incubation at 37 ℃ for 90min, washing the coated plate for 3 times and 5 min/time by PBST solution (each experiment is provided with 3 holes of negative, positive and blank controls), adding OPD substrate solution with 100 mu L of holes, placing the coated plate in a room temperature for 15min for observation, adding 50 mu L of stop solution into each hole to stop reaction and measure the OD value of each hole after obvious color change appears in the positive control holes, selecting two fusion cells with the largest OD value, cloning and expanding culture by a limiting dilution method;
respectively taking the culture supernatant of the fused cells as a primary antibody, using abcam goat anti-mouse IgG (HRP-labeled) as a secondary antibody, using verified rGP73 as an antigen, using abcam anti-human GP73 monoclonal antibody as a reference primary antibody, and verifying that the two fused hybridoma cells are positive to human GP73 through WB;
④ purification of monoclonal antibody:
collecting two fused cell culture supernatants, respectively slowly dropwise adding saturated ammonium sulfate with the same volume into the supernatants, and standing at 4 deg.C for more than 3 hr; centrifuging at 4 deg.C at 4200r/min for 30min, discarding supernatant, and dissolving precipitate with 200mL PBS (pH 7.4); adding saturated ammonium sulfate with a total volume of 33%, standing at 4 deg.C for 3 hr, and centrifuging at 4 deg.C at 4200r/min for 30 min; the pellet after centrifugation was dissolved in 200mL PBS and further filled into a 10kD dialysis bag; finally, the membrane is placed in an environment of 4 ℃, PBS dialysate (pH7.4) with 20 times volume is adopted for dialysis for 12h for desalting, and the antihuman rGP73 monoclonal antibodies are obtained and are respectively marked as antihuman rGP73 monoclonal antibody 1 and antihuman rGP73 monoclonal antibody 2;
⑤ recombinant anti-human rGP73 monoclonal antibody was demonstrated:
when the purchased rGP73 protein was used as an antigen, the monoclonal antibody prepared above was used as a primary antibody, and a rabbit anti-mouse IgG (HRP-labeled) antibody from abcam was used as a secondary antibody for WB, a positive band was produced at 73 kD. As shown in fig. 1, two monoclonal antibodies were used to verify rGP73 that a positive band appears, which indicates that the two monoclonal antibodies were successfully prepared (in fig. 1, positive fragments appear in lanes 2 and 3, and a positive fragment also appears in lane 4, indicating that both prepared monoclonal antibodies bind to GP73 antigen, and a positive band does not appear in lane 1, indicating that the monoclonal antibody binds to antigen specifically);
(2) preparation of latex microspheres of cross-linked anti-human rGP73 monoclonal antibody 1 and latex microspheres of cross-linked anti-human rGP73 monoclonal antibody 2:
crosslinking the anti-human rGP73 monoclonal antibody 1 obtained in the step (1) on a polystyrene latex microsphere by using a polystyrene latex microsphere with the diameter of 80-120 nm and adopting a chemical crosslinking method to obtain a latex microsphere of a crosslinked anti-human rGP73 monoclonal antibody 1;
crosslinking the anti-human rGP73 monoclonal antibody 2 obtained in the step (1) on a polystyrene latex microsphere by using a polystyrene latex microsphere with the diameter of 80-120 nm and adopting a chemical crosslinking method to obtain a latex microsphere of a crosslinked anti-human rGP73 monoclonal antibody 2;
(3) preparation of GP73 latex enhanced immunoturbidimetry kit
① formulation reagent R1:
according to the component content of the reagent R1, mixing the components in the same container, and uniformly mixing to obtain a reagent R1;
② formulation reagent R2:
mixing the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 1, the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 2 and the rest of other component substances prepared in the step (2) in the same container according to the component content of the reagent R2, and uniformly mixing to prepare a reagent R2;
③ formulation of GP73 calibrator:
mixing rGP73 and the rest other components in the same container according to the component content of the GP73 calibrator, and uniformly mixing to obtain the GP73 calibrator.
Example 5
This example is a method of determining the kit for the latex-enhanced immunoturbidimetry-based GP73 determination in examples 1-3 above:
the analysis method comprises a two-point end point method;
the reaction direction is ascending reaction;
the calibration mode is Logit-Log (4P);
the measuring wavelength is 600 nm;
the measuring temperature is 37 ℃;
sample reagent R1 reagent R2 ═ 10:180:60(μ L)
The testing steps are as follows: sucking 10 μ L of sample, adding 180 μ L of reagent R1, and incubating at 37 deg.C for 5 min; add 60. mu.L of reagent R2, read absorbance A1 after 1min, read absorbance A2 after 3min, calculate Δ A.
The calibration method comprises the following steps: 6 point calibration, adopting Hitachi 7180 full-automatic biochemical analyzer (or other brands and models), detecting, setting the concentration of the calibrator as follows: 0. 18.7, 55.7, 166.7, 500 and 1500 ng/mL. And calculating the GP73 content in the sample according to the calibration value and the delta A.
Example 6
This example is intended to evaluate the kit for the determination of GP73 based on latex enhanced immunoturbidimetry in the above examples:
(1) linear correlation verification
The reagent prepared by the formula of the embodiment 3 is compared with the existing GP73 detection kit approved by the State food and drug administration for detection, 100 clinical serum samples are detected, the detection result is shown in the table 1, and the correlation curve of the kit disclosed by the invention and other existing GP73 detection reagents sold on the market is obtained, which is shown in the figure 2. The detection result shows that the linear correlation curve of the two kits is that y is 1.0011+0.0857X, and the correlation coefficient R is2The greater correlation between the two is illustrated at 0.9996.
TABLE 1 alignment of the linear correlation of the kit of the invention with the on-market GP73 detection kit
Figure BDA0002282071620000211
Figure BDA0002282071620000221
(2) Linear range verification
The concentrations of rGP73 and physiological saline are 1500ng/mL, 500ng/mL, 166.7ng/mL,The concentrations of the respective test samples were measured using the kit of the present invention, and the linear regression equation was calculated using the dilution concentration as an independent variable and the measurement result as a dependent variable, and the relative deviation of the measurement results was calculated, as shown in Table 2, for the test samples of 56.7ng/mL, 18.7ng/mL and 0ng/mL (saline control). The results showed that the linear regression equation between the assay and the diluted concentration was 1.011+3.2175X, see FIG. 3, correlation coefficient R2The linear relation is good when the value is 1.000, and the linear range can reach 1500 ng/mL.
TABLE 2 validation of the Linear Range of the kit of the invention
Figure BDA0002282071620000231
(3) Accuracy verification
And taking one portion of traceable high-value serum quality control and one portion of traceable low-value serum quality control, detecting for 10 times by using the kit, taking a mean value, and comparing with a quality control target value. The result shows that the detection value has smaller relative deviation than the target value and higher accuracy. See table 3.
TABLE 3 accuracy verification results of the kit of the invention
Figure BDA0002282071620000232
Figure BDA0002282071620000241
(4) Precision verification
Taking high value and low value of clinical serum samples detected by the kit on sale, continuously detecting the same serum sample for 10 times by using the kit, and calculating the coefficient of variation of the kit. The precision detection data are shown in the following table 4, and the detection results show that the variation coefficients of the kit are smaller and respectively 0.34% and 1.73% when the kit is used for detecting high-value samples and low-value samples, and the precision is better.
TABLE 4 results of the precision verification of the kit of the present invention
Figure BDA0002282071620000242
(5) Verification of sensitivity and specificity
The imported indirect ELISA method GP73 detection kit is adopted to detect and screen 50 parts of positive serum and 50 parts of negative serum, a latex enhanced immunoturbidimetry kit prepared by anti-GP 73 polyclonal antibody sold in the market, a latex enhanced immunoturbidimetry kit prepared by anti-monoclonal antibody sold in the market and the kit of the invention are selected to synchronously detect the 100 parts of serum samples, the sensitivity and specificity of each kit are calculated by taking the imported indirect ELISA method detection kit as a gold standard according to the judgment standard of each kit, and the results are shown in Table 5. The result shows that the kit has higher sensitivity and specificity compared with two kits sold in the market. The invention has the outstanding advantages that: compared with a monoclonal antibody preparation kit, the kit has higher sensitivity, and the kit prepared by more cloned antibodies has higher specificity, thereby greatly improving the accuracy of clinical detection and meeting the requirement of clinical detection.
TABLE 5 comparison of sensitivity and specificity of the kit of the present invention with commercially available kits
Figure BDA0002282071620000251
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims (10)

1. A kit for measuring GP73 based on a latex enhanced immunoturbidimetry is characterized by comprising two liquid components of a reagent R1 and a reagent R2 which are independent of each other, and comprises the following components in parts by weight:
reagent R1:
Figure FDA0002282071610000011
the solvent is purified water;
reagent R2:
Figure FDA0002282071610000012
the solvent is purified water.
2. The kit for determining GP73 based on latex-enhanced immunoturbidimetry according to claim 1, comprising the following components in parts by weight:
reagent R1:
Figure FDA0002282071610000021
the solvent is purified water;
reagent R2:
Figure FDA0002282071610000022
the solvent is purified water.
3. The kit for determining GP73 based on latex-enhanced immunoturbidimetry according to claim 1, further comprising a GP73 calibrator comprising the following components in respective amounts:
Figure FDA0002282071610000023
Figure FDA0002282071610000031
the solvent is purified water.
4. The kit for determining GP73 based on latex-enhanced immunoturbidimetry according to claim 3, wherein the GP73 calibrator comprises the following components in parts by weight:
Figure FDA0002282071610000032
the solvent is purified water.
5. The latex-enhanced immunoturbidimetry-based GP73 kit of claim 3, wherein rGP73 in the GP73 calibrator is recombinant human GP73 protein.
6. The kit for measuring GP73 based on latex enhanced immunoturbidimetry according to claim 1, wherein the anti-human rGP73 monoclonal antibody 1 in the latex microsphere of cross-linked anti-human rGP73 monoclonal antibody 1 and the anti-human rGP73 monoclonal antibody 2 in the latex microsphere of cross-linked anti-human rGP73 monoclonal antibody 2 are two anti-human rGP73 monoclonal antibodies having rGP73 epitope recognition function.
7. The kit for determining GP73 based on latex enhanced immunoturbidimetry according to any one of claims 1 to 6, wherein the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 1 are obtained by the following method: crosslinking an anti-human rGP73 monoclonal antibody 1 to a polystyrene latex microsphere by using the polystyrene latex microsphere with the diameter of 80-120 nm by adopting a chemical crosslinking method;
the method for obtaining the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 2 comprises the following steps: and (2) crosslinking the anti-human rGP73 monoclonal antibody 2 to the polystyrene latex microspheres by using the polystyrene latex microspheres with the diameters of 80-120 nm and adopting a chemical crosslinking method.
8. The kit for determining GP73 based on latex enhanced immunoturbidimetry according to claim 7, wherein the specific preparation method of the anti-human rGP73 monoclonal antibody 1 and the anti-human rGP73 monoclonal antibody 2 comprises the following steps:
① immunization of mice:
2mL of rGP73 antigen of 1mg/mL is taken and fully emulsified with an equivalent amount of Freund's adjuvant to form stable emulsion which is marked as emulsified antigen; injecting 0.3mL of emulsified antigen into the abdominal cavity of each mouse, and carrying out primary immunization; after 14 days, booster immunization with emulsified antigen was performed once; on 28 days, the immunization was boosted once again; on day 42, booster immunization was performed again with neat antigen that was not emulsified; removing eyeball and blood, and collecting spleen at 45 days;
② preparation of fused cells:
recovering SP2/0 cells and screening by using 8-Ag selective medium; spleen was prepared as single suspension cells, washed twice with PBS and resuspended, counted separately, 1X 10 in volume8Spleen cells and 1X 107Adding the suspension of SP2/0 cells into the same 50mL centrifuge tube, and supplementing incomplete culture solution to 30 mL; after fully and uniformly mixing, centrifuging for 15min under the condition of 1500 r/min;
the supernatant was aspirated and the deposited fused cells were suspended in 100mL of HAT medium; then, cells were seeded into 5 96-well flat-bottom cell plates, 0.2mL per well; after inoculation, the cells were incubated at 37 ℃ with 5% CO2Culturing under the condition of saturated humidity; when the fused cells grow to half of the plate holes, amplifying the monoclonal cells to a 24-hole plate for continuous culture, replacing the monoclonal cells with a complete culture medium of 20% fetal calf serum for culture, and collecting the culture supernatant of the fused cells;
③ screening positive fusion cell strain by indirect ELISA:
coating a polystyrene micropore plate with 10 mu g/mL rGP73 antigen, wherein each hole is 100 mu L, and then sealing for 60min at the temperature of 37 ℃ by using a sealing solution containing 5% calf serum; then, washing with PBST washing solution containing 0.05% Toween for 3 times, 5 min/time;
adding 100 mu L of fusion cell culture supernatant prepared in the step ② into the coated plate, placing the coated plate in a wet box for incubation at 37 ℃ for 60min, washing the coated plate for 3 times and 5 min/time by PBST (basic fibroblast growth factor) solution, adding 100 mu L of goat anti-mouse HRP-IgG with the dilution multiple of 1:5000 into the coated plate, placing the coated plate in the wet box for incubation at 37 ℃ for 90min, washing the coated plate for 3 times and 5 min/time by PBST solution, adding 100 mu L/hole of OPD substrate solution, placing the coated plate at room temperature for 15min for observation, adding 50 mu L of stop solution into each hole after obvious color change appears in the holes, stopping reaction, measuring the OD value of each hole, selecting two fusion cells with the largest OD value, cloning and carrying out expanded culture by a limiting dilution method;
respectively taking the culture supernatant of the fusion cells as a primary antibody, using goat anti-mouse IgG marked by HRP as a secondary antibody, using verified rGP73 as an antigen, using the existing anti-human GP73 monoclonal antibody as a reference primary antibody, and verifying that the two fusion hybridoma cells are positive to anti-human GP73 through WB;
④ purification of monoclonal antibody:
collecting two fused cell culture supernatants, respectively slowly dropwise adding saturated ammonium sulfate with the same volume into the supernatants, and standing at 4 deg.C for more than 3 hr; centrifuging at 4 deg.C at 4200r/min for 30min, discarding supernatant, and dissolving precipitate with 200mL PBS; adding saturated ammonium sulfate with a total volume of 33%, standing at 4 deg.C for 3 hr, and centrifuging at 4 deg.C at 4200r/min for 30 min; the pellet after centrifugation was dissolved in 200mL PBS and further filled into a 10kD dialysis bag; finally, the membrane is placed in an environment of 4 ℃, PBS dialysate with 20 times volume is adopted for dialysis for 12h for desalting, and the antihuman rGP73 monoclonal antibodies are obtained and are respectively marked as antihuman rGP73 monoclonal antibody 1 and antihuman rGP73 monoclonal antibody 2;
⑤ recombinant anti-human rGP73 monoclonal antibody was demonstrated:
rGP73 protein is used as an antigen, the prepared monoclonal antibody is used as a primary antibody, HRP-labeled rabbit anti-mouse IgG is used as a secondary antibody for preparing WB, and the antigen has a positive band at 73 kD; the two monoclonal antibodies are used for verifying rGP73 that a positive band appears, namely, the two monoclonal antibodies are successfully prepared.
9. A method for the preparation of a kit for the latex-enhanced immunoturbidimetry-based GP73 assay according to any of claims 1 to 8, comprising the steps of:
(1) preparation of anti-human rGP73 monoclonal antibody 1 and anti-human rGP73 monoclonal antibody 2:
① immunization of mice:
2mL of rGP73 antigen of 1mg/mL is taken and fully emulsified with an equivalent amount of Freund's adjuvant to form stable emulsion which is marked as emulsified antigen; injecting 0.3mL of emulsified antigen into the abdominal cavity of each mouse, and carrying out primary immunization; after 14 days, booster immunization with emulsified antigen was performed once; on 28 days, the immunization was boosted once again; on day 42, booster immunization was performed again with neat antigen that was not emulsified; removing eyeball and blood, and collecting spleen at 45 days;
② preparation of fused cells:
recovering SP2/0 cells and screening by using 8-Ag selective medium; spleen was prepared as single suspension cells, washed twice with PBS and resuspended, counted separately, 1X 10 in volume8Spleen cells and 1X 107Adding the suspension of SP2/0 cells into the same 50mL centrifuge tube, and supplementing incomplete culture solution to 30 mL; after fully and uniformly mixing, centrifuging for 15min under the condition of 1500 r/min;
the supernatant was aspirated and the deposited fused cells were suspended in 100mL of HAT medium; then, cells were seeded into 5 96-well flat-bottom cell plates, 0.2mL per well; after inoculation, the cells were incubated at 37 ℃ with 5% CO2Culturing under the condition of saturated humidity; when the fused cells grow to half of the plate holes, amplifying the monoclonal cells to a 24-hole plate for continuous culture, replacing the monoclonal cells with a complete culture medium of 20% fetal calf serum for culture, and collecting the culture supernatant of the fused cells;
③ screening positive fusion cell strain by indirect ELISA:
coating a polystyrene micropore plate with 10 mu g/mL rGP73 antigen, wherein each hole is 100 mu L, and then sealing for 60min at the temperature of 37 ℃ by using a sealing solution containing 5% calf serum; then, washing with PBST washing solution containing 0.05% Toween for 3 times, 5 min/time;
adding 100 mu L of fusion cell culture supernatant prepared in the step ② into the coated plate, placing the coated plate in a wet box for incubation at 37 ℃ for 60min, washing the coated plate for 3 times and 5 min/time by PBST (basic fibroblast growth factor) solution, adding 100 mu L of goat anti-mouse HRP-IgG with the dilution multiple of 1:5000 into the coated plate, placing the coated plate in the wet box for incubation at 37 ℃ for 90min, washing the coated plate for 3 times and 5 min/time by PBST solution, adding 100 mu L/hole of OPD substrate solution, placing the coated plate at room temperature for 15min for observation, adding 50 mu L of stop solution into each hole after obvious color change appears in the holes, stopping reaction, measuring the OD value of each hole, selecting two fusion cells with the largest OD value, cloning and carrying out expanded culture by a limiting dilution method;
respectively taking the culture supernatant of the fusion cells as a primary antibody, using goat anti-mouse IgG marked by HRP as a secondary antibody, using verified rGP73 as an antigen, using the existing anti-human GP73 monoclonal antibody as a reference primary antibody, and verifying that the two fusion hybridoma cells are positive to anti-human GP73 through WB;
④ purification of monoclonal antibody:
collecting two fused cell culture supernatants, respectively slowly dropwise adding saturated ammonium sulfate with the same volume into the supernatants, and standing at 4 deg.C for more than 3 hr; centrifuging at 4 deg.C at 4200r/min for 30min, discarding supernatant, and dissolving precipitate with 200mL PBS; adding saturated ammonium sulfate with a total volume of 33%, standing at 4 deg.C for 3 hr, and centrifuging at 4 deg.C at 4200r/min for 30 min; the pellet after centrifugation was dissolved in 200mL PBS and further filled into a 10kD dialysis bag; finally, the membrane is placed in an environment of 4 ℃, PBS dialysate with 20 times volume is adopted for dialysis for 12h for desalting, and the antihuman rGP73 monoclonal antibodies are obtained and are respectively marked as antihuman rGP73 monoclonal antibody 1 and antihuman rGP73 monoclonal antibody 2;
⑤ recombinant anti-human rGP73 monoclonal antibody was demonstrated:
rGP73 protein is used as an antigen, the prepared monoclonal antibody is used as a primary antibody, HRP-labeled rabbit anti-mouse IgG is used as a secondary antibody for preparing WB, and the antigen has a positive band at 73 kD; the two monoclonal antibodies are used for verifying rGP73 that a positive strip appears, namely the two monoclonal antibodies are successfully prepared;
(2) preparation of latex microspheres of cross-linked anti-human rGP73 monoclonal antibody 1 and latex microspheres of cross-linked anti-human rGP73 monoclonal antibody 2:
crosslinking the anti-human rGP73 monoclonal antibody 1 obtained in the step (1) on a polystyrene latex microsphere by using a polystyrene latex microsphere with the diameter of 80-120 nm and adopting a chemical crosslinking method to obtain a latex microsphere of a crosslinked anti-human rGP73 monoclonal antibody 1;
crosslinking the anti-human rGP73 monoclonal antibody 2 obtained in the step (1) on a polystyrene latex microsphere by using a polystyrene latex microsphere with the diameter of 80-120 nm and adopting a chemical crosslinking method to obtain a latex microsphere of a crosslinked anti-human rGP73 monoclonal antibody 2;
(3) preparation of GP73 latex enhanced immunoturbidimetry kit
① formulation reagent R1:
according to the component content of the reagent R1, mixing the components in the same container, and uniformly mixing to obtain a reagent R1;
② formulation reagent R2:
mixing the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 1, the latex microspheres of the cross-linked anti-human rGP73 monoclonal antibody 2 and the rest of other component substances prepared in the step (2) in the same container according to the component content of the reagent R2, and uniformly mixing to prepare a reagent R2;
③ formulation of GP73 calibrator:
the GP73 calibrator contained the following components and corresponding contents:
Figure FDA0002282071610000081
the solvent is purified water;
the GP73 calibrator was prepared by mixing rGP73 and the remaining other ingredients in the same vessel according to the ingredient content of the GP73 calibrator.
10. Use of a kit for the latex-enhanced immunoturbidimetry based GP73 assay according to any of claims 1 to 8, comprising the following specific steps:
(1) sucking 10 μ L of sample, adding 180 μ L of reagent R1, and incubating at 37 deg.C for 5 min;
(2) adding 60 μ L reagent R2, and incubating at 37 deg.C;
(3) after incubation for 1min, reading a light absorption value A1, after incubation for 3min, reading a light absorption value A2, and calculating delta A;
the calibration method is 6-point calibration, a full-automatic biochemical analyzer is adopted for detection, and the concentrations of calibrators are set as follows: 0. 18.7, 55.7, 166.7, 500 and 1500 ng/mL; and (4) according to the calibration value, calculating the GP73 content in the sample according to the Delta A.
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