CN111751534A - Glypican 3 detection kit and application thereof - Google Patents
Glypican 3 detection kit and application thereof Download PDFInfo
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Abstract
A glypican 3 detection kit and application thereof, wherein the kit comprises: (a) a first antibody: GPC3 monoclonal antibody working solution, wherein the GPC3 monoclonal antibody is mouse-derived GPC3 antibody or human-derived GPC3 antibody; (b) secondary antibody: HRP marks goat anti-mouse IgG antibody working solution; (c) DAB color development liquid A buffer solution and DAB color development liquid B buffer solution; (d) hematoxylin cell staining solution; (e) tissue antigen repair fluid; (f) 2-3% by volume of H2O2(ii) a The detection kit has the characteristics of high sensitivity, high specificity and high accuracy, and can distinguish canceration tissues from normal tissues and canceration cells and normal cells in the canceration tissues; gThe PC3 shows negative expression staining in normal liver cells and positive expression in cancer cell cytoplasm and cell membrane, effectively distinguishes liver cancer cells from normal cells, and provides reference basis for clinical diagnosis and treatment.
Description
Technical Field
The invention relates to the technical field of molecular biology, medical immunology and in-vitro serological diagnosis, in particular to a glypican 3 detection kit and application thereof, and the clinical applicability of an antibody is verified by establishing the kit which can be used for clinical pathological diagnosis.
Background
Hepatocellular Carcinoma (HCC) is the fifth most common cancer with high worldwide incidence, and the number of deaths per year in our country is about 11 thousands of people with liver cancer, accounting for 45% of the deaths in liver cancer worldwide. Surgical removal of tumors is currently the best and most rapid method of treating HCC. However, most patients lose the best treatment opportunity at the time of treatment, and only 10-20% of patients can be resected by an early operation, and even if the patients are resected by the operation, the recurrence rate is high. Therefore, early detection, early diagnosis and early treatment of HCC are the key to improve the survival rate of patients and are also the key to prognosis and treatment.
Glypican 3(glypican-3, GPC3), the gene of which is located in human chromosome X26q1, 900kb in length, 1740bp in GPC3 open reading frame, encodes 580 amino acids, and has a molecular weight of 66 kD. The protein encoded by GPC3 gene belongs to Heparan Sulfate Proteoglycans (HSPGs), is anchored on the cell surface by glycosyl-phosphatidyl protein (CPI), and can be combined with growth factors and the like to regulate the behaviors of cell growth, reproduction, differentiation, adhesion, migration and the like.
The research proves that the GPC3 protein is highly expressed in liver cancer tissues, but is not expressed or is rarely expressed in non-cancer tissues, the content of GPC3 can be detected in serum, the positive rate of GPC3 of liver cancer patients is over 90 percent by an immunohistochemical method, the expression rate of other benign liver diseases and normal liver tissues is extremely low, and the specificity performance of the GPC3 protein is over 96 percent. In addition, the detection rate of GPC3 in serum was significantly higher for HCC than AFP, which were 76.3% and 31.3%, respectively. Therefore, the diagnosis rate of GPC3 on HCC is obviously higher than that of AFP, and especially the GPC3 plays an important role in the diagnosis of AFP-negative early liver cancer.
GPC3 is used as a new biological index, no detection method aiming at the GPC is provided clinically at present, the application in monitoring the occurrence and development of tumors is still blank, but the blank is difficult to break through, the GPC3 protein is difficult to express in an in vitro recombination mode, the preparation of antibodies is influenced, a cell line capable of stably expressing the GPC3 protein is constructed, a mouse is immunized by using the protein expressed by the cell line, and a hybridoma cell line capable of expressing monoclonal antibodies with higher titer is screened and prepared. Since hybridoma cells are at risk of losing high specific activity, or are poorly conditioned or even dead. Corresponding sequences can be obtained through sequencing of the hybridomas, and antibodies can be stably obtained in a recombinant protein mode; therefore, the developer or the producer does not need to reserve the antibody by using the hybridoma cell as a carrier, and can store, transmit, communicate and identify the antibody in the form of a base sequence. After the clone of the GPC3 monoclonal antibody was modified by sequencing treatment, the constructed humanized antibody recombinant expression vector was used to express a large amount of antibody in a stable cell line verified by the company.
Disclosure of Invention
The invention aims to provide a glypican 3 detection kit and application thereof, and aims to solve the technical problem that liver cancer diagnosis in the prior art is insensitive.
In order to achieve the purpose, the invention adopts the following technical scheme:
a glypican 3 detection kit, characterized in that the kit contains:
(a) a first antibody: GPC3 monoclonal antibody working solution, wherein the GPC3 monoclonal antibody is mouse GPC3 antibody or human GPC3 antibody, and both antibodies are prepared in the laboratory;
(b) secondary antibody: HRP marks goat anti-mouse IgG antibody working solution;
(c) DAB color development liquid A buffer solution and DAB color development liquid B buffer solution;
(d) hematoxylin cell staining solution;
(e) tissue antigen repair fluid;
(f) 2-3% by volume of H2O2；
Further preferably, the preparation method of the mouse GPC3 antibody comprises the following steps:
(a) animal immunization: purifying GPC3 protein by affinity chromatography and an ion exchange layer, concentrating to 5mg/ml, mixing and emulsifying a GPC3 protein antigen and Freund's adjuvant in equal volume, and then immunizing BALb/c mice at multiple points under the back skin for 0 day, 14 days, 28 days and 35 days, wherein each mouse is 0.2mg, 0 day immunization is Freund's complete adjuvant, blood is collected after one week of last immunization, the antibody titer is detected by indirect ELISA, and the selected mice are subjected to abdominal cavity impact by hepatitis B vaccine stock solution after one week of last immunization, namely, the standby fusion mice;
(b) cell fusion: restoring and culturing SP2/0 cell strain, performing expanded culture 3 days before fusion, removing RPMI1640 cell culture solution 1 day before fusion, adding culture solution again, preparing mouse spleen cell suspension from spleen cells of the standby fusion mouse selected in the step (a), and fusing with SP2/0 cell stored in the laboratory;
(c) large-scale preparation of monoclonal antibodies: the ELISA method is used for determining that the supernatant fluid selects a hole with high positive, the positive hole cell is transferred into a 24-hole plate for amplification culture, and subcloning is timely carried out for 3 times to obtain a cell line stably secreting the antibody; ascites of the cell-immunized mouse is obtained and purified to obtain the GPC3 monoclonal antibody.
Further, the animal immunization specifically comprises: after GPC3 protein purification, monitoring the wavelength at 280nm, collecting an elution peak, taking a small amount of eluted protein liquid for electrophoretic analysis, dialyzing the rest with 0.85% NaCl solution at 4 ℃ for 48h, then concentrating to 5mg/ml with an ultrafiltration membrane, mixing and emulsifying a glypican 3 protein antigen and Freund's adjuvant in equal volume, and then carrying out subcutaneous multipoint immunization on Bal b/c mice at the back for 0 day, 14 day, 28 day and 35 day, 0.2 mg/mouse, collecting blood after the last immunization for one week, detecting the antibody titer in serum by indirect ELISA, diluting the serum by 104The OD value at time of doubling is 1.089, the selected mice are impacted in the abdominal cavity by hepatitis B vaccine stock solution after one week of the last immunization, and the spleen of the mice is taken 3 days later for cell fusion.
Further preferably, the cell fusion specifically comprises:
(a) recovering the SP2/0 cell strain and preparing a mouse spleen cell suspension: recovering and culturing SP2/0 cell strain before cell fusion, performing amplification culture 3 days before cell fusion, removing RPMI1640 cell culture solution 1 day before cell fusion, and adding culture solution again to complete preparation of SP2/0 cells; killing the immune mice, and preparing a mouse splenocyte suspension by a conventional method;
(b) cell mixing preparation: respectively adding a proper amount of incomplete DMEM culture solution into counting results of splenocytes and SP2/0 cells, shaking and uniformly mixing SP2/0 cells, blowing and beating the splenocytes uniformly by a pipette, combining the splenocytes and SP2/0 cells in a 50ml centrifuge tube according to a ratio of 1:2, uniformly mixing, adding the incomplete DMEM culture solution into the centrifuge tube to 50ml, centrifuging for 5 minutes, emptying supernatant, tapping the bottom of the fusion tube to loosen and uniformly the precipitated cells, and placing the centrifuge tube in 37 ℃ water bath to prepare fusion;
(c) cell fusion: slowly dripping 50% PEG4000lml preheated at 37 ℃ into a mixed cell tube by using a dropper, rotating a centrifugal tube while dripping to ensure that cells are stored in a uniformly mixed state, standing for 90s, immediately and slowly adding 15ml of serum-free DMEM medium at 37 ℃, centrifuging for 5 min, discarding supernatant, adding DMEM complete culture solution, mixing, adding suspension into 96-hole cell culture plates respectively, culturing in a 37 ℃ incubator at 100 mu l/hole, adding HAT culture solution into the next day cell plate at 100 mu l/hole, changing HAT culture solution once every 3 days, observing whether hybridoma appears, changing HT culture medium after two weeks, and observing the growth condition of fused cells.
The application of the glypican 3 detection kit is characterized in that the kit is used for clinical liver cancer diagnosis.
Compared with the prior art, the invention has the following characteristics and beneficial effects:
the practical expression and purification of the invention is carried out, the GPC3 antibody constructs an immunohistochemical kit which can be used for pathological diagnosis, the clinical application performance of the kit in liver cancer diagnosis is verified by detecting clinical pathological sections, and the result shows that the kit related by the invention has excellent specificity in liver cancer diagnosis, can be used for effectively eliminating false positive results of liver cancer diagnosis, and has good clinical applicability.
The detection kit has the characteristics of high sensitivity, high specificity and high accuracy, and can distinguish canceration tissues from normal tissues and canceration cells and normal cells in the canceration tissues; GPC3 shows negative expression and staining in normal liver cell and positive expression in cancer cell cytoplasm and cell membrane, and can distinguish liver cancer cell from normal cell effectively to provide reference for clinical diagnosis and treatment.
Drawings
FIG. 1 is a color representation of a healthy liver GPC3 immunohistochemical pathological section;
FIG. 2 is a color representation of liver GPC3 immunohistochemical pathological section of a liver cirrhosis patient;
FIG. 3 is a first color rendering diagram of liver GPC3 immunohistochemical pathological section of liver cancer patient;
FIG. 4 is a color representation of liver GPC3 immunohistochemical pathological section of liver cancer patient showing color development two;
FIG. 5 is a color representation of liver GPC3 immunohistochemical pathological section of liver cancer patient;
FIG. 6 is a fourth color representation of liver GPC3 immunohistochemical pathological section of liver cancer patient.
Detailed Description
In order to make the technical means, innovative features, objectives and functions realized by the present invention easy to understand, the present invention is further described below.
The examples described herein are specific embodiments of the present invention, are intended to be illustrative and exemplary in nature, and are not to be construed as limiting the scope of the invention. In addition to the embodiments described herein, those skilled in the art will be able to employ other technical solutions which are obvious based on the disclosure of the claims and the specification of the present application, and these technical solutions include technical solutions which make any obvious replacement or modification for the embodiments described herein.
The kit related by the invention is an immunohistochemical staining detection kit for liver cancer, and specifically comprises:
1) a first antibody: GPC3 monoclonal antibody working solution, including a monoclonal antibody against mouse GPC3 and a monoclonal antibody against human GPC 3;
2) secondary antibody: HRP marks goat anti-mouse IgG antibody working solution;
3A) DAB color development liquid A buffer solution;
3B) DAB color development liquid B buffer solution;
4) hematoxylin cell staining solution;
5) tissue antigen repair fluid;
preparation of an anti-mouse GPC3 antibody: the method comprises the steps of preparing a stable cell line capable of expressing glypican 3 protein, purifying the protein by adopting affinity chromatography and ion exchange chromatography, monitoring the wavelength to be 280nm, collecting an elution peak, taking a small amount of eluted protein liquid for electrophoretic analysis, and dialyzing the rest by using 0.85% NaCl solution at 4 ℃ for 48 hours. Concentrating with ultrafiltration membrane to 5mg/ml, mixing and emulsifying glypican-3 protein antigen and Freund's adjuvant (Freund's complete adjuvant for 0 day immunization, Freund's incomplete adjuvant for 14 days, 28 days and 35 days) at equal volume, immunizing Balb/c mouse with subcutaneous multiple spots on back for 0 day, 14 days, 28 days and 35 days, 0.2 mg/mouse, collecting blood after last immunization for one week, detecting antibody titer by indirect ELISA, diluting serum for 104The OD value at time of doubling is 1.089, the selected mice are impacted in the abdominal cavity by hepatitis B vaccine stock solution after one week of the last immunization, and the spleen of the mice is taken 3 days later for cell fusion.
SP2/0 cell line was cultured by recovery before cell fusion, and the culture was expanded 3 days before cell fusion, and RPMI1640 cell culture medium (Gibco) was removed 1 day before cell fusion, and the culture medium was added again to prepare SP2/0 cells. Meanwhile, the immunized mice are sacrificed and a mouse splenocyte suspension is prepared according to a conventional method. According to counting results of the splenocytes and SP2/0 cells, a proper amount of incomplete IMDM culture solution (Gibco) is respectively added, the SP2/0 cells are shaken and uniformly mixed, and the splenocytes are uniformly blown and beaten by a pipette. Then, the spleen cells and SP2/0 cells were combined in a 50ml centrifuge tube at a ratio of 1:2, and mixed well. Incomplete MDM medium was added to 50ml, centrifuged for 5 min and the supernatant was decanted. And (3) slightly tapping the bottom of the fusion tube to enable the precipitated cells to be loose and uniform, and putting the centrifugal tube in a water bath at 37 ℃ to prepare fusion. 50% PEG4000lml preheated at 37 ℃ is slowly dripped into the mixed cell tube by a dropper, and the centrifugal tube is rotated while dripping, so that the cells are stored in a uniform state. Immediately after standing for 90s, 15ml of serum-free MDM medium (Gibco) were slowly added, and centrifuged at 37 ℃ for 5 minutes, and the supernatant was discarded. MDM complete medium (Gibco) was added, mixed and the suspension was added to 96-well cell culture plates, 100. mu.l/well, and cultured in a 5% CO2 incubator at 37 ℃. Day 2 plates were plated with HAT medium (MDM contains 1 × HAT (Sigma))100 μ l/well. HAT culture medium was changed every 3 days to observe the appearance of hybridomas, and HT medium (MDM containing 1 × HT (Sigma)) was changed two weeks later to observe the growth of fused cells.
And observing the growth condition of the hybridoma cells from the seventh day after cell fusion, and sucking out supernatant for antibody ELISA detection when the hybridoma cells grow to a bottom area of a hole of 1/10 or more. Transferring the positive hole cells into a 24-hole plate for amplification culture and timely subcloning. The cell line stably secreting antibody was obtained by 3 subcloning, named GPC3-FX, and was frozen in time. The cells are used for immunizing a mouse to obtain ascites, and the ascites is purified to obtain the glypican 3(GPC3) monoclonal antibody.
Preparation of an anti-human GPC3 antibody: the cell line from which the glypican 3 protein (GPC3) antibody has been obtained was subjected to sequencing by a third-party organization to obtain the gene sequences of the variable regions of the heavy and light chains of the antibody. The heavy chain variable region sequence was spliced to a human IgG1Fc fragment and the light and recombinant heavy chain DNA sequences were synthesized separately by Jinzhi, Suzhou. Splicing the gene sequence of the heavy chain variable region and the gene sequence of the light chain variable region with the Fc gene sequence of a human antibody to prepare a recombinant humanized GPC3 antibody heavy chain sequence VH-hFc-GPC 3;
designing NheI and EcoRI restriction sites at two ends of the synthetic gene fragment, and taking pUC57 as a vector to obtain plasmids carrying recombinant genes, namely pUC57-VL-GPC3 and pUC57-VH-hFc-GPC3 respectively;
preparation of humanized recombinant plasmid: the recombinant pUC57-VL-GPC3 plasmid and pUC57-VH-hFc-GPC3 plasmid were digested with vector pCDNA3.1/ZEO (+) by Nhe I and EcoR I, respectively, and VL-GPC3 fragment, VH-hFc-GPC3 fragment and pCDNA3.1/ZEO (+) vector fragment were recovered and ligated with T4Ligase to give recombinant plasmids pCDNA3.1/ZEO (+) -VL-GPC3 and pCDNA3.1/ZEO (+) -VH-hFc-GPC3, respectively.
The construction of the humanized recombinant plasmid of the present invention is characterized in that: the Fc gene sequence of the humanized antibody in the step (1) is a human IgG1Fc gene sequence.
The present invention provides use of a humanized recombinant plasmid for constructing a cell line stably expressing the recombinant humanized GPC3 antibody according to claim 1, wherein the humanized recombinant plasmid comprises: the method comprises the following steps:
A. transfecting and screening positive clones;
co-transfecting the recombinant plasmids pCDNA3.1/ZEO (+) -VL-GPC3 and pCDNA3.1/ZEO (+) -VH-hFc-GPC3 of claim 4 with cationic liposome into Chinese hamster ovary cells CHO-K1, and then selecting a stably transfected cell line integrating the GPC-3 antibody;
B. pressure culture of aminomethylpterin MTX;
b, digesting all positive clone cells formed in the step A by using pancreatin, inoculating the positive clone cells into a cell culture dish, and performing mixed culture to obtain a cell culture dish capable of being cultured at 10 DEG C-6A mixed clone of recombinant CHO-K1 cells grown normally at mol/L aminomethylpterin MTX was designated CHO/hFc-GPC 3.
The application of the cell strain is characterized in that: for use in the preparation of a recombinant humanized GPC3 antibody, the humanized GPC3 antibody of claim 1.
The method for producing the humanized GPC3 antibody of the present invention is characterized in that: the method comprises the following steps:
C. carrying out amplification culture on the monoclonal and harvesting the protein;
selecting a cell strain CHO/hFc-GPC3 with the highest expression level to culture in a CD CHO culture medium, changing to a serum-free culture medium when the cells are full, collecting cell culture solution after 1-2 days, and continuously collecting the solution for 3-5 times; adopting Protein ASepharose 4Fast Flw separation medium to carry out affinity chromatography, and purifying expression Protein; a humanized recombinant glypican 3 protein (GPC3) antibody was obtained.
The specific application mode of the glypican 3 detection kit comprises the following steps:
paraffin section staining procedure:
1. dewaxing and hydrating:
1) dewaxing: slicing the dried paraffin into fresh dimethylbenzene, soaking for 10 minutes, and replacing the dye vat for 2 times;
2) hydration: the slices are hydrated by descending alcohol, absolute ethyl alcohol is used for 5 minutes, 95% ethyl alcohol is used for 2 times (each time is 2 minutes), and 85% ethyl alcohol is used for 2 minutes; 2 minutes with 75% ethanol, washing with tap water, 2 minutes with deionized water and 2 times;
2. endogenous catalase clearance: incubating 2-3% hydrogen peroxide (diluted with 0.01M PBS) at room temperature for 5-10 min to eliminate the effect of endogenous catalase, and washing the slices with distilled water for 3 times;
3. antigen retrieval: heating 0.01M citric acid buffer solution (pH6.0, reagent 5) to 100 deg.C in beaker, placing into slice, maintaining 95-100 deg.C, performing thermal restoration for 2-3 min (or heating sodium citrate buffer solution in beaker to 100 deg.C, performing microwave restoration in microwave oven for 70 s × 2 times), cooling antigen restoration solution to room temperature naturally or indirectly cooling to room temperature via cold water bath, washing specimen with 0.01M PBS for 3 times, each for 1-3 min.
In the case of antigen retrieval, the following points should be noted.
1) After restoration, the buffer solution needs to be naturally cooled, the slices can be taken out after washing with tap water, and the quenching can cause crystallization or antigen blocking.
2) The amount of buffer must be such that all sections are soaked, and the citrate buffer cannot be reused.
3) If the reagent is a micro concentrated solution, the solution attached to the inner cover and the tube wall is separated to the bottom by low-speed centrifugation before use.
4. And (3) performing primary anti-incubation, namely wiping excess liquid around the tissue of the slide with absorbent paper, marking the specimen area with immunohistochemical strokes, adding a drop (20-50 microliters) of primary anti-working solution into the stroke area, and incubating for 2 hours at room temperature or incubating for 60 minutes at 37 ℃. The primary antibody is mouse GPC3 antibody, but human GPC3 antibody can be used, and the specific working concentration needs to be adjusted according to the purity and concentration of the antibody in each batch.
5. Washing: the specimens were washed 3 times with 0.01M PBS, each for 1-3 min.
6. And (3) secondary antibody incubation: excess fluid around the slide tissue was wiped dry with absorbent paper, a drop of secondary antibody working fluid (20-50 μ l) was added to the circled area, and incubated for 10 min at room temperature or 37 ℃.
7. Washing: the specimens were washed 3 times with 0.01M PBS, each for 1-3 minutes.
8. Color development:
1) removing the PBS liquid, wiping the PBS liquid with absorbent paper and slicing the PBS liquid;
2) dripping 1-2 drops of DAB color development liquid which is prepared freshly and 50-100 microliters (reagents 3A and B are prepared according to a proportion), developing for 5-10 minutes in a dark place at room temperature, observing a dyeing result under an optical microscope, and avoiding over-deep color development; the sections were rinsed with tap water to stop the color development.
9. And (3) counterstaining, namely adding 100 mu L of hematoxylin (reagent 4) for counterstaining, incubating for about 5 minutes, washing for three times by water, performing rapid fading treatment by using 1% hydrochloric acid ethanol for 30 seconds, and washing for 3 minutes by using deionized water. According to the intensity of the hematoxylin staining solution and the length of incubation time, the contrast staining result causes the cell nucleus to present a reaction from light blue to dark blue, and the judgment of correct results can be influenced by over-staining or insufficient staining.
10. Dewatering, transparent and sealing sheet
1) Soaking the slices in 70% ethanol for 2 min; soaking the slices in 80% ethanol for 2 min;
2) soaking the slices in 90% ethanol for 2 min; soaking the slices in 95% ethanol for 2 min;
3) soaking the slices in anhydrous ethanol for 2min, and replacing the dye vat once again; placing the slices into xylene, soaking for 2min, and replacing the dye vat once again;
4) the slices were sealed with neutral resin glue, examined under a microscope, and photographed.
(II) frozen section or cell staining procedure:
1. pretreatment of frozen sections or cells for staining:
1) drying the frozen slices by a fan at room temperature; cultured cells may be grown as smears or patches.
2) Fixing: fixing 4% paraformaldehyde or 10% formalin at room temperature for 30 min; washing with deionized water for 3min × 3. The rest steps are the same as the 3-10 steps in the paraffin section staining procedure.
FIGS. 1 to 6 are color representations of liver slices prepared by the above method, wherein for the convenience of observation, substantial examination reference data are submitted at the same time of filing the application, color representations 1 to 6 respectively correspond to FIGS. 1 to 6 of the drawings of the specification, and FIG. 1 is a color representation of a healthy liver GPC3 immunohistochemical reaction pathological section; FIG. 2 is a color representation of liver GPC3 immunohistochemical pathological section of a liver cirrhosis patient; FIGS. 3 to 6 are color development graphs of liver GPC3 immunohistochemical pathological sections of liver cancer patients, and it can be seen from FIG. 2 that liver cells of liver cirrhosis patients cannot be stained by GPC3 antibody; from fig. 3-6, it can be seen that the cytoplasm and cell membrane of liver cells of liver cancer patients are stained brick red by the specificity of the GPC3 antibody, and from fig. 2-6 and fig. 1, the kit is used for pathological diagnosis of liver cancer, has clear color development and clear contrast, and can be widely applied to scientific research and clinical diagnosis.
And (3) evaluating the performance of the immunohistochemical detection kit:
1. clinical performance verification
The GPC3 immunohistochemical kit produced by the invention is used for clinically detecting histopathological sections of 228 liver cancer patients, 40 liver cirrhosis patients and 100 liver tissue normal patients, and 5 liver cancer specimens, 5 liver cirrhosis specimens and 5 normal control specimens are repeatedly stained in a laboratory. Staining was repeated 3 times per specimen using a random double-blind method. All specimen staining results showed 100% reproducibility; 5 liver cancer specimens, 5 cirrhosis specimens, and 5 normal control specimens were stained in 5 days in 3 laboratories. The dyeing is repeated by adopting a random double-blind method, and all specimen dyeing results show 100 percent of repeatability; the laboratory repeatability tests were conducted between laboratories with different geographical locations at 3. 10 liver cancer specimens, 10 cirrhosis specimens and 10 normal control specimens were randomly double-blindly assigned and stained. All specimen staining results showed 100% reproducibility. The results are shown below:
TABLE 1 clinical test results of GPC3 immunohistochemical kit
The sources of the reagent and the medicine of the invention are listed as follows:
chinese hamster ovary cells (CHO/K1)-) Available from American Type Culture Collection (ATCC);
10% fetal bovine serum was purchased from Hangzhou ilex bioengineering, Inc.;
eukaryotic expression vectors pCDNA3.1(+), pCDNA3.1/ZEO (+) purchased from Invitrogen, USA;
containing human IgG1The heavy chain constant region (CH) and light chain constant region (CL) vectors pGEM-TEASY/CH and pGEM-TEASY/CL are constructed and stored in the laboratory;
lipofectamine 2000, a cationic liposome, was purchased from Invitrogen, USA;
g418, hypoxanthine, thymine and glycine are all Gibco products;
the BCA-100 protein quantitative kit is derived from Beijing saichi organisms, and has a product cargo number of 300001-B;
sequencing of glypican 3 protein (GPC3) antibody cell lines was accomplished by Nanjing Kinsley organisms;
QuaCell CHO CD04 medium (suspension), cat #: a11004, brand: zhongshan kang Tian Cheng He Biotechnology GmbH;
protein a Sepharose 4Fast Flow separation media was purchased from Amersham Bioscience;
the HRP-labeled goat anti-mouse IgG antibody is prepared in the laboratory of the company;
DAB color developing solution is prepared in laboratories of the company;
hematoxylin cell staining solution is prepared in laboratories of the same company;
the tissue antigen repair liquid is prepared for laboratories of the company.
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, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (5)
1. A glypican 3 detection kit, characterized in that the kit contains:
(a) a first antibody: GPC3 monoclonal antibody working solution, wherein the GPC3 monoclonal antibody is mouse-derived GPC3 antibody or human-derived GPC3 antibody;
(b) secondary antibody: HRP marks goat anti-mouse IgG antibody working solution;
(c) DAB color development liquid A buffer solution and DAB color development liquid B buffer solution;
(d) hematoxylin cell staining solution;
(e) tissue antigen repair fluid;
(f) 2-3% by volume of H2O2。
2. The glypican 3 detection kit according to claim 1, wherein the preparation method of the murine GPC3 antibody comprises the following steps:
(a) animal immunization: purifying GPC3 protein by affinity chromatography and an ion exchange layer, concentrating to 5mg/ml, mixing and emulsifying a GPC3 protein antigen and Freund's adjuvant in equal volume, and then immunizing BALb/c mice at multiple points under the back skin for 0 day, 14 days, 28 days and 35 days, wherein each mouse is 0.2mg, 0 day immunization is Freund's complete adjuvant, blood is collected after one week of last immunization, the antibody titer is detected by indirect ELISA, and the selected mice are subjected to abdominal cavity impact by hepatitis B vaccine stock solution after one week of last immunization, namely, the standby fusion mice;
(b) cell fusion: restoring and culturing SP2/0 cell strain, performing expanded culture 3 days before fusion, removing RPMI1640 cell culture solution 1 day before fusion, adding culture solution again, preparing mouse spleen cell suspension from spleen cells of the standby fusion mouse selected in the step (a), and fusing with SP2/0 cell stored in the laboratory;
(c) large-scale preparation of monoclonal antibodies: the ELISA method is used for determining that the supernatant fluid selects a hole with high positive, the positive hole cell is transferred into a 24-hole plate for amplification culture, and subcloning is timely carried out for 3 times to obtain a cell line stably secreting the antibody; ascites of the cell-immunized mouse is obtained and purified to obtain the GPC3 monoclonal antibody.
3. The glypican 3 detection kit as claimed in claim 2, wherein the animal immunization specifically comprises: after GPC3 protein is purified, the monitoring wavelength is 280nm, an elution peak is collected, a small amount of eluted protein liquid is taken for electrophoresis analysis, the rest is dialyzed for 48h at 4 ℃ by 0.85% NaCl solution, then, ultrafiltration membrane is used for concentration to 5mg/ml, glypican 3 protein antigen and Freund's adjuvant are mixed and emulsified in equal volume, then, Balb/c mice are immunized subcutaneously and multiply at the back for 0 day, 14 day, 28 day and 35 day, 0.2 mg/mouse is collected after one week of last immunization, the antibody titer in serum is detected by indirect ELISA, the OD value is 1.089 when the serum is diluted by 104 times, the selected mice are impacted in abdominal cavity by hepatitis B vaccine stock solution after one week of last immunization, and the spleen of the mice is taken after 3 days for cell fusion.
4. The glypican 3 detection kit as claimed in claim 2, wherein the cell fusion specifically comprises:
recovering the SP2/0 cell strain and preparing a mouse spleen cell suspension: recovering and culturing SP2/0 cell strain before cell fusion, performing amplification culture 3 days before cell fusion, removing RPMI1640 cell culture solution 1 day before cell fusion, and adding culture solution again to complete preparation of SP2/0 cells; killing the immune mice, and preparing a mouse splenocyte suspension by a conventional method;
cell mixing preparation: respectively adding a proper amount of incomplete IMDM culture solution into counting results of splenocytes and SP2/0 cells, shaking and uniformly mixing SP2/0 cells, blowing and beating the splenocytes uniformly by a pipette, combining the splenocytes and SP2/0 cells into a 50ml centrifuge tube according to a ratio of 1:2, uniformly mixing, adding the incomplete MDM culture solution into the centrifuge tube to 50ml, centrifuging for 5 minutes, emptying supernatant, tapping the bottom of the fusion tube to ensure that precipitated cells are loose and uniform, and placing the centrifuge tube in a 37 ℃ water bath to prepare fusion;
cell fusion: slowly dripping 50% PEG4000lml preheated at 37 ℃ into a mixed cell tube by using a dropper, rotating a centrifugal tube while dripping to ensure that cells are stored in a uniformly mixed state, standing for 90s, immediately and slowly adding 15ml of 37 ℃ serum-free MDM culture medium, centrifuging for 5 minutes, removing supernatant, adding MDM complete culture solution, mixing, adding the suspension into 96-hole cell culture plates respectively, culturing in a 37 ℃ incubator at 100 ul/hole, adding HAT culture solution into the cell plates on the next day at 100 ul/hole, changing the HAT culture solution once every 3 days, observing whether hybridoma appears, changing the HT culture medium after two weeks, and observing the growth condition of fused cells.
5. The use of the glypican 3 detection kit as claimed in any one of claims 1 to 4 for clinical liver cancer diagnosis.
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| CN202010596422.5A CN111751534A (en) | 2020-06-28 | 2020-06-28 | Glypican 3 detection kit and application thereof |
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| CN202010596422.5A CN111751534A (en) | 2020-06-28 | 2020-06-28 | Glypican 3 detection kit and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117607440A (en) * | 2023-11-28 | 2024-02-27 | 中拓生物有限公司 | Phosphatidylinositol proteoglycan 3 protein assay kit and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090116210A (en) * | 2008-05-06 | 2009-11-11 | 한국생명공학연구원 | A method for dignosing cancer and a diagnostic kit using detection of glypican-3 |
| CN102621324A (en) * | 2012-04-05 | 2012-08-01 | 武汉康迈生物技术有限公司 | Kit for immunohistochemical staining detection of colorectal cancer and application |
| CN111187351A (en) * | 2020-04-14 | 2020-05-22 | 北京瀚梅生物科技有限公司 | Liver cancer detection kit |
-
2020
- 2020-06-28 CN CN202010596422.5A patent/CN111751534A/en not_active Withdrawn
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090116210A (en) * | 2008-05-06 | 2009-11-11 | 한국생명공학연구원 | A method for dignosing cancer and a diagnostic kit using detection of glypican-3 |
| CN102621324A (en) * | 2012-04-05 | 2012-08-01 | 武汉康迈生物技术有限公司 | Kit for immunohistochemical staining detection of colorectal cancer and application |
| CN111187351A (en) * | 2020-04-14 | 2020-05-22 | 北京瀚梅生物科技有限公司 | Liver cancer detection kit |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117607440A (en) * | 2023-11-28 | 2024-02-27 | 中拓生物有限公司 | Phosphatidylinositol proteoglycan 3 protein assay kit and preparation method and application thereof |
| CN117607440B (en) * | 2023-11-28 | 2024-05-14 | 中拓生物有限公司 | Phosphatidylinositol proteoglycan 3 protein assay kit and preparation method and application thereof |
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