CN108330107B - Hybridoma cell strain, CD68 monoclonal antibody, preparation method and application - Google Patents
Hybridoma cell strain, CD68 monoclonal antibody, preparation method and application Download PDFInfo
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- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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Abstract
The invention relates to a hybridoma cell strain, a CD68 monoclonal antibody, a preparation method and application, wherein the monoclonal antibody adopts purified CD68 recombinant extracellular region protein as an immunogen immune animal body; fusing myeloma cells SP2/0 with B lymphocytes of the immunized animal body to obtain hybridoma cells; screening specific hybridoma positive clones, cloning the positive clones, and screening out hybridoma cells stably secreting CD68 monoclonal antibody. Has the characteristics of strong specificity, high affinity, good stability and high potency.
Description
Technical Field
The invention relates to the technical field of biomedicine, and more particularly relates to a hybridoma cell strain capable of producing a CD68 monoclonal antibody, a CD68 monoclonal antibody, a preparation method and application of the antibody.
Background
CD68 is a glycoprotein with a molecular weight of 110KDa and is associated with lysosomal particles. CD68 is a macrophage marker in many tissues of the human body, including bone marrow, spleen, intestinal lamina propria, alveoli, etc. Peripheral blood mononuclear cells also showed positive responses, and bone marrow precursor cells and peripheral blood granulocytes were also studied. In recent years, CD68 has been reported in literature as a marker of tissue cells, and is mainly used for research on macrophages and acute and chronic myeloid leukemia and tumors derived from tissue cells, such as malignant fibrous histiocytoma.
With the increasingly deep research on tumor immunology, it is known that cells infiltrating the tumor site can better reflect the immune function of the tumor host. Infiltrating cells in tumor tissue are dominated by lymphocytes and macrophages, designated tumor-infiltrating lymphocytes and tumor-associated macrophages (TAMs). Research shows that the level of the TAM content is related to the intensity of the tumor metastasis capacity, and the tumor progression stage can be judged according to the TAM content. Tumor-associated macrophages (TAMs) are important regulators of inflammation and tumor-associated activity, exhibit tumor-promoting properties in the tumor microenvironment, secrete growth factors and matrix proteases, can be used as an index for judging tumor prognosis, and can also be another target in tumor treatment besides traditional treatment targets. At present, the relation of TAM in tumor tissues of colorectal cancer, breast cancer, lung cancer and the like is reported, and the TAM can help tumor diagnosis and treatment.
Immunohistochemistry (IHC) is a staining technique in which an antibody specifically binds to an antigen component in a tissue or a cell to be recognized, a tracer (e.g., fluorescein, enzyme, biotin, heavy metal ion, etc.) labeled on the antibody is developed by a chemical reaction, and the antigen (polypeptide and protein) on a tissue section or a cell sheet is qualitatively and locally stained. In recent years, with the development of immunohistochemical technology and the successful development of various specific antibodies, more importantly, the understanding of tumor molecular mechanisms is deepened, and the application value of immunohistochemistry in molecular diagnosis and identification of tumors is generally accepted.
In immunohistochemistry, CD68 is a macrophage marker in many tissues of the human body and is used to detect tumor angiogenesis. At present, the CD68 antibody at home and abroad mainly comprises a polyclonal antibody, and the monoclonal antibody has high preparation cost and poor stability, so that the stable, high-specificity and high-activity CD68 monoclonal antibody for immunohistochemical detection has important significance for clinical diagnosis and scientific research.
Disclosure of Invention
The invention aims to solve the problems and aims to provide a hybridoma cell strain capable of generating a CD68 monoclonal antibody, a CD68 monoclonal antibody, a preparation method and application of the antibody.
In order to achieve the purpose, the invention adopts the following technical scheme:
on one hand, the embodiment of the invention discloses a hybridoma cell strain capable of producing a CD68 monoclonal antibody, which is preserved in China center for type culture Collection in 2018, 3, 9 and with the preservation number of CCTCC NO: C201823.
in another aspect, the embodiment of the present invention also discloses a method for preparing the CD68 monoclonal antibody, which comprises:
step (1), using purified CD68 recombinant extracellular region protein as immunogen to immunize animal Balb/c mouse;
fusing myeloma cells SP2/0 with B lymphocytes of the immunized animal body to obtain hybridoma cells;
screening specific hybridoma cell positive clones, carrying out cell cloning on the positive clones, screening hybridoma cells which stably secrete a CD68 monoclonal antibody, and carrying out screening on the specific hybridoma cell positive clones by an ELISA method;
and (4) obtaining the CD68 monoclonal antibody.
Further, in the step (4), the obtaining of the CD68 monoclonal antibody comprises:
inoculating the hybridoma cells which stably secrete the CD68 monoclonal antibody into an animal body, producing an ascites antibody, and purifying to obtain a CD68 monoclonal antibody; or
And culturing the hybridoma cells stably secreting the CD68 monoclonal antibody in vitro, and separating and purifying a culture solution to obtain the CD68 monoclonal antibody.
The embodiment of the invention also provides the CD68 monoclonal antibody prepared by the method.
The embodiment of the invention also provides the application of the CD68 monoclonal antibody in immunohistochemical detection.
Further, when the monoclonal antibody is applied to immunohistochemical detection, the CD68 monoclonal antibody which is optimally combined with the CD68 antigen aiming at the tissue markers of giant cell tumor, colorectal cancer and lung cancer of the bone is screened out by immunohistochemical staining of tumor tissues.
The monoclonal antibody type of the invention is identified to be IgG2b by an ELISA method, immunohistochemical staining is carried out on giant cell tumor paraffin sections of bones, and the CD68 antibody with the most obvious positive result under the same concentration is compared to be considered as the CD68 antibody which is best combined with the CD68 antigen.
The specificity of the CD68 monoclonal antibody is verified by immunohistochemical staining and contrast staining of commercial antibody and the CD68 optimal monoclonal antibody screened by the invention on colorectal cancer and lung cancer tissues under the same condition respectively.
The best CD68 monoclonal antibody screened by the invention is 17E11, the staining effect is best under the same condition, and the positive rate is highest. The invention has the same immunohistochemical staining specificity to the same tissue under the same condition as the commercial antibody, and has high accuracy.
The invention has the beneficial effects that:
1. the invention uses recombinant CD68 protein as immunogen to immunize Balb/c mice, and adopts classical cell fusion technology to obtain hybridoma cells which stably secrete CD68 monoclonal antibody. The secreted CD68 monoclonal antibody has the advantages of immunohistochemical staining of giant cell tumor, colorectal cancer and lung cancer tissues of bones, clear staining background and no non-specific staining, and proves that the monoclonal antibody has high recognition capability of CD68 protein and can detect various tumor tissues with high expression of CD68 protein.
2. When the CD68 monoclonal antibody and the like are applied to immunohistochemical detection and screening, the specificity and the sensitivity are high, compared with the immunohistochemical result of a common monoclonal antibody on the market, the positive strength of the monoclonal antibody is higher than that of a common antibody on the market, the monoclonal antibody accords with the clinical detection standard, and the diagnosis accuracy can be improved.
Drawings
FIG. 1 shows the partial positive results of immunohistochemistry of 17-E11 of the present invention and antibodies from Picea beijinensis in giant cell tumor, colorectal cancer and lung cancer tissues.
FIG. 2 shows Western blot (immunoblotting) to verify the specificity of the 17-E11 monoclonal antibody of the present invention, M is protein Marker, and CD68 recombinant protein (Beijing Yinqiao: 11192-H08B1) can be specifically detected in WB by using the purified 17-E11 monoclonal antibody.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The preservation information of the hybridoma cell strain capable of stably producing the CD68 monoclonal antibody disclosed by the invention is as follows:
and (3) classification and naming: anti-human CD68 hybridoma cell line 17-E11(IgG2 b);
the preservation unit: china center for type culture Collection;
address: wuhan university in Wuchang Lojia mountain in Wuhan city, Hubei province;
the preservation date is as follows: year 2018, month 3, day 9;
the preservation number is as follows: CCTCC NO: c201823;
culture name and identifying characteristics noted: anti-human CD68 hybridoma cell line 17-E11(IgG2 b).
The preparation method of the CD68 monoclonal antibody disclosed by the embodiment of the invention comprises the steps of immunizing a Balb/c mouse by taking purified CD68 recombinant extracellular domain protein as immunogen, fusing myeloma cells SP2/0 and spleen B lymphocytes of the Balb/c mouse by a cell fusion technology, and screening and cloning by an indirect ELISA method to obtain a positive hybridoma cell line stably secreting the CD68 monoclonal antibody. The hybridoma cells are inoculated in mice to prepare ascites, and the ascites is purified by protein A/G column affinity chromatography to obtain the CD68 monoclonal antibody. Main reagents and consumables:
EXAMPLE 1 preparation of CD68 monoclonal antibody
1) Preparation of immunogens
The eukaryotic expression CD68 protein is used as immunogen, the eukaryotic expression CD68 protein is human engineering cell expression CD68 protein (purchased from Beijing Shenzhou Yinqiao, the cargo number is 11192-H08B1), a female Balb/c mouse is immunized, and spleen lymphocytes are obtained for hybridoma fusion experiments.
2) Immunogen immune Balb/c mice
The purified recombinant CD68 protein was solubilized and, after determination of the protein content, diluted to 1.0mg/mL with 10mmol/LPBS at pH 7.4. 7 female Balb/c mice of 6-8 weeks of age and equal body weight were selected. For the first immunization, CD68 protein and Freund's complete adjuvant with the same volume are emulsified and mixed to be used as immunogen; carrying out the second immunization and the third immunization every two weeks, and changing the adjuvant into Freund incomplete adjuvant; 3 days before fusion, the mixture is boosted, CD68 is used as immunogen, adjuvant is not added, and the mixture is injected into abdominal cavity. The amount of each immunization was 50 ug/mouse, and the injection volume was unchanged.
3) Determination of serum titer of immune Balb/c mice
Diluting CD68 protein to optimum working concentration of 2 mug/mL, adding enzyme label plate according to 100 mug/well, coating overnight at 4 deg.C, washing 5min × 5 times, and finally inverting the plate on absorbent paper to dry the washing liquid in the well. Collecting 20 μ l blood from tail vein 7 days after the third immunization, centrifuging at 2000rpm for 30min, mixing the supernatant with 1 μ l antibody diluent 999 μ l, diluting at 1: 1000-1: 1024000 times, adding diluted serum to be detected at 100 μ l/hole, diluting the serum 1:100 before the immunization of mouse as negative control, using the antibody diluent as blank control, acting at 37 deg.C for 1h, and washing for 5min × 5 times; diluting horse radish peroxidase labeled goat anti-mouse antibody at a ratio of 1:6000, acting at 100 μ l/well at 37 deg.C for 1h, washing for 5min × 5 times; adding substrate color developing solution into 100 μ l/hole, and standing at room temperature in dark for 3-5 min; and (3) observing the result by adding the stop solution into 50 mu l/well, recording the reading of 450mm by using an enzyme-linked immunosorbent assay (ELISA), carrying out withering on a blank control well, measuring the OD value of each well, and selecting the well with the highest serum titer of more than 1:128000 for cell fusion.
4) Preparation of spleen B lymphocytes and SP2/0 cell suspension, feeder cells
Preparation of spleen B lymphocytes: taking an immunized female Balb/c mouse, removing an eyeball, bleeding and killing the mouse, taking out a spleen by aseptic operation, soaking the spleen in a clean PBS solution, adding a 10mL PBS solution into a culture dish, placing a 200-mesh cell sieve, taking out the spleen, placing the spleen in the cell sieve, grinding the spleen by using an inner core of an injector, occasionally washing the cell sieve by the PBS solution, enabling spleen cells to pass through meshes and suspend in the solution, transferring the spleen cells into a 10mL centrifuge tube, horizontally centrifuging at 1500rpm for 10min, and removing a supernatant. Adding 2mL of erythrocyte lysate, resuspending, and acting at 37 deg.C for 2 min; the supernatant was discarded by centrifugation, washed once with PBS, and the cells were resuspended, mixed well with 10mL of incomplete medium and counted.
Preparation of SP2/0 cell suspension: SP2/0 cells were removed from liquid nitrogen, quickly placed in a 37 ℃ water bath, shaken until the cell solution was completely dissolved, centrifuged horizontally at 1000rpm for 10min, the supernatant was discarded, 5mL of the complete medium was resuspended, the cell suspension was transferred to a 50mL flask and incubated in a 37 ℃ 5% CO2 incubator. After the cells grow well, screening the cells by using a selective medium containing 8-AG for one week; the cells were passaged once 2 days before fusion, so that the cells were in logarithmic growth phase on the day of fusion. Cells were harvested on the day of fusion and centrifuged, supernatant was discarded, and after the pellet was washed with incomplete medium, 10mL of incomplete medium was resuspended and counted.
Preparation of feeder cells: taking an uninmmunized female Balb/c mouse, removing an eyeball, bleeding and killing the mouse, soaking and sterilizing the mouse for 5min by using 75% ethanol, cutting off the peritoneum, flushing the abdominal cavity by using a precooled incomplete culture medium, and collecting a washing liquid to be sucked into a 50mL centrifuge tube. The peritoneal cavity was washed 3 times repeatedly, the washes were collected, centrifuged horizontally at 1000rpm for 10min at room temperature, the supernatant was removed, and 10mL of incomplete medium was used to resuspend the cells and counted.
6) Cell fusion
The myeloma cell suspension and the spleen B lymphocyte suspension are mixed in a 50mL centrifuge tube according to the cell number of 1:7, 30mL of serum-free RPMI-1640 culture medium (Roswell Park mental Institute 1640) is added, the mixture is fully mixed, the mixture is centrifuged at 1500rpm for 10min, the supernatant is discarded, and the tube bottom is flicked to loosen the cell mass into paste. Sucking 1mL of the pre-warmed PEG1450 solution with a dropper, adding the solution into the cells slowly along the tube wall at a position of about 2cm from the centrifugal tube opening within about 1min, and rotating the centrifugal tube while adding the solution to mix the cells gently (without blowing). Standing the cells for 1min, gradually adding 29mL of 1640 medium (30 mL of PEG and medium) containing no serum and pre-warmed at 37 deg.C for terminating fusion, and rotating the centrifuge tube while mixing the cells gently for about 15 min; standing for 5min, centrifuging for 5min at 1500rmp, discarding supernatant, adding 10ml fusion 1640 culture medium, gently resuspending cells, inoculating the fusion cells to 96-well cell culture plates with feeder cells laid thereon at a concentration of 100. mu.l/well, reserving 6 wells for each culture plate, inoculating SP2/0 cells as HAT negative control, and culturing in 5% CO2 incubator at 37 ℃. After 48 hours of culture, 100. mu.l of fresh 2 XHAT selection medium was added to each well. After fusion, 100 mul of 1 XHAT selective medium can be supplemented every 4-5 days, and clone groups can be seen in 2-5 weeks.
7) Screening for specific hybridomas
After culturing for 10 days, detecting the grown clone cell hole as a fusion positive hole under a microscope, and calculating the fusion rate. Detecting culture supernatant by adopting an indirect ELISA method, and screening positive clones: the plate was coated with CD68 protein (2ug/mL), 50. mu.l/well, washed overnight at 4 ℃ 5 min. times.5 times. Draining the liquid, adding 0.2% PBST (phosphate buffer solution with pH7.4 containing 0.2% Tween) and 50 μ l cell supernatant at 50 μ l/well, taking PBST as blank control, SP2/0 cell culture supernatant as negative control, taking immune serum of mouse as positive control, incubating at 37 deg.C for 1h, and washing for 5min × 5 times; mu.l of 0.1% PBST diluted enzyme-labeled goat anti-mouse IgG antibody (1: 3000) was added to each well, incubated at 37 ℃ for 30min, and washed 5 min. times.5 times. Draining off liquid, adding a freshly prepared substrate color developing agent according to 100 mul/hole, reacting for 3-5min at room temperature in a dark place, adding a stop solution according to 50 mul/hole to terminate the reaction, and detecting the absorbance value of 450nm by an enzyme-linked immunosorbent assay. And determining the positive clone as the positive clone with OD value more than 2.1 times higher than that of the negative control, performing subclone culture on the positive clone by adopting a limiting dilution method, continuously performing cloning screening for more than three times until a high-specificity anti-CD 68 monoclonal hybridoma cell strain is obtained, and performing expanded culture.
The result is that CD68 protein expressed by artificial process cells is used as immunogen, 7 Balb/c mice are immunized in sequence and fused for 7 times, CD68 antibody 19 strains with high affinity are obtained by screening in sequence, hybridoma cell strains secreting CD68 monoclonal antibodies are obtained by screening 3 times of cloning detection, CD68 monoclonal antibodies can be stably secreted after subculture for more than 2 months, and finally the hybridoma cell strains are frozen in a liquid nitrogen tank.
8) Preparation of ascites with CD68 monoclonal antibody
After screening out positive clones, cloning and culturing the positive wells by a limiting dilution method, obtaining hybridoma cell clones capable of stably producing high-titer monoclonal antibodies through two to three rounds of cloning and culturing, gradually transferring the hybridoma cell clones into a 24-well plate, and performing amplification culture in a 5% CO2 incubator at 37 ℃ until the hybridoma cell clones are frozen and stored in liquid nitrogen. After 6-8 weeks old mice were treated with paraffin oil for about 10 days, 5X 10 injections were administered to each mouse5Each hybridoma cell, 10-14 days, was used to obtain ascites fluid for antibody purification by way of extraction or killing of the mice.
9) Purification of antibodies in ascites
Collecting ascites, centrifuging at 3000rpm/min for 15min to remove cell components (or solid matter formed in the freezing process, taking clear ascites at the upper layer, adding pH7.2 barbiturate buffer solution saline (VBS0.04mol/l barbiturate, 0.15mol/l NaCl, 0.8mmol/lMg2+, 0.3mmol/lCa2+) in equal amount for dilution, then adding 150mg of silicon dioxide powder into each 10ml of diluted ascites, mixing, incubating the suspension at room temperature for 30min, shaking occasionally, centrifuging at 3000g for 20min, and removing lipid by the method to obtain clarified monoclonal antibody ascites.
(1) Configuring Buffer required for antibody purification:
binding Buffer (solution a): 100mmol/L sodium phosphate, 100mmol/L sodium citrate, pH7.0;
elution Buffer (solution B): 100mmol/L sodium phosphate, 100mmol/L sodium citrate, pH3.0;
assembly Buffer (liquid C): 1mmol/L Tris-HCl, pH9.0.
(2) Mixing the monoclonal antibody ascites and the solution A in a ratio of 1:10, and filtering with a 0.45um filter membrane for sample loading.
(3) Selecting a HiTrap rProtein A HP column, inoculating an AKTA Explorer, and balancing pipelines by A, B liquid respectively;
(4) and (4) loading the sample from the tube A, eluting the purification column by using the solution B, and collecting an elution peak to an EP tube added with the solution C in advance. If the pH value of the eluent is low, adjusting the pH value of the eluted protein to 7.0-8.0 by using liquid C to prevent denaturation, measuring the content of the protein in the tube by using a spectrophotometer, and subpackaging and freezing the CD68 monoclonal antibody for storage.
10) Monoclonal antibody typing
The subclass identification was carried out using a Monoclonal Antibody subclass identification kit (Mouse Monoclonal Antibody Isotyping Reagents, ISO2-1KT, available from Sigma, USA) according to the instructions. The result shows that the CD68 monoclonal antibody type is IgG2b type.
11) ELISA for identifying specificity of CD68 monoclonal antibody
CD68 protein was diluted to the optimal working concentration of 2. mu.g/mL, ELISA plates were added at 100. mu.l/well, coated overnight at 4 ℃ and washed 5min X5 times the next day without antigen solution. Diluting CD68 monoclonal antibody at a ratio of 1:1000 to 1:1024000, adding into corresponding wells at a ratio of 100 μ l/well, performing yin and yang control and blank control, acting at 37 deg.C for 1h, and washing for 5min × 5 times; diluting HRP-labeled goat anti-mouse secondary antibody at a ratio of 1:3000, allowing the secondary antibody to act at a concentration of 100. mu.l/well for 40min at 37 ℃, and washing for 5min × 5 times; adding substrate color developing solution into 100 μ l/hole, and standing at room temperature in dark for 3-5 min; the result was observed at 50. mu.l/well with stop buffer and 450mm reading was recorded using an ELISA. The result shows that the CD68 monoclonal antibody has better specificity.
EXAMPLE 2 screening for optimal CD68 monoclonal antibodies binding to CD68 antigen
The technical scheme of immunohistochemistry (enzyme-labeled antibody indirect method) is that a group of CD68 antibodies obtained are subjected to immunohistochemical staining on a same tissue continuous section under the same condition, antibodies with the strongest specificity and the highest titer are screened out from the screened antibodies, and the expression condition of the CD68 monoclonal antibody on tumor tissues is detected.
1) Paraffin sections of giant cell tumor tissue of bone, which are routinely dewaxed and hydrated: baking at 65 deg.C for 30min, and soaking in xylene for 15min × 2 times; soaking in anhydrous ethanol for 10min × 2 times; sequentially soaking in 95% ethanol, 85% ethanol, and 75% ethanol for 5 min; washing with distilled water for 5min × 3 times;
2) antigen retrieval:
adding 500mL of citric acid repair liquid (pH6.0) into a stainless steel pressure cooker, covering the pressure cooker with a pressure cooker cover without locking, placing the slices on a high-temperature-resistant plastic frame, placing the pressure cooker in the pressure cooker after the repair liquid is boiled, locking the pressure cooker cover to pressurize, starting timing after steam is supplied, removing a heat source after 2min30s, washing with tap water to cool until a small valve falls, opening the cover, and naturally cooling to room temperature. PBS immersion wash 5min X3 times.
The preparation method of the citric acid repair liquid comprises the following steps:
solution A (0.1mol/L trisodium citrate): weighing 14.7g of trisodium citrate, and adding 500mL of distilled water for dissolving;
solution B (0.1mol/L citric acid): weighing 4.2g of citric acid, and adding 200mL of distilled water for dissolving;
taking 41mL of the solution A and 9mL of the solution B, and adding distilled water to a constant volume of 500mL to obtain 0.01mol/L citrate buffer solution (pH6.0).
3) Cutting into slices, and blocking in 3% H2O 2-methanol thermostat at 37 deg.C for 30 min; PBS immersion washing for 5min multiplied by 3 times;
preparation method of 3% H2O 2-methanol: 20mL of methanol and 30% H2O220mL were weighed out, and distilled water was added to the solution to a volume of 200 mL.
4) Wiping tissue, forming pen circle, adding goat serum albumin dropwise, sealing at 37 deg.C for 30min, and pouring off without washing;
5) dripping different CD68 monoclonal antibodies with the same dilution degree, simultaneously taking PBS as a negative control, and staying overnight at 4 ℃; rewarming for 30min in a thermostat at 37 ℃ the next day, and soaking and washing for 5min by 3 times with PBS;
6) throwing off redundant liquid, dropwise adding a goat anti-mouse secondary antibody marked with HRP, and incubating for 30min in a thermostat at 37 ℃; PBS immersion washing for 5min multiplied by 3 times;
7) dripping DAB color development liquid (prepared as before), controlling dyeing under a mirror, and stopping flushing with tap water; d
8) Counterstaining cell nucleus with hematoxylin, performing alcohol differentiation with hydrochloric acid, and returning ammonia water to blue;
9) dehydrating and transparent: sequentially soaking in 75% ethanol, 85% ethanol, and 95% ethanol for 3 min; soaking in anhydrous ethanol for 10min × 2 times; soaking in xylene for 15min × 2 times;
10) and (5) sealing the neutral gum.
Paraffin sections of giant cell tumor tissue of bone were stained and the CD68 antibody with the most pronounced effect at the same concentration and dilution was considered to be the CD68 antibody that binds best to CD68 antigen.
FIG. 1 shows that, the prepared CD68 monoclonal antibodies were all stained with bone giant cell tumor paraffin tissue sections with the same dilution, the CD68 monoclonal antibody of the invention can identify the giant cell of the histiocyte, and the monoclonal antibody corresponding to the screening result 17-E11 has the best staining effect and the highest positive rate under the same conditions.
Example 3 comparison of specificity of immunohistochemical detection of CD68 monoclonal antibody
Selecting and purchasing a monoclonal antibody of CD68 of China Beijing Chinese fir, adopting an instruction and groped 17E11 optimal conditions to respectively stain the same tumor tissue sections, comparing staining conditions, and determining whether the specificity is consistent, wherein the specific operations are as follows:
1) paraffin tissue sections of giant cell tumor of bone, colorectal cancer and lung cancer tissues, and conventional dewaxing and hydration of the paraffin sections: baking the slices in a thermostat at 65 ℃ for 30min, and soaking in xylene for 15min twice; soaking in anhydrous ethanol for 10min twice; sequentially soaking in 95% ethanol, 85% ethanol, and 75% ethanol for 5 min; washing with distilled water for 5min × 3 times;
2) antigen retrieval: 0.01M citric acid repair solution (pH6.0), high pressure repair for 2min30 s; washing with distilled water for 5min × 3 times;
3) blocking in 3% H2O 2-methanol at 37 deg.C for 30 min; PBS immersion washing for 5min multiplied by 3 times;
4) wiping tissue, making pen-shaped circle, adding goat enclosed serum dropwise, sealing at 37 deg.C for 30min, pouring off, and washing-free;
5) respectively dripping the monoclonal antibody of China fir company and the CD68 monoclonal antibody corresponding to the 17E11 of the invention, simultaneously taking PBS as negative control, and staying overnight at 4 ℃; rewarming for 30min at 37 deg.C the next day, and washing with PBS for 5min × 3 times;
6) throwing off redundant liquid, dropwise adding enzyme-labeled goat-anti-mouse secondary antibody, and acting at 37 ℃ for 30 min; PBS immersion washing for 5min multiplied by 3 times;
7) dripping DAB color developing solution, controlling dyeing under a mirror, and stopping running water;
8) counterstaining cell nucleus with hematoxylin, performing alcohol differentiation with hydrochloric acid, and returning ammonia water to blue;
9) dehydrating and transparent: sequentially soaking in 75% ethanol, 85% ethanol, and 95% ethanol for 3 min; soaking in anhydrous ethanol for 10min × 2 times; soaking in xylene for 15min × 2 times;
10) and (5) sealing the neutral gum.
The results are shown in fig. 1, and the positive results of the immunohistochemical staining of the monoclonal antibody of CD68 show that the monoclonal antibody of CD68 corresponding to 17E11 of the present invention has a staining effect similar to that of the monoclonal antibody of CD68 of sequoia, and the results of the staining region and the staining location are consistent, so that the monoclonal antibody of CD68 corresponding to 17E11 of the present invention can specifically recognize tumor-associated macrophages, and thus, the monoclonal antibody of CD68 of the present invention has a good effect and specificity of recognizing CD68 protein, and is a monoclonal antibody specific to CD 68.
The immunohistochemical experiments of the invention on giant cell tumor of bone, colorectal cancer and lung cancer are respectively carried out in the above embodiments, and the histochemical results show that the invention has good expression effects in different tumors, which indicates that the antibody provided by the invention can be applied to immunohistochemical detection, well detect the expression quantity and expression position of CD68 in tumor tissues, and is convenient for directly interpreting the location and quantification of histiocyte and tumor-related macrophages in tumor tissues from an immunohistochemical chart, thereby judging the growth stage of the tumor.
Example 4 Western blot (immunoblotting) to confirm the specificity of the 17E11 monoclonal antibody of the present invention
Recombinant CD68 eukaryotic protein (Beijing Yinqiao, 11192-H08B1) is selected for immunoblotting experiments, and the specific operation is as follows: the loading of protein was 4ug, 10% polyacrylamide gel electrophoresis, wet or semi-dry transfer of gel protein to PVDF membrane, 5% milk powder solution (0.5% PBST configuration) and shaking table closure at room temperature for 1 h. The antibody is diluted in a ratio of 1:1000, incubated at 4 ℃ overnight or at room temperature for 1h, PBST is washed for 15min multiplied by 3 times, and a secondary antibody (prepared by a milk powder solution) is incubated at room temperature for 1 h. And (3) washing the membrane again, adding an ECL luminescent reagent, and collecting chemiluminescence image data according to operation gel Imaging (Chemi DocTM MP Imaging System of BIO-RAD company in America), wherein the result is shown in figure 2, and the 17E11 monoclonal antibody disclosed by the invention can be used for identifying eukaryotic protein and has strong specificity.
In conclusion, the CD68 monoclonal antibody generated by the hybridoma cell strain disclosed by the invention has the characteristics of strong specificity, high affinity, good stability and high titer:
1) high accuracy and high recognition ability. The purified CD68 monoclonal antibody disclosed by the invention is used for immunohistochemical staining of giant cell tumor, colorectal cancer and lung cancer of bones respectively, and the contrast staining result shows that the antibody disclosed by the invention has no non-specific staining, and the positive contrast ratio has no obvious difference, so that the antibody disclosed by the invention is proved to have high recognition capability on CD68 protein. The antibody disclosed by the invention has higher sensitivity, accurate cell positioning and more reliable detection result, is suitable for IHC application with strict requirements, and can obviously improve the diagnosis accuracy.
2) Strong specificity and good stability. The CD68 antibody is IgG2b monoclonal antibody. Compared with polyclonal antibodies, the monoclonal antibodies have higher specificity and uniformity, and are not easy to cause problems of nonspecific background and cross reaction. Although some technical companies are dedicated to the development and production of monoclonal antibodies, the quality of the monoclonal antibodies still varies, and the stability of the monoclonal antibodies is not easy to control. The experimental result proves that the staining positive strength of the CD68 monoclonal antibody is higher than that of the general antibody on the market, the specificity and the affinity of the monoclonal antibody are detected by continuous immunohistochemical storage at 4 ℃, the positive rate is not obviously changed, and the antibody has good stability.
3) Low cost, high titer and easy popularization and application. The invention is self-developed and prepared, and screens out a cell line for stably producing the CD68 monoclonal antibody. The titer of the purified antibody is high, the dilution (1: 100-1: 200) recommended by common antibodies at home and abroad and the high dilution (1: 800-1: 3200) of the antibody are used for dyeing the continuous sections of the same tissues, and the contrast dyeing process is adopted, so that the antibody has shorter development time, and the development time is averagely within 20 s; compared with a dyeing result, the background is clearer, the signal to noise ratio is reduced, and the research and clinical detection efficiency is improved. According to investigation, researchers and clinical immunohistochemistry workers are better to use foreign imported primary antibodies such as Abcam and CST, but the purchase period is long in time consumption and expensive, domestic commonly-used ready-to-use antibodies are concerned, but the titer is strong, and the matched use of the immunohistochemical kit is limited. The experimental result of the embodiment proves that the CD68 monoclonal antibody can have the advantages that the antibody does not have, the stable cell line is produced in a standardized way, the difference among batches is small, the repeated inspection is guaranteed, and the clinical detection standard is met.
The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that any modification, variation and improvement made by those skilled in the art without departing from the spirit of the present invention shall be included in the protection scope of the present invention.
Claims (4)
1. A hybridoma cell strain capable of producing a CD68 monoclonal antibody is characterized by being preserved in China center for type culture Collection in 2018, 3 and 9 months and being CCTCC NO: C201823.
2. a CD68 monoclonal antibody secreted by the hybridoma cell line of claim 1.
3. Use of the CD68 monoclonal antibody of claim 2 for the preparation of an immunohistochemical pathological diagnostic agent.
4. The use of the CD68 monoclonal antibody according to claim 3, wherein the CD68 monoclonal antibody that is optimal for binding to the CD68 antigen is selected by immunohistochemical staining of tumor tissues in immunohistochemical detection of giant cell tumor, colorectal cancer and lung cancer tissues.
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