CN105349497B - Hybridoma cell strain of anti-alpha-antitrypsin polyclonal antibody - Google Patents
Hybridoma cell strain of anti-alpha-antitrypsin polyclonal antibody Download PDFInfo
- Publication number
- CN105349497B CN105349497B CN201510792392.4A CN201510792392A CN105349497B CN 105349497 B CN105349497 B CN 105349497B CN 201510792392 A CN201510792392 A CN 201510792392A CN 105349497 B CN105349497 B CN 105349497B
- Authority
- CN
- China
- Prior art keywords
- antitrypsin
- mouse
- cells
- alpha
- hybridoma cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a hybridoma cell strain secreting an anti-human alpha-antitrypsin polyclonal antibody and a preparation method thereof. The invention takes a conjugate obtained by coupling bovine serum albumin BSA and human alpha-antitrypsin as an antigen to immunize a Balb/c mouse, takes spleen cells of the Balb/c mouse to be fused with SP2/0 myeloma cells, uses HAT culture medium to carry out selective culture, selects fused cells of the spleen cells of the mouse and mouse hybridoma cells SP2/0, and finally identifies a hybridoma cell strain secreting the anti-human alpha-antitrypsin polyclonal antibody by an ELISA method. The hybridoma cell strain prepared according to the invention is preserved in China center for type culture Collection with the preservation number of CCTCC C2015194.
Description
Technical Field
The invention relates to a hybridoma cell strain and a preparation method thereof, in particular to a hybridoma cell strain of an anti-human alpha-antitrypsin polyclonal antibody and a preparation method thereof.
Background
Alpha-antitrypsin (alpha-antitrysin) is also known as an alpha-1-protease inhibitor. It is the most important protease inhibitor in human plasma, and accounts for more than 90% of the inhibition capacity of total plasma protease. It can inhibit various serine endopeptidases (such as Neutrophil Elastase (NE), trypsin, plasmatin, and thrombin, and has the main effects of protecting normal cells and organs of organism from protease damage, inhibiting infection and inflammation, and maintaining the balance of internal environment. Antitrypsin in plasma is produced mainly by the liver, and other things such as the intestine, kidney, spleen, etc. can also be produced in small amounts, and this extrahepatic synthesized antitrypsin plays an important role in the regulation of local tissue damage. Alpha-antitrypsin is a major inhibitor of serum albumin-dissolving enzymes (e.g., trypsin) in humans.
An imbalance in the protease-antiprotease system plays an important role in the pathogenesis of several inflammatory lung diseases such as emphysema, pulmonary Cystic Fibrosis (CF), Acute Respiratory Distress Syndrome (ARDS), and acute and chronic bronchitis. Anti-alpha-antitrypsin is the most important protease inhibitor in the lower respiratory tract, and in order to restore the protease-antitrypsin balance, anti-alpha-antitrypsin substitution or supplement treatment not only becomes a main treatment method for congenital anti-alpha-antitrypsin deficiency, but also opens up a new way for the treatment of other inflammatory lung diseases.
Besides basic research, the polyclonal antibody against human alpha-antitrypsin can also be used for preparing high-purity human alpha-antitrypsin and preparing a human alpha-antitrypsin detection reagent. Anti-human alpha-antitrypsin antibody can be used for detecting anti-human alpha-antitrypsin coagulation activity and antigen content. At present, no report for developing human plasma anti-alpha-antitrypsin polyclonal antibody is seen at home, and no high-purity anti-human alpha-antitrypsin product exists, but the anti-human alpha-antitrypsin polyclonal antibody must be prepared firstly by preparing high-purity anti-human alpha-antitrypsin and anti-human alpha-antitrypsin detection reagents.
Thus, there is a need in the art for polyclonal antibodies against human α -antitrypsin.
Disclosure of Invention
In order to solve the problems, the invention provides a hybridoma cell strain secreting an anti-human alpha-antitrypsin polyclonal antibody and a preparation method thereof.
In a first aspect, the present invention provides a method for producing a hybridoma cell line that secretes an anti-human α -antitrypsin polyclonal antibody (also sometimes simply referred to herein as "the method of the present invention"), the method comprising the steps of:
1) preparing immune antigen: coupling human alpha-antitrypsin and bovine serum albumin by a homotype bifunctional protein cross-linking agent to obtain a human alpha-antitrypsin-bovine serum albumin conjugate;
2) immunizing a mouse: immunizing Balb/c mice by adopting a small-dose multiple immunization scheme and utilizing the human alpha-antitrypsin-bovine serum albumin conjugate obtained in the step 1) to act on an immunizing antigen;
3) cell fusion: fusing SP2/0 mouse myeloma cells with spleen cells of the immune Balb/c mouse obtained in the step 2), and selectively culturing in an RPMI-1640 medium containing HAT;
4) selective culture of hybridoma cells: after the cells fused in the step 3) are selectively cultured in HAT medium, selecting fused cells of mouse spleen cells and mouse hybridoma cells SP 2/0; and
5) and (3) identification: detecting the culture supernatant of the fused hybridoma obtained in the step 4), and identifying a hybridoma cell strain secreting the anti-human alpha-antitrypsin polyclonal antibody.
In a second aspect, the present invention provides a hybridoma cell line (hereinafter sometimes referred to as "hybridoma cell line of the present invention") that secretes a polyclonal antibody against human α -antitrypsin, which is prepared by the method of the present invention.
In a preferred embodiment, a hybridoma cell line produced by the method of the invention is deposited with the chinese type culture collection on day 11/6 of 2015 at the address: the preservation number of the preservation center of Wuhan university in Wuchang district, Wuhan city, Hubei province is CCTCC C2015194, and the culture name is as follows: hybridoma cell strain alpha-antiprysin-P-01.
Detailed Description
Defining:
"protein crosslinking agent" is a small molecule compound having one or more specific groups (-NH) at each end of the molecule2-COOH, -HS, etc.) can be coupled to the active groups on the biomolecules, respectively, to bind these molecules together. Protein crosslinkers can be divided into three classes of homobifunctional crosslinkers (also referred to as "homocrosslinkers"), heterobifunctional crosslinkers (also referred to as "heterocrosslinkers"), and photoactivated crosslinkers (also referred to as "photoreactive crosslinkers").
The two molecular ends of the homobifunctional protein cross-linking agent have the same reactive terminal or activation reactive group. Commonly used homobifunctional crosslinkers are well known in the art, non-limiting examples of which include, but are not limited to, NHS ester crosslinkers such as N-hydroxysuccinimide (NHS), dithiobis (succinimidyl propionate) (DSP) and 3,3 '-dithiobis (succinimidyl propionate sulfosuccinate) (DTSSP), octanedioic succinate (DSS) and butadiene-styrene copolymer (BS), disuccinimidyl tartrate (DST) and Sulfo-DST, bis (2- [ succinimidyloxycarbonyloxy) ethyl) sulfone (BSOCOES) and Sulfo-BSOCOES, ethyleneglycol bis [ succinimidyl succinate ] (EGS) and Sulfo-EGS, octanedioic glutarate (DSG), N' -disuccinimidyl carbonate (DSC); carbodiimides such as 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride (EDC); imino ester type crosslinking agents such as dimethyl Diimine (DMA), dimethyl imino ester (DMP), dimethyl benzimide ester (DMS), di-t-butyl peroxide (DTBP); mercapto-reactive cross-linking agents such as 1, 4-bis (3 '- [ 2' -dithiopyridine ] propanoylamino) butane (DPDPDPPB); 1, 5-difluoro-2, 4-dinitrobenzene (DFDNB) which is a difluorobenzene derivative, bis (4-fluoro-3-nitrophenyl) sulfoxide (DFDNPS), and the like; photoreactive crosslinking agents such as bis- [ β - (azidosalicylamido) ethyl ] disulfide (BASED) and the like; aldehyde crosslinking agents such as formaldehyde, glutaraldehyde, and the like; 1, 4-butanediol glycidal as a diepoxide; hydrazide crosslinking agents such as adipic acid dihydrazide, carbohydrazide and the like; double derivative crosslinking agents such as diazotized o-tolidine, double diazotized benzidine, and the like; and dialkyl halides and the like.
The photoreactive crosslinking agent is a crosslinking agent having a photoreactive group. The activating reactive group of the homobifunctional crosslinking agent may also be a photoreactive group.
The types of partial homobifunctional protein cross-linkers, reactive groups, protein binding groups, photoactivity, etc. are listed in table 1.
TABLE 1
"antibody" (antibody) refers to immunoglobulin produced by plasma cells differentiated from B lymphocytes or memory cells under antigen stimulation and specifically combined with corresponding antigen by immune system of body.
The invention provides a hybridoma cell strain secreting an anti-human alpha-antitrypsin polyclonal antibody and a preparation method thereof.
In one embodiment, the method of the invention comprises the steps of:
1) preparing immune antigen: coupling human alpha-antitrypsin and bovine serum albumin by a homotype bifunctional protein cross-linking agent to obtain a human alpha-antitrypsin-bovine serum albumin conjugate;
2) immunizing a mouse: immunizing Balb/c mice by adopting a small-dose multiple immunization scheme and utilizing the human alpha-antitrypsin-bovine serum albumin conjugate obtained in the step 1) to act on an immunizing antigen;
3) cell fusion: fusing SP2/0 mouse myeloma cells with spleen cells of the immune Balb/c mouse obtained in the step 2), and selectively culturing in an RPMI-1640 medium containing HAT;
4) selective culture of hybridoma cells: after the cells fused in the step 3) are selectively cultured in HAT medium, selecting fused cells of mouse spleen cells and mouse hybridoma cells SP 2/0; and
5) and (3) identification: detecting the culture supernatant of the fused hybridoma obtained in the step 4), and identifying a hybridoma cell strain secreting the anti-human alpha-antitrypsin polyclonal antibody.
Any homobifunctional protein crosslinking agent known in the art may be employed in the methods of the present invention, preferably a carbodiimide-based protein crosslinking agent, more preferably EDC.
In the invention, the animal immunization adopts a small-dose multiple immunization scheme, and a mouse is taken as an example, the immunization is carried out 3-4 times by using a conventional dose of 100-200 mug/mouse. One specific example of an immunization protocol is: immunizing 7-week-old female Balb/c mice with immune antigen, mixing 100 mu g of immune antigen with Freund's complete adjuvant according to a volume ratio of 1:1, emulsifying to obtain a water-in-oil state, injecting the emulsified complete antigen into the immunized mice at the back of the neck and multiple points subcutaneously, emulsifying with the same antigen and the same volume of Freund's incomplete adjuvant after 2 weeks, performing boosting immunization on 100 mu g of the mice, performing third immunization after 2 weeks, performing the dose and method as the second immunization, performing the last impact immunization on the mice before cell fusion, and performing intraperitoneal injection on 100 mu g of immune antigen.
In the method of the present invention, the method of cell fusion is not limited, and methods and procedures known in the art may be employed.
In the method of the present invention, the method of antibody detection is not limited, and detection methods known in the art may be used, for example, ELISA method may be used.
By the method, hybridoma cell strains secreting the anti-human alpha-antitrypsin polyclonal antibody can be screened.
By way of example, an exemplary preparation procedure for hybridoma cell lines secreting polyclonal antibodies against human α -antitrypsin of the present invention is provided below:
1) preparation of immune antigen:
dissolving 1mg human alpha-antitrypsin in 0.25ml LDMSO solution, adding EDC, shaking vigorously at room temperature in dark for 2 hr, reacting at 4 deg.C overnight, slowly dropping the above solution in BSA solution, shaking at room temperature in dark for 2 hr, and reacting at 0.05mol L-1Dialyzing in PBS buffer for 72 hours in the dark to obtain the conjugate of human alpha-antitrypsin and carrier protein BSA.
2) Mouse immunization:
using human alpha-antitrypsin-BSA conjugate as an immunizing antigen, immunizing a 7-week-old female Balb/c mouse, mixing 100 mu g of the antibody per mouse with Freund's complete adjuvant in a volume ratio of 1:1, emulsifying to obtain a water-in-oil state, injecting the emulsified complete antigen into the Balb/c mouse at the neck and back of the neck and in a multi-point subcutaneous manner, emulsifying with the same antigen and the same volume of Freund's incomplete adjuvant after 2 weeks, performing boosting immunization on 100 mu g of the antibody per mouse, performing third immunization after 2 weeks, performing the same method as the second immunization, performing the last impact immunization on the mouse before cell fusion, and performing intraperitoneal injection on 100 mu g of the antibody per mouse.
3) Cell fusion and selective culture of hybridoma cells:
uniformly mixing SP2/0 mouse myeloma cells and immune Balb/c mouse spleen cells according to the proportion of 1:5-1:10, centrifuging for 7 minutes at 1300rpm, placing the mixture in a 40 ℃ metal bath for preheating, adding 1mL 50% PEG4000 preheated to 40 ℃ in 45s by using a 1mL suction pipe while gently shaking, then adding 30mL 1640 incomplete culture medium preheated to 37 ℃ in 90s, standing for 10 minutes at room temperature, centrifuging for 5 minutes at 1000rpm, removing supernatant, adding 20mL 1640 culture medium containing 20% fetal calf serum and HAT for resuspension, subpackaging the mixture onto a culture plate with existing feeder cells, culturing in a 5% carbon dioxide incubator for 7 days, observing the growth condition of the cells, replacing 1/2 culture medium by using the 1640 culture medium containing 20% fetal calf serum and HT, and replacing the culture medium with 20% fetal calf serum and HT1640 culture medium after 14 days.
4) Identification of hybridoma cells secreting anti-human α -antitrypsin antibodies:
the antibody was diluted to a protein content of 2. mu.g/ml with 0.05M carbonate coating buffer pH 9.6. 50 μ L of the solution was added to the reaction well of each polystyrene plate overnight at 4 ℃. The next day, the well was discarded, and the wells were washed 3 times with wash buffer for 3 minutes each time (washing, the same below). To the reaction well of each polystyrene plate was added 300. mu.L of 5% skim milk powder (BD Co.) in PBS, incubated at 37 ℃ for 2 hours, and then washed. Adding 50 μ L of diluted sample to be tested into the coated reaction wells, incubating at 37 deg.C for 1 hr, and washing (simultaneously making blank wells, negative control wells and positive control wells). To each reaction well, 50. mu.L of an enzyme-labeled antibody (diluted as described above) diluted with a diluent was added, incubated at 37 ℃ for 1 hour, and then washed. To each reaction well, 50. mu.L of a temporarily prepared TMB substrate solution was added, and color development was carried out at 37 ℃ for 15 minutes. The reaction was terminated by adding 50. mu.L of 2M sulfuric acid to each reaction well. On a white background, the results were directly observed with the naked eye: the darker the color in the reaction well, the stronger the positive degree, and the colorless or very light negative reaction, which is indicated by "+" and "-" signs according to the color. As an alternative determination method, OD values can be detected on an ELISA detector at 450nm and 630 dual wavelengths, the OD value of each well is detected after the blank control well is zeroed, and if the OD value is more than 2.1 times of the specified negative control OD value, the positive control is obtained.
In a preferred embodiment, the hybridoma cell line prepared by the method of the present invention is named α -antiprypsin-P-01 and deposited at the chinese type culture collection on 11/6 of 2015 at the address: the preservation number of the Wuhan university preservation center in the Wuchang area of Wuhan city, Hubei province is CCTCC C2015194.
The preparation method of the hybridoma strain alpha-antiprysin-P-01 comprises the following steps:
human alpha-antitrypsin (DMSO) solution was prepared and activated by adding 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC). The solution is slowly dripped into Bovine Serum Albumin (BSA) solution for coupling the human alpha-antitrypsin and the BSA, and a reaction product is dialyzed by Phosphate Buffer Solution (PBS) to obtain a conjugate of the human alpha-antitrypsin and the carrier protein BSA. Detecting tail venous blood of the immunized mouse by using an enzyme-linked immunosorbent assay after four times of immunization, and fusing splenocytes of the mouse with the serum titer of more than 1:10000 with myeloma cells SP 2/0; culturing with selective culture medium (HAT, available from SIGMA) containing hypoxanthine (H), aminopterin (A) and thymidine (T) and RPMI-1640 (available from HYCLONE) containing 20% fetal bovine serum, and forming colonies seven days after fusion; human alpha-antitrypsin antigen is used for coating, and a hybridoma cell strain which stably secretes the anti-human alpha-antitrypsin polyclonal antibody is obtained through enzyme-linked immunosorbent assay (ELISA) identification and is named as alpha-antiprypsin-P-01. The hybridoma cell line is 1 × 106Injecting Balb/c female mice which are sensitized by liquid paraffin in advance and are 8-10 weeks old into the abdominal cavity, collecting ascites after 10-14 days, detecting the ascites to be positive by enzyme-linked immunosorbent assay, and proving that the female mice can identify human alpha-antitrypsin by a western blot method. The polyclonal antibody subtype secreted by the hybridoma cell strain alpha-antitrypsin-P-01 is IgM which is determined by a mouse polyclonal antibody subtype typing kit.
Examples
1. Materials and instruments
1.1 cell lines, tissue specimens and animals
a. Myeloma cells (SP 2/0): derived from ATCC (American type culture Collection).
Balb/c mice: purchased from the animal platform of Qinghua university.
1.2 Primary reagents
a. Freund's complete adjuvant, Freund's incomplete adjuvant, HAT, HT, TMB, PEG (molecular weight 4000),
EDC: purchased from Sigma company;
hrp-labeled goat anti-mouse IgG: purchased from Gibco corporation;
rpm 1640 medium: purchased from Hyclone;
d. cellulose acetate membrane (NC): purchased from Hybond corporation;
e. fetal bovine serum: purchased from PAN company;
f. mouse polyclonal antibody subclass typing kit: purchased from Thermo sauce;
g. coating buffer solution: every 500mL of deionized water is added with 0.795g of sodium carbonate and 1.465g of sodium bicarbonate, and the pH value is 9.6.
h. Substrate buffer: every 500mL of deionized water is added with 9.2g of disodium hydrogen phosphate dodecahydrate and 2.55g of citric acid, and the pH value is 5.0.
TMB (Tetramethylbenzidine) use solution 0.5mL of TMB (10mg/5mL of anhydrous ethanol)
Substrate buffer (pH5.5)10mL of 30% H2O20.01μL；
j. Incomplete 1640 culture solution: 15mL of 7.5% sodium bicarbonate was added to 500mL of the stock solution of 1640 culture solution.
k. Complete 1640 culture solution: 20mL of fetal bovine serum was added to 80mL of incomplete 1640 culture medium.
Antibody dilutions: 0.1g BSA per 100mL PBS was added.
m. wash buffer: 0.5mL of Tween-20 was added per 100mL of PBS.
And n.PEG preparation liquid: each 8.9mL of incomplete 1640 medium was supplemented with 1mL of DMSO and 0.1mL of 7.5% sodium bicarbonate.
1.3 Main instruments
a. A carbon dioxide incubator: heraeus corporation;
b. superclean bench: suzhou clean plant instrumentation;
c. and (3) inverting the microscope: leica corporation;
d.37 ℃ constant-temperature incubator: the Yao city oriental electrical instrument factory;
e. polystyrene plastic plate (ELISA plate) 96 holes, cell culture plate (96 holes and 24 holes): NEST Corp;
f. a centrifuge: eppendorf Co Ltd
g. A water purifier: milipore Corp
h. Micro vertical plate electrophoresis tank: Bio-Rad Ltd
i. An enzyme-labeling instrument: TECAN Inc
2. Method of producing a composite material
2.1 cell culture: myeloma cells SP2/0 were cultured in a complete 1640 culture medium, placed in a 5% carbon dioxide cell incubator at 37 ℃ and subjected to liquid change 1 time every other day and passage 1 time for 3-4 days.
2.2 antigen preparation: 1mg of human alpha-antitrypsin was dissolved in 0.25mL of DMSO solution, 2mmol of EDC was added to the solution, and the solution was vigorously shaken at room temperature for 2 hours in the absence of light, and then reacted at 4 ℃ overnight. Coupling of human α -antitrypsin: the above solution was slowly added dropwise to BSA solution (2mg protein in 1mL of 0.1mol L)-1Sodium bicarbonate solution), adding a magnetic rotor, placing on a magnetic oscillator, and oscillating for 2h at room temperature in a dark place; the reaction product is added in 0.05mol L-1Dialyzing in PBS buffer solution at 4 ℃ for 72h in the dark, and replacing the dialyzate once every 5h to obtain the conjugate of the human alpha-antitrypsin and the carrier protein BSA.
2.3.1 immunization of mice
The coupled antigen immunizes 3 Balb/c mice (4-8 weeks old, female) by the following specific steps:
a. first immunization: 100 mu g/mouse of human alpha-antitrypsin-BSA is added with equal volume of Freund's complete adjuvant, mixed well and injected into Balb/c mouse with 1mL subcutaneous multiple points, 0.2 mL/point; at 2 week intervals.
b. And (3) second immunization: the dose and route were as above, this time with an equal volume of Freund's incomplete adjuvant, at 2 weeks intervals.
c. And (3) third immunization: the same dose as above, this time with the same volume of Freund's incomplete adjuvant, 10 days later, the tail venous blood of the immunized mice was collected (after collection, the mice were placed in a refrigerator at 4 ℃ overnight, and the supernatant was left for use 10 minutes after centrifugation at 2000 rpm the next day), and the serum titer was determined by ELISA.
d. The fourth immunization: the dose is the same as above, and adjuvant is not added, and the immunization is directly carried out by intraperitoneal injection.
2.3.2ELISA method for detecting the serum titer of the immunized mice:
a. the day before detection, diluting the antigen human alpha-antitrypsin to 15 mu g/mL by using a coating buffer solution, adding the diluted antigen human alpha-antitrypsin into an enzyme-linked immunosorbent plate, wherein each hole is 100 mu L, and placing the enzyme-linked immunosorbent plate in a refrigerator at 4 ℃ for overnight;
b. pouring out the liquid in the coated enzyme-linked plate holes the next day, washing with a washing buffer solution for 3 times, and drying each time;
c. adding 100 μ L/well of serum to be tested (serum: PBS is 1: 100; 1: 1000; 1: 2000; 1: 4000; 1: 8000; 1:16000) diluted with PBS in gradient, and placing in a constant temperature water bath box at 37 deg.C for 1 hr with serum of nonimmunized Balb/c mouse as negative control;
d. pouring out the liquid in the holes of the enzyme-linked plate, washing for 3 times by using a washing buffer solution, and drying by beating each time;
e. adding 100 mu L of goat anti-mouse IgG antibody (diluted by an antibody diluent at a ratio of 1:5000) marked by HRP into each hole, and placing the mixture in a constant-temperature water bath box at 37 ℃ for 1 hour;
f. pouring out the liquid in the holes of the enzyme-linked plate, washing for 3 times by using a washing buffer solution, and drying by beating each time;
g. color development: to each reaction well, 50. mu.L of a temporarily prepared TMB substrate solution was added at 37 ℃ for 15 minutes.
h. And (4) judging a result: and (5) detecting by using a microplate reader.
And judging that the three immunized mice generate antibodies aiming at the human alpha-antitrypsin by an ELISA detection result, and taking the mouse with the highest OD value (the titer is more than 10000) to perform the subsequent steps.
2.3.3 preparation of feeder cells (macrophages from the abdominal cavity of mice were taken the day before fusion):
7-week-old Balb/c female mice were used; killing by pulling the neck, soaking in 75% alcohol for 5 minutes for disinfection, cutting the abdominal skin with sterile scissors to expose the peritoneum, injecting 8mL of incomplete 1640 culture solution each time with a sterile syringe, repeatedly washing, sucking out the washing solution, putting into a 50mL centrifuge tube (about 40mL in total), and centrifuging at 1300rpm for 5 minutes; discarding the supernatant, resuspending the pellet in complete 1640 medium, adjusting the cell count to 4X 105Adding into 24-well cell culture plate at 500 μ L/well, placing at 37 deg.C and 5% of twoAnd (5) culturing the cells in a carbon oxide cell culture box.
2.3.4 cell fusion
a. Observing the SP2/0 state of myeloma cells (the requirement is optimal in logarithmic growth phase), and preheating the prepared 50% PEG and 20mL of incomplete 1640 culture solution in a 37 ℃ cell culture box; (50% PEG preparation: 50% PEG is prepared in the day before the fusion and put in a refrigerator at 4 ℃ for standby, 1g of PEG is put in a small penicillin bottle and put in an autoclave for autoclaving to obtain about 1mL of liquid, and 1mL of PEG preparation liquid is added in the liquid to obtain 50% PEG);
b. SP2/0 was collected in a 50mL sterile centrifuge tube and counted (total number of cells required was 1X 10)6One), 1300rpm for 7 minutes;
c. preparing a sterile plate, a screen and a penicillin bottle, killing BlAB/c mice immunized four times by pulling the neck, and soaking the mice in 75% alcohol for 5 minutes;
d. uniformly mixing SP2/0 mouse myeloma cells and immune Balb/c mouse spleen cells according to the ratio of 1:5-1:10, centrifuging at 1300rpm for 7 minutes, discarding supernatant, and tapping the bottom of a tube with a palm to loosen and uniformly obtain cells;
e. preheating in 40 deg.C metal bath, adding 1mL 50% PEG4000 preheated to 40 deg.C with 1mL suction tube within 45s, and gently oscillating while adding;
f. then adding 30mL of 1640 incomplete culture medium preheated to 37 ℃ into 90s, and standing for 10 minutes at room temperature;
g.1000rpm for 5 minutes, discarding the supernatant, adding 20mL 1640 medium containing 20% fetal calf serum and HAT, resuspending, subpackaging to existing feeder cells culture plate, and culturing in 5% carbon dioxide incubator.
2.3.5 Selective culture of hybridomas
After 24 hours of fusion, the selection culture solution is added, and the specific process is as follows:
a. after 24 hours of fusion, adding HAT 1.6mL and HT 0.4mL into 20mL of complete 1640 culture solution, mixing uniformly, adding into a 24-well plate, and adding into 500 mu L/well;
b. changing the culture medium every 5 days, sucking 1500 μ L of the total 1640 culture medium from each well of the 24-well plate, and adding the total 1640 culture medium to be changed (each 60mL of the total 1640 culture medium is composed of 0.6mL of HAT and 0.6mL of HT);
c. the medium was changed every 7 days, 1500. mu.L of each well of the 24-well plate was aspirated, and the complete 1640 medium to be changed was added (1.2 mL of HT per 60mL of complete 1640 medium).
After 3 to 7 days from the c step, the cells in the culture well from which the cell colony grew in the 24-well plate were collected and subjected to the subsequent steps.
2.3.6 identification of hybridoma cells secreting polyclonal antibodies against human alpha-antitrypsin
The hybridoma obtained in 2.3.5 was cultured, and the culture supernatant was subjected to the following steps:
a. coating: the antibody was diluted to a protein content of 2. mu.g/mL with 0.05M carbonate coating buffer pH 9.6. 50 μ L of the solution was added to the reaction well of each polystyrene plate overnight at 4 ℃. The next day, the well was discarded, and the wells were washed 3 times with wash buffer for 3 minutes each time (washing, the same below).
b. And (3) sealing: to the reaction well of each polystyrene plate was added 300. mu.L of skim milk powder (BD Co.) containing 5% (P BS), incubated at 37 ℃ for 2 hours, and then washed.
c. Sample adding: adding 50 μ L of diluted sample to be tested into the coated reaction wells, incubating at 37 deg.C for 1 hr, and washing (simultaneously making blank wells, negative control wells and positive control wells).
d. Adding an enzyme-labeled antibody: 50. mu.L of an enzyme-labeled antibody (diluted as described) diluted with a diluent was added to each reaction well. Incubate 1 hour at 37 ℃ and wash.
e. Adding a substrate solution for color development: to each reaction well, 50. mu.L of a temporarily prepared TMB substrate solution was added at 37 ℃ for 15 minutes.
f. And (3) terminating the reaction: 50. mu.L of 2M sulfuric acid was added to each reaction well.
g. And (4) judging a result: the results can be observed directly with the naked eye on a white background: the darker the color in the reaction well, the stronger the positive degree, and the colorless or very light negative reaction, which is indicated by "+" and "-" signs according to the color. Or detecting OD values on an ELISA detector at 450nm and 630 dual wavelengths, zeroing the blank control wells, and detecting the OD value of each well, wherein if the OD value is more than 2.1 times of the specified negative control OD value, the wells are positive.
i. As a result: the culture supernatant was detected by a microplate reader as positive.
2.3.7 amplification culture and cryopreservation of hybridomas
a. And (3) amplification culture: and (3) transferring the polyclonal cells with positive supernatant into a 24-well plate for culture (1 hole is transferred into 1 hole), dividing the cells into 2 holes in the 24-well plate after 3 days, dividing the cells into 4 holes after 3 days, and transferring the cells with 4 holes into a 50mL cell culture bottle for expanded culture after 2 days. One of the hybridoma cells is named as alpha-antiprysin-P-01.
b. Freezing and storing the hybridoma cells: when the bottom area of the flask is about 70% full of cells in a 50mL flask → the cells in the flask are cut and beaten by a straw to be completely suspended, the cell suspension is transferred into a 50mL sterile centrifuge tube and counted, the cell suspension is centrifuged at 1200 rpm for 6 minutes → the supernatant is discarded, the cell frozen stock solution (the cell frozen stock solution component: 50% fetal bovine serum; 40% incomplete 1640 culture solution; 10% DMSO) is used for resuspension and precipitation, and the cell density is adjusted to be 1 × 107L → subpackaging in a freezing tube, 1 mL/branch → placing in a refrigerator at-70 ℃ overnight → storing the frozen cells in liquid nitrogen the next day.
2.3.8 Mass production of polyclonal antibodies
Taking hybridoma cell strain alpha-antiprypsin-P-01 as an example, 10 Balb/c female mice aged 8-10 weeks are pre-sensitized by 0.3mL of liquid paraffin per intraperitoneal injection, and hybridoma cell strain alpha-antiprypsin-P-01 is collected after 1 week and is treated by 1 × 106Injecting the pre-sensitized mice into abdominal cavity at a dose of one mouse (diluted to 1mL by equivalent physiological saline), observing that the mice have obvious abdominal distension, slow action, poor appetite and disordered hair after 10-14 days, collecting ascites, killing the mice by pulling the neck, sucking the ascites into a centrifuge tube by a clean dropper, centrifuging for 2000 r/min, and keeping the speed for 5 min.
As a result: the ascites were all positive by ELISA.
2.4 characterization of polyclonal antibody subclasses
A mouse polyclonal antibody subclass typing kit (Thermo Co.) was used, and the detailed procedures were as described in the specification.
As a result: the polyclonal antibody subtype secreted by the hybridoma cell strain alpha-antiprysin-P-01 is IgM through the determination of a kit.
2.5 Western blotting (Western blotting)
2.5.1 sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE)
a. Preparing separation glue and lamination glue according to a conventional method;
b. mixing a certain amount of protein sample with equal volume of 2 XSDS loading buffer (100mmol/l Trisbase, 200mmol/l dithiothreitol, 4% SDS, 0.2 bromFinland, 20% Glycerol), and denaturing at 100 deg.C for 5 min;
c. loaded into discrete polyacrylamide gel sample wells at 100ug per lane;
d. performing electrophoresis at constant voltage of 50V (lamination gel)/100V (separation gel) until bromophenol blue reaches the bottom edge of the gel;
e. the gel was removed and used for Western blotting.
2.5.2Western blotting
SDS-PAGE gel was soaked in transfer buffer (25mmol/l Tris base, 192 mmol/l Dlycin, 20% formaldehyde) for 10-20 min;
b. assembling the cassette type transfer tower according to the sequence of 3 pieces of filter paper, gel, NC membrane and 3 pieces of filter paper, and loading the cassette type transfer tower into a transfer device in the direction of the NC membrane to the anode;
c. under the condition of room temperature, the steady flow is 0.8mA/cm2Electrotransfer for 90 minutes;
d. dyeing the NC membrane by using ponceau dye liquor, marking the positions of each strip with the standard protein molecular weight by using a marking pen, and washing off ponceau by using deionized water;
sealing the NC membrane with 10% skimmed milk powder at room temperature for 2 hours;
f. incubated overnight at 4 ℃ with antibodies diluted in 5% skim milk powder (freshly prepared polyclonal antibody 1:1000, anti-. beta. -actin 1:5000), and shaken for 4X 15 minutes in TBST (10mmol/l Tris base, 150mmol/l NaCl, 0.05% Tween20, pH 8.0);
g. incubated with HRP-labeled goat anti-mouse IgG diluted in 4% skim milk powder for 4 hours at room temperature;
TBST was shaken for a total of 4 times, 15 min/time;
i. equivalently mixing the solution A + B in the ECL color development system, and dripping the solution A + B to an NC membrane;
j. and (6) imaging.
k. As a result: the detection of ascites generated by the hybridoma alpha-antiprysin-P-01 through intraperitoneal injection of mice by a WesternBlot method proves that the polyclonal antibody generated by the hybridoma cell strain can identify the human alpha-antitrypsin.
Claims (1)
1. A hybridoma cell strain secreting an anti-human alpha-antitrypsin polyclonal antibody, which is characterized in that: the hybridoma cell strain is named as alpha-antiprysin-P-01, is preserved in a China Center for Type Culture Collection (CCTCC) on 11/6/2015, has a preservation number of CCTCC C2015194, and the polyclonal antibody subtype secreted by the hybridoma cell strain alpha-antiprysin-P-01 is IgM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510792392.4A CN105349497B (en) | 2015-11-17 | 2015-11-17 | Hybridoma cell strain of anti-alpha-antitrypsin polyclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510792392.4A CN105349497B (en) | 2015-11-17 | 2015-11-17 | Hybridoma cell strain of anti-alpha-antitrypsin polyclonal antibody |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105349497A CN105349497A (en) | 2016-02-24 |
| CN105349497B true CN105349497B (en) | 2019-12-20 |
Family
ID=55325561
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510792392.4A Active CN105349497B (en) | 2015-11-17 | 2015-11-17 | Hybridoma cell strain of anti-alpha-antitrypsin polyclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105349497B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107942055A (en) * | 2017-11-22 | 2018-04-20 | 南宁科城汇信息科技有限公司 | The ELISA method of transcript profile and protein science in a kind of liver cancer biological process |
| CN113049805B (en) * | 2021-03-22 | 2022-09-23 | 深圳市亚辉龙生物科技股份有限公司 | Antibody compound, preparation method thereof and detection kit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5399684A (en) * | 1982-05-20 | 1995-03-21 | Washington Research Foundation | DNA sequences expressing mammalian alpha-1-antitrypsin |
-
2015
- 2015-11-17 CN CN201510792392.4A patent/CN105349497B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| α1 -抗胰蛋白酶的研究进展;权燕敏;《现代农业科学》;20090531;第16卷(第5期);14-16 * |
| 小分子抗原人工合成进展;王建华 等;《应用化学》;20110430;第28卷(第4期);第369页第2节第1段，第370页第3节第2段 * |
| 抗α1-抗胰蛋白酶单克隆抗体的制备及临床应用价值;王赛西 等;《第二军医大学学报》;19891231;第10卷(第4期);第369页 材料方法部分，第370页结果部分以及第371页讨论部分第1段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105349497A (en) | 2016-02-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107475203B (en) | H7 avian influenza virus monoclonal antibody and application | |
| CN111848779B (en) | Chicken complement receptor 2(ChCR2) monoclonal antibody and application thereof | |
| CN105349497B (en) | Hybridoma cell strain of anti-alpha-antitrypsin polyclonal antibody | |
| CN112979800A (en) | Preparation method of monoclonal antibody for detecting phosphorylation tau protein pairing | |
| CN110818778B (en) | Antigen, monoclonal antibody, polyclonal antibody and method for preparing Listeria monocytogenes monoclonal antibody | |
| CN105349496B (en) | The hybridoma cell strain of anti-macroglobulin monoclonal antibody | |
| CN105349495B (en) | The hybridoma cell strain of anticoagulin IX monoclonal antibody | |
| CN105400769B (en) | The hybridoma cell strain of anticoagulin VIII monoclonal antibody | |
| CN101235086B (en) | Recombination human rabies viruses resisting antibody | |
| CN101921336B (en) | Monoclonal antibody against human alpha1 acid glycoprotein and preparation method thereof | |
| CN107188961A (en) | A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof | |
| CN105349524B (en) | The hybridoma cell strain of anticoagulin VII polyclonal antibody | |
| CN108004214B (en) | Homer2 monoclonal antibody and application thereof | |
| CN110452883B (en) | Avian leukosis P27 protein monoclonal antibody and preparation method and application thereof | |
| CN113603770A (en) | Novel coronavirus nucleoprotein antibody and application thereof | |
| CN104448000B (en) | Monoclonal antibody of anti-turbot serum immune globulin M and preparation method and application | |
| CN111961002A (en) | Pyraclostrobin hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| CN105368786B (en) | Secrete the hybridoma cell strain of 1 type hepatitis A virus hypotype monoclonal antibody of duck | |
| CN113999304B (en) | Anti-enterobacterin monoclonal antibody mAb4 and application thereof in enterobacterin detection | |
| CN116445419B (en) | 11dhTxB2 monoclonal antibody, preparation method and application thereof | |
| CN114752569B (en) | Hybridoma cell strain 8D3, monoclonal antibody and application thereof | |
| CN109182364A (en) | A kind of polyclonal antibody and its preparation method and application of specific recognition albumin A | |
| CN116813768B (en) | Panda TSH beta monoclonal antibody, hybridoma cell strain and application thereof | |
| CN117417453B (en) | Anti-chicken IgY antibody, antibody composition and application thereof | |
| CN116144601B (en) | Hybridoma cell strain secreting anti-peste des petits ruminants virus H protein extracellular region monoclonal antibody and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |