CN112011517B - Panda LBP monoclonal antibody hybridoma cell strain and application thereof - Google Patents

Abstract

The invention discloses a panda LBP monoclonal antibody hybridoma cell strain and application thereof, and belongs to the technical field of LBP concentration detection in panda blood, urine and other samples. The invention provides giant panda LBP monoclonal antibody hybridoma cell strains LBP _ panda _1 and LBP _ panda _2, wherein the preservation numbers are CCTCC NO: c202084 and CCTCC NO: C202085. the invention utilizes cell fusion technology to establish hybridoma cell strain secreting anti-LBP monoclonal antibody, collects and purifies cell strain culture supernatant, obtains high specificity LBP monoclonal antibody to be applied to western blot protein detection, and is the positioning and tissue expression information of LBP protein, and the giant panda LBP protein is detected through enzyme linked immunosorbent assay (ELISA), and a detection technology system is established and is simultaneously used as one of giant panda inflammation state judgment indexes. And the use of high specific monoclonal antibodies to LBP receptor ligands, antagonists of LBP receptor. The lipopolysaccharide binding protein antibody is applied to scientific research experiments such as IHC, WB, IF, IP, ELISA and the like.

Description

Panda LBP monoclonal antibody hybridoma cell strain and application thereof

Technical Field

The invention belongs to the technical field of panda blood/urine LBP concentration detection, and particularly relates to a panda LBP monoclonal antibody hybridoma cell strain and application thereof.

Background

Lipopolysaccharide (LPS), also known as endotoxin, is the major component of the outer membrane of gram-negative bacteria. LPS binds to the LPS Binding Protein (LBP), which causes uncontrolled inflammatory response and decreased barrier function of immune defense, leading to systemic inflammatory response syndrome, septic shock, acute lung injury or even multiple organ dysfunction syndrome. It is synthesized in the form of a single-chain polypeptide in hepatocytes, and can be synthesized in extrahepatic tissues under stress, but in a small amount. LBP is expressed mainly in the liver, but also in tissues such as kidney, lung, small intestine epithelium, etc., and plays an important role in local tissue inflammatory responses. The concentration of LBP can be increased 100 to 1000-fold under the stimulation of inflammatory factors. LBP can catalyze LPS to be combined with CD14, LBP protein is relatively conservative, and panda LBP has 80% of sequence similarity with human LBP protein and 89.32% of sequence similarity with dog LBP protein. Therefore, the detection of the panda LBP is helpful for researching the panda inflammatory reaction mechanism, but no rapid and effective method for detecting the panda LBP exists at present, and the level of the panda LBP cannot be accurately detected.

Disclosure of Invention

The invention aims to provide a panda LBP monoclonal antibody hybridoma cell strain and application thereof, so as to solve the problem that the prior art cannot accurately detect the LBP level of a panda.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

giant panda LBP monoclonal antibody hybridoma cell strains LBP _ panda _1 and LBP _ panda _2 with the preservation numbers of CCTCC NO: c202084 and CCTCC NO: C202085. panda LBP monoclonal antibody hybridoma cell strains LBP _ panda _1 and LBP _ panda _2 are preserved in China type culture Collection (CCTCC) at 7, 8 months in 2020 at the preservation addresses: wuhan university in China.

The panda LBP monoclonal antibody hybridoma cell strain LBP _ panda _1 or LBP _ panda _2 is used for producing the anti-panda LBP monoclonal antibody.

The anti-panda LBP monoclonal antibody is produced by a panda LBP monoclonal antibody hybridoma cell strain LBP _ panda _1 or LBP _ panda _2, and the subtypes are IgG 1.

The preparation method of the anti-panda LBP monoclonal antibody comprises the following steps:

(1) according to the amino acid sequence of the panda LBP subunit in NCBI database, the sequence is shown as SEQ ID NO: 1, selecting the polypeptide shown as SEQ ID NO: 2, and coupling with KLH protein to be used as immunogen LBP-KLH for producing specific antibody;

(2) injecting the immunogen LBP-KLH in the step (1) and an adjuvant into an abdominal cavity of a mouse for immunizing the mouse for multiple times until the serum titer reaches the standard;

(3) carrying out cell fusion on myeloma cells and spleen cells by adopting a conventional method to obtain hybridoma cells;

(4) detecting the hybridoma cells, screening out a plurality of hybridoma cells with the highest antibody positive, and taking cell secretion of the hybridoma cells for antibody subtype identification;

(5) and (4) culturing the hybridoma cells of which the antibody subtype is identified as the IgG1 monoclonal antibody in a large quantity, freezing and storing the cells after the cells grow well, collecting cell secretion, and purifying by a column to obtain the anti-panda LBP monoclonal antibody of which the antibody subtype is identified as IgG 1.

The preparation method of the panda LBP monoclonal antibody hybridoma cell strains LBP _ panda _1 and LBP _ panda _2 comprises the following steps:

(1) taking an anti-panda LBP monoclonal antibody with an antibody subtype identified as IgG1, and coupling and marking the monoclonal antibody into LBP-HRP;

(2) then, the corresponding antibody subtype is identified as an anti-panda LBP monoclonal antibody of IgG1, the plate is coated according to the method of coating the ELISA plate, the concentration of the coated antibody is 2ug/ml, and pairwise matching screening is carried out on the coated antibody and the corresponding LBP-HRP;

(3) through ELISA detection, two groups of best-matched LBP antibodies and LBP-HRP are obtained by screening, namely two strains of corresponding hybridoma cell strains LBP _ panda _1 and LBP _ panda _2 of the anti-panda LBP monoclonal antibody are obtained by screening.

Compared with the prior art, the invention has the following beneficial effects:

1. according to the invention, a cell fusion technology is utilized to establish a hybridoma cell strain secreting an anti-LBP monoclonal antibody, the cell strain culture supernatant is collected and purified, and the obtained high-specificity LBP monoclonal antibody is applied to protein detection such as ELISA, western blot and the like, so that the detection specificity of the panda LBP is improved, and the panda LBP level is accurately detected;

2. the invention carries out more in-depth research on the positioning and tissue expression information of LBP protein, enzyme-linked immunosorbent assay (ELISA) protein detection, and the ligand, antagonist and anti-antagonist of LBP receptor by using the LBP high-specificity monoclonal antibody;

3. the invention further understands LBP secretion and gene, provides new data for revealing physiological rule, and the screened antibody can be applied to IHC, wB, IF, IP, ELISA and other scientific research experiments.

Drawings

FIG. 1 is a graph of LBP polypeptide dilution scale by gradient.

Detailed Description

The present invention is further illustrated by the following examples, which include, but are not limited to, the following examples.

Example 1

The embodiment provides a preparation method of an anti-panda LBP monoclonal antibody, which comprises the following steps:

1. design and synthesis of specific polypeptide fragments:

according to the amino acid sequence of the panda LBP subunit in NCBI database, the sequence is shown as SEQ ID NO: 1, selecting the polypeptide shown as SEQ ID NO: 2, and was coupled to KLH protein as an immunogen for producing a specific antibody (LBP-KLH) at a concentration of 1mg/ml, and the amino acid synthesis part was assigned to Shanghai Biotech.

2. Antigen immunization of mice:

experimental mouse preparation: the Balb/c female mice of 8-10 weeks old are 3 mice, and are adapted to the environment for 1 week.

Antigen treatment: sterilized glass syringes, disposable three-way tubes, disposable syringes were prepared prior to immunization. The antigen was first drawn into the syringe using a sterile glass syringe (total 1000. mu.l, four doses) and the adjuvant was then drawn into the syringe using a sterile glass syringe (total 1000. mu.l, four doses); finally, connecting two glass syringes to a three-way pipe for emulsification, mixing and emulsifying antigen LBP-KLH and an adjuvant according to the proportion of 1: 1 completely (emulsification is about 5 minutes until the water-in-oil state is achieved), and finally transferring the emulsified mixed solution to a disposable syringe.

Immunizing a mouse: the purpose of immunizing a mouse is achieved by injecting the emulsified mixed solution into the abdominal cavity of the mouse, and the tail blood of the mouse is collected after multiple immunizations, and serum is separated to detect the immune titer until the serum titer reaches the standard. The first immunization adjuvant is Freund complete adjuvant, the injection dosage is half of the first immunization, and Freund incomplete adjuvant is adopted for emulsification. If the antigen concentration is high and the volume is small, the antigen can be diluted by sodium chloride and then mixed and emulsified with adjuvant 1: 1. First-stage immunization, intraperitoneal injection, 250 μ l (antigen amount is 150 ug/mouse). Two weeks later a second immunization, i.e., intraperitoneal injection, was performed, one at 250 μ l (75 ug/antigen from the second immunization followed by a one week interval between immunizations).

And (3) detecting the immune titer: and (3) collecting blood from the tail of the mouse 3 to 4 days after the third immunization, centrifuging the blood at 12000rpm for 8min, taking serum to perform an ELISA experiment to determine the titer, preparing for fusion after the titer reaches the standard, continuing the immunization until the titer is not high enough, immunizing for 3 to 6 times under normal conditions, and determining whether to strengthen the immunization according to the determination result.

Coating an ELISA plate:

(1) the storage concentration of the polypeptide is 1.0mg/ml, the required coating working concentration is 2ug/ml, and each plate is 5.5ml, so that 11 mul of 1.0ml/ml polypeptide and 5.5ml coating solution are mixed uniformly when preparing 1ELISA plate (96-well plate), then 50 mul/well is added into the ELISA plate, sealed by a sealing plate film after completion, and put into a refrigerator at 4 ℃ for overnight (the time for coating the ELISA plate each time should be relatively fixed, and a plurality of ELISA plates should be coated in the same batch to reduce errors).

(2) The above 96-well plate was washed 3 to 5 times with a plate washer and then the liquid in the plate was blotted.

(3) 2.5% BSA blocking solution was prepared from the coating solution, 200. mu.l/well of the blocking solution was added, and the mixture was sealed with a sealing plate membrane and allowed to stand at room temperature for half a day.

(4) The liquid in the board was spun off and patted dry on absorbent paper.

(5) Quickly vacuumizing by a vacuum packaging machine, marking, and storing in a refrigerator at-20 ℃ for later use.

Mouse serum titer assay (ELISA):

(1) taking serum: the tail of the mouse is sampled with about 10 drops of blood, the blood is centrifuged at 12000rpm for 8min, the serum is taken and marked, and the serum to be detected is diluted to a certain multiple.

TABLE 1 serum dilution table

Dilution factor	Amount of serum (. mu.l)	Diluent (PBS/. mu.l)	Final volume (μ l)
				100	2	198	160
500	40 (100 times dilution from the previous tube)	160	150
				2000	50	150	120
5000	80	120	100
				10000	100	100	100
20000	100	100	100
				40000	100	100	200

(2) Sample adding: the serum to be tested (1 well for each dilution of serum) and 1 well for negative control were added to the corresponding wells, respectively, at 50. mu.l/well.

(3) And (3) incubation: the plates were sealed with a sealing plate and incubated in a 37 ℃ incubator (200 rpm horizontal shaking) for 30 minutes.

(4) Washing: carefully remove the sealing film, wash 3-5 times with a plate washer or manually, and press the liquid in the plate as dry as possible.

(5) Adding double antibodies: goat anti-mouse HRP was diluted 5000-fold at 50. mu.l/well.

(6) And (3) incubation: the plates were sealed with a sealing plate and incubated in a 37 ℃ incubator for 30 minutes.

(7) Washing: carefully remove the sealing film, wash 3-5 times with a plate washer or manually, and dry the liquid in the plate as much as possible.

(8) Color development: adding 100 μ l of TMB into each well, mixing by gentle shaking, incubating in incubator, observing, and developing for 6-10 min.

(9) And (3) colorimetric determination: adding 50 μ l of stop solution (1M HCl) into each well, gently shaking and mixing, setting a reading at a wavelength of 450nm by using an enzyme-labeling instrument, storing the result and analyzing.

3. Cell fusion

3.1 Resuscitation of SP2 cells

First, a cell culture experiment is performed strictly according to aseptic operating specifications, and 5-10ml of sterile PBS buffer solution is added into a 15ml centrifuge tube in advance. Taking out the frozen SP2 cells (myeloma cells) from liquid nitrogen, and rapidly thawing in a water bath at 37 ℃ to loosen the cells; adding cells into PBS, mixing, centrifuging at 1000rpm for 5min, discarding supernatant, washing SP2 cells with PBS twice, suspending cells with 1ml of 10% DMEM complete culture solution prepared in advance, transferring cells into DMEM culture bottle, marking, observing cell density under microscope, and placing the culture bottle at 37 deg.C and 5% CO₂Culturing in an incubator.

Passage of SP2 cells

When the bottom of the culture bottle is about 80% full of cells, the cells can be passaged. Blowing off the cells by using a gun head, transferring the culture solution into a 15ml centrifugal tube, centrifuging at 1000r/min for 5 minutes; discarding the supernatant, adding 5-10ml PBS, gently blowing and mixing, centrifuging again and discarding the supernatant, and repeating the PBS washing step for 2 times. After washing, 2ml of 10% DMEM complete culture solution is added for cell resuspension, and a proper amount of cells are taken out to be placed into a culture bottle and placed into a carbon dioxide incubator for subculture. (sufficient passages for SP2 cells for cell fusion)

3.3 preparation of immune splenocytes

(1) Mice sacrificed

After blood is collected in the orbit of the mouse, the mouse is centrifuged at 12000r/min for 8min, and serum is subpackaged and stored at minus 20 ℃ for later use. The cervical vertebrae were then cut off to kill the mice (without injuring the internal organs), and the mice were soaked in 75% ethanol.

(2) Taking the spleen of a mouse

The experiment is carried out strictly according to the aseptic operating specifications of cell culture, and after the mice are sacrificed, the mice are soaked in 75% alcohol for 5-10 minutes, placed in a 9cm sterile plate and placed in a position (in an ultra-clean bench) which is favorable for self operation, and then are dissected. The spleens of the mice were removed by aseptic manipulation and the adipose tissue on the surface of the spleens was removed as much as possible.

(3) Spleen cell suspension preparation

Washing spleen with PBS for 3-5 times, placing the spleen in a sterile plate, shearing the spleen as much as possible with an ophthalmic scissors, adding PBS for washing by blowing, filtering with a 70-micron cell filter sieve, collecting separated spleen cell suspension, centrifuging for 5 minutes at 1000r/min, and discarding supernatant; washing with 5-10ml PBS at 1000r/min, and centrifuging for 5 min; repeating the steps for three times, adding 2ml of incomplete culture medium (DMEM) to resuspend the cells after washing is finished, taking 10 mu l of the cells to dilute 100 times or 1000 times for cell counting, and placing the rest cells in a water bath kettle at 37 ℃ for standby.

3.4 preparation of SP2 cell suspension

Sucking cell sap from pre-cultured SP2 cells by using a pipette gun to blow the bottom of the bottle to suspend most of the cells, transferring all the cell sap into a 50ml centrifugal tube for 1000r/min, and centrifuging for 5 minutes; discarding the supernatant, adding PBS 5-20ml, mixing, centrifuging at 1000r/min for 5min, washing twice, and adding 2ml DMEM incomplete culture solution to resuspend the cells after washing; 10 mul of the cells were diluted 100-fold or 1000-fold for cell counting, and the remaining cells were placed in a 37 ℃ water bath for future use.

3.5 cell fusion

(1) After counting the cells, myeloma cells are mixed with spleen cells in a ratio of 1: 4 (between 1: 10 and 1: 2); centrifuging at 600rpm for 3 min; the supernatant was discarded. Lightly flicking the bottom of the centrifugal tube to loosen and disperse the precipitated cells into cell suspension.

(2) Slowly adding 0.4-0.7ml of 50% PEG solution preheated at 37 deg.C in advance (the amount of PEG is increased or decreased according to the amount of cells) within 1min while slightly shaking and tapping. Standing for 1min after the addition. Under the action of polyethylene glycol (PEG), various lymphocytes can be fused with myeloma cells to form hybridoma cells.

(3) Slowly adding 10ml DMEM incomplete culture solution preheated at 37 ℃ at a constant speed to stop the action of PEG, tapping while dripping, rotating the centrifuge tube, and standing for 2min after adding.

(4) Centrifuging at 800rpm for 5min, and discarding the supernatant; the residual PEG was removed by washing 2 times with 5-10ml of PBS or DMEM incomplete medium.

(5) After centrifugation, the supernatant was discarded and the cells were resuspended in 40ml HAT selection medium (39.2ml 10% DMEM complete medium +0.8ml HAT).

(6) Adding the fused cells into a 96-well cell culture plate (if macrophages are prepared, sucking the cell culture solution, adding 100 mu l of incomplete culture solution, standing for 5-10min, sucking the culture solution, and finally adding the fused cells into the macrophage culture plate), and adding 200 mu l of the fused cells into each well; place the plates in CO₂Culturing in an incubator.

3.6 selection culture

The fourth day of culture HAT medium was changed by the semiliquid method: the supernatant in the 96-well plate was pipetted 100. mu.l per well using a pipette, discarded, and 100. mu.l of HAT medium (HAT medium formulation: 10% complete medium: HAT 1: 50) was added to 100. mu.l per well, 200. mu.l of 50 XHAT per plate being required.

On the seventh day of culture, the HT medium was changed by the semiliquid method, 100. mu.l of the supernatant was aspirated from each well of the 96-well plate using a sample gun, discarded, and 100. mu.l of HT medium (HT medium formulation: 10% complete medium: HT 1: 50) was added to each well, and 200. mu.l of 50 XHT was added to 9.8ml of complete medium per plate.

3.7 detection of antibodies in culture

Detecting the hybridoma cells, and screening out antibody positive hybridoma cells: after the cells are cultured for about 7 days, obvious clone cells in the holes can be seen, and when the clone cells grow enough (about 10-13 days), culture solution can be sucked to detect whether the cells secrete antibodies.

(1) The LBP polypeptide coated ELISA plate is taken out from a refrigerator, after the temperature is recovered, 100 mu l/hole of cell secretion which is determined in advance to be detected is sucked and added to the coated plate (the cell is not polluted), marking is carried out in advance, two holes are reserved to be used as negative and positive controls, 10% DMEM complete culture solution is used for the negative controls, and serum which is diluted by 10000 times (serum which is collected by the eye socket of the mouse before fusion is used for the positive controls).

(2) And (3) incubation: the plate was sealed with a sealing plate and then placed in an incubator at 37 ℃ with horizontal shaking at 200rpm for 1 hour.

(3) Washing: carefully remove the sealing film, wash 3-5 times with a plate washer or manually, and finally press the liquid dry as much as possible.

(4) Adding double antibodies: goat anti-mouse HRP was diluted 5000-fold in 50. mu.l per well.

(5) And (3) incubation: the plates were sealed with a sealing plate and then incubated in a 37 ℃ incubator for 30 minutes.

(6) Washing: carefully remove the sealing film, wash 3-5 times with a plate washer or manually, and finally press the liquid dry as much as possible.

(7) Color development: adding 100 μ l of color-developing agent TMB into each well, gently shaking and mixing, and developing in an incubator for 6-10 min.

(8) And (3) colorimetric determination: add 50. mu.l of stop solution (1M HCl) to each well, mix them by gentle shaking, set the wavelength of microplate reader at 450nm, and measure OD value.

And (3) selecting a cell hole with a high OD value (at least 3 times of that of a negative control) to expand to the porous culture, repeating the ELISA test to detect the antibody in the cell secretion, and finally selecting 9 groups of cells capable of keeping the high OD value to perform a subsequent limiting dilution experiment.

3.8 limiting dilution screening of monoclonal cells

(1) Counting of positive well cells: suspending cells in the holes, transferring the cells to a 15ml centrifuge tube (rotating while blowing to suspend the cells), and supplementing 10% DMEM complete culture solution to 2 ml; after cell counting, the cell fluid containing only 1000 cells was taken for the next experiment.

(2) The cell fluid was added to 200ml of complete culture medium preheated at 37 ℃ and mixed well, and then added to a 96-well plate (the sample addition tank was gently shaken while adding the cells so as to distribute the cells uniformly, thereby containing a single cell per 200. mu.l), 200. mu.l/well, and ten pieces of 96-well plates in total.

(3) Place the plates in CO₂Culturing in an incubator.

(4) After 4-5 days of culture, small cell clones were visualized on an inverted microscope, and the growth of the cells was observed and wells with only single clones were recorded.

(5) On days 4-7 of culture, wells with single cells recorded were subjected to a half-liquid exchange, and 100. mu.l/well of 10% complete medium was added.

(6) On days 8-10, cell clones were visualized with naked eyes, antibody detection was performed in time, wells with single cells growing and better growth were tested for culture medium (ELISA assay), and antibody positive wells were recorded. Finding out the hole with strong single clone positive, continuously enlarging and culturing, taking cell secretion as antibody subtype identification, and judging the cell strain antibody subtype.

3.9 subtype identification of cell line antibody (ELISA)

Subtype identification was performed using the mouse monoclonal antibody Ig class/subclass identification using ELIsA kit. Kit information: beijing bo olong immuno-technology Limited, cat #: BF16001 standard: 2 × 96 well plates.

The experimental steps are as follows:

(1) the kit was first brought to room temperature and the wash solution was then made to working concentration with pure water (1 part concentrated wash plus 19 parts pure water).

(2) And taking out the enzyme label plate. 8 wells were required for each sample, 8 wells for positive control and 8 wells for negative control.

(3) Adding 50 mul/hole of sample diluent into a sample detection hole, then adding 50 mul of cell culture supernatant into an enzyme-labeled microporous plate, and adding 8 holes into each sample; positive control and negative control were added to 8 wells each without the addition of the dilution, 100. mu.l/well. The plate was incubated for 30 minutes at 37 ℃ with the sealing film.

(4) Discarding the liquid in the plate, washing the plate for 5 times, and then drying the plate on a water-absorbing material without fiber. Each of 8 wells of each sample was filled with 100. mu.l of each of 8 enzyme-labeled secondary antibodies (lgG1, IgG2a, IgG2b, IgG3, IgM, IgA, Kappa, and Lambda), and the same was applied to each well of the universal positive and negative controls. The plate was incubated for 30 minutes at 37 ℃ with the sealing film.

(5) And (5) absorbing the liquid in the plate, washing the plate for 5 times, and then patting the plate on a water-absorbing material without fiber. Add 50. mu.l each of developer A and developer B to each well and replace a new sealing plate film to incubate for 20 min at 37 ℃ in the dark.

(6) It will be seen that the liquid in the wells turns blue, and the enzyme-labeled secondary antibody corresponding to the blue wells is the Ig class or subclass of the specimen. After the reaction of the reaction stop solution (50 mul/hole) is stopped, the wavelength is measured by an enzyme-labeling instrument at 450nm, the positive control OD value is generally not less than 0.8, the negative control OD value is generally not more than 0.15, and the positive judgment standard: the OD value of the sample is larger than OD +0.15, and the negative OD is lower than 0.05 calculated as 0.05.

As a result: if monoclonal antibody is used, only one hole of six samples of IgG1, IgG2a, IgG2b, IgG3, lgM and lgA is developed, namely only one subtype is developed, and if a plurality of holes are developed, the selected cells secrete non-monoclonal antibody and the screening is continued. Both Kappa and Lambda belong to the light chains of immunoglobulins. The type on each light chain can only belong to one of the two types, but not both.

The results of subtype identification are shown in Table 2 below.

TABLE 2 subtype identification results

As shown in the table above, the LBP antibody of group 9 only has IgG1 color in the six indexes of IgG1, IgG2a, IgG2b, IgG3, IgM and IgA, four groups of positive strongest monoclonal antibody of LBP antibody 1/LBP antibody 2/LBP antibody 4/LBP antibody 9 and corresponding hybridoma cells are selected for the subsequent cell strain screening test.

3.10 expansion culture of cell lines

(1) Batch culture: the wells in which the subtype was identified and the monoclonal antibody was obtained were transferred to a 24-well plate (cell suspension was suspended by pipetting while rotating, and complete transfer was performed) and cultured in 1000. mu.l of 10% complete medium.

(2) The growth of the cells was observed, after a relatively large number of cells had grown, titer measurement was performed, and cells with high titer were transferred and cultured in small culture flasks (cells in a 24-well plate were first blown to suspend the cells, and then transferred to culture flasks with a sample gun, supplemented with 7-9ml of 10% complete medium).

(3) And observing the growth condition of the cells, and transferring the cells to a large culture bottle for culture after the cells grow well. One small flask was transferred to two large flasks for culture (cell passaging).

(4) The cell can be transferred to 5-10 large culture bottles for culture, the cells are frozen after growing, and cell secretion is collected for the next antibody column-passing purification test.

3.11 antibody purification

The column used was Pierce^TM Protein G Agarose。

(1) A sample to be subjected to column chromatography (cell culture solution or serum) was prepared, and the sample was taken out from the refrigerator and thawed at room temperature (cell culture solution of 9 cells was selected and subjected to column chromatography).

(2) The column is packed, and the volume of the column is 2-5 ml.

(3) And (3) column washing: washed with 25ml PBS (five column volumes of PBS).

(4) And (3) purification: 5ml of the sample to be purified was then added continuously to the column and the sample passed through the column was collected.

(5) Washing: washed with 40ml PBS (eight column volumes of PBS) and eluted with about 20ml glycine (10 minutes after addition of glycine).

(6) The filtered liquid was collected in 1ml per tube using EP tubes (5. mu.l 1M Tris per tube, pH adjusted).

(7) The concentration was initially determined by Coomassie light and collection was not continued if blue did not change.

(8) And (3) column washing: rinse with 20ml PBS.

(9) Protection of the column: 5ml of PBS and 2% NaN were added₃Protecting the column bed, and storing at 4 ℃.

(10) The collected samples were added to 10000MWCO centrifuge tubes.

(11) Concentrating by centrifugation at 5000rpm for 15min until only 1-2ml of liquid remains.

(12) The antibody concentration was determined.

(13) The 9 antibodies were separately dispensed and stored at-80 ℃.

3.12 cell cryopreservation

(1) After the identified hybridoma cell lines LBP _ panda _ monoclonal antibody LBP _ hybridoma cell lines LBP _ panda _1 and LBP _ panda _2 were stably cultured, the cells in the culture flasks were aspirated to suspend the cells in the culture medium (the cells were generally suspended in the culture medium or grown adherently), and the cells were transferred to a 15ml or 50ml centrifuge tube.

(2) Centrifuge at 1000rpm for 5 minutes.

(3) Wash twice with PBS: the supernatant in the centrifuge tube is sucked out, discarded, PBS is added, mixed evenly, centrifuged for 1000 rpm/5 min, and finally 10ml of PBS is added for suspension to estimate the cell amount.

(4) Diluting 10 microliter suspension cells by 100 times, counting cells, and freezing to obtain cells with density of at least 1 × 10⁶And/ml, the living cell rate is more than 80 percent.

(5) Centrifuging the suspended cells, sucking out the supernatant with a pipette, adding a proper amount of a cryopreservation solution (9 ml of fetal bovine serum +1ml of DMS0, mixing by inversion, filtering for later use) into the cells, and mixing.

(6) Finally, the cells were added to the cryopreservation tubes, 1ml of cell sap per tube. Placing the cells into a freezing storage box, placing the cells into liquid nitrogen for long-term storage after placing the cells for 24 hours at minus 80 ℃.

4. The screening of the anti-panda LBP monoclonal antibody hybridoma cell strain with good specificity and high sensitivity by an ELISA detection method comprises the following steps:

(1) 4 groups of anti-panda LBP monoclonal antibodies with the antibody subtype being identified as IgG1 are taken and respectively coupled with a labeled LBP-HRP, and the step is finished by Shanghai Biometrics company.

(2) The ELISA plate coated with 4 groups of anti-panda LBP monoclonal antibodies (the concentration of the coated antibody is 2ug/ml) is pairwise paired with 4 corresponding LBP-HRPs, and the pairing with good ELISA reaction specificity and sensitivity is screened by adjusting the dilution times of the LBP-HRPs, wherein, the detection positive control sample is 50 mul/hole and 5 mul/ml LBP polypeptide, and the PBS 50 mul/hole is used as the negative control.

(3) Through ELISA detection, two groups of best-matched LBP antibodies and LBP-HRP are obtained by screening, namely two strains of corresponding hybridoma cell strains LBP _ panda _1 and LBP _ panda _2 of the anti-panda LBP monoclonal antibody are obtained by screening.

In this example, two anti-panda LBP monoclonal antibody hybridoma cell strains LBP _ panda _1 (accession number: CCTCC NO: C202084) and LBP _ panda _2 (accession number: CCTCC NO: C202085) with good specificity and high sensitivity were obtained by screening 9 screened cells with high positive titer again.

Example 2

This embodiment provides a method for detecting LBP concentration in panda serum, which includes the following steps.

(1) ELISA plates were coated with screening-determined LBP antibody.

(2) The LBP polypeptide fold gradient dilution was used as a standard curve, and 50. mu.l/well was added to the plate. LBP polypeptide gradient dilution concentration of 25ug/ml, 12.5ug/ml, 6.25ug/ml, 3.125ug/ml, 1.5625ug/ml, 0.78125ug/ml, 0.390625ug/ml, standard curve is shown in figure 1. The standard curve shows that the antibody has a significant linear relationship with the antibody concentration in the range of 0.39-25 dilution times.

(3) And (3) detecting a panda individual serum sample: 50 ul/well of serum (if the concentration is detected to be too high and exceeds the detection range, the sample needs to be diluted and then detected again).

(4) And (3) incubation: the plates were sealed with a sealing plate and incubated in a 37 ℃ incubator for 30 minutes.

(5) Washing: carefully removing the sealing film, washing with a plate washing machine or manually for 3-5 times, and finally drying the water as much as possible.

(6) Adding LBP-HRP: LBP-HRP was diluted 500-fold with PBS, 50. mu.l/well.

(7) And (3) incubation: the plates were sealed with a sealing plate and incubated in a 37 ℃ incubator for 30 minutes.

(8) Washing: carefully removing the sealing film, washing with a plate washing machine or manually for 3-5 times, and finally drying the water as much as possible.

(9) Color development: after dilution of the developer TMB with sodium citrate buffer, 100. mu.l/well were incubated in an incubator at 37 ℃ and all samples developed for 10 min.

(10) And (3) colorimetric determination: after the color development is finished, adding 50 mu l/hole (1M HCl) of stop solution, quickly finishing the operation of adding the stop solution, lightly shaking and uniformly mixing, setting the wavelength of an enzyme-labeling instrument to be 450nm, and measuring the value of each hole.

Based on the determined OD values, the concentration of LBP protein in serum was calculated (Table 3).

TABLE 3 detection results of serum drawn by bears

The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or changes made within the spirit and scope of the main design of the present invention, which still solve the technical problems consistent with the present invention, should be included in the scope of the present invention.

SEQUENCE LISTING

<110> research base for breeding pandas in Chengdu province

<120> giant panda LBP polypeptide monoclonal antibody hybridoma cell strain and application thereof

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 481

<212> PRT

<213> NCBI database panda LBP subunit amino acids

<400> 1

Met Arg Ala Leu Ala Gly Ala Leu Leu Ser Leu Leu Leu Arg Ala Leu

1 5 10 15

Leu Thr Ser Thr Pro His Ala Leu Gly Ala Asn Pro Gly Leu Val Ala

20 25 30

Arg Ile Thr Asp Lys Gly Leu Glu Tyr Val Ala Arg Glu Gly Ser Val

35 40 45

Ala Leu Gln Lys Glu Leu Leu Arg Ile Thr Leu Pro Asp Phe Thr Gly

50 55 60

Asp Phe Lys Ile Lys Pro Phe Gly Arg Gly His Tyr Glu Phe His Ser

65 70 75 80

Leu Ser Leu His Ser Cys Glu Leu Arg Gly Ser Ala Leu Thr Pro Leu

85 90 95

Pro Gly Gln Gly Leu Ser Leu Thr Ile Ser Asp Ser Phe Val Arg Val

100 105 110

Gln Gly Glu Trp Lys Val Arg Lys Ala Phe Val Lys Leu His Gly Ser

115 120 125

Phe Asp Val Gln Val Lys Gly Ile Thr Ile Ser Val Asn Leu Leu Leu

130 135 140

Gly Arg Glu Pro Ser Gly Arg Pro Thr Val Thr Ala Ser Gly Cys Ser

145 150 155 160

Ser His Ile Arg Asp Val Glu Val Asp Val Ser Gly Asp Leu Gly Trp

165 170 175

Leu Leu Asn Leu Phe His Asn Gln Ile Glu Ser Lys Phe Arg Arg Met

180 185 190

Leu Glu Ser Lys Ile Cys Glu Met Leu Gln Asn Ser Val Thr Ser Asp

195 200 205

Leu Arg Pro Tyr Leu Gln Thr Leu Pro Val Thr Thr Glu Ile Asp Ser

210 215 220

Phe Ala Asn Ile Asp Tyr Ser Leu Met Glu Ala Pro Arg Ala Thr Ala

225 230 235 240

Gln Met Leu Asp Val Met Phe Lys Gly Glu Ile Phe Asn Arg His His

245 250 255

Tyr Ser Pro Val Thr Phe Leu Ala Pro Val Met Asn Leu Pro Glu Gln

260 265 270

His Asp Arg Met Val Tyr Phe Ala Ile Ser Asp Tyr Val Phe Asn Thr

275 280 285

Ala Ser Leu Val Tyr His Glu Leu Gly Tyr Met Asn Phe Ser Ile Thr

290 295 300

Asp Asp Met Val Pro Pro Ser Ser Asn Ile Arg Leu Thr Thr Lys Ser

305 310 315 320

Phe Arg Pro Phe Val Pro Arg Leu Ala Lys Leu Tyr Pro Asn Met Asn

325 330 335

Leu Glu Leu Gln Gly Ala Met Ala Ser Ala Pro Phe Leu Asn Phe Ser

340 345 350

Pro Gly Asn Leu Ser Ser Thr Pro Leu Ile Asp Ile Glu Ala Phe Val

355 360 365

Leu Leu Pro Ser Ser Val Arg Glu Pro Val Phe Arg Leu Gly Val Ala

370 375 380

Thr Asn Met Ser Ala Met Leu Thr Phe Asn Thr Ser Lys Ile Thr Gly

385 390 395 400

Leu Leu Lys Pro Gly Lys Ile Gln Val Glu Leu Lys Glu Ser Lys Val

405 410 415

Gly Val Phe Asn Val Ala Leu Leu Glu Gly Leu Leu Asn Tyr Tyr Ile

420 425 430

Leu Ser Thr Leu Tyr Pro Lys Val Asn Glu Lys Leu Ala Glu Gly Phe

435 440 445

Pro Leu Pro Leu Leu Lys Asp Ile Arg Leu Tyr Asp Pro Val Leu Glu

450 455 460

Ile His Lys Asp Phe Leu Phe Leu Gly Thr Asn Leu Gln Tyr Met Arg

465 470 475 480

Ala

<210> 2

<211> 50

<212> PRT

<213> Artificial Synthesis

<400> 2

Leu Leu Gly Arg Glu Pro Ser Gly Arg Pro Thr Val Thr Ala Ser Gly

1 5 10 15

Cys Ser Ser His Ile Arg Asp Val Glu Val Asp Val Ser Gly Asp Leu

20 25 30

Gly Trp Leu Leu Asn Leu Phe His Asn Gln Ile Glu Ser Lys Phe Arg

35 40 45

Arg Met

50

Claims (3)

1. Giant panda LBP monoclonal antibody hybridoma cell strain LBP _ panda _1 or LBP _ panda _2, the preservation numbers are CCTCC NO: c202084 and CCTCC NO: C202085.

2. use of the panda LBP monoclonal antibody hybridoma cell strain LBP _ panda _1 or LBP _ panda _2 of claim 1 for producing anti-panda LBP monoclonal antibodies.

3. An anti-panda LBP monoclonal antibody produced by the panda LBP monoclonal antibody hybridoma cell strain LBP _ panda _1 or LBP _ panda _2 according to claim 1, wherein the subtypes are all IgG 1.

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