CN114752569A

CN114752569A - Hybridoma cell strain 8D3, monoclonal antibody and application thereof

Info

Publication number: CN114752569A
Application number: CN202210519043.5A
Authority: CN
Inventors: 李炎鑫; 刘全国; 史喜菊; 徐立伟; 侯颖
Original assignee: China Customs Science And Technology Research Center
Current assignee: China Customs Science And Technology Research Center
Priority date: 2022-05-12
Filing date: 2022-05-12
Publication date: 2022-07-15
Anticipated expiration: 2042-05-12
Also published as: CN114752569B

Abstract

The invention provides a hybridoma cell strain 8D3, a monoclonal antibody and application thereof, and belongs to the technical field of Rana virus detection. The hybridoma cell strain 8D3 is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is CGMCC No. 45105. The hybridoma cell strain 8D3 can efficiently and stably secrete the monoclonal antibody, the specificity of the monoclonal antibody obtained by secretion is good, prion protein can be specifically identified, and pathogens of mad cow disease and scrapie can be distinguished.

Description

Hybridoma cell strain 8D3, monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of Ruan virus detection, and particularly relates to a hybridoma cell strain 8D3, a monoclonal antibody and application thereof.

Background

Prions are causative agents of transmissible spongiform encephalopathies in animals and humans. Due to normal PrP (PrP)^c) Conversion to the pathogenic PrP (PrPSc), thus triggering a series of degenerative diseases of the nervous system, which are termed "transmissible spongiform encephalopathies", also known as prion diseases, on the basis of their infectivity and on the basis of their common neuropathological changes. Prion diseases share the common characteristic of causing chronic degenerative diseases of the lethal central nervous system, with typical pathological features including neuronal death, vacuolar degeneration, glial and stellate cell proliferation, PrPSc (abnormal form of prion) accumulation and amyloid plaque formation, many cavities in the brain and central nerves, spongiform changes, and lesions that are usually bilaterally symmetrical. The clinical symptoms mainly include central nervous symptoms such as dementia or hyperesthesia, ataxia, tremor, etc., such as scrapie and mad cow disease. The incubation period is long, ranging from months to years and even decades, but the disease course is rapidly accelerated once clinical symptoms appear, death is realized within weeks to months, and the death rate reaches 100%.

Currently, the evaluated TSE diagnostic method in the European Union is the same method, detects PrPsc in brain tissue, can be used for detecting cattle and sheep, judges mad cow disease if the brain detection of cattle is positive, and judges scrapie if the brain detection of sheep is positive, but cannot distinguish the mad cow disease from the scrapie. Therefore, a monoclonal antibody which can be used for diagnosing prion diseases and distinguishing the pathogens of mad cow disease and scrapie is urgently needed.

Disclosure of Invention

In view of the above, the present invention aims to provide a hybridoma cell line 8D3, which can secrete a monoclonal antibody specifically recognizing prion protein, and can be used for mad cow disease scrapie caused by prion and distinguishing the pathogens of mad cow disease and scrapie.

In order to achieve the above purpose, the invention provides the following technical scheme:

a hybridoma cell strain 8D3, wherein the hybridoma cell strain 8D3 is preserved in China center for type culture Collection with the preservation number of CGMCC No. 45105.

Preferably, the hybridoma cell line 8D3 uses OvPrP (89-104aa) -KLH polypeptide conjugate as immunogen, and hybridoma cell fusion technology is adopted to obtain the hybridoma cell line 8D 3.

Preferably, the OvPrP (89-104aa) -KLH polypeptide conjugate is formed by coupling a ovine PrP polypeptide and a carrier protein, a keyhole limpet hemocyanin.

The invention also provides a monoclonal antibody, which is secreted and produced by the hybridoma cell strain 8D 3.

Preferably, the ascites titer of the monoclonal antibody is 1:10⁵。

Preferably, the monoclonal antibody specifically recognizes prion proteins.

The invention also provides application of the hybridoma cell strain 8D3 and/or the monoclonal antibody in preparation of a product for detecting prion diseases.

Preferably, the prion diseases comprise mad cow disease and/or scrapie.

The invention also provides application of the hybridoma cell strain 8D3 and/or the monoclonal antibody in preparation of products for distinguishing pathogens of mad cow disease and scrapie disease.

Preferably, the method for distinguishing pathogens of mad cow disease and scrapie comprises: treating the infected tissue with proteinase K, electrophoresis, transfer printing to obtain monoclonal antibody, and immunological detection to obtain monoclonal antibody 8D3 only used in combination with scrapie PrP^scReact without reacting with mad cow disease PrP^scAnd (4) reacting.

The invention has the following beneficial effects:

the hybridoma cell strain 8D3 can efficiently and stably secrete the monoclonal antibody, the specificity of the monoclonal antibody obtained by secretion is good, prion protein can be specifically identified, and pathogens of mad cow disease and scrapie can be distinguished.

Deposit description

The hybridoma cell strain 8D3 (anti-nutritional PrP protein specific fragment mouse hybridoma cell strain) provided by the invention is preserved in the China general microbiological culture Collection center (CGMCC), the preservation time is 2022 years, 3 months and 1 day, the preservation address is Beijing West Lu No. 1 in the morning area of the Yangyang, and the preservation number is CGMCC No.45105 at the institute of microbiology of China academy of sciences.

Drawings

FIG. 1 is SDS-PAGE of the purification of monoclonal antibody of strain 8D3, in which 1 is low molecular weight protein Marker, 2 is purified ascites (1ul), 3 is purified ascites (2ul), 4 is ascites (1ul), and 5 is ascites (2 ul);

FIG. 2 is a graph showing the measurement of single antibody ELISA titer in hybridoma 8D3 culture supernatant and ascites fluid;

FIG. 3 shows the results of western-blot detection of 8D3 strains of ascites, recombinant PrP antigen, normal goat brain tissue and the like, wherein 1 is recombinant PrP, 2 is protein molecular weight Marker, 3 is goat brain homogenate treated by proteinase K, 4 is goat brain homogenate, and 5 is hamster brain tissue infected by scrapie 263K;

FIG. 4 shows the PrPres band patterns of BSE and Scrapie-infected tissues after proteinase K treatment with different antibodies, the left is the monoclonal antibody 6H4 reaction, the right is the monoclonal antibody 8D3 reaction, where M is molecular weight Marker, 1 is Scrapie 263K-infected hamster brain tissue, and 2 is BSE C57 BL-infected mouse brain tissue;

FIG. 5 shows Western blot assay results of samples after enrichment of brain tissue of mice infected with scrapie through sodium phosphotungstate precipitation at a ratio of 1:10 and at a ratio of 1:20, wherein 1, 3 and 5 are diluted at a ratio of 1:10 after no protease K treatment (Pk-), 7 and 9 are diluted at a ratio of 1:20 after no protease K treatment (Pk-), 2, 4 and 6 are diluted at a ratio of 1:10 after protease treatment (PK +), and 8 and 10 are diluted at a ratio of 1:20 after protease treatment (PK +).

Detailed Description

The invention provides a hybridoma cell strain 8D3, wherein the hybridoma cell strain 8D3 is preserved in China center for type culture Collection with the preservation number of CGMCC No. 45105. The preservation time of the hybridoma cell strain 8D3 is 2022 years, 3 months and 1 day, the preservation address is Beijing, Chaoyang, district, Xilu No. 1, institute of microbiology, China academy of sciences.

In the present invention, the hybridoma cell line 8D3 is preferably obtained by using OvPrP (89-104aa) -KLH polypeptide conjugate (polypeptide sequence: GGGGWGQGGSHSQWNK) as an immunogen and hybridoma cell fusion technology to obtain the hybridoma cell line 8D 3.

In the present invention, the OvPrP (89-104aa) -KLH polypeptide conjugate is preferably formed by conjugation of an ovine PrP polypeptide and a carrier protein, the keyhole limpet hemocyanin.

In the invention, in the process of obtaining the hybridoma cell strain by using OvPrP (89-104aa) -KLH polypeptide conjugate as an immunogen and adopting a hybridoma cell fusion technology, the immune animal is preferably a Balb/c mouse. The specific method of the hybridoma cell fusion technology is not particularly limited, and any conventional hybridoma cell fusion technology in the art can be adopted.

The monoclonal antibody is identified as an IgG 2b subclass by a Southern Biotech kit, a light chain is a kappa chain, and the monoclonal antibody is preferably obtained by inoculating hybridoma cell strain cells to an abdominal cavity of an experimental animal to generate ascites, and the experimental animal is preferably a Balb/c mouse. The serum titer of the hybridoma cell culture is 1:10⁴Ascites titer is 1:10⁵。

In the present invention, the monoclonal antibody preferably specifically recognizes prion protein.

The invention also provides application of the hybridoma cell strain 8D3 and/or the monoclonal antibody in preparation of products for detecting prion diseases.

In the present invention, the prion disease preferably includes mad cow disease and/or scrapie.

The invention also provides application of the hybridoma cell strain 8D3 and/or the monoclonal antibody in preparation of products for distinguishing pathogens of mad cow disease and scrapie.

In the present invention, the method for distinguishing the pathogens of mad cow disease and scrapie comprises: treating the infected tissue with proteinase K, electrophoresis, transfer printing to obtain monoclonal antibody, and immunological detection to obtain monoclonal antibody 8D3 only used in combination with scrapie PrP^scReact without reacting with mad cow disease PrP^scAnd (4) reacting.

In the invention, the product preferably comprises an immunological diagnostic reagent, a test strip and a kit, and the kit preferably comprises an enzyme-linked immunosorbent assay diagnostic kit and a colloidal gold kit.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Establishment of hybridoma cell line 8D3

1. Synthesis of immunogens

The ovine PrP polypeptide (ovPrP 89-104) was synthesized by Biotechnology (Shanghai) GmbH, the synthetic peptide sequence: GGGGWGQGGSHSQWNK (see sequence listing for details), and coupling the carrier protein keyhole liposome cytocyanin (haemocyanin, KLH) with glutaraldehyde, wherein the purity is more than 95%, and obtaining the OvPrP (89-104aa) -KLH polypeptide conjugate.

2. Immunization of mice

Synthetic OvPrP (89-104aa) -KLH polypeptide conjugates were used as immunizing antigens to immunize 3 individual 6-7 week old Balb/c mice (purchased from the institute of Chinese academy), with the routes, doses and adjuvants for each immunization shown in Table 1. The former three immunizations are separated by 4 weeks each time, the blood serum titer is measured by an indirect ELISA method after 12-15 days of four-immunization after tail breaking by a small amount of blood collection, and the mice with high titer are taken for strengthening immunization and fusion.

TABLE 1 immunization of animals

The highest ELISA titer of the anti-sheep prion protein polypeptide positive serum reaches 1:1.6 multiplied by 10 days after four-immunization⁵. The results show that OvPrP (89-104aa) -KLH polypeptide conjugate has good antigenicity and the immunization scheme is reasonable and feasible.

3. Preparation of feeder cells

The non-immunized Balb/c mice were blood-collected and sacrificed 1 day before the fusion (serum was isolated as a negative control and stored at-20 ℃ for future use), soaked in 75% alcohol for 5min, and fixed on a sterile wax tray of an ultraclean bench. The abdominal skin was carefully cut open, the skin separated and the peritoneum exposed. And (3) sucking a proper amount of DMEM nutrient solution by using a sterile syringe, injecting the DMEM nutrient solution into the abdominal cavity of the mouse, and slightly vibrating the abdominal wall of the mouse to ensure that cells in the abdominal cavity fully enter the nutrient solution. The nutrient solution was aspirated back and added to a sterile centrifuge tube and centrifuged at 1000rpm for 10 min. Suspending the precipitate with 1% HAT-containing nutrient solution, mixing, adding two 96-well cell culture plates (100 ul/well), and placing in CO₂Culturing in an incubator for later use. Feeder cells are typically prepared 1-2 days prior to fusion.

4. Preparation of SP2/0 cells

SP2/0 cells were grown in 100mL cell flasks 24-48 hours prior to confluency. On the day of fusion, well-formed, logarithmically growing SP2/0 cells were selected, gently blown off the vial wall, collected in a 50ml centrifuge tube, centrifuged at 1000rpm for 10min, resuspended in DMEM nutrient solution and counted for future use.

5. Preparation of immune spleen cells

On the third day after the boosting, the immunized mice were collected and sacrificed by blood sampling from the eyeballs (serum was isolated as a positive control and stored at-20 ℃ for future use). The sacrificed mice were immersed in 75% alcohol for 5min and transferred to a clean bench. The spleen of the mouse is taken out aseptically, put into a dish containing 5-10ml of DMEM nutrient solution, and rinsed gently. The spleen was transferred to another dish containing about 20ml of DMEM nutrient solution, the nutrient solution was aspirated by a sterile syringe and inserted into one end of the spleen, DMEM solution was injected, and the spleen was repeatedly washed several times until the spleen became white in color. The washed splenocytes were transferred into a 50ml centrifuge tube, centrifuged at 1000rpm for 10min, the supernatant was discarded, and the pellet was resuspended and counted for further use.

6. Fusion

Respectively suck 10⁸Spleen cells and 2X 10⁷The suspension of individual myeloma cells was added to a 50ml centrifuge tube and gently mixed. Centrifuge at 1000rpm for 8min, and discard the supernatant as much as possible. By fingersLightly flicking the bottom of the tube to loosen and mix the settled cells into paste.

1ml of DMEM solution is taken up in 1g of sterilized PEG4000 (previously thawed in a water bath) and treated with 7.5% NaHCO₃Adjusting pH to 7.5, and mixing. Uniformly rotating the centrifuge tube by one hand, sucking 1ml of the prepared PEG4000 solution by the other hand, slowly adding the solution along the wall of the rotated centrifuge tube, controlling the addition to be finished within 60s, and standing for 60 s.

The action of PEG4000 was stopped by adding 25ml of DMEM solution as follows: 1ml of DMEM solution was added at 1 minute, 4ml of DMEM solution was slowly added at 2 minutes, and then the remaining DMEM solution was slowly added over 3 minutes.

The fused cells were centrifuged at 800rpm/min for 8min, the supernatant was discarded, and 40ml of 1% HAT medium was added and gently aspirated to mix the precipitated cells.

Adding the suspension into 96-well plate containing feeder cells, placing CO in each well at 100ul₂Culturing in an incubator.

Cell growth was recorded daily, half the selection medium was changed with 1% HAT on the fourth day after confluency, and the total selection medium was changed with 1% HT on the eighth day.

7. Establishment of ELISA screening method

Respectively establishing an indirect ELISA method which takes synthetic peptide as a coating antigen, conjugate hyperimmune mouse serum as a positive control and nonimmune mouse serum as a negative control, and jointly screening positive clone cells capable of secreting specific antibodies. Establishing sheep brain homogenate (normal cellular PrP) indirect ELISA method according to the above method, and detecting positive supernatant screened by synthetic peptide indirect ELISA method again to eliminate false positive hole.

Determination of optimal reaction conditions

And determining the optimal coating concentration of the antigen and the optimal reaction concentration of negative and positive controls by adopting a matrix method, and determining the dilution of the goat anti-mouse enzyme-labeled secondary antibody according to a product specification.

And (3) judging standard: the ratio of the positive OD value to the negative OD value is more than 2.1, the experiment is established, the positive OD value is about 1.0, the negative OD value is less than 0.1, and the antigen coating dilution and the negative/positive serum dilution are the best when the ratio of the positive OD value to the negative OD value is the maximum.

Indirect ELISA reaction procedure

(1) Coating: the antigen was diluted with coating solution at the optimum coating concentration determined and added to each well in an amount of 100. mu.l (200 ng) at 4 ℃ overnight

(2) Washing: the plate was drained and washed 3 times with 350. mu.l/well of wash solution for 3 minutes.

(3) And (3) sealing: add 100. mu.l of blocking solution to each well and work at 37 ℃ for 60 minutes.

(4) Washing: as above.

(5) Adding a primary antibody: the cell supernatant stock solution to be detected and the negative and positive serum diluted to the optimal reaction concentration by the antibody diluent are added into each hole by 50 mu l and acted for 60 minutes at 37 ℃.

(6) Washing: as above.

(7) Adding an enzyme-labeled secondary antibody: the enzyme-labeled goat anti-mouse IgG was diluted with an antibody diluent at a working concentration (1:10,000) and added to each well in an amount of 100. mu.l, and allowed to react at 37 ℃ for 60 minutes.

(8) Washing: as above.

(9) Adding a substrate: add 100. mu.l of freshly prepared substrate solution to each well and allow to act at 37 ℃ for 15 min.

(10) And (3) terminating the reaction: add stop solution 50. mu.l per well.

(11) Reading: measurement of OD in each well₄₅₀。

OD of different concentrations (μ g/well) of coating antigen_450nmThe values are detailed in table 2.

TABLE 2 OD of different concentrations (. mu.g/well) of antigen coated_450nmValue of

The + number is positive serum; no. -is negative serum.

According to table 2, the optimal coating concentration of antigen was determined to be 0.2 ug/well and the optimal dilution of antibody was 1: 5000.

8. Screening for Positive hybridoma cells

When the fused cells grew to cover the bottom of the wells 1/4-1/3, all culture supernatants with cell growth were screened by the established ELISA method. Selecting cells which are detected to be positive for amplification culture or selecting strong positive holes for cloning, wherein cells which have stronger reaction with normal cellular PrP are selected to be cloned. And 3 days later, detecting the negative hole by ELISA, discarding the negative hole if the negative hole is still negative, and further detecting and culturing the positive hole by the same method as the above method. In addition, cell strains which react only with the recombinant bovine prion protein and react strongly with the cell supernatant are also cloned and expanded simultaneously.

After fusion by the PEG method, 2 cell culture plates with 96 holes are inoculated, cell clone growth can be seen on day 3, 168 holes with hybridoma cells grow are included, the fusion rate is 87.5%, screening by the established ELISA method is carried out on day 10 after fusion, 36 holes are included for positive secreted antibody, and the positive rate is 21.4%. Meanwhile, normal brain homogenate is used as a coating antigen to eliminate false positive holes. 3 strong positive holes are selected and cloned for 3 times to obtain 3 hybridoma cell strains 5H9, 8A5 and 8D3 which can stably secrete anti-recombinant PrP protein antibody, wherein only 8D3 can specifically recognize normal cell type sheep prion protein.

9. Cloning and cryopreservation of Positive hybridoma cells

By limiting dilution

Feeder cells were prepared the day before cloning and the procedure was as described above. And (3) gently sucking the cell in the cell hole which is detected to be strong positive by using a suction pipe to suspend and uniformly disperse the cell, sucking the cell into 1ml of DMEM (DMEM) containing 20% fetal calf serum, and taking a small amount of suspension to count the cell. Positive hybridomas were diluted to 10/ml, 5/ml, 2/ml, 1/ml with 1% HT selection medium and individually plated in 96-well feeder cells at 100 ul/well in 32 wells per dilution.

Cell growth was observed and recorded daily. On the fourth day, half of the 1% HT selection medium was changed, and on the eighth day, the whole of the 1% HT selection medium was changed, and the supernatant was examined. Positive wells with only one clone growing were selected for cloning again until the positive wells reached 100%.

And (5) obtaining a monoclonal cell strain capable of stably secreting the antibody, and then performing amplification culture and freezing storage in time.

Cells were selected in logarithmic growth phase and gently blown down the bottle wall. The cell suspension was centrifuged at 1000rpm for 10min and the supernatant was discarded. Suspending the cell sediment by using the freezing medium, subpackaging in cell freezing tubes with 1.5 ml/tube, covering and sealing, and marking the cell code and the freezing date. The frozen tube is placed at 4 ℃ for 4 hours, transferred to-80 ℃ overnight, and finally transferred into a liquid nitrogen tank for long-term storage.

Example 2

Preparation of monoclonal antibodies

1. Recovery of hybridoma cells

And taking out the freezing tube from the liquid nitrogen tank, quickly putting the tube into a water bath at 37 ℃, and shaking to melt the tube as soon as possible. Centrifuge at 1000rpm for 10min and discard the supernatant. Suspending the precipitate with 20% serum DMEM solution, adding into cell bottle, and placing in CO₂Culturing in an incubator. The next day, the cells were cultured in DMEM containing 20% serum.

2. The monoclonal antibody can be prepared by in vitro culture method and/or in vivo induced ascites method

In vitro culture method

The established hybridoma cell line 8D3 was expanded and cultured continuously until all cells were dead. Collecting cell culture supernatant, centrifuging at 1000rpm for 10min, and storing the supernatant at-20 deg.C for use.

In vivo induced ascites method

Selecting Balb/c producing mice, and injecting sterilized paraffin oil into the abdominal cavity of each mouse at a dose of 0.5 ml. After 10 days, the expanded hybridoma cells were blown down, centrifuged at 1000rpm for 10min, the supernatant was discarded, resuspended in 5ml of DMEM and counted. Each mouse was inoculated with hybridoma cells in the abdominal cavity 10⁶And (4) respectively. After 7-10 days, the abdomen of the mouse is obviously enlarged, and ascites is collected. The ascites fluid is centrifuged at 5000rpm for 10min, the supernatant is collected and stored at-20 ℃ for later use.

Example 3

Identification of monoclonal antibodies

1. Immunoglobulin subclass and light chain species identification of monoclonal antibodies

Identified with southern Biotech kit. The specific method comprises the following steps:

(1) coating: the antibody provided in the kit is diluted to a concentration of 5-10 ug/ml and 100 ul/well with a phosphate buffer solution with a pH value of 9.6, and is kept overnight at 4 ℃.

(2) Washing: washed three times with PBS containing 0.05% Tween-20.

(3) And (3) sealing: blocking with 1% bovine serum albumin in PBST, 200 ul/well, and incubating at 37 ℃ for 1 h.

(4) Washing: as above.

(5) Sample adding: hybridoma cell culture supernatant was added at 100 ul/well and incubated at 37 ℃ for 1 h.

(6) Washing: as above.

(7) Adding an enzyme: alkaline phosphatase-labeled immunoglobulins, subclasses, light chains and heavy chains were diluted with 1% bovine serum albumin-containing PBST to appropriate concentrations and added to an ELISA plate at 100 ul/well, incubated at 37 ℃ for 1 h.

(8) Washing: as above.

(9) Color development: diluting the substrate to 1mg/ml (5 mg tablets provided by the kit are added into 5ml substrate buffer solution), adding the substrate into a reaction hole, performing 100 ul/hole, and measuring the OD value at 450nm by using an enzyme-labeling instrument when developing for 10-20 min.

And (4) judging standard: the highest OD was the McAb subclass.

Hybridoma cell 8D3 was of IgG 2b subclass and the light chain was kappa as determined by Southern Biotech kit.

2. Purification of monoclonal antibodies in ascites fluid

Ascites purification (by octanoic acid-saturated ammonium sulfate method)

(1) 10ml of ascites was taken, and 20ml of 0.06M sodium acetate-acetic acid buffer (pH4.0) was added thereto with stirring.

(2) After adjusting the pH of the ascites to 4.8 with 1M sodium hydroxide, 0.33 ml of n-octanoic acid was added with stirring, and the mixture was stirred in an ice bath for 30 min.

(3) Centrifuging the ascites at 10000rpm for 30min, removing the precipitate, adjusting the pH value of the supernatant to 7.2 with 1M sodium hydroxide, slowly adding equal volume of saturated ammonium sulfate while stirring, centrifuging at 10000rpm for 30min after stirring for 30min, removing the supernatant, and dissolving the precipitate in 5.5ml of PBS.

(4) Slowly dropping 4.5ml saturated ammonium sulfate, stirring for 30min, centrifuging at 10000rpm for 30min, dissolving the precipitate in 2.2ml PBS, transferring into dialysis bag, and dialyzing at 4 deg.C for 48 hr. Collecting and storing at-20 ℃.

And detecting the purification effect of the ascites monoclonal antibody by an SDS-PAGE method.

A small amount of each of the purified and unpurified ascites fluids was subjected to SDS-PAGE, and the concentrations of the concentrated gel and the separation gel were 5% and 12%, respectively, and electrophoresis was carried out at constant pressure of 150V for about one hour until bromophenol blue migrated to the edge of the gel. Carefully take the gel down and put it into Coomassie brilliant blue staining solution, stain for half an hour, decolour until the protein band is clear. The gel imaging system observes the recorded results.

As shown in FIG. 1, after 8D3 monoclonal antibody is purified by the octanoic acid-saturated ammonium sulfate method, SDS-PAGE is carried out, and compared before and after purification, most of the impure proteins in the ascites are removed, so that a better purification effect is obtained. The monoclonal antibody has a heavy chain molecular weight of about 50KD and a light chain molecular weight of about 25 KD.

3. Determination of single-antibody ELISA titer in hybridoma cell culture supernatant and ascites

Hybridoma cell culture supernatants were diluted 10-fold from 1:10 and ascites fluid was diluted 10-fold from 1:100, and their titers were determined by established ELISA methods, as detailed in FIG. 2.

As can be seen from fig. 2, the ELISA titers of the monoclonal antibodies obtained from hybridoma cell 8D3 were 1:10⁴In ascites the single antibody ELISA titer was 1:10⁵。

4. Stability assay for monoclonal antibodies

Freezing hybridoma cell 8D3 for 3 months, recovering, culturing, collecting cell supernatant, and measuring the stability of monoclonal antibody secreted by hybridoma cell and monoclonal antibody 10 secreted by hybridoma cell by indirect ELISA method⁴The ELISA values were judged positive at dilution and are shown in Table 3.

TABLE 3 stability assay for mAb 8D3

As shown in Table 4, the monoclonal antibody titer secreted by the hybridoma cell line 8D3 reached 1:10⁴Can stably secrete anti-sheep PrPThe antibody of (1).

5. Western-Blot analysis

SDS-PAGE electrophoresis

And sequentially adding the processed low-molecular-weight protein Marker, the normal brain homogenate and the purified bovine prion protein into each lane, performing 120V stabilized electrophoresis for about 1.5 hours until bromophenol blue migrates to the edge of the gel, and finishing the electrophoresis.

Transfer printing

And adopting an immersion method. The sandwich of filter paper, glue, PVDF membrane and filter paper is placed conventionally and the current is stabilized at 200mA for 1 hour. The Marker was cut and stained with Coomassie blue.

Immunoassay

Sealing the blotting membrane with 5% skimmed milk powder for 1 hour, adding 2500 times diluted ascites for acting for 1 hour, washing for 3 times and 10 minutes each time, adding 1:5000 times diluted horse radish peroxidase labeled goat anti-mouse IgG for acting for 1 hour at 37 ℃, washing for 3 times and 10 minutes each time, soaking the membrane in a newly prepared ECL luminescent reagent, incubating, developing and fixing.

The immunoblotting test is mainly divided into three parts, namely SDS-PAGE electrophoresis, membrane transfer and antibody detection, wherein the membrane transfer efficiency can affect the later test, if the membrane transfer time is too long, nonspecific strips on the PVDF membrane can be increased, the specificity of the antibody detection test can be affected, if the membrane transfer time is too short, the content of target strips on the PVDF membrane can be reduced, the sensitivity of the antibody detection test can be affected, and the membrane transfer efficiency can be improved by properly increasing the content of methanol in a transfer buffer solution; in the antibody detection test, the concentration of the ascites or the enzyme-labeled secondary antibody is too high, so that the non-specificity of the reaction result is enhanced, in order to optimize the reaction conditions and reduce the non-specific reaction in the test, the ascites of the purified 8D3 monoclonal antibody is diluted by 1:1000, 1:2500, 1:5000 and 1:10000 times, and the enzyme-labeled secondary antibody is diluted by 1:5000 and 1:10000 times, so that the result shows that the optimal reaction conditions are that the ascites and the enzyme-labeled secondary antibody are diluted by 1:2500 and 1:10000 times respectively.

As shown in FIG. 3, the reaction results of ascites due to strain 8D3 with recombinant PrP antigen, normal sheep brain tissue and the like. The 8D3 monoclonal antibody ascites is combined with protein with molecular weight of 33-35KD in normal sheep brain tissue and reacts with specific protein with 27-33KD in itch 263K infected hamster brain tissue. Therefore, it can be determined that the monoclonal antibody 8D3 is a specific antibody against ovine PrP protein.

Example 4

Establishment of method for identifying mad cow disease and scrapie by hybrid Westernblot

1. Preparation of the experiment

Monoclonal antibody 6H4, murine anti PrP IgG1, working concentration 1:5000 dilution (tube can be centrifuged to avoid liquid sticking to the bottle wall or cap)

Alkaline phosphatase-labeled goat anti-mouse antibody IgG-AP, working concentration 1:2500 dilution (to avoid liquid sticking to the bottle wall or cap, the tube can be centrifuged)

Anti-sheep PrP specific fragment monoclonal antibody 8D31:2500 dilution

CDP-Star concentrate, Alkaline Phosphatase Substrate (APS) (12.5mM or 25mM) Roche

Control sample (BSEPrP)^scSample), one vial of normal PrPc and molecular weight marker (97/66/45/30/20/14KD) in gel-like buffer, mixed before use, e.g. flicked.

2. Sample preparation

(1) Sample numbering

(2) All samples can be traced by completing the detection record.

(3) In 2 BII the biosafety cabinet was cutting tissue.

(4) The tissue was trimmed on the cutting plate and excess blood was aspirated with a paper towel. The cut sheets and tissues were sterilized with 2% bleach for one hour and rinsed. The instrument is put into NaOH with 2N for soaking for 2h and washed. After the bleach has been emitted, the paper towel is placed in an open biohazard bag in a biosafety cabinet and autoclaved at 135 ℃ for 30 minutes.

(5) A piece of tissue is cut from the medullary part of the medulla oblongata under a 2 BII cover, and the piece of tissue is placed in a pre-weighed homogenizer tube, the piece of tissue should be between 0.45-0.70g (preferably 0.5-0.55 g).

(6) A10-fold volume of 1 Xhomogenization buffer (W/V) was added to the homogenization tube in which the tissue mass was placed, and the lid was closed.

(7) The slurry was automatically homogenized for 45 seconds using a FASTH or MediFASTH homogenizer.

(8) 1.5mL of 10% homogenization buffer was transferred to a 2.0mL microcentrifuge tube.

(9) Centrifuge at 5000rpm (1130g) for 5 minutes at 10 ℃. Or stored at-20 deg.C (this tube is the master tube).

3. Hybrid western blot method

(1) Using a sample applicator, 100. mu.L of the supernatant was transferred from the mother tube to the bottom of a new 2ml microcentrifuge tube.

(2) Add 10. mu.L proteinase K and 10. mu.L digestion buffer and blow and mix well (8-10 times). The final concentration of proteinase K was 50. mu.g/ml.

(3) The lid was closed and placed in an incubation heater and heated at 50 ℃ for 40 minutes.

Preparing a 17-well 12% NuPAGE gel during incubation, carefully pulling out a comb, tearing off white plastic foil at the bottom of the gel, placing the gel into an electrophoresis tank under the reference numeral (1-6) in the left well, washing the gel well with SDS-MOPS to prepare for sample loading, adding the MOPS to a distance of 3-5cm from the gel tank, adding 1 Xtransfer buffer into the transfer tank, and opening a cooling device to pre-cool the transfer buffer.

(4) After incubation, the enzymatic digestion reaction was stopped by adding 10. mu.L of digestion stop solution.

(5) Add 100. mu.L of PAGE loading buffer to the digestion tube, and mix by pipetting.

(6) Incubate at 100 ℃ for 10 minutes in an incubation heater (old samples previously incubated at 96 ℃ were heated to 65 ℃ for 2 minutes).

(7) Centrifuge at 14000rpm for 5 minutes.

(8) And adding 12 mu L of sample supernatant into a 17-hole 12% NuPAGE gel, and sequentially adding a molecular weight Marker, BSE C57BL infected mouse brain tissue, itch 263K infected hamster brain tissue, a blank (pre-stained Marker), the molecular weight Marker, BSE C57BL infected mouse brain tissue and itch 263K infected hamster brain tissue from left to right. The molecular weight Marker only needs to be added by 1 mu L.

(9) Add 1 XSDS-MOPS running buffer to the inner chamber to ensure that the buffer covers the gel tips.

(10) Add 500. mu.L of antioxidant and run at 200V until the staining line is 1-2cm from the bottom of the gel. (about 30 minutes).

(11) Simultaneously with electrophoresis, the PVDF membrane was prepared, cut to size, placed in a plastic tray, soaked in methanol for a few seconds, decanted, equilibrated in 1 Xtransfer buffer for at least 10 minutes, and pre-cooled 1 Xtransfer buffer was added to the transfer bath.

(12) In a container containing 1 Xthe transfer buffer, a sponge was wetted, a filter paper was placed over the sponge to wet the filter paper, and a PVDF membrane was placed over the filter paper, and a plate number was written on the top left corner of the membrane to ensure that there was sufficient buffer in the container so that the membrane did not dry when the gel was placed.

(13) The plastic frame of the NuPAGE glue was slit with a guillotine, the top part of the glue containing the holes was cut off, and the part above the bottom end of the dyed thread was cut off. A cut is made at the corner where the glue is located to help orient the glue to the film.

(14) The glue is placed on the film as shown to ensure that there are no air bubbles between the glue and the film which could interfere with the transfer.

(15) On the gel was placed the moistened filter paper and then a second sponge.

(16) Carefully transfer the transfer sandwich into the transfer box to avoid air bubbles in the sandwich.

(17) The transfer was carried out at 140-150V for 1 hour at 4 ℃ with continuous cooling and mixing.

(18) After transfer, the transfer film was removed from the sandwich and placed in a plastic incubator and rinsed with TBST for 2 times, 1 minute each.

(19) The TBST was decanted off, an appropriate amount of PVDF blocking buffer was added depending on the size of the membrane, and the membrane was shaken on a plate shaker at room temperature for 60 minutes.

(20) The PVDF blocking buffer was decanted and the PVDF membrane was cut in half from the middle (open channel), half for 6H4 immunoreaction and half for murine anti-ovine PrP-specific antibody 8D3 immunoreaction. Preparing a proper amount of primary antibody by using TBST according to the size of the membrane, 1) adding 2 mu L of 6H 41: 5000 into 10mL of 1-time confining liquid; 2) murine anti-ovine PrP-specific antibody 8D3, 4. mu.L, was added to 10mL of 1-fold blocking solution and the membrane incubated overnight at 4 ℃.

(21) The membranes were washed 4 times with 50ml TBST for 7 minutes each.

(22) The last TBST wash was decanted, appropriate secondary antibodies (AP, goat anti mouse AP) were prepared from TBST according to the size of the membrane, and the membrane was incubated on a shaker at room temperature for 60 minutes.

(23) The membranes were washed 4 times with 50ml TBST for 7 minutes each.

(24) The last TBST wash was decanted, 50ml of one-fold luminescence buffer was made up with deionized water, an appropriate amount of luminescence buffer was added to another separate tube, and the tube was shaken on a membrane for 5 minutes.

(25) The appropriate amount of CDP-Star (12.5 mM; 50X) or (25 mM; 100X) was added to the decanted portion of the luminescence buffer and stored in the dark until use.

(26) The luminescence buffer is poured off, and the membrane is taken out of the plastic incubation box and placed on a plastic or organic glass sheet. An appropriate amount of diluted CDP-Star solution was applied uniformly to the top for 5 minutes at room temperature to ensure that the entire membrane was wet throughout the process.

(27) The remaining luminescence buffer was removed from the film with a soft tissue (without completely drying the film), immediately covered with a plastic preservative film, with creases and air bubbles removed from under the plastic film, and placed in a film cassette.

(28) The membrane is exposed to X-ray film (Hyperfilm) in a dark room until a strong signal of the positive control and a membrane background or proteinase K band appear, (approximately 4-8 minutes), and the exposure time can be longer or shorter for optimal visible signal appearance. The film can also be placed on a clean plastic film and placed on a chemiluminescent imager to detect the optical signal using a CCD-camera detection system. The basic principle is to stain a gel electrophoresis-treated cell or biological tissue sample with a specific antibody. Information on the expression of a specific protein in the analyzed cell or tissue is obtained by analyzing the location and depth of staining.

The brain tissue of mouse infected by BSE C57BL, the brain tissue of hamster infected by 263K, and the brain tissue of mouse treated by proteinase K, electrophoresis, transfer printing and immunological detection are shown in FIG. 4, and BSEPrP^scThe sample reacted strongly with monoclonal antibody 6H4 but very weakly or not with monoclonal antibody 8D3, scrapie PrP^scThe samples showed strong reactions with both mAbs 6H4 and 8D 3. And the typical three bands of PrPsc appear, namely, a double glycosylation band, a single glycosylation band and an aglycosylation band from top to bottom, and the molecular weight is distributed between 17 and 30 KDa. And the density of the three strips: double glycosylation zone>Mono-glycosylation band>Aglycosylated band, scrapie PrP^scThe molecular weight of the aglycosylation zone is slightly greater than BSEPrP^sc. Therefore, the monoclonal antibody 8D3 prepared by the invention can obviously identify scrapie PrP^scAnd mad bovine disease PrP^sc。

4. PrP enrichment by sodium phosphotungstate precipitation^scIs established by

Following the hybridization western blot procedure, 20% brain homogenate was prepared and 0.6mL was added to the same volume of 4% Sarkosyl solution (4% Sarkosyl in PBS, pH7.4), incubated at 37 ℃ for 10 minutes, then DNAse I (100. mu.g/mL) was added, incubated at 37 ℃ for 30 minutes, centrifuged at 4000rpm (1538g) for 10 minutes, centrifuged, and 1mL of the supernatant was added to proteinase K to a final concentration (50. mu.g/mL) and incubated at 50 ℃ for 40 minutes. After digestion with protease, a final concentration of 0.4% sodium phosphotungstate solution (4%, W/V, 170mM MgCl, PBS, pH7.4) was added, incubated with shaking at 37 ℃ for 30 minutes, centrifuged at 13000rpm for 30 minutes, the supernatant was discarded, and the precipitate was suspended in 12. mu.L of water. Then 12. mu.L of NuPAGE sample buffer is added, the mixture is heated for 10 minutes at 100 ℃, and finally electrophoresis, transfer printing and immunological detection are carried out according to a method of hybridizing western blot.

FIG. 5 shows a typical PrPsc band pattern, and the scrapie 263K infected hamster brain tissue is significantly more strongly signal precipitated with sodium phosphotungstate even after 1:20 dilution than without sodium phosphotungstate precipitation.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> research center of China customs science and technology

<120> hybridoma cell strain 8D3, monoclonal antibody and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gly Gly Gly Gly Trp Gly Gln Gly Gly Ser His Ser Gln Trp Asn Lys

1 5 10 15

Claims

1. A hybridoma cell strain 8D3, wherein the hybridoma cell strain 8D3 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 45105.

2. The hybridoma cell line 8D3, wherein the hybridoma cell line 8D3 is derived from OvPrP (89-104aa) -KLH polypeptide conjugate as an immunogen by hybridoma cell fusion technology to obtain hybridoma cell line 8D 3.

3. The hybridoma cell line 8D3, according to claim 2, wherein the OvPrP (89-104aa) -KLH polypeptide conjugate is formed by conjugation of an ovine PrP polypeptide and a carrier protein, a keyhole lipid hemocyanin.

4. A monoclonal antibody secreted by the hybridoma cell line 8D3 according to any one of claims 1 to 3.

5. The monoclonal antibody of claim 4, wherein the monoclonal antibody has an ascites titer of 1:10⁵。

6. The monoclonal antibody of claim 4, wherein the monoclonal antibody specifically recognizes a prion protein.

7. Use of the hybridoma cell line 8D3 according to any one of claims 1 to 3 and/or the monoclonal antibody according to any one of claims 4 to 6 for the preparation of a product for detecting prion diseases.

8. The use of claim 7, wherein the prion disease comprises mad cow disease and/or scrapie.

9. Use of the hybridoma cell line 8D3 according to any one of claims 1 to 3 and/or the monoclonal antibody according to any one of claims 4 to 6 for the preparation of a product for differentiating pathogens of mad cow disease and scrapie.

10. The use according to claim 9, wherein the method of differentiating between the pathogens mad cow disease and scrapie comprises: treating the infected tissue with proteinase K, electrophoresis, transfer printing to obtain monoclonal antibody, and immunological detection to obtain monoclonal antibody 8D3 only used in combination with scrapie PrP^scReact without reacting with mad cow disease PrP^scAnd (4) reacting.