CN113005096A - Hybridoma cell strain secreting anti-serine phosphorylation tau protein monoclonal antibody - Google Patents

Abstract

The invention belongs to the field of immunodetection, and discloses two hybridoma cell strains secreting anti-serine phosphorylation Tau protein monoclonal antibodies for secreting p-Tau³⁹⁶ _, ⁴⁰⁴The hybridoma cell strain of the monoclonal antibody is preserved in China center for type culture Collection with the preservation numbers of CCTCC NO: c2020219, CCTCC NO:C2020218. the present invention provides 2 strains of secreted novel p-Tau³⁹⁶ _, ⁴⁰⁴Hybridoma cell strain of monoclonal antibody, and method for preparing specific recognition human p-Tau by using hybridoma cell strain³⁹⁶ _, ⁴⁰⁴And the monoclonal antibody of the aggregate thereof, solves the problem of the lack of the current simultaneous targeting of p-Tau³⁹⁶ _, ⁴⁰⁴The difficulty of antibodies. The two monoclonal antibodies can be further applied to the preparation of the p-Tau with high specificity and sensitivity³⁹⁶ _, ⁴⁰⁴The development of a specific targeting p-Tau³⁹⁶ _, ⁴⁰⁴The immunological treatment of the (B) can slow down the disease process and fill up the current targeting p-Tau³⁹⁶ _, ⁴⁰⁴Blank immunological therapy of (1).

Description

Hybridoma cell strain secreting anti-serine phosphorylation tau protein monoclonal antibody

Technical Field

The invention belongs to the field of immunodetection, and particularly relates to two hybridoma cell strains secreting anti-serine phosphorylation Tau protein monoclonal antibodies, namely two Tau proteins (p-Tau) capable of secreting anti-human 396 and 404 site serine phosphorylation^396,404) Hybridoma cell strain of monoclonal antibody.

Background

Alzheimer's Disease (AD) is a progressive degenerative disease of the nervous system with occult onset. Clinically characterized by global dementia manifestations of memory impairment, aphasia, disuse, agnosia, impairment of visuospatial skills, executive dysfunction and alterations in personality and behavior, the etiology of most alzheimer's disease remains unknown, with genetic differences being confirmed in only 1 to 5% of cases. Alzheimer's disease is the most common cause of dementia, and 60% to 80% of dementia cases worldwide are caused by alzheimer's disease. Alzheimer's disease is highly prevalent in the elderly, and its incidence increases exponentially with age. With the increasing trend of global aging, prevention and treatment of alzheimer's disease has become the most important public health problem in the world. With the increasing global aging, the incidence of alzheimer's disease is steadily rising, and about 3400 million patients with alzheimer's disease are globally present by 2018 as reported by the DRG database under the korui wei-an flag (Decision Resources Group). By 2028, patients with alzheimer's disease are also expected to increase by 38%. By 2020, more than 100 million new patients with Alzheimer's disease in China are treated every year, and the number of total patients with Alzheimer's disease approaches 750 million. With the aging population structure, the total number of Alzheimer's disease patients in China is expected to exceed 1000 thousands by 2029 years, and the 10-year composite growth rate reaches 4%. This increase is particularly significant in the population of cities in china, and the 10-year compound growth rate will reach 6%.

The alzheimer disease still lacks an effective early diagnosis method and treatment means, and brings great economic burden to the family and the society of patients. Therefore, early diagnosis of Alzheimer's Disease (AD) is highly desirable. Pathological features of AD disease include plaques containing amyloid peptide beta and neurofibrillary tangles (NFTs) containing tau. For decades, the amyloid hypothesis has been dominant and targeted drugs developed based on amyloid strategies are mostly less than ideal from a clinical outcome perspective. Many researchers now begin reviewing tau protein and investigating whether it is an important biological driver of alzheimer's disease. Amyloid accumulates widely in the brain, sometimes even in people without any symptoms, while tau concentrates right where brain atrophy is most severe and where it helps explain differences in patients' symptoms, e.g. in areas related to language and memory. tau foci are more strongly associated with cognitive loss in AD patients, whereas phosphorylated tau proteins, neurofibrillary tangles and loss of synapses and neurons are strongly associated with memory impairment. Thus, pathological processes directed to tau protein may be more beneficial than treatment directed to a β in ameliorating clinical symptoms. At present, researchers have found about 84 phosphorylation sites on tau proteins, with phosphorylation at tau proteins 181, 199, 231, 396,404 being more common. Currently, the more studied phosphorylated Tau proteins include p-Tau¹⁸¹、p-Tau²³¹、p-Tau¹⁹⁹And p-Tau is³⁹⁶，p-Tau⁴⁰⁴There are few studies. Monoclonal antibodies and detection kits thereof targeting 396 and 404 site serine phosphorylated tau protein are not reported. In view of this, development of high quality anti-p-Tau^396,404The monoclonal antibody has great significance for early diagnosis and detection and targeted therapy of AD.

Disclosure of Invention

Against the current lack of specific targeting of p-Tau^396,404Antibody, high specificity and sensitivity p-Tau^396,404The detection kit and the specific targeting p-Tau^396,404The present invention has been made in view of the above problems, and an object of the present invention is to provide a secretory anti-p-Tau protein^396,404Hybridoma cell strain of monoclonal antibody and application thereof, and two novel secreted anti-p-Tau strains are provided^396,404Hybridoma cell strain of monoclonal antibody, and the hybridoma cell strain can be used for preparing the monoclonal antibody capable of specifically recognizing human p-Tau^396,404The monoclonal antibody of monomer and aggregate solves the problem of the lack of the prior monoclonal antibody which targets p-Tau simultaneously^396,404The difficulty of antibodies. The two monoclonal antibodies can be further applied to the preparation of the p-Tau with high specificity and sensitivity^396,404The development of a specific targeting p-Tau^396,404The immunological treatment of the (B) can slow down the disease process and fill up the current targeting p-Tau^396,404Blank immunological therapy of (1). The monoclonal antibody prepared by the invention has the characteristics of strong specificity, high titer and good stability, and can be applied to p-Tau^396,404In the detection reagents with different forms, the method can also be used for developing antibody medicines for treating senile dementia and related vaccines.

To achieve the above object, according to one aspect of the present invention, there is provided a method for secreting p-Tau^396,404The hybridoma cell strain of the monoclonal antibody is preserved in China center for type culture Collection with the preservation number of CCTCC NO: c2020219; wherein, p-Tau^396,404Representing Tau protein phosphorylated by serine at sites 396 and 404 of human.

According to another aspect of the present invention, there is provided a method for secreting p-Tau^396,404The hybridoma cell strain of the monoclonal antibody is preserved in China center for type culture Collection with the preservation number of CCTCC NO: c2020218; wherein, p-Tau^396,404Representing Tau protein phosphorylated by serine at sites 396 and 404 of human.

According to the inventionIn still another aspect of the present invention, the present invention provides the above-mentioned method for secreting human p-Tau^396,404Preparation of human p-Tau by using hybridoma cell strain of monoclonal antibody^396,404The application of the monoclonal antibody.

According to still another aspect of the present invention, the present invention provides the above-mentioned method for secreting human p-Tau^396,404Preparation of p-Tau by using hybridoma cell strain of monoclonal antibody^396,404Monomers, or p-Tau^396,404The use in an aggregate detection reagent; wherein, the p-Tau^396,404The monomer is a single protein formed by Tau protein phosphorylated by serine at sites 396 and 404 of human; the p-Tau^{396 ,404}The aggregate is protein with different molecular weights obtained by mutual folding of Tau protein phosphorylated by serine at position 396 and 404 of human.

According to still another aspect of the present invention, the present invention provides the above-mentioned method for secreting human p-Tau^396,404Preparation of p-Tau by using hybridoma cell strain of monoclonal antibody^396,404Monomers, or p-Tau^396,404The application in an aggregate detection kit; wherein, the p-Tau^396,404The monomer is a single protein formed by Tau protein phosphorylated by serine at sites 396 and 404 of human; the p-Tau^396,404The aggregate is protein with different molecular weights obtained by mutual folding of Tau protein phosphorylated by serine at position 396 and 404 of human.

According to a final aspect of the present invention, the present invention provides the above-mentioned method for secreting human p-Tau^396,404The application of the hybridoma cell strain of the monoclonal antibody in preparing the antibody medicament for treating the senile dementia.

As a further optimization of the invention, the antibody drug for treating senile dementia is particularly used for inhibiting and eliminating p-Tau protein formed by phosphorylation of serine at sites 396 and 404 of human beings^396,404Monomers and aggregates.

Through the technical scheme, compared with the prior art, the two secreted anti-human 396,404 site serine phosphorylated Tau proteins (p-Tau) in the invention^396,404) Hybridoma cell lines Hustaumab-3G5 and Hustaumab-4B1 of monoclonal antibody, and preparation of hybridoma cell lines using the sameObtaining specific recognition human p-Tau^396,404And monoclonal antibodies to aggregates thereof, and can further act against these human p-Tau^396,404The monoclonal antibody is applied subsequently, and the current simultaneous targeting of p-Tau is filled^396,404Antibody blank.

The 2 hybridoma cell strains can be used for preparing anti-human p-Tau through secretion^396,404Monoclonal antibody, and specific recognition p-Tau obtained therefrom^396,404The monoclonal antibody of the monomer and the aggregate can be further applied to the preparation of p-Tau^{396 ,404}Detection reagents (e.g. different forms of p-Tau)^396,404Detection kit, etc.), and can also be used for inhibiting p-Tau^396,404Monomers or p-Tau^396,404Folding into neurofibrillary tangle, preparing antibody medicine to eliminate neurofibrillary tangle, and treating senile dementia. The prepared antibody drug and related vaccine can inhibit and eliminate the formation of neurofibrillary tangles by phosphorylated tau protein and eliminate the neurofibrillary tangles.

The two monoclonal antibodies based on the invention can prepare p-Tau with high specificity and sensitivity^396,404The detection kit realizes early detection of AD diseases and fills the current p-Tau^396,404And detecting the blank of the kit. In addition, the specific targeting p-Tau can be further developed based on the developed two monoclonal antibodies^396,404The immunological treatment of the traditional Chinese medicine can slow down the disease process and fill up the current lack of the target p-Tau^396,404Blank immunological therapy of (1).

Drawings

In FIG. 1, A is synthetic human p-Tau^396,404Protein SDS-PAGE results; b in FIG. 1 is SDS-PAGE result after purification of monoclonal antibody 3G5,4B 1; c in FIG. 1 is 2 strains of anti-human p-Tau^396,404Detecting the ELISA activity of the monoclonal antibody; d in FIG. 1 is 3G5,4B1 for detecting artificially synthesized human p-Tau^396,404Performing Western blot result; FIG. 1 shows that E is 3G5,4B1 detects natural p-Tau secreted in brain of APP/PS1 transgenic mouse^396,404And (5) Western blot result.

FIG. 2 shows the results of the specific detection of mAb 3G5,4B 1.

In FIG. 3(a) Is 3G5 p-Tau^396,404Affinity results for the polypeptide; FIG. 3 shows that (B) is 4B1 vs. p-Tau^{396 ,404}Affinity results for the polypeptide.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

Example 1: anti-human p-Tau^396,404Preparation of specific monoclonal antibodies

For ease of reference hereinafter, the meanings of the major abbreviations are now explained as follows:

p-Tau^396,404monomers, i.e. p-Tau^396,404monomer: is a single protein formed by Tau protein phosphorylated by serine at the position 404 of 396 of human source.

p-Tau^396,404Aggregates, i.e. p-Tau^396,404oligomer: the human Tau protein, phosphorylated at position 404 serine, is obtained by folding over each other in human source 396.

1. Preparation of antigens

A Tau protein (p-Tau) phosphorylated by serine at the 396 and 404 sites of human origin and synthesized by a method known in the prior art^396,404) Dissolved in TBS solution to a final concentration of 1 mg/mL. As a result: SDS-PAGE results showed that the synthesized polypeptide was of good purity and the electrophoretic bands were uniform (A in FIG. 1).

2. Preparation of mouse monoclonal antibody

2.1 immunization of mice

Mixing 100 mu L of prepared antigen with equivalent volume of Freund's complete adjuvant, and performing primary immunization; carrying out second immunization 14 days after the first immunization, and mixing 100 mu L of prepared antigen with equivalent volume of Freund incomplete adjuvant for immunization; the third immunization was identical to the second, with 14 days intervals. After the third immunization, 100. mu.L of the prepared antigen was taken for tail vein booster immunization.

3.2 cell fusion screening of Positive clones

3 days after the booster immunization, splenocytes from the mice were collected and fused with SP2/0 cells, the fused cells were cultured in HAT medium containing 20% serum, and the HT medium was replaced after one week; performing first ELISA detection 12 days after cell fusion, and performing first subcloning on a positive hole; carrying out the first subcloning for 14 days, carrying out the second ELISA detection, and carrying out the second subcloning on the positive hole; subcloning for 14 days for the second time, and performing ELISA detection for the third time; if the ELISA detection results are positive after 3 times of subcloning, the monoclonal antibody becomes a monoclonal antibody, and the monoclonal antibody is frozen and stored in liquid nitrogen after the amplification culture.

3.3 preparation and purification of ascites

3 female Balb/c mice of 6-7 weeks old are injected with 0.5mL of sterile liquid paraffin into the abdominal cavity, and each mouse is injected with 10 percent of sterile liquid paraffin into the abdominal cavity after one week⁸(ii) individual hybridoma cells; after one week, collecting ascites; purification of ascites was performed using a protein a Sepharose column.

As a result: after cell fusion, 2 strains of anti-human p-Tau were successfully selected^396,404The monoclonal antibody cell strains are named as Hustaumab-3G5 and Hustaumab-4B 1. They are respectively preserved in China Center for Type Culture Collection (CCTCC), wherein the preservation number of the 3G5 strain is as follows: CCTCC No: c2020219, class name: the hybridoma cell strain Hustaumab-3G5 has the preservation time as follows: year 2020, 11, 10; the accession number of the 4B1 strain is: CCTCC No: c2020218, class name: the hybridoma cell strain Hustaumab-4B1 has the preservation time as follows: year 2020, 11, 10; the preservation units are as follows: china center for type culture Collection; the preservation address is: wuhan university Collection, Lodoku mountain, Wuchang, Wuhan, Hubei province. 2 monoclonal antibodies were successfully purified using protein A Sepharose, and the SDS-PAGE result is shown as B in FIG. 1.

3.4 identification of biological Properties of monoclonal antibodies

3.4.1 identification of monoclonal antibody Activity

3.4.1.1 ELISA detection

Coating antigen: antigen was diluted to 10. mu.g/mL with TBS, then 100. mu.L of antigen was added per well and coated overnight at 4 ℃;

sealing: after coating, discarding the liquid in the wells, washing with TBS-T for 3 times, adding 200 μ L of blocking solution into each well, and blocking at 37 deg.C for 2 h;

adding a primary antibody: after the sealing, the liquid in the wells was discarded, and after washing with TBS-T for 3 times, 100. mu.L of monoclonal antibody (1:1000) diluted with skim milk was added to each well and reacted at 37 ℃ for 2 hours;

adding enzyme-labeled secondary antibody: after the reaction is finished, liquid in the holes is discarded, TBS-T is used for washing for 3 times, 100 mu L of goat anti-mouse enzyme-labeled secondary antibody (1:6000) diluted by blocking solution is added into each hole, and the reaction is carried out for 1h at 37 ℃;

color development: after the reaction is finished, liquid in the hole is discarded, TBS-T is used for washing for 3 times, 100 mu L of TMB is added into each hole, and the reaction is carried out for 15min at 37 ℃;

and sixthly, terminating: add 50. mu.L of 2M H per well₂SO₄The reaction was terminated and the OD450nm absorbance was measured.

As a result: performing ELISA activity detection on the prepared monoclonal antibody ascites; the ELISA results showed (C in FIG. 1) that 2 monoclonal antibodies were able to react with p-Tau ascites^396,404And (4) reacting.

3.4.1.2 Western blot detection

(1) Loading: 12% SDS-PAGE was prepared and each well was loaded with 1. mu. g p-Tau^396,40430 μ g of APP/PS1 brain tissue slurry.

(2) Film transfer: after the gel was applied, the glass plate was removed and the gel was washed with ultrapure water. Then according to a sandwich structure, placing a spongy cushion-filter paper-NC membrane-gel-filter paper-spongy cushion-black sea-mat plate on the positive white backing plate.

(3) And (3) sealing: after the membrane transfer is finished, the NC membrane is carefully taken out by using clean tweezers, a certain volume of TBS-T is added at 80r/min, and the membrane is washed for 5 min. After washing, 10mL of blocking solution was added and blocked at 37 ℃ for 2 h. TBS-T was added and the wash was performed three times for 5min each.

(4) Adding a primary antibody: the blocking solution was discarded, and the monoclonal antibody ascites (1:1000) was added and incubated at 37 ℃ for 2 h.

(5) Adding a secondary antibody: after the primary antibody was recovered, the membrane was washed 5 times for 5min with TBS-T on a horizontal shaker at 80 r/min. Then, HRP-labeled goat anti-mouse IgG (1:5000) was added and incubated at 37 ℃ for 1 hour at 80 r/min.

(6) Color development: after recovery of the secondary antibody, the membrane was washed 5 times for 5min each with TBS-T on a horizontal shaker at 80 r/min. And then adding equal volume of ECL A solution and B solution, uniformly mixing, uniformly dripping on a membrane, and observing the result by color and luminescence under an imager.

As a result: western blot results show that (D in figure 1) 2 monoclonal antibody ascites can be combined with the synthesized p-Tau^396,404Reacting and specifically recognizing p-Tau in APP/PS1 brain tissue grinding fluid^396,404(E in FIG. 1).

3.4.2 identification of monoclonal antibody specificity

3.4.2.1 ELISA detection

Coating antigen: adding P-Tau^396,404pT231-Ala, pT231-PIP, pT231-DMP, npT231, ZIKV-NS1, BSA, skim milk were diluted to 10. mu.g/mL with TBS, and 100. mu.L of antigen was added to each well and coated overnight at 4 ℃;

sealing: after coating, discarding the liquid in the wells, washing with TBS-T for 3 times, adding 200 μ L of blocking solution into each well, and blocking at 37 deg.C for 2 h;

adding a primary antibody: after the sealing, the liquid in the wells was discarded, and after washing with TBS-T for 3 times, 100. mu.L of monoclonal antibody (1:1000) diluted with skim milk was added to each well and reacted at 37 ℃ for 2 hours;

adding enzyme-labeled secondary antibody: after the reaction is finished, liquid in the holes is discarded, TBS-T is used for washing for 3 times, 100 mu L of goat anti-mouse enzyme-labeled secondary antibody (1:6000) diluted by blocking solution is added into each hole, and the reaction is carried out for 1h at 37 ℃;

color development: after the reaction is finished, liquid in the hole is discarded, TBS-T is used for washing for 3 times, 100 mu L of TMB is added into each hole, and the reaction is carried out for 15min at 37 ℃;

as a result: the prepared monoclonal antibody ascites is only specific and p-Tau^396,404(ii) reactive, non-reactive with other control antigens; the results of ELISA-specific detection are shown in FIG. 2.

3.4.3 monoclonal antibody affinity detection

(1) Coating antigen: p-Tau is reacted with^396,404Diluting to 10 μ g/mL with coating solution, adding 100 μ L/well enzyme label plate at 4 deg.CCoating overnight.

(2) And (3) sealing: after coating, the wells were discarded, washed 3 times with TBS-T, and 200. mu.L of blocking solution was added to each well and blocked at 37 ℃ for 2 h.

(3) Addition of antiserum: after blocking was complete, the wells were drained, washed 3 times with TBS-T, 100. mu.L of antibody (10-20000ng) diluted in a blocking medium gradient was added to each well, and the 96-well plates were incubated at 37 ℃ for 2 h.

(4) Adding enzyme-labeled secondary antibody: after 2h, the wells were drained and washed 3 times with TBS-T, and 100. mu.L of HRP-labeled goat anti-mouse IgG secondary antibody (1:5000) was added to each well. The 96-well plate was left to react at 37 ℃ for 1 h.

(5) Color development: after 1h, the well was drained, washed 3 times with TBS-T, and 100. mu.L of a color developing solution for soluble TMB substrate was added to each well and reacted at 37 ℃ for 15 min.

(6) And (4) terminating: add 50. mu.L of 2M H per well₂SO₄The reaction was terminated and the OD450nm absorbance was measured.

The results are as follows: the Kd values of 3G5,4B1 were 2.65. + -. 0.4nM, 0.16. + -. 0.022nM, as shown in FIG. 3 (a) and FIG. 3 (B).

Where the invention has not been described in detail (e.g. reagents employed, processing operations performed, etc.), reference may be made directly to the relevant prior art.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (7)

1. A hybridoma cell line for secreting p-Tau ^396,404 monoclonal antibody, preserved in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: C2020219; wherein, p-Tau ^396,404 represents human 396,404 site Serine phosphorylated tau protein. 2. A hybridoma cell line for secreting p-Tau ^396,404 monoclonal antibody, preserved in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: C2020218; wherein, p-Tau ^396,404 represents human 396,404 site Serine phosphorylated tau protein. 3. Use of the hybridoma cell line for secreting human p-Tau ^396,404 monoclonal antibody according to claim 1 or 2 in preparing human p-Tau ^396,404 monoclonal antibody. 4. The application of the hybridoma cell line for secreting human p-Tau ^396,404 monoclonal antibody according to claim 1 or 2 in the preparation of p-Tau ^396,404 monomer or p-Tau ^396,404 aggregate detection reagent; wherein, The p-Tau ^396,404 monomer is a single protein formed by human 396,404 serine phosphorylated Tau proteins; the p-Tau ^396,404 aggregates are obtained by mutual folding of human 396,404 serine phosphorylated Tau proteins proteins of different molecular weights. 5. The application of the hybridoma cell line for secreting human p-Tau ^396,404 monoclonal antibody according to claim 1 or 2 in the preparation of p-Tau ^396,404 monomer or p-Tau ^396,404 aggregate detection kit; wherein , the p-Tau ^396,404 monomer is a single protein formed by human 396,404 serine phosphorylated Tau proteins; the p-Tau ^396,404 aggregates are human 396,404 Serine phosphorylated Tau proteins are obtained by mutual folding proteins of different molecular weights. 6. The application of the hybridoma cell line for secreting human p-Tau ^396,404 monoclonal antibody according to claim 1 or 2 in the preparation of an antibody drug for the treatment of Alzheimer's disease. 7. application as claimed in claim 6, it is characterised in that the described antibody drug for the treatment of senile dementia is specifically for suppressing and clearing human 396, 404 site serine phosphorylated Tau protein to form p-Tau ^396,404 monomer and Aggregates.

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