CN107118277B

CN107118277B - Monoclonal antibody

Info

Publication number: CN107118277B
Application number: CN201710565929.2A
Authority: CN
Inventors: 何杨; 杨剑峰; 高峰青
Original assignee: Suzhou Bosai Biological Medical Co ltd
Current assignee: Suzhou Bosai Biological Medical Co ltd
Priority date: 2017-07-12
Filing date: 2017-07-12
Publication date: 2020-12-04
Anticipated expiration: 2037-07-12
Also published as: CN107118277A

Abstract

The invention relates to the technical field of biology, in particular to a monoclonal antibody. The monoclonal antibody 3G3 binds to FVII and is dependent on the presence of calcium ions. The subtype of the 3G3 antibody is identified by adopting an antibody subtype identification test strip of Roche company, and the result proves that the subtype of the 3G3 monoclonal antibody is IgG1 and Kappa type. Recombinant human factor VII was successfully purified using Sepharose 4B conjugated with monoclonal antibody 3G3, the molecular weight of the protein of interest was shown to be 60kDa, consistent with expectations. The affinity constant of the 3G3 monoclonal antibody was determined. The result shows that the monoclonal antibody 3G3 provided by the invention has better affinity with the blood coagulation factor FVII.

Description

Monoclonal antibody

Technical Field

The invention relates to the technical field of biology, in particular to a monoclonal antibody.

Background

The coagulation cascade is the central link in the hemostatic process. When the coagulation pathway is deficient, bleeding time (bleeding time) is prolonged and the amount of bleeding is increased. Coagulation factors are protein molecules involved in the coagulation process, and in the physiological case, most of the coagulation factors exist in the form of inactive zymogens, which are sequentially activated by enzymolysis to be converted into active proteases when tissues are damaged, and finally, the zymogens are converted into thrombin, so that soluble fibrinogen is converted into insoluble fibrin clots.

Factor VII belongs to the group of vitamin K dependent coagulation factors, which are synthesized by the liver and mainly function to form a complex with tissue factor, thereby initiating the extrinsic coagulation pathway.

Hemophilia a and hemophilia B are hereditary hemorrhagic diseases linked to the X chromosome, characterized by deficiencies in coagulation factors viii (fviii) and ix (fix), respectively. Such patients are at risk of massive and spontaneous bleeding after trauma. World health organization statistics show that the incidence of hemophilia a is about 1/5000 newborn boys and the incidence of hemophilia B is about 1/20000 newborn boys. The traditional treatment for hemophilia is coagulation factor supplementation, i.e., intravenous infusion of plasma-derived or genetically recombinant FVIII or FIX, correction of coagulation factor abnormalities, and reduction of bleeding tendency. However, about 25% of patients with hemophilia a produce FVIII inhibitors (antibodies) and about 4% of patients with hemophilia B produce FIX inhibitors, rendering coagulation factor supplementation less effective or even ineffective. Many patients die from uncontrolled bleeding; the problem of severe joint damage due to bleeding is still quite severe; the hemostasis of patients with inhibitors during surgery is a particular problem. FVIIa can bypass the coagulation pathway dependent on FVIII and FIX through an alternative pathway, activating FX to produce FXa, promoting the conversion of a large amount of prothrombin to thrombin, forming a "thrombin burst". Recombinant FVIIa is used to treat spontaneous or surgical bleeding in patients with congenital hemophilia, a congenital factor VII deficiency, in the presence of FVIII or FIX inhibitors.

The human coagulation factor VII antibody can be used for affinity chromatography purification and detection of recombinant coagulation factor VII. However, in general, the antibody-mediated affinity chromatography requires acid elution, and one of the effects is that the sample buffer in the purification process is changed, which complicates the purification process; furthermore, low pH is highly likely to affect the activity of the sample. The calcium ion-dependent antibody can be separated from the original sample buffer solution by adding a calcium ion chelating agent EDTA, so that the adverse effects caused by acid elution are avoided. Therefore, there is an urgent need for the autonomous development of calcium ion-dependent anti-human factor VII monoclonal antibodies.

Disclosure of Invention

Accordingly, the present invention provides a monoclonal antibody. The monoclonal antibody can be specifically combined with coagulation factor VII, and has better affinity.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a monoclonal antibody which is dependent on calcium ions and specifically binds to coagulation factor VII, and the monoclonal antibody is prepared from a monoclonal antibody with a preservation number of CCTCC No: c2012158.

The preparation method of the monoclonal antibody comprises the following steps: fully mixing commercial recombinant human activated blood coagulation factor VII (NovoSeven) with equal volume of Freund complete adjuvant, emulsifying, injecting subcutaneously at multiple points on the back of Balb/c mice, mixing NovoSeven with equal volume of Freund incomplete adjuvant, emulsifying, injecting subcutaneously at multiple points on the back of mice for boosting immunization once after 4 weeks, injecting NovoSeven mice tail vein for immunization shock after 8 weeks, taking out spleen after 4 days, separating out spleen cells, mixing with mouse myeloma cells SP2/0 according to the cell number ratio of 10:1, adding fusion agent PEG1500 into cell precipitation for fusion after centrifugation, transferring into a 96-well plate, screening by using selective medium containing 1% HAT, half-changing liquid every 2 days, taking cell culture supernatant for ELISA detection and screening after 10-14 days.

For screening calcium-dependent anti-FVII antibodies, NovoSeven (1. mu.g/ml) coated ELISA plates were primed with TBS with/without 10mM EDTA, followed by cell culture supernatant, incubated at 37 ℃ for 1 hour, washed 5 times with TBST (0.1% tween-20), followed by horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody, incubated at 37 ℃ for 1 hour, washed 5 times with TBST (0.1% tween-20), followed by TMB substrate development, stopped with 1N sulfuric acid, and read OD450 on a microplate reader. A hybridoma cell, 3G3, which bound strongly positive in the EDTA-free supernatant but significantly inhibited antibody binding by the addition of 10mM EDTA was obtained (table 1, fig. 1).

The results indicate that binding of the 3G3 antibody to FVII is dependent on the presence of calcium ions; while the other hybridoma, 3D7, was still able to bind FVII in the presence of EDTA, indicating that 3D7 binds FVII independent of calcium ion. Further, 3G3 hybridoma cells were monocloned by limiting dilution method, and 3G3 cells were cultured in an expanded state and then frozen for seed preservation.

Purification of monoclonal antibodies: injecting Pristane0.5 ml/cell into abdominal cavity 5-7 days before injecting hybridoma, injecting 3G3 cell into abdominal cavity, collecting ascites of mouse by abdominal puncture 10-14 days later, standing at 37 deg.C for half an hour, and standing at 4 deg.C overnight. The following day, the blood clots were removed by centrifugation, 3 volumes of phosphate buffer were added to the ascites fluid, centrifuged, and the supernatant was purified by protein G column after filtration through a filter.

Identification of monoclonal antibodies: the subtype of the 3G3 antibody was identified using an antibody subtype identification test strip from Roche, and the results confirmed that the subtype of the 3G3 monoclonal antibody was IgG1, Kappa type (FIG. 2).

The invention also provides a hybridoma cell strain, which has a preservation number of CCTCC No: C2012158.

the invention also provides the application of the monoclonal antibody in preparing a tool for purifying the coagulation factor VII. In some embodiments of the invention, the purification is a nucleophilic chromatography purification.

The invention also provides a purification tool comprising the monoclonal antibody. In some embodiments of the invention, the purification is a nucleophilic chromatography purification.

The invention also provides application of the monoclonal antibody in preparing a detection tool for detecting the coagulation factor VII. In some embodiments of the invention, the detection means is a kit, chip or strip.

In addition, the invention also provides a detection tool which comprises the monoclonal antibody.

In some embodiments of the invention, the detection means is a kit, chip or strip.

The invention provides a hybridoma cell strain (with the preservation number of CCTCC No: C2012158) and a monoclonal antibody 3G3 produced by the hybridoma cell strain. The invention also provides application of the monoclonal antibody in preparing a tool for purifying the coagulation factor VII, application of the monoclonal antibody in preparing a detection tool for detecting the coagulation factor VII, and the detection tool containing the monoclonal antibody. The monoclonal antibody provided by the invention

Binding of 3G3 to FVII is dependent on the presence of calcium ions.

The subtype of the 3G3 antibody is identified by adopting an antibody subtype identification test strip of Roche company, and the result proves that the subtype of the 3G3 monoclonal antibody is IgG1 and Kappa type.

The monoclonal antibody provided by the invention is used for purifying the coagulation factor VII in a solid phase manner, and the result shows that the recombinant human coagulation factor VII can be successfully purified by using Sepharose 4B coupled with the monoclonal antibody 3G3, and the molecular weight of the target protein is shown as 60kDa and is consistent with the expectation.

The affinity constant of the 3G3 monoclonal antibody was determined using the Biacore X100 protein interaction system, coated on a biosensing chip with commercial recombinant coagulation factor FVII (NovoSeven). The results showed that the affinity constant of 3G3 antibody was 1.3X 10^-9And M. The monoclonal antibody 3G3 provided by the invention is proved to have very good affinity with the blood coagulation factor FVII.

When the monoclonal antibody provided by the invention is used for purifying the blood coagulation factor FVII, the calcium ion-dependent monoclonal antibody 3G3 can be added into the original sample buffer solution to separate the antigen and the antibody, so that the adverse effect caused by acid elution is avoided.

Biological preservation Instructions

Hybridoma cell line Anti-F7-3G 3: and (3) classification and naming: the hybridoma cell strain Anti-F7-3G3 is preserved in China Center for Type Culture Collection (CCTCC) at 11/8/2012, wherein the address of the CCTCC is in the university school of Wuhan in Lojia mountain of Wuchang city, Hubei, and the preservation number is CCTCC No. C2012158.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows Elisa screening of calcium ion-dependent anti-human FVII antibodies;

FIG. 2 shows the subtype identification of the 3G3 antibody;

FIG. 3 shows the identification of affinity chromatographically purified recombinant factor VII by Coomassie Brilliant blue staining following SDS-PAGE electrophoresis; NR is non-reducing condition; r: reducing conditions;

FIG. 4 shows the affinity constants of the 3G3 monoclonal antibody; wherein line 1 represents 150 nM; line 2 shows 75 nM; line 3 shows 37.5 nM; line 4 shows 18.75 nM; line 5 shows 9.375 nM.

Detailed Description

The invention discloses a monoclonal antibody, which can be realized by appropriately improving process parameters by persons skilled in the art with reference to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The raw materials and reagents used in various embodiments of the monoclonal antibodies provided herein are commercially available.

The invention is further illustrated by the following examples:

example 1 animal immunization and hybridoma screening

Fully mixing and emulsifying 100 mu g of commercial recombinant human activated blood coagulation factor VII (NovoSeven) with equal volume of Freund complete adjuvant, then injecting the mixture into Balb/c mice at multiple points under the back skin, after 4 weeks, taking 100 mu g of NovoSeven and equal volume of Freund incomplete adjuvant, after emulsification, injecting the mixture into multiple points under the back skin, boosting immunity once, after 8 weeks, taking 100 mu g of NovoSeven mice at tail vein, injecting immune shock, after 4 days, taking out spleen, separating out spleen cells, mixing the spleen cells with mouse myeloma cells SP2/0 according to the cell number ratio of 10:1, after centrifugation, adding 1ml of fusion agent PEG1500 into cell precipitates for fusion, transferring the mixture into a pore plate 96, screening the mixture by using selective medium containing 1% HAT, half-changing the solution every 2 days, and taking cell culture supernatant for ELISA detection and screening 10-14 days. To screen for calcium-dependent anti-FVII antibodies, NovoSeven (1. mu.g/ml) coated ELISA plates were primed with 50. mu.l TBS with/without 10mM EDTA, then with 50. mu.l cell culture supernatant, incubated at 37 ℃ for 1 hour, washed 5 times with TBST (0.1% tween-20), followed by horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody, incubated at 37 ℃ for 1 hour, washed 5 times with TBST (0.1% tween-20), followed by TMB substrate for color development, stopped with 1N sulfuric acid, and read OD450 with a microplate reader. A hybridoma cell 3G3 (table 1, fig. 1) which bound strongly positive in the EDTA-free supernatant and significantly inhibited antibody binding by the addition of 10mM EDTA was obtained, showing that 3G3 antibody binds to FVII dependent calcium ions; while the other hybridoma, 3D7, was still able to bind FVII in the presence of EDTA, indicating that 3D7 binds FVII independent of calcium ion. Further, 3G3 hybridoma cells were monocloned by limiting dilution method, and 3G3 cells were cultured in an expanded state and then frozen for seed preservation.

TABLE 1 EDTA inhibition of binding of 3G3 antibody to factor VII

Group of	OD450
		3G3	2.144
3D7	2.238
		3G3+EDTA	0.173
3D7+EDTA	2.231

Example 2 purification of anti-human factor VII monoclonal antibodies

Injecting Pristane0.5 ml/piece into abdominal cavity 5-7 days before injection of hybridoma, and collecting 5 × 10⁶3G3 cells were injected into abdominal cavity, ascites from mice were collected by abdominal puncture 10-14 days later, left for half an hour at 37 ℃ and left overnight at 4 ℃. The following day, the blood clots were removed by centrifugation, 3 volumes of phosphate buffer were added to the ascites fluid, centrifuged at 13000rpm for 30 minutes, and the supernatant was filtered through a 0.4 μm filter and purified by a protein G column.

Example 3 antibody subtype identification

The subtype of the 3G3 antibody was identified using an antibody subtype identification test strip from Roche, and the results confirmed that the subtype of the 3G3 monoclonal antibody was IgG1, Kappa type (FIG. 2).

Example 4 immobilization of antibodies for purification of factor VII

Preparation of 3G3 monoclonal antibody-coupled Sepharose 4B gel: weighing 0.268G of cyanogen bromide activated Sepharose 4B dry gel, adding 10ml of precooled 1mM hydrochloric acid to swell for 30 minutes, centrifuging, adding 15ml of precooled 1mM hydrochloric acid to wash precipitates, centrifuging, washing the gel once by using a coupling buffer solution, adding 6mg of 3G3 monoclonal antibody, gently mixing at room temperature and uniformly and reacting for 2 hours, centrifuging, washing free 3G3 antibody by using the coupling buffer solution, then blocking by using 0.1MTris-Hcl (pH8.0), and reacting for 1 hour at room temperature. The column was packed by washing with a low pH washing buffer (0.1M sodium acetate, 0.5M sodium chloride, pH3.5) and a high pH washing buffer (0.1M sodium bicarbonate, 0.5M sodium chloride, pH8.3) in this order, and repeatedly washing 4 times. Storing at 4 ℃.

Transfection of CHO cells of recombinant factor VII, serum-free medium CD-CHO (Invitrogen) containing 10. mu.g/ml vitamin K1 was used 24 hours after transfection, after continuous culture for 7 days, the supernatant was collected, high-speed centrifugation was performed to remove insoluble particles, 0.22 μm filtration was performed, the obtained product was added to a 3G3-Sepharose 4B gel column, unbound impurity proteins were removed by washing, the target protein was eluted with TBS buffer containing 10mM EDTA, and the eluted product was collected, 2ml was collected for each tube, and 5 tubes were collected in total. Adding the eluted product into an isovolumetric reduced type sample loading buffer solution, boiling for 5 minutes, cooling, loading, performing 10% SDS-PAGE electrophoresis, staining with Coomassie brilliant blue, decoloring, and observing the purification effect of the blood coagulation factor. As a result: it can be seen that recombinant human factor VII was successfully purified using Sepharose 4B conjugated with 3G3 monoclonal antibody, and the molecular weight of the protein of interest was shown to be 60kDa, in agreement with the expectation (FIG. 3).

Example 5 monoclonal antibody 3G3 affinity assay for calcium ion dependent FVII

The affinity constant of the 3G3 monoclonal antibody was determined using the Biacore X100 protein interaction system, coated on a biosensing chip with commercial recombinant coagulation factor FVII (NovoSeven).

Experimental materials:

CM5 chip: GE, cat No. BR 100012;

Running buffer：10mM HEPES,150mMNaCl,5mMEDTANa2,0.05％P20,pH7.4；

amino coupling kit: GE, cargo number: 2060499, respectively;

sample preparation: 3G3 monoclonal antibody and 1 each of recombinant human factor VII (NovoSeven).

The experimental method comprises the following steps:

1. coupling of

The coupling conditions were optimized according to isoelectric point and protocol of BiacoreX100control soft, and sodium acetate was selected as the coupling dilution buffer for slope optimization. NovoSeven samples were diluted to 25. mu.g/ml with this buffer and coupled to CM5 chips. The preset coupling level was 1500 RU.

2. KD test

The 3G3 monoclonal antibody was diluted with Running buffer at pH7.4 to a range of concentrations of 0nM, 9.375nM, 18.75nM, 37.5nM, 75nM, 150 nM. The binding time was set at 180s, the dissociation time was set at 15min, and 50mM Gly-HCl (pH1.7) was used as the regeneration buffer. The on-board test was performed according to the protocol of BiacoreX100control soft.

3. Data processing

Analysis was performed using biacore X100 self-contained analysis software. The data was interpreted as follows:

ka: binding constant, reaction and binding speed, and the larger the value, the faster the binding.

Kd: dissociation constant, reaction dissociation speed, smaller value and slower dissociation.

KD: KD ═ KD/Ka, affinity constant, how strongly the reactive antigen antibody binds, the smaller the number, the stronger the affinity.

The experimental results are as follows:

1. coupling of

Finally selecting sodium acetate with pH4.5 as a coupling dilution buffer according to the isoelectric point characteristics and the combination slope of the antigen. The coupling level was 1500 RU.

2. Affinity results: see fig. 4. The results showed that the affinity constant of 3G3 antibody was 1.3X 10^-9M。

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.

Claims

1. A monoclonal antibody which specifically binds to coagulation factor VII and is calcium ion dependent, and is prepared from a monoclonal antibody with a preservation number of CCTCC No: c2012158; the monoclonal antibody has an affinity constant of 1.3X 10^-9M。

2. A hybridoma cell strain with a preservation number of CCTCC No: c2012158, which produces monoclonal antibodies with an affinity constant of 1.3X 10^-9M; the monoclonal antibodies are calcium ion dependent.

3. Use of a monoclonal antibody according to claim 1 for the preparation of a tool for the purification of factor VII.

4. Use according to claim 3, wherein the purification is affinity chromatography purification.

5. A purification tool comprising the monoclonal antibody of claim 1.

6. The purification tool of claim 5, wherein the purification is affinity chromatography purification.

7. Use of a monoclonal antibody according to claim 1 for the preparation of a detection means for the detection of factor VII.

8. The use of claim 7, wherein the detection means is a kit, chip or strip.

9. A detection kit comprising the monoclonal antibody of claim 1.

10. The test kit of claim 9, wherein the kit is a kit, chip or strip.