Hybridoma cell strain 9C1, PLGF-1 monoclonal antibody, preparation method therefor and application thereof

Abstract

Provided are a hybridoma cell strain 9C1, a PLGF-1 monoclonal antibody, a preparation method therefor and an application thereof. The PLGF-1 monoclonal antibody is secreted by the monoclonal antibody

Description

18 07 25 FIELD OF THE PRESENT DISCLOSURE

[0001] The present invention relates to the field of bioengineering, in particular to a hybridoma cell strain 9C1, a PLGF-1 monoclonal antibody and application thereof. BACKGROUND

[0002] During remodeling of uterine spiral artery, syncytiotrophoblast cells secrete placental growth factors (PLGF-1), which promotes angiogenesis and improves blood perfusion. In normal pregnancy, the serum level of PLGF-1 in healthy pregnant women is low during pregnancy week 5-15, and the expression of PLGF-1 increases rapidly within week 15-26, then reaches a peak at week 28-30. Afterwards, with the gradual maturing and aging of the placenta, the PLGF-1 level decreases significantly. The significant reduction of the PLGF-1 level in the presence of placentation defects will result in placental dysplasia, causing placental insufficiency, so it is difficult to provide sufficient nutrients and oxygen to the fetus, which may lead to a series of diseases, such as miscarriage, gestational hypertension, preeclampsia (PE), fetal growth restriction and premature birth, etc., and in severe cases, also may lead to related complications, such as stillbirth, eclampsia, HELLP syndromes and placental abruption. Compared with the traditional diagnosis methods, the decrease of PLGF-1 concentration occurs 9-11 weeks earlier than the arising of hypertension and proteinuria, and is mainly demonstrated in the concentration within five weeks before the disease onset.

[0003] At present, the methods for PLGF-1 detection include enzyme-linked immunosorbent assay and microfluidic fluorescence immunoassay, while high requirements for the relevant detection antibodies have been put forward due to the low concentration of PLGF-1 in serum. Therefore, it is urgent to develop an anti-human placental growth factor-1 antibody that can be detected rapidly and sensitively, and is promising to be used in clinical diagnosis. 18 07 25 SUMMARY

[0004] An object of the present invention is to provide a hybridoma cell strain 9C1, which is collected in China Center for Type Culture Collection (CCTCC) (Address: Wuhan University in Wuhan, China) on September 11,2019 with a collection number of CCTCC NO: C2019216.

[0005] A further object of the present invention is to provide a monoclonal antibody of human placental growth factor-1, i.e., a PLGF-1 monoclonal antibody, secreted by the hybridoma cell strain 9C1 (collection number, CCTCC NO: C2019216), wherein the heavy chain has the amino acid sequence shown in SEQ ID NO.1 of the sequence listing, and the light chain has the amino acid sequence shown in SEQ ID NO.2. The subtype of the monoclonal antibody is lgG1, k.

[0006] Another object of the present invention is to provide a use of the hybridoma cell strain 9C1 in the preparation of the PLGF-1 monoclonal antibody of the invention.

[0007] Still another object of the present invention is to provide a detection reagent comprising the PLGF-1 monoclonal antibody of the invention.

[0008] Also described herein is a preparation method for a PLGF-1 monoclonal antibody, which comprises the following steps:

[0009] (1) antigen preparation: obtaining a PLGF-1 recombinant protein by expressing in Escherichia coli;

[0010] (2) mice immunization: selecting 6-week-old BALB / C mice, and immunizing the selected mice by using the PLGF-1 recombinant protein obtained in Step (1) as an antigen;

[0011] (3) culture of mouse myeloma cells SP2 / 0: culturing myeloma cells SP2 / 0 from at least one of the mice in Step (2), and maintaining the cells in good growth status;

[0012] (4) cell fusion: inducing cell fusion of splenic lymphocytes of mice in Step (2) and mouse myeloma cells SP2 / 0 in Step (3) by a cell fusion method using polyethylene glycol(PEG), and culturing the fused cells by using peritoneal macrophages of the BALB / C mice as feeder cells; 18 07 25

[0013] (5) screening of hybridoma cells: culturing the culture obtained in Step (4), collecting a cell culture supernate for antibody identification, and screening for positive hybridoma cells;

[0014] (6) cloning of hybridoma cells: cloning the positive hybridoma cells selected in Step (5) by limiting dilution method, and repeating the cloning several times until the positive rate of the wells for the hybridoma cells reaches 100%;

[0015] (7) induction of ascites: injecting pristane to at least one of the BALB / C mice obtained in Step (2) intraperitoneally, inoculating with the positive hybridoma cells obtained in Step (6), and collecting ascites for centrifugation; and

[0016] (8) purification of monoclonal antibody: purifying the monoclonal antibody in the ascites collected in Step (7).

[0017] The method may specifically include:

[0018] (1) antigen preparation: obtaining the PLGF-1 recombinant protein by expressing in Escherichia coli, wherein the PLGF-1 recombinant protein contains 132 amino acids, and has a GenBank registration number of P49763.2; and optionally, the preparation of this protein is completed by Shanghai Puxin Biotechnology Co., Ltd.;

[0019] (2) mice immunization: selecting 6-week-old BALB / C mice, and immunizing the mice by using the PLGF-1 recombinant protein obtained in Step (1) as an antigen, wherein each mouse is immunized through intramuscular injection on the inner thigh with 100 pg of the antigen peptide mixed with an adjuvant, and immunized again after 21 days, then used for cell fusion 3 days later from being booster-immunized for the third time;

[0020] (3) culture of mouse myeloma cells SP2 / 0: culturing mouse myeloma cells SP2 / 0, and maintaining the cells in good growth status for hybridoma cell fusion;

[0021] (4) cell fusion: using peritoneal macrophages of the BALB / C mice as feeder cells, seeding the BALB / C mice peritoneal macrophages into a 96-well culture plate and culturing for one day with a HAT medium containing 20% calf serum on the day before the cell fusion wherein a polyethylene glycol(PEG) cell fusion method is used; killing the mice prepared in Step (2) to obtain splenic lymphocytes; collecting the mouse myeloma cells SP2 / 0 in Step (3); mixing and centrifuging the above two cells, and mediating cell 18 07 25 fusion with PEG; diluting the fused cells with a cell culture medium, and seeding them into the culture plate of the feeder cells, then culturing at 37°C in a 5% CO2 incubator;

[0022] (5) screening of hybridoma cells: culturing the above-obtained culture, suctioning a supernate of the cell culture for antibody identification in case of the cell colonies growing to a suitable size, and screening for the positive clones;

[0023] (6) cloning of hybridoma cells: cloning the positive hybridoma cells by a limiting dilution method, wherein the cells of a certain dilution density are seeded into a 96-well cell culture plate such that only one cell grows per well; taking a culture supernate from each well that has formed cell colonies for enzyme-linked immunosorbent assay (ELISA), and screening and identifying the positive clones; repeating the cloning several times until the positive rate of the wells for the hybridoma cells reaches 100%;

[0024] (7) induction of ascites: injecting each of the BALB / C mice intraperitoneally with 0.5 ml of pristine one week before inoculation of the hybridoma cells; then inoculating each of the mice with 5x106 well-grown positive hybridoma cells, collecting and centrifuging the ascites 10 days later, and determining the antibody potency; and

[0025] (8) purification of monoclonal antibody: purifying the monoclonal antibody in the ascites by using a Protein G affinity purification method.

[0026] Optionally, after Step (8), the method further includes: (9) subtype identification of the monoclonal antibody.

[0027] Further, an example of the step (9) may be: diluting the purified monoclonal antibody appropriately and then assaying by a Serotec's immunoglobulin typing kit for mouse monoclonal antibody.

[0028] Furthermore, the operation in Step (9) is performed by strictly following the kit instructions, and the monoclonal antibody secreted by the hybridoma cell strain 9C1 belongs to the subtype lgG1, k, and is highly specific and sensitive to the PLGF-1 antigen.

[0029] The present invention has the following beneficial effects: the hybridoma cell 9C1 of the present invention secretes the PLGF-1 monoclonal antibody of the present invention with high efficiency and stability, and grows well after recovery from liquid nitrogen cryopreservation without decline in the antibody secretion; and the PLGF-1 18 07 25 monoclonal antibody of the present invention is highly specific and sensitive to the PLGF-1 antigen, and can be used in preparation of detection reagents for qualitative and quantitative detections in clinic and laboratory, with the detection results providing a laboratory diagnostic basis for clinical practice, so they both have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 shows an electrophoresis pattern of recombinant human PLGF-1 (rHuPLGF-1), where 1 indicates a protein Marker, and 2 and 3 indicate rHuPLGF-1 proteins.

[0031] FIG. 2 shows the sensitivity of a PLGF-1 monoclonal antibody detected by an indirect ELISA method.

[0032] FIG. 3 shows the accuracy of a PLGF-1 monoclonal antibody detected by a chemiluminescence method. THE EMBODIMENTS

[0033] The exemplary embodiments will be described in detail herein, and examples thereof are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings indicate the same or similar elements. The implementations described in the following exemplary embodiments are not all the implementations consistent with the present invention. On the contrary, they are simply examples of methods consistent with some aspects of the present invention, as specified in the appended claims.

[0034] The present invention will be further explained below in conjunction with the embodiments and the accompanying drawings. The following examples are only used to illustrate the present invention, rather than limit the implementation scope of the present invention.

[0035] Example 1

[0036] Preparation of a PLGF-1 monoclonal antibody includes the following steps:

[0037] (1) antigen preparation: a PLGF-1 recombinant protein comprising 132 amino 18 07 25 acids (GenBank registration number: P49763.2), which is obtained by expressing in Escherichia coli., has a molecular weight of about 15 kDa as shown in the result of SDS-PAGE (FIG. 1), and its preparation has been completed by Shanghai Puxin Biotechnology Co., Ltd.;

[0038] (2) mice immunization: 6-week-old BALB / C mice are selected and immunized by using the PLGF-1 recombinant protein obtained in Step (1) as an antigen, wherein each mouse is immunized through intramuscular injection on the inner thigh with 100 pg of the antigen peptide mixed with an adjuvant, and immunized again after 21 days, then used for cell fusion 3 days later from being booster-immunized for the third time;

[0039] (3) culture of mouse myeloma cells SP2 / 0: mouse myeloma cells SP2 / 0 are cultured and maintained in good growth status for hybridoma cell fusion;

[0040] (4) cell fusion: peritoneal macrophages of the BALB / C mice, which are used as feeder cells, are seeded into a 96-well culture plate and cultured for one day with a HAT medium containing 20% calf serum on the day before the cell fusion wherein a PEG cell fusion method is used; the mice prepared in Step (2) are killed to obtain splenic lymphocytes; the mouse myeloma cells SP2 / 0 in Step (3) are collected; the above two cells are mixed and centrifuged, then induced to fuse by PEG; the fused cells are diluted with a cell culture medium, seeded into the culture plate of the feeder cells, and then cultured at 37°C in a 5% CO2 incubator;

[0041] (5) screening of hybridoma cells: the above-obtained culture are cultured, and a supernate of the cell culture is suctioned out for antibody identification and screening of positive clones when the cell colonies grow to a suitable size;

[0042] (6) cloning of hybridoma cells: the positive hybridoma cells are cloned by a limiting dilution method, wherein the cells of a certain dilution density are seeded into a 96-well cell culture plate such that only one cell grows per well; a culture supernate is taken from each well that has formed cell colonies for enzyme-linked immunosorbent assay (ELISA), so as to screen and identify the positive clones; the cloning is repeated several times until the positive rate of the wells for the hybridoma cells reaches 100%;

[0043] (7) induction of ascites: each of the BALB / C mice is injected intraperitoneally with 0.5 ml of pristine one week before inoculation of the hybridoma cells; then each of 18 07 25 the mice is inoculated with 5* 106 well-grown positive hybridoma cells; the ascites is collected and centrifuged 10 days later, and the antibody potency is determined;

[0044] (8) purification of monoclonal antibody: the monoclonal antibody in the ascites is purified by using a Protein G affinity purification method; and

[0045] (9) subtype identification of monoclonal antibody: the purified monoclonal antibody is diluted appropriately and then assayed by a Serotec's immunoglobulin typing kit for mouse monoclonal antibody strictly following the kit instructions, and the monoclonal antibody secreted by the hybridoma cell strain 9C1 belongs to the subtype IgG 1, k, and is highly specific and sensitive to the PLGF-1 antigen.

[0046] The hybridoma cell strain 9C1 has been collected in China Center for Type Culture Collection (CCTCC) (Address: Wuhan University in Wuhan, China) on September 11, 2019 with the collection number of CCTCC NO: C2019216.

[0047] Example 2

[0048] In this example, the specificity and sensitivity of the PLGF-1 monoclonal antibody is detected by an indirect ELISA method including the following steps:

[0049] (1) performing gradient dilution of a PIGF-1 recombinant protein (by double dilution method from 1000 ng / ml) with a carbonate buffer solution (CBS), then adding the diluted protein solutions to a 96-well ELISA plate (100 pl / well) and coating overnight at 4°C;

[0050] (2) washing with PBST (phosphate buffer solution containing Tween-20) three times, 5 min for each time;

[0051] (3) adding a blocking solution (PBS containing 5% bovine serum albumin) and blocking at 37°C for 1 h, then washing with PBST;

[0052] (4) adding the antibody, which is gradient-diluted (by double dilution method from 500 ng / ml) with PBS (phosphate buffer solution), 100 pl per well; using PBS as a negative control and incubating at 37°C for 1 h , then washing with PBST;

[0053] (5) adding an HRP-labeled sheep anti-murine secondary antibody, 100 pl per well, and incubating at 37°C for 45 min, then washing with PBST; and

[0054] (6) adding a TMB chromogenic solution and incubating at 37°C for 15 min, then ending with a sulfuric acid solution(2M) and reading with a microplate reader (450 nm).

[0055] The results are shown in FIG. 2 and Table 1. In this experiment, when the antibody concentration is 500 ng / ml, the recombinant protein at a concentration of 32.2 ng / ml has an OD value that is still shown as a clear positive signal. Tested for a single concentration of recombinant protein, 9C1 shows significant sensitivity at a concentration of >250 ng / ml. 18 07 25

[0056] Table 1 OD value Antibody n. concentration (ng / mL) RecombinantX protein concentration (ng / mIK 500 250 125 62.5 31.25 0 1000 2.75 2.184 1.43 0.974 0.657 0.125 500 2.49 1.779 1.125 0.882 0.575 0.12 250 1.894 1.27 1.04 0.684 0.455 0.109 125 1.178 1.021 0.627 0.445 0.326 0.1 62.5 0.713 0.597 0.34 0.251 0.193 0.097 31.2 0.376 0.31 0.198 0.147 0.13 0.087 15.6 0.269 0.187 0.149 0.122 0.116 0.099 0 0.071 0.073 0.065 0.066 0.07 0.056

[0057] The accuracy of the PLGF-1 monoclonal antibody is detected by a chemiluminescence method including the following steps:

[0058] (1) the 9C1 monoclonal antibody is labeled with biotin, and a PLGF commercial antibody (Human PIGF Antibody (R&D systerms, catalog number AF-264-PB)) is labeled with acridinium ester;

[0059] (2) magnetic beads are diluted with a diluent to 1 mg / ml;

[0060] (3) the labeled antibodies are diluted with an antibody diluent to 500 ng / ml respectively;

[0061] (4) the antibodies and the magnetic beads are loaded into a kit separately;

[0062] (5) maternal blood samples are assigned with values using a Roche human placental growth factor (PIGF) electrochemiluminescence kit;

[0063] (6) the kit is inserted into a fully automatic chemiluminescence instrument, and the blood samples are inserted into the sample slot; and

[0064] (7) a one-step method is set for sample detection.

[0065] The results are shown in Table 2 and FIG. 3, and the resulting linear equation is y=1150.1x-2509.1, R2=0.9906.

[0066] Table 2 18 07 25 Sample Nos. pigf concentration (pg / mi) Detection results 6 1197 1336969 5 902.4 1112826 4 669.5 743862 3 500.7 604021 2 392.8 419783 1 368 383987 0 0 16169

[0067] In making the present invention, an indirect ELISA method included a step in which the hybridoma cell culture supernate obtained after the cell fusion reacts with a PLGF-1 antigen in a 96-well ELISA plate, a horseradish peroxidase-labeled sheep anti-murine antibody is added, and a light absorption value of each well is measured at a working wavelength of 450 nm, thus a hybridoma cell strain that secrets the anti-PLGF-1 monoclonal antibody is obtained by such screening and labeled as 9C1. After an immunological detection and screening method, the hybridoma cells that secret the anti-PLGF-1 monoclonal antibody are screened out, wherein the secreted antibody is the anti-PLGF-1 monoclonal antibody.

[0068] According to the present invention, the monoclonal antibody prepared for PLGF-1 provides higher accuracy, specificity and sensitivity for the detection of PLGF-1 protein, and the application of this antibody lays a foundation for functional research of PLGF-1 and the development of PLGF-1-based diagnostic reagents.

[0069] The present application has been described in detail in combination with specific embodiments and exemplary examples, but these descriptions cannot be understood as limitations on the present application. Those skilled in the art will LO CXI 18 07 understand that the protection scope of the present application shall comply with the appended claims. 18 07 25

Claims

1. A hybridoma cell strain 9C1, which is collected in China Center for Type Culture Collection and has the collection number of CCTCC NO: C2019216, wherein a PLGF-1 monoclonal antibody is produced from the hybridoma cell strain 9C1 and wherein the heavy chain of the monoclonal antibody has the amino acid sequence as shown in SEQ ID NO.1, and the light chain of the monoclonal antibody has the amino acid sequence as shown in SEQ ID NO.2.

2. A PLGF-1 monoclonal antibody, which is produced from the hybridoma cell strain 9C1 according to claim 1 that has the collection number of CCTCC NO: C2019216, wherein the heavy chain of the monoclonal antibody has the amino acid sequence as shown in SEQ ID NO: 1 and the light chain of the monoclonal antibody has the amino acid sequence as shown in SEQ ID NO: 2.

3. Use of the hybridoma cell strain 9C1 according to claim 1 in the preparation of a PLGF-1 monoclonal antibody, wherein the heavy chain of the monoclonal antibody has the amino acid sequence as shown in SEQ ID NO: 1 and the light chain of the monoclonal antibody has the amino acid sequence as shown in SEQ ID NO: 2.

4. Use of the PLGF-1 monoclonal antibody according to claim 2 in the preparation of an anti-human PLGF-1 detection reagent, wherein the anti-human PLGF-1 detection reagent comprises the PLGF-1 monoclonal antibody according to claim 2.

5. A detection reagent, comprising the PLGF-1 monoclonal antibody according to claim 2.

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