CN112279917B

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Info

Publication number: CN112279917B
Application number: CN202010484563.8A
Authority: CN
Inventors: 孟逊
Original assignee: Puzhong Discovery Pharmaceutical Technology Shanghai Co ltd
Current assignee: Puzhong Discovery Pharmaceutical Technology Shanghai Co ltd
Priority date: 2020-06-01
Filing date: 2020-06-01
Publication date: 2024-01-05
Anticipated expiration: 2040-06-01
Also published as: CN112279917A

Abstract

The invention relates to a monoclonal antibody, in particular to a monoclonal antibody of a mouse anti-cell surface glycoprotein CD138, which can be applied to capturing tumor cells, and comprises the following steps: s1, protein QC; s2, animal immunization; s3, serum detection and screening; s4, fusion and screening; s5, subcloning and screening; s6, preparing an antibody supernatant; s7, antibody verification. The monoclonal antibody of the mouse anti-cell surface glycoprotein CD138, which can be applied to tumor cell capture, has the characteristics of high affinity, high specificity, multiple application scenarios and the like, and can be applied to living cell enrichment.

Description

Monoclonal antibody of mouse anti-cell surface glycoprotein CD138 capable of being applied to tumor cell capture

Technical Field

The invention relates to a monoclonal antibody, in particular to a monoclonal antibody of a mouse anti-cell surface glycoprotein CD138, which can be applied to tumor cell capture.

Background

CD138 is a multimeric proteoglycan, belongs to a member of the adhesion molecule integrin transmembrane-binding proteoglycan family, is mainly distributed on the surfaces of endothelial cells, epithelial cells, fibroblasts and keratinocytes, can be combined with a series of ligands such as cell adhesion molecules, matrix molecules, growth factors, enzyme inhibitors and the like through a heparin sulfate side chain on the molecular surfaces of the members, and can regulate the interaction between cells and microenvironment in a co-receptor mode, thus participating in the regulation of a series of physiological processes such as tissue organ differentiation and development, angiogenesis, tissue regeneration and the like, and being related to proliferation, adhesion and migration of the cells. The soluble CD138 molecule can be used as an antagonist or an agonist of basic fibroblast growth factor in tissue injury repair, participates in the injury repair process, and has the effect of inhibiting tumor cell proliferation. Along with the deep research, the expression of CD138 molecules is found to have important roles in the growth, metastasis and other processes of malignant tumor cells in the air, can be used as an index for judging tumor prognosis, has important significance for knowing clinical diagnosis and immunotherapy, has few antibody products taking CD138 as a target carrier in the market at present, and has no data to indicate that the CD138 molecules can be used for enriching living tumor cells. While there is a lack of effective antibody tools for this research area.

Disclosure of Invention

The invention aims to provide a tool for researching targeted proteins, and the deeper research on protein expression changes and protein network patterns of different cancer patients and different stages of cancers is realized.

The technical scheme adopted by the invention is as follows: a monoclonal antibody of a mouse anti-cell surface glycoprotein CD138 applicable to tumor cell capture, which comprises the following steps:

s1, protein QC: purchasing commercial proteins;

s2, animal immunization: selecting 6 Balb/c mice for antigen immunization, monitoring serum titers of the Balb/c mice to determine optimal immunized mice, performing boosting for 3 to 4 times after primary immunization, detecting titer by taking serum of the mice after boosting, screening out qualified mice with one impact for fusion, and continuing boosting for one to two times until the titer is highest for fusion;

s3, serum detection and screening: immunized mice are subjected to orbital blood collection, and the serum titer is detected by ELISA, so that the qualified serum titer is determined;

s4, fusion and screening: fusing the whole spleen and 1/2 lymph nodes with a myeloma SP2/0 cell line, paving fused cells on 4 384-well plates for culture, collecting supernatants of all the wells, screening immune proteins by ELISA, performing microscopic examination on positive Kong Zhuaidao-well plates with cells for continuous culture, collecting supernatants of all the wells after a few days of growth, detecting reaction with soluble fragment detection antigen by ELISA, further detecting protein binding of different dilutions of positive wells, and performing affinity sequencing on 20 parent clones with highest affinity for each fusion immunogen into subclones;

s5, subcloning and screening: subcloning by limiting dilution method and ELISA screening to obtain monoclonal hybridoma cells, spreading 96-well plates on the cells, culturing to cover the bottom of about 1/6, ELISA detecting the reaction of each well supernatant on immune antigen, taking two wells with high OD value and good cell state to enter the next round of subcloning, repeating the steps until the positive rate of cell strains in the wells is 100%, obtaining monoclonal cell strains at this time, immediately expanding and culturing all positive cells after the last round of subcloning, freezing one part for later use, and preparing the other part of supernatant or ascites;

s6, preparing antibody supernatant: injecting the finally obtained monoclonal cell strain into an F1 mouse through the abdomen for antibody production, purifying the generated ascites by using Protein A/G, and using the ascites for subsequent detection;

s7, antibody verification: ELISA, western blotting, co-immunoprecipitation, mass spectrometry, antibody chip and other verification are performed on the obtained monoclonal antibody cell strain to determine the most effective antibody.

As a preferred technical scheme of the invention: the Balb/c mice in the S2 are 8-12 week old Balb/c mice.

As a preferred technical scheme of the invention: and the serum titer gridlines in the S3 are serum titers of more than 10K.

As a preferred technical scheme of the invention: the fusion process in S4 is optimized PEG fusion.

As a preferred technical scheme of the invention: the number of cells per well on 4 384 well plates in S4 is 10² ～10⁴ 。

As a preferred technical scheme of the invention: the ELISA verification in S7 specifically comprises the following steps: coating an antibody to be detected on a 96-hole ELISA plate, incubating, washing, blocking the skimmed milk overnight, washing with PBS, and preserving at 4 ℃ for later use; then, antigen incubation and PBS washing are carried out, a control is arranged at the same time, his-HRP and TMB are added for color reaction, and the enzyme label instrument reads.

As a preferred technical scheme of the invention: the antibody chip in the S7 is specifically: the anti-CD 138 antibody and the control antibody are spotted on a glass sheet with NC film as a matrix by using a chip spotter to form antibody spots with the diameter of 100um, the whole protein of A375 is subjected to biotin labeling, incubated on an antibody chip according to the concentration of 2ug/ml, incubated for half an hour at room temperature, gently washed three times by PBS, incubated by CY3-SA fluorescent secondary antibody, washed three times by PBS, and the chip is scanned by using a GenePix fluorescent chip scanner with 523 nm.

As a preferred technical scheme of the invention: the monoclonal antibody variable region sequencing result of the mouse anti-cell surface glycoprotein CD138 is as follows:

VHF1

EVKLEESGGGLVQPGGSMELSCVAS

HYAESVKGRFTISRDDSKNSVYLQMNNLRTEDTGIYYC

DIKMTQSPSSMYASLGERVTITCKAS

RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC

the monoclonal antibody of the mouse anti-cell surface glycoprotein CD138, which can be applied to tumor cell capture, has the characteristics of high affinity, high specificity, multiple application scenarios and the like, and can be applied to living cell enrichment.

Drawings

FIG. 1 is a graph showing the results of antibody flow cytometry in a preferred embodiment of the present invention;

FIG. 2 is a graph showing the killing result of cancer cells according to the preferred embodiment of the present invention;

FIG. 3 is a graph showing the results of an antibody chip test in a preferred embodiment of the present invention.

Detailed Description

It should be noted that, under the condition of no conflict, the embodiments and features in the embodiments may be combined with each other, and the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Referring to FIG. 1, a preferred embodiment of the present invention provides a monoclonal antibody of the mouse anti-cell surface glycoprotein CD138 for tumor cell capture, which comprises the following steps:

s1, protein QC: purchasing commercial proteins; specifically, the purchase in this embodiment is the goods number of the ACRO company: protein SD 1-H5228.

S2, animal immunization: selecting 6 Balb/c mice for antigen immunization, monitoring serum titers of the Balb/c mice to determine optimal immunized mice, performing boosting for 3 to 4 times after primary immunization, detecting titer by taking serum of the mice after boosting, screening out qualified mice with one impact for fusion, and continuing boosting for one to two times until the titer is highest for fusion; in particular, optimized adjuvants and immunization methods are capable of producing high affinity antibodies (IgG subtypes) against most antigens.

s6, preparing antibody supernatant: the finally obtained monoclonal cell strain, in this example, 7 monoclonal cell strains were finally obtained, which were injected into F1 mice via the abdomen for antibody production, and the ascites produced was purified with Protein a/G and used for subsequent detection;

s7, antibody verification: ELISA, western blotting, co-immunoprecipitation, mass spectrometry, antibody chip and the like are carried out on the obtained 7 monoclonal antibody cell strains, and the most effective antibody is determined.

In this embodiment: the Balb/c mice in the S2 are 8-12 week old Balb/c mice.

In this embodiment: and the serum titer gridlines in the S3 are serum titers of more than 10K.

In this embodiment: the fusion process in S4 is optimized PEG fusion.

In this embodiment: the number of cells per well on 4 384 well plates in S4 is 10² ～10⁴ 。

In this embodiment: the ELISA verification in S7 specifically comprises the following steps: and (3) coating the antibody to be detected on a 96-well ELISA plate, incubating, washing, blocking with skimmed milk overnight, washing with PBS, and preserving at 4 ℃ for later use. Antigen incubation (gradient dilution), PBS wash, and control were set. His-HRP (antigen with His tag) was added, TMB was developed for reaction, and the microplate reader was read.

In this example, the inventors verified the affinities of the 8 cell lines obtained by ELISA affinity verification of S7 as follows:

nM	100	50	25	12.5	6.25	3.125	1.57	0.78	0.4	0.2	0.1	NC	affinity nM
														2O5	3.166	3.229	3.243	2.935	2.908	2.730	2.120	1.573	0.886	0.511	0.256	0.055	0.2
2K19	3.900	3.779	3.900	3.900	3.955	3.779	2.746	1.882	1.298	0.511	0.263	0.058	0.2
														2B14	2.719	2.538	2.445	2.170	1.593	0.917	0.571	0.236	0.132	0.068	0.057	0.056	1.57
3F3	3.900	3.954	3.887	3.829	3.733	3.778	3.454	3.025	2.171	1.218	0.536	0.076	0.1
														1H16-B	3.390	3.286	3.131	2.955	2.716	2.587	2.181	1.948	1.333	0.952	0.509	0.056	0.1
1F5	2.720	2.289	1.972	1.477	1.062	0.714	0.505	0.271	0.163	0.111	0.093	0.068	1.57
														1J13	3.900	3.760	3.812	3.673	3.569	3.414	2.929	2.044	1.135	0.615	0.277	0.056	0.2

It should be added that in this example we also performed flow cytometry validation of antibodies and cancer cell killing experiments, respectively.

As shown in fig. 1 in particular, the flow cytometry validation of antibodies was: RPMI-8226 cells were used as the detection cell line, washed with PBS, digested with EDTA, transferred to a centrifuge tube, centrifuged to discard the supernatant, resuspended in PBS, goat serum blocked at room temperature for 1 hour, primary antibody incubation, working concentration 50ug/ml, fluorescent secondary antibody incubation, working concentration 1:300. the results show that the antibody can be applied to flow cytometry.

As shown in fig. 2, the cancer cell killing experiment is: preparing 100ul of cell RPMI-8226 suspension in a 96-well plate, pre-culturing for 24 hours, adding 10ul of anti-CD 138 antibodies with different dilution gradients into a culture plate, adding mouse secondary antibodies coupled with small molecule drugs, culturing for 3 days, adding 10ul of CCK-8 solution into each well, incubating for 1-4 hours, and measuring the absorbance at 450nm by using an enzyme-labeled instrument. Experimental results show that the antibody has good killing effect on cancer cells RPMI-8226.

In this embodiment: the antibody chip in the S7 is specifically: the anti-CD 138 antibody and the control antibody are spotted on a glass sheet with NC film as a matrix by using a chip spotter to form antibody spots with the diameter of 100um, the whole protein of A375 is subjected to biotin labeling, incubated on an antibody chip according to the concentration of 2ug/ml, incubated for half an hour at room temperature, gently washed three times by PBS, incubated by CY3-SA fluorescent secondary antibody, washed three times by PBS, and the chip is scanned by using a GenePix fluorescent chip scanner with 523 nm.

The experimental results are shown in FIG. 3, and the experimental results show that the anti-CD 138 (clone 1H 16-B) has obvious enrichment binding effect on the target protein, the fluorescence intensity is high, and the antigen-antibody binding reaction of the control antibody does not occur.

In this embodiment: the monoclonal antibody variable region sequencing result of the mouse anti-cell surface glycoprotein CD138 is as follows:

VHF1

EVKLEESGGGLVQPGGSMELSCVAS

HYAESVKGRFTISRDDSKNSVYLQMNNLRTEDTGIYYC

DIKMTQSPSSMYASLGERVTITCKAS

RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC

it will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Sequence listing

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