CN114276457A

CN114276457A - Monoclonal antibody of anti-human immunoglobulin kappa light chain, preparation method thereof, immunodetection reagent and application

Info

Publication number: CN114276457A
Application number: CN202111667681.3A
Authority: CN
Inventors: 翟晋豫; 张楠; 靳冉; 王迎利; 柴素真; 王鑫; 秦志洁; 陈明涛; 李卫娟; 齐华; 李三华
Original assignee: Henan Celnovtebio Biotechnology Inc
Current assignee: Henan Celnovtebio Biotechnology Inc
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2022-04-05

Abstract

The invention belongs to the field of monoclonal antibodies, and particularly relates to an anti-human immunoglobulin kappa light chain monoclonal antibody, a preparation method thereof, an immunodetection reagent and application. The monoclonal antibody comprises VHCDR1, VHCDR2 and VHCDR3 which have amino acid sequences shown in SEQ ID NO. 1-3, and VLCDR1, VLCDR2 and VLCDR3 which have amino acid sequences shown in SEQ ID NO. 4-6. The invention provides a diagnostic reagent for evaluating lymphoma or lymph node reactive hyperplasia, and the diagnostic reagent can replace imported reagents, realize the localization of the diagnostic reagent and greatly reduce the diagnostic cost. In the aspect of reagent application, compared with the L1C1 monoclonal antibody widely used internationally, the monoclonal antibody of the anti-human immunoglobulin kappa light chain has consistent specificity evaluation, the using concentration of the antibody is lower, and stronger staining effect and better background can be achieved at 0.05 mu g/mL.

Description

Monoclonal antibody of anti-human immunoglobulin kappa light chain, preparation method thereof, immunodetection reagent and application

Technical Field

The invention belongs to the field of monoclonal antibodies, and particularly relates to an anti-human immunoglobulin kappa light chain monoclonal antibody, a preparation method thereof, an immunodetection reagent and application.

Background

The molecular weight of the antibody light chain is about 25kD and is composed of about 214 amino acid residues. Light chains are classified into kappa and lambda chains, and abs can be classified into two types, namely kappa and lambda. The type of the two light chains on a natural Ab molecule is always the same, but antibody molecules with kappa or lambda chains, respectively, may be present in the same body.

The light chain of each Ab in five Ab types (IgM, IgG, IgA, IgD, IgE) can have kappa chain or lambda chain, and the functions of the two light chains are not different. The proportion of the two types of light chains in organisms of different species is different, and the normal human serum immunoglobulin kappa: λ is approximately 2:1, and in mice is 20: 1. Kappa: abnormalities in the lambda ratio may reflect abnormalities in the immune system.

The recognition of immunoglobulin kappa light chains can be used for differential diagnosis of lymphoma and lymph node reactive hyperplasia. The clone number of the anti-human immunoglobulin kappa light chain monoclonal antibody which is widely used internationally is L1C1, and the imported reagent is expensive, so that the diagnosis cost of pathological diagnosis is high.

Disclosure of Invention

The invention aims to provide an anti-human immunoglobulin kappa light chain monoclonal antibody, which has consistent specificity evaluation compared with an imported reagent L1C1, can achieve stronger staining at lower antibody concentration and shows better affinity.

The second objective of the invention is to provide a preparation method of the above monoclonal antibody against human immunoglobulin kappa light chain.

The third objective of the invention is to provide an immunoassay reagent containing the above monoclonal antibody against human immunoglobulin kappa light chain.

The fourth purpose of the invention is to provide the application of the monoclonal antibody and the immunodetection reagent in immunohistochemistry.

In order to achieve the purpose, the invention adopts the technical scheme that:

a monoclonal antibody against human immunoglobulin kappa light chain comprising VHCDR1, VHCDR2 and VHCDR3 having the amino acid sequences shown in SEQ ID Nos. 1-3, and VLCDR1, VLCDR2 and VLCDR3 having the amino acid sequences shown in SEQ ID Nos. 4-6.

The monoclonal antibody of the anti-human immunoglobulin kappa light chain provides a diagnostic reagent for evaluating lymphoma or lymph node reactive hyperplasia, and the diagnostic reagent can replace an imported reagent, so that the localization of the diagnostic reagent is realized, and the diagnostic cost is greatly reduced. In the aspect of reagent application, compared with the L1C1 monoclonal antibody widely used internationally, the monoclonal antibody of the anti-human immunoglobulin kappa light chain has consistent specificity evaluation, the using concentration of the antibody is lower, and stronger staining effect and better background can be achieved at 0.05 mu g/mL.

Preferably, the anti-human immunoglobulin kappa light chain monoclonal antibody comprises an amino acid sequence as set forth in SEQ ID NO: 7, and the amino acid sequence is as shown in SEQ ID NO: 8, or a light chain variable region. The heavy chain and light chain variable regions adopt the sequences, and the application effect can be further ensured.

A method for preparing a monoclonal antibody against a kappa light chain of human immunoglobulin, comprising the steps of:

(1) immunizing rabbits with immunoglobulin kappa light chain immunogen, collecting rabbit peripheral blood, collecting mononuclear cells of the rabbit peripheral blood, and separating antigen specific B lymphocytes;

(2) and (2) aiming at the antigen specific B lymphocyte obtained in the step (1), culturing, identifying and screening out positive cell pores, further obtaining heavy chain and light chain genes of the antibody, amplifying target genes of the heavy chain and the light chain of the antibody, and recombining and expressing to obtain the rabbit-derived monoclonal antibody.

The preparation method of the monoclonal antibody of the anti-human immunoglobulin kappa light chain takes rabbits as immune animals to prepare the rabbit-derived monoclonal antibody, and has good reproducibility and strong operability. The adoption of B cell separation and culture facilitates early discovery of excellent antibody cell strains, can greatly reduce subsequent expression analysis work, and shorten the research and development period of enterprises.

In order to further increase the expression level of the antibody, preferably, in step (2), a signal region capable of increasing the expression level is added to the 5' end of each of the heavy chain and light chain genes of the antibody, and then the amplification is performed.

Further preferably, the nucleotide sequence of the signal region is as set forth in SEQ ID NO: shown at 9.

In order to obtain the target gene conveniently by a PCR amplification method, preferably, in the step (2), the upstream and downstream primers for amplifying the target gene of the heavy chain of the antibody are respectively shown as SEQ ID NO: 10. SEQ ID NO: 11 is shown in the figure; the upstream primer and the downstream primer of the target gene of the amplified antibody light chain are respectively shown as SEQ ID NO: 12. SEQ ID NO: shown at 13.

An immunoassay reagent containing the monoclonal antibody against human immunoglobulin kappa light chain.

Based on the monoclonal antibodies described above, suitable immunoassay reagents may be constructed according to techniques well known in the art. Taking an immunohistochemical detection reagent as an example, the immunohistochemical detection reagent can be prepared by matching TBS buffer solution, protective protein BSA, preservative and the like.

The monoclonal antibody and the immunodetection reagent of the anti-human immunoglobulin kappa light chain are applied to immunohistochemistry.

Experiments prove that the monoclonal antibody has good effect in the aspect of immunohistochemical detection of the human immunoglobulin Kappa light chain, and can replace an imported reagent L1C1 for use, so that the diagnosis cost is greatly reduced.

Drawings

FIG. 1 shows the results of immunohistochemical staining of the self-developed antibody E37 in tonsil tissues according to an embodiment of the present invention;

FIG. 2 is the result of immunohistochemical staining of control antibody L1C1 in tonsil tissues;

FIG. 3 shows the results of immunohistochemical staining of the self-developed antibody E37 in lymphoma tissues according to the example of the present invention;

FIG. 4 shows the result of immunohistochemical staining of the control antibody L1C1 in tonsil tissues.

Detailed Description

The following describes the practice of the present invention in detail with reference to specific examples.

EXAMPLE 1 monoclonal antibodies against human immunoglobulin kappa light chains

In the monoclonal antibody against human immunoglobulin kappa light chain of the present embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7, the amino acid sequence of the light chain variable region is shown as SEQ ID NO: shown in fig. 8.

The heavy chain variable region comprises VHCDR1, VHCDR2 and VHCDR3 having the amino acid sequences shown in SEQ ID Nos. 1-3, and the light chain variable region comprises VLCDR1, VLCDR2 and VLCDR3 having the amino acid sequences shown in SEQ ID Nos. 4-6.

EXAMPLE 2 preparation of monoclonal antibodies against human immunoglobulin kappa light chain

The preparation method of the monoclonal antibody against human immunoglobulin kappa light chain of the present embodiment includes the following steps:

(1) rabbit immunization: 5 immunogens (immunogens I, II, III, IV and V are MSH6 (mismatch repair protein MSH6), MUM1 (interferon regulatory factor MUM1), p57 (cancer suppressor factor p57 protein), CK7 (cell keratins 7) and Kappa (immunoglobulin Kappa light chain)) and an immune chaperone (CSF2 protein; immune effect is improved) are added into a water-soluble adjuvant to obtain the immune reagent, the concentration of the immune chaperone in the immune reagent is controlled to be 50 mu g/mL, and the immune doses of the 5 immunogens are all 100 mu g/mL. And then, immunizing a New Zealand white rabbit by using an immunological reagent, and performing immunogen impact immunization on the rabbit after the evaluation of the immune effect reaches the preset stage so as to mobilize the rabbit body to quickly activate B lymphocytes aiming at the target immunogen.

The CSF2 protein can be prepared by a method comprising the following steps:

1) first, the full-length gene of the native CSF2 protein, including the promoter and the stop codon, was determined by consulting the Uniprot database.

2) The CSF2 gene is constructed on the carrier of mammal expression system such as pcDNA3.1 by using traditional gene amplification and carrier construction method, and it is necessary to ensure that CSF2 does not contain other non-self gene (such as label protein gene) after successfully constructed.

3) A target vector (pcDNA3.1-CSF2) with correct sequencing construction is transfected into a rabbit-derived mammal expression system such as rabbit kidney cell RK-13 and the like which is verified to have no related protein expression by WB by using a transient expression method. Expression levels were detected using the conventional WB approach.

4) After the expression level meets the requirement, an anti-CFS 2 antibody is coupled to a purification matrix by using an immunoaffinity chromatography means, a target solution which is crushed and subjected to supernatant centrifugation and filtration is purified by using the method by using the binding characteristic of an antigen and an antibody, the eluted CFS2 is stored at pH according to isoelectric point regulation, and is stored under the condition of a PBS buffer system for split charging at-80 ℃.

When animals are immunized, the animals are immunized in a leg muscle mode, and the period is two months. After the immunization is finished, the E (kappa) protein is used for carrying out antibody titer detection on the blood collected from the marginal ear vein of the rabbit so as to obtain a detection result of 6 multiplied by 10⁴The above results are qualified. After the titer reached the desired titer, the rabbits were given a challenge immunization with the E (kappa) immunogen and 10mL of blood was collected via the marginal ear vein on day 3.

(2) Rabbit peripheral blood collection and PBMCs separation, B lymphocyte separation and culture: PBMCs cells (namely peripheral blood mononuclear cells; can be separated by using a commercial rabbit peripheral blood lymphocyte separation kit) are separated from peripheral blood of the rabbit after the shock immunization by using lymphocyte separation liquid, and then negative screening is carried out by using B lymphocyte surface markers CD4, CD8, CD14, CD28 and CD80 to remove cells such as T, mononuclear cells and the like, so that the abundance of the B lymphocytes is improved.

And then selecting B lymphocytes expressing the recognition immunogen by using goat anti-rabbit IgG labeled by a fluorescent dye FITC.

And marking immunogens I, II, III, IV and V respectively by utilizing a commercial biotin marking kit according to the instructions, wherein the immunogens are expressed by the molar weight of 1: 1: 1: 1:1, mixing, uniformly mixing with a commercial avidin labeled coupled Fluorescein (FITC) reagent, and incubating for 1h at the temperature of 2-8 ℃ (shaking and stirring are needed in the process) to obtain the biotin-avidin screening reagent. Storing the incubated biotin-avidin screening reagent in a split-charging mode at the temperature of 2-8 ℃, wherein the biotin-avidin screening reagent is stored according to the cell amount (1 multiplied by 10) when being used⁶Cells/5 μ L) were added. And (3) performing positive screening on the B lymphocytes again by using the biotin-avidin screening reagent to obtain a B lymphocyte population for identifying various immunogens.

(3) B lymphocyte culture supernatant ELISA detection: and (3) carrying out plating culture on the selected B lymphocyte population, paving 1-2 cells/hole into a 96 plate containing trophoblast cells in advance, simultaneously adding IL-2 and IL-10 cytokines for co-culture, respectively carrying out ELISA detection by using 5 immunogens (immunogen I, immunogen II, immunogen III, immunogen IV and immunogen V) after the culture time reaches 7-14 days, selecting serum collected by the rabbit before immunization as a negative control hole, and selecting a positive hole by defining the positive hole as 2.1 times larger than the negative control hole. Through the steps, antibodies respectively aiming at the immunogens can be screened, and cell positive holes for identifying various immunogens can be obtained through one-time sorting culture.

And detecting the supernatant by using an indirect ELISA detection method, and coating an ELISA detection plate with E (kappa) protein to screen out cell culture wells secreting the target antibody.

(4) Obtaining cDNA, amplifying heavy and light chain gene and sequencing: and (3) carrying out cell lysis on the antibody cell strain hole obtained by screening, extracting RNA, obtaining cDNA by an RT-PCR method, respectively amplifying heavy and light chain genes by a PCR amplification method, and determining a sequence by sequencing. RT-PCR was performed using reverse transcription kit instructions.

After obtaining the target genes of the heavy chain and the light chain of the antibody. The signal region is added to the 5' upstream of the target gene by means of PCR. The nucleotide sequence of the signal region is shown as SEQ ID NO: shown at 9. And then designing upstream and downstream primers of the heavy chain and the light chain of the antibody to amplify the target gene, thereby increasing a section of signal region at the upstream of the target gene and conveniently improving the expression quantity of the target gene at the later stage. The upstream primer and the downstream primer for amplifying the heavy chain target gene are respectively shown as SEQ ID NO: 10. SEQ ID NO: 11 is shown in the figure; the upstream primer and the downstream primer of the amplified light chain target gene are respectively shown as SEQ ID NO: 12. SEQ ID NO: shown at 13.

The PCR amplification cycles were set as: i, 5min at 95 ℃; II: 10s at 95 ℃; III, at 56 ℃ for 10 s; IV: 60s at 72 ℃; II to IV: circulating for 35 times; 72 ℃ C: and 5 min. 1% nucleic acid electrophoresis of the target band and gel cutting recovery of the target fragment, the operation referred to Axygen gel cutting recovery kit instructions for operation.

(5) Constructing heavy chain antibody recombinant plasmids and light chain antibody recombinant plasmids: the strain pTT5 positive DH5 alpha was expanded and subjected to plasmid (pTT5 plasmid) extraction and double digestion (EcoRI and BamHI). Plasmid extraction was performed according to the operational requirements of the Axygen plasmid extraction kit instructions. The double enzyme digestion reaction system is as follows: pTT5 plasmid 2 μ g, EcoRI 1.5 μ L, BamHI1.5 μ L, Buffer 5 μ L, ddH2O 40 μ L; the enzyme digestion condition is 37 ℃ water bath, and the enzyme digestion time is 2 h. And (3) performing nucleic acid electrophoresis on the system subjected to enzyme digestion, and cutting gel to recover the double-enzyme digestion plasmid fragment, wherein the operation refers to the specification of the Axygen gel cutting recovery kit for operation.

The target gene fragment and the double enzyme digestion plasmid fragment are subjected to homologous recombination by utilizing a seamless cloning kit, and the steps are carried out according to the operation requirements of the instruction manual of the near-shore seamless cloning kit (cargo number: NR 005-01A).

DH 5. alpha. competent cells were transformed after seamless recombination and plated with ampicillin resistant LB plates. Incubate overnight at 37 ℃. And 5 monoclonals are picked for gene sequencing on the second day, and the heavy-light chain plasmid is extracted by amplification culture with correct sequencing result.

(6) Plasmid transfection: and (3) respectively carrying out operation on the high-concentration plasmids obtained by amplification culture according to the operation instruction of the PEI transfection reagent, wherein the heavy chain and the light chain of the antibody are expressed according to the molar mass of 1:1, plasmid was diluted with CHO (or 293F) cell basal medium, while an equal volume of this medium diluted PEI, plasmid: PEI 1: 2, W/W. Transfection into CHO cells (or 293F cells) cultured to logarithmic growth phase. After culturing for 48h, detecting the titer of the antibody secreted by the supernatant by an indirect ELISA method, and determining a cell line with relatively high expression level.

(7) Antibody engineering expression and purification: and performing amplification culture on the determined cell line to 500mL of culture medium, adjusting the growth state of the cells to a logarithmic growth phase, transfecting plasmids, culturing for 48-72 h, and collecting the cells. And (3) centrifuging the supernatant to remove cells, performing affinity chromatography purification by a Protein G column, eluting the antibody obtained by the binding adsorption by using a citric acid buffer solution with the pH value of 3.0, collecting an eluent, and quickly neutralizing the eluent to the pH value of 7.2-7.4 by using a Tris-HCl solution with the pH value of 8.8. The purified antibody is then dialyzed and concentrated to a concentration of 1mg/mL or more.

Example 3 immunoassay reagent

The immunoassay reagent of this example was an immunohistochemical detection reagent, and a working solution of monoclonal antibody was constructed on the basis of the monoclonal antibody of example 1, wherein the concentration of the monoclonal antibody was 0.05. mu.g/mL, the solvent was TBS buffer solution, and the working solution contained BSA (bovine serum albumin) at a concentration of 10mg/mL and preservatives Proclin-300 and Proclin-950, and the preservatives were added in a ratio of 1:1000 (V/V).

Example 4 application in immunohistochemistry

The monoclonal antibody against human immunoglobulin kappa light chain of example 1 (E37) was applied to immunohistochemical staining with parallel comparison of the imported mab L1C1 as follows:

1. different paraffin tissue samples are sliced at 3 mu m and baked for 2h at 65 ℃.

2. Dewaxing and hydrating: paraffin sections were processed as follows: xylene 15 min-absolute ethanol 5 min-90% ethanol 5 min-80% ethanol 5 min-70% ethanol 5min, purified water soaking for 5 min.

3. Antigen retrieval: boiling with EDTA solution of pH9.0 for 20min, naturally cooling for 5min, cooling with tap water, cooling, taking out, slicing, soaking in pure water for 5min, and washing and soaking in TBS for 5min for 2 times.

4. Adding peroxidase blocking agent 100 μ L/sheet, incubating at room temperature for 5min, and washing and soaking in TBS for 5min for 2 times.

5. Primary antibody incubation: adding the above working solution for preparing antibody, incubating at 37 deg.C for 30min, washing with TBS and soaking for 2 times, 5 min/time.

6. And (3) secondary antibody incubation: first, 100. mu.L of secondary antibody was added dropwise, incubated at 37 ℃ for 30min, and washed and soaked in TBS for 2 times, 5 min/time.

7. Color development: TBS solution was removed, 100. mu.L of DAB staining solution was added dropwise, incubated at room temperature for 5min, and soaked in purified water for 2 times and 5 min/time.

8. Counterdyeing: adding 100 μ L hematoxylin complex staining solution dropwise, incubating at room temperature for 3min, and soaking in purified water for 2 times, 5 min/time.

9. And (3) dehydrating and transparency: dehydrating with conventional gradient ethanol, and making xylene transparent.

10. And (5) observing a neutral gum sealing sheet.

The results of immunohistochemical staining of self-developed antibody E37 in tonsil tissues are shown in FIG. 1. The results of immunohistochemical staining of the control antibody L1C1 are shown in FIG. 2.

The immunohistochemical staining results for self-studied antibody E37 in lymphoma control tissues are shown in FIG. 3. The immunohistochemical staining results for the control antibody L1C1 are shown in FIG. 4.

By combining the immunohistochemical staining results, compared with the control antibody L1C1 (antibody concentration of 0.8. mu.g/mL), E37 has better affinity, and the antibody concentration of 0.05. mu.g/mL can achieve stronger staining effect and better background.

The results of comparison of the two antibodies in terms of specificity of tissue evaluation are shown in table 1 below. The evaluation tissues include tonsil tissue, lymphoma tissue, tonsil, appendix (acute gangrenous appendicitis), placenta, normal spleen, normal colon, liver-differentiated hepatocellular carcinoma, pancreas, lung (bronchus, alveolus), normal kidney, thyroid (follicular), mixed germ cell tumor, ovarian serous carcinoma, right ovarian cyst, pheochromocytoma, malignant melanoma of left forearm, and urothelial carcinoma, glioblastoma, synovial sarcoma, mammary fibroadenoma, solitary fibroma, serous carcinoma, ureter-small cell tumor, embryonic tumor, spindle cell tumor, diffuse astrocytoma, acute suppurative appendicitis, cervix, thymus, diffuse large B-cell lymphoma, seminoma, thymoma, schwannoma, cervical-squamous cell carcinoma, etc., in total, 62 were evaluated.

TABLE 1 tissue evaluation statistics for two antibodies

As can be seen from table 1, there was no significant difference between the two antibodies in the evaluation of the yin-yang identity.

<110> Henan Sainur Biotechnology Ltd

<120> monoclonal antibody of anti-human immunoglobulin kappa light chain, preparation method thereof, immunoassay reagent and application

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Claims

1. A monoclonal antibody against human immunoglobulin kappa light chain, comprising VHCDR1, VHCDR2 and VHCDR3 having the amino acid sequences shown in SEQ ID Nos. 1 to 3, and VLCDR1, VLCDR2 and VLCDR3 having the amino acid sequences shown in SEQ ID Nos. 4 to 6.

2. The anti-human immunoglobulin kappa light chain monoclonal antibody of claim 1, comprising an amino acid sequence as set forth in SEQ ID NO: 7, and the amino acid sequence is as shown in SEQ ID NO: 8, or a light chain variable region.

3. A method for preparing an anti-human immunoglobulin kappa light chain monoclonal antibody as claimed in claim 1 or 2, comprising the steps of:

4. The method of claim 3, wherein in step (2), a signal region capable of increasing the expression level is added to the 5' end of each of the genes of the heavy and light chains of the antibody, followed by said amplification.

5. The method of claim 4, wherein the signal region has the nucleotide sequence set forth in SEQ ID NO: shown at 9.

6. The method of claim 5, wherein in step (2), the primers upstream and downstream of the target gene of the amplified heavy chain are represented by SEQ ID NO: 10. SEQ ID NO: 11 is shown in the figure; the upstream primer and the downstream primer of the target gene of the amplified antibody light chain are respectively shown as SEQ ID NO: 12. SEQ ID NO: shown at 13.

7. An immunoassay reagent comprising the anti-human immunoglobulin kappa light chain monoclonal antibody according to claim 1 or 2.

8. The use of the monoclonal antibody against human immunoglobulin kappa light chain according to claim 1 or 2, the immunodetection reagent according to claim 7 for immunohistochemistry.