Anti-pit-1 protein monoclonal antibody and preparation method and application thereof

Abstract

The present application relates to a kind of monoclonal antibody that can identify human Pit-1 antigen, its preparation method and its use in immunodetection.The above technical scheme synthesizes the amino acid of 372-448 of Pit-1 protein as antigen peptide, obtains immunogen after coupling KLH.The Pit-1-KLH protein is immunized to mouse, is fused to cell, screening and subcloning, obtain mouse hybridoma cell strain 4G5, and the monoclonal antibody secreted by the cell strain anti Pit-1 protein.The antibody obtained in the present application has high specificity, sensitivity, can specifically recognize the cell expressing Pit-1 protein, is suitable for immunological detection, especially immunohistochemical detection.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, and in particular to an anti-Pit-1 protein monoclonal antibody, its preparation method, and its application. Background Technology

[0002] Pituitary transcription factor 1 (Pit-1), also known as growth hormone factor-1 (GHF-1), is an important member of the POU gene family and has been officially named "POUIF1" internationally. Encoded by the Pit-1 gene, it is a transcription factor specifically expressed in the anterior pituitary gland with important functions. It was first discovered in 1991 by researchers such as MeCORMICK. The Pit-1 gene promoter contains a TATA region element, which is essential for tissue-specific expression of the Pit-1 gene. The Pit-1 gene is located on the short arm of chromosome 3 (3PH) and contains 6 exons and 5 introns. Pit-1 consists of 291 amino acid residues, including three functional regions: the N-terminal region, the C-terminal region, and the POU protein region.

[0003] The Pit1 gene is closely related to the development of the anterior pituitary gland and the expression of related genes during the embryonic period. Pit-1 affects the development of three types of pituitary cells: thyroid-stimulating hormone (TSH), prolactin (PRL), and growth hormone (CH). It can specifically recognize and bind to target genes such as TSH, PRL, and GH, regulating and activating target gene transcription. Pit1 has important value in the diagnosis and pathological classification of pituitary adenomas, such as growth hormone cell adenomas and thyroid-stimulating hormone cell adenomas. Summary of the Invention

[0004] The inventors provide a monoclonal antibody against Pit-1 protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1; and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.2.

[0005] Furthermore, the monoclonal antibody clone number is 4G5.

[0006] Furthermore, the monoclonal antibody is a mouse IgG2a subtype monoclonal antibody.

[0007] The inventors have also provided a method for preparing a monoclonal antibody against Pit-1 protein, which involves selecting the amino acid sequence (positions 372-448) shown in SEQ ID NO.3 of Pit-1 protein, adding a cysteine ​​residue to its carboxyl terminus, and then conjugating it with the carrier protein KLH as an immunogen.

[0008] The inventors also provided the use of the above-mentioned monoclonal antibody in the immunoassay of Pit-1 protein.

[0009] Furthermore, the immunoassay includes immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

[0010] The inventors have finally provided an immunohistochemical detection reagent for Pit-1 protein, wherein the immunohistochemical detection reagent contains an anti-Pit-1 protein monoclonal antibody whose amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1 and whose amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0011] Unlike existing technologies, the beneficial technical effects of this invention are as follows: The above technical solution selects amino acids 372-448 of the Pit-1 protein as the antigenic peptide. After conjugation with KLH, the resulting immunogen is used to immunize mice. Through cell fusion, screening, and subcloning, a mouse hybridoma cell line 4G5 that efficiently secretes anti-Pit-1 protein monoclonal antibodies is obtained, along with the anti-Pit-1 protein monoclonal antibody secreted by this cell line. The antibody obtained by this method has high specificity and sensitivity, and can specifically recognize cells expressing Pit-1 protein, making it suitable for immunological detection, especially immunohistochemical detection. Attached Figure Description

[0012] Figure 1 The image shows a comparison of immunohistochemical staining results for a pituitary tumor. The left image shows Pit-1 of the present invention, and the right image shows commercially available Pit-1. Detailed Implementation

[0013] To explain in detail the technical content, structural features, objectives, and effects of the technical solution, the following description is provided in conjunction with specific embodiments and accompanying drawings.

[0014] Example 1: Preparation of Immunogen

[0015] I. Immunogen Preparation

[0016] Sequence and secondary structure analysis was performed based on the protein sequence with accession number NP_005140.1 in the NCBI database. The full-length Pit-1 protein has a molecular weight of approximately 74 kDa, consisting of 448 amino acids. Based on the protein's secondary structure and surface accessibility parameters predicted using the online server http: / / www.cbs.dtu.dk / services / NetSurfP / , and through analysis of its antigenicity index, amino acid sequence positions 372-448 (SEQ ID NO.3) were selected as the antigen for chemical synthesis. To facilitate coupling, a cysteine ​​residue was added to the carboxyl terminus of the peptide to provide thiol coupling.

[0017] II. Coupling and Purification of Peptides

[0018] Select the Maleamide-Activated Keyhole Hemocyanin Kit from Thermo Scientific (catalog number: 77653) and follow the instructions provided. For the peptide to be coupled, first detect the free sulfhydryl groups in the peptide using Ellman's reagent (Thermo Scientific, catalog number: 22582): Add 100 μL of Ellman's reagent stock solution to a 96-well plate, then add 10 μL of peptide solution. Measure the UV absorbance at λ = 412 nm using a Nano Drop spectrophotometer. If the OD value is > 0.15, proceed to the next step; if the OD value is < 0.15 or > 0.05, add more peptide until the requirement is met; if the OD value is < 0.05, return to the peptide synthesis step for quality control. At the start of coupling, 200 μL of deionized water was added to each mcKLH package to prepare a 10 mg / mL KLH solution. 2 mg of hapten was dissolved in 500 μL of Imject EDC coupling buffer. 500 μL of peptide solution was added to 200 μL of carrier protein solution. 1 mL of deionized water was added to one package of EDC (10 mg). The mixture was slowly shaken until completely dissolved. 50 μL of the solution was added to the mcKLH peptide solution. After reacting for 2 hours, uncoupled cross-linking agents and salts were removed by desalting column treatment to obtain Pit-1-KLH.

[0019] Example 2: Establishment of hybridoma cell lines

[0020] I. Immunity

[0021] The cross-linked peptides from Example 1 were emulsified with Freund's complete adjuvant (Sigma, F5881) and used to immunize ICR mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). Each mouse was injected subcutaneously into the abdomen at 6 points, with a dose of 60 μg / mouse. A booster immunization was administered every 14 days, with the antigen emulsified using Freund's incomplete adjuvant (Sigma, F5506), at a dose of 30 μg / mouse. Seven days after the third booster immunization, the polyclonal antibody titer against the immunogen in mouse serum was detected by indirect ELISA (wavelength 450 nm). Mice with the highest titer were given a tail vein injection for pulse immunization, with the antigen mixed with physiological saline at a dose of 50 μg / mouse.

[0022] II. Cell Fusion

[0023] Aseptically prepare a suspension of immunocompromised mouse spleen cells and mix it with mouse myeloma cells sp2 / 0 (ATCC Number CRL-8287) at a ratio of 5:1. Centrifuge at 1500 rpm for 5 min. Discard the supernatant and place the centrifuge tube in a 37°C water bath. Slowly add 1 mL of PEG1500 (Roche) over 1 minute while stirring the cells. After incubating in warm water for 1 min, add 10 mL of serum-free IMDM (Sigma), mix well, and centrifuge at 1000 rpm for 5 min. Discard the supernatant and add 10 mL of serum (PAA) to carefully agitate the cells. Add 5 mL of thymocytes mixed with 10×HAT (Sigma) and mix well. Add 25 mL of semi-solid culture medium containing 2.1% nitrocellulose (Sigma) and mix thoroughly. Then pour the mixture evenly into 20 cell culture dishes. Place the cell culture dishes in a humidified chamber and incubate at 37°C in a 5% CO2 incubator.

[0024] III. Cloning and ELISA screening of positive hybridoma cells

[0025] Seven days after fusion, the clonal cell clusters reached a suitable size and density. Under a dissecting microscope, round, solid, and large clonal clusters were aspirated and transferred into 96-well plates containing pre-prepared culture medium, and incubated at 37°C in a 5% CO2 incubator. After three days, the cell mass occupied approximately two-thirds of the bottom area. 100 μL of the supernatant was collected and screened using ELISA with immunogen and synthetic peptides, respectively. Positive clones were completely replaced with 200 μL of complete culture medium containing feeder cells and 1% HT (Sigma). Two days later, a second ELISA screening was performed, and positive clones were transferred to 24-well plates containing pre-prepared culture medium (containing feeder cells and HT). Five days later, 100 μL of the supernatant was collected for a third ELISA screening. Positive clones were successively transferred to 6-well plates and cell culture flasks for expansion culture and cryopreservation.

[0026] Example 3: Preparation of Monoclonal Antibodies by Ascites Induction Method

[0027] I. Preparation of Ascites

[0028] Cells in the logarithmic growth phase were washed with serum-free medium and suspended, and counted at approximately 5 × 10⁶ cells. 5 1 mL of the suspended cells were injected intraperitoneally into mice that had been sensitized with paraffin oil. Ascites fluid was collected starting 7 days later. The collected ascites fluid was centrifuged at 4000 rpm for 10 min at 4°C. The middle ascites fluid was carefully aspirated and collected in centrifuge tubes and stored at 4°C or -20°C.

[0029] II. Purification of Monoclonal Antibodies

[0030] The antibody was purified from ascites fluid using HiTrap rProtein A FF (GE) affinity chromatography according to the manufacturer's instructions. Purity was determined by SDS-PAGE gel chromatography, and concentration was determined by the Bradford method. The purified antibody was stored at -20°C.

[0031] Example 4: Characterization of Monoclonal Antibody

[0032] I. Subclass Identification

[0033] Dilute the coated goat anti-mouse IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) with 100 mM PBS (pH 7.4) to a final concentration of 0.5 μg / mL. Add 100 μL to each well and incubate overnight at 4°C. Empty the liquid and wash three times with PBS containing 0.05% Tween (PBS-T). Add 200 μL of blocking buffer (PBS containing 2% BSA and 3% sucrose) to each well and incubate at 37°C for 1 h. Empty the liquid and wash three times with PBS-T. Add 0.1 mL of hybridoma supernatant to each well and incubate at 37°C for 1 h. Empty the liquid and wash three times with PBS-T. Add 0.1 mL of HRP-labeled goat anti-mouse (κ,λ) antibody (1:1000) or HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech) (1:2000) to each well and incubate at 37°C for 1 h. Pour off the liquid and wash three times with PBS-T. Add 50 μL of citrate buffer (pH 4.0) containing 0.15% ABTS (Southern Biotech) and 0.03% H2O2 to each well for colorimetric reaction. Measure the OD value at 405 nm within 10–20 min.

[0034] The results showed that the monoclonal antibody of the present invention is a murine monoclonal antibody of type IgG2a.

[0035] II. Determination of Affinity Constant

[0036] The Pit-1 peptide prepared in Example 1 was used at a coating concentration of 2 μg / mL, 100 μL / well, and coated overnight at 4°C, followed by washing three times with PBS-T. 200 μL of blocking buffer was added to each well, and the mixture was blocked at 37°C for 2 h, followed by washing three times with PBS-T. The monoclonal antibody purified in Example 3 was serially diluted 2-fold starting at 1:200, with the last well containing a blank control. The mixture was incubated at 37°C for 1 h, followed by washing three times with PBS-T. HRP-labeled goat anti-mouse secondary antibody was diluted 1:20000, 100 μL per well, and incubated at 37°C for 1 h, followed by washing three times with PBS-T. 100 μL of citrate-phosphate buffer containing 0.1% TMB (Sigma) and 0.03% H2O2 was added to each well for color development for 10 min, and the reaction was terminated by adding 50 μL of 0.5M sulfuric acid solution. The absorbance was measured at 450 nm using a microplate reader. Plot the curve of OD value corresponding to antibody dilution factor, find the dilution factor A corresponding to half of the maximum binding OD value, and calculate the affinity constant of the antibody as 9.6 × 10⁻⁶ using the following formula. 8 .

[0037]

[0038] III. Monoclonal antibody reaction specificity and application efficacy

[0039] The Pit-1 peptide prepared in Example 1 was used to detect the recognition specificity of the monoclonal antibody of the present invention by immunoblotting, followed by 12% polyacrylamide gel electrophoresis. The gel protein band was transferred to a PVDF membrane (Millipore) using a Bio-Rad electrotransfer system according to standard procedures. The membrane was incubated overnight at 4°C in TBS-T blocking buffer containing 5% skim milk powder. The Pit-1 monoclonal antibody purified in Example 3 (1:1000 dilution) was added and incubated overnight at 4°C. After washing the membrane with TBS-T, goat anti-mouse secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) diluted 1:5000 was added, and the membrane was incubated at room temperature for 1 hour. After washing again with TBST, ECL ultrasensitive chromogenic solution (Beijing Pulilai Gene Technology Co., Ltd.) was added, and chemiluminescence image data was acquired using a ChemiDocMP multicolor fluorescence imaging system (Bio-Rad).

[0040] Example 5: Determination of the variable region sequence of the antibody:

[0041] Freshly cultured hybridoma cells were collected, and the supernatant was used to verify antigen-binding properties. This confirmed that the cell line used for cloning could indeed secrete the required antibodies. After confirmation, 10 cells were collected by centrifugation. 6The above hybridoma cells were used. Total RNA was extracted from hybridoma cells using the Trizol method. 9 μL of total RNA was added to 2.5 μL of oligo(dT)12–18 primer (10 mM) and 5 μL of dNTPs. The mixture was thoroughly incubated at 70°C for 5 minutes, then placed on ice for 5 minutes, or denatured using the reverse transcriptase used. Subsequently, 5 μL of RT buffer (5X), 2.5 μL of DTT (0.1 M), and 1 μL of reverse transcriptase were added, and the reaction was carried out at 42°C for 1 hour. The reaction was terminated by incubation at 70°C for 15 minutes, and the obtained cDNA was stored at -20°C. The first-strand cDNA was then subjected to PCR amplification. 25 pmol of each primer was added to a 50 μL reaction system. The primer sequences for amplifying the heavy and light chain variable regions were designed and synthesized according to the mouse monoclonal antibody primer sequences in the book *Recombinant Antibodies* (Science Press, 2005), edited by Shen Beifen.

[0042] The remaining dNTPs and buffer were added as usual. Finally, 1 μL of cDNA template and 1 U of hot-start Taq DNA polymerase were added. The PCR amplification program was set to 94℃ for 40 seconds, 52℃ for 40 seconds, and 72℃ for 40 seconds, for 20 to 25 cycles, with a final extension at 72℃ for 3 minutes. The product could be stored at 4℃ for later use or directly electrophoresed. 20 μL of the PCR product was taken for electrophoretic analysis. Separation was performed on a 1.5% agarose gel. The length of the light chain (κ light chain) was between 320-340 bp, and the length of the heavy chain was between 340-370 bp. If products specific to this region were found, the gel was excised and recovered, cloned into a T vector or expression vector, and sequenced.

[0043] Example 6. Immunohistochemical tissue microarray staining and identification

[0044] I. Chip fabrication process

[0045] Each sample was first stained with hematoxylin and eosin (HE) to determine the tumor location. Tissue microarrays were fabricated using a fully automated tissue microarray system from 3DHISTECH. The fabricated tissue microarray blocks were placed in a wax block mold and placed in a 68°C oven for 10 minutes to fuse the tissue core with the recipient wax block. The mold was then gently removed from the oven, and the semi-molten paraffin was allowed to cool at room temperature for about 30 minutes, followed by freezing at -20°C for 6 minutes. The tissue microarray blocks were then removed from the mold and sectioned or stored at 4°C for later use. After trimming, serial sections were prepared with a thickness of 3 μm. The serial sections were then immersed in 40% alcohol and allowed to spread naturally. The separated sections were then transferred to 50°C warm water for 30 seconds for further spreading. The sections were mounted on poly-L-lysine-treated slides, and the fabricated tissue microarrays were baked in a 68°C oven for 2 hours. After removal, they were cooled to room temperature and stored at -4°C.

[0046] II. IHC staining and analysis

[0047] The slides were dewaxed three times with xylene for 6 minutes each time, then hydrated in a gradient of 100%, 100%, 95%, and 85% ethanol for 3 minutes each time, and finally rinsed with tap water. Antigen retrieval was performed, and the slides were then placed in a humidified chamber and rinsed with PBS for 3 x 3 minutes. Incubation with 3% H2O2 for 10 minutes was followed by PBS rinsing for 3 x 3 minutes. The slides were then dehydrated, and a suitable diluted primary antibody (the initial dilution should be designed based on the antibody concentration) was added. Incubation was performed at room temperature (25°C) for 1 hour, followed by PBS rinsing for 3 x 3 minutes. Secondary antibody was added and incubated at room temperature for 15-30 minutes, followed by PBS rinsing for 3 x 3 minutes. The PBS was discarded, and the slides were developed with freshly prepared DAB chromogenic solution for 3-10 minutes. Counterstaining with hematoxylin for 25 seconds and then blue staining with PBS for 30 seconds was performed. Dehydration was then performed sequentially with an ethanol gradient of 85% (3 minutes), 95% (3 minutes), 100% (3 minutes), and 100% (3 minutes). Finally, the slides were cleared with xylene for 3 minutes and mounted with neutral resin.

[0048] Immunohistochemical staining results are categorized as positive or negative. Positive expression must occur at a specific antigenic site on the cell or tissue to be considered positive. When tissue staining distribution is clear and cell localization is accurate, the staining results are further subdivided based on differences in staining intensity, as follows:

[0049] 1. The sample is weakly positive; it is marked as "+";

[0050] 2. The sample is moderately positive; marked as "++";

[0051] 3. The sample is highly positive; it is marked as "+++".

[0052] 4. If the sample is negative, mark it as "-".

[0053] III. Data Statistics

[0054] 1. Tumor tissue microarray detection results:

[0055] The antibody Pit-1 (4G5) and the commercially available antibody Pit-1 (G-2) were used to simultaneously detect pituitary tumors in 45 patients, and the results were compared. The immunohistochemical results of Pit-1 were statistically analyzed. The entire trial was a double-blind design, and the statistical results are shown in the table below:

[0056]

[0057] The results showed that the anti-Pit-1 protein monoclonal antibody prepared in this invention had accurate staining localization, clear staining without nonspecific staining, and a clean background. Its positive rate was comparable to that of commercially available antibodies, but its positive intensity was higher. Specifically, the staining intensity of the Pit-1 protein monoclonal antibody secreted by one 4G5 cell line was significantly higher than that of the commercially available Pit-1, indicating higher sensitivity and effective avoidance of false negative results.

[0058] Figure 1 Comparison of immunohistochemical staining results for a pituitary tumor (left: Pit-1 of the present invention, right: commercially available Pit-1).

[0059] 2. Results of microarray analysis of normal tissue:

[0060] The normal tissue microarray includes 30 types of normal tissue samples, primarily selected from fresh, promptly fixed surgical specimens; each tissue type includes three different case samples. The 30 normal tissue types include: brain, heart, cerebellum, esophagus, adrenal glands, stomach, ovary, small intestine, pancreas, colorectal region, parathyroid glands, liver, pituitary gland, salivary glands, testes, kidneys, thyroid gland, prostate, breast, uterus, spleen, bladder, tonsils, skeletal muscle, thymus (in young children), skin, bone marrow, peripheral nerves, lungs, and mesothelial cells. Simultaneous detection of this antibody (4G5) and commercially available antibodies on the normal tissue microarray showed consistent positive and negative results, indicating that the specificity of this antibody in normal tissues is comparable to that of commercially available antibodies.

[0061] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Unless otherwise specified, an element defined by the phrase "comprising..." or "including..." does not exclude the presence of additional elements in the process, method, article, or terminal device that includes said element. Additionally, in this document, "greater than," "less than," "exceeding," etc., are understood to exclude the stated number; "above," "below," "within," etc., are understood to include the stated number.

[0062] It should be noted that although the above embodiments have been described herein, this does not limit the scope of patent protection of the present invention. Therefore, any changes and modifications made to the embodiments described herein based on the innovative concept of the present invention, or equivalent structural or procedural transformations made using the content of the present invention's specification and drawings, directly or indirectly applying the above technical solutions to other related technical fields, are all included within the scope of patent protection of the present invention.

Claims

1. A monoclonal antibody against Pit-1 protein, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is a mouse IgG2a subtype monoclonal antibody.

3. A Pit-1 protein immunoassay reagent, characterized in that, The immunoassay reagent contains the anti-Pit-1 protein monoclonal antibody as its active ingredient, as described in claim 1.

4. The immunoassay reagent according to claim 3, characterized in that, The immunoassays include immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

Images