Anti-Pit-1 protein monoclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to a monoclonal antibody capable of recognizing a human Pit-1 antigen, a preparation method of the monoclonal antibody and application of the monoclonal antibody in immunodetection. According to the technical scheme, the 372-448 site amino acid of the Pit-1 protein is chemically synthesized to serve as the antigen peptide, and the immunogen is obtained after the antigen peptide is coupled with the KLH. The Pit-1-KLH protein is used for immunizing a mouse, and through cell fusion, screening and subcloning, a mouse hybridoma cell strain 4G5 and an anti-Pit-1 protein monoclonal antibody secreted by the cell strain are obtained. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing Pit-1 protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and in particular to an anti-Pit-1 protein monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Pituitary transcription factor 1 (Pit-1). Also known as growth hormone factor-1 (GHF-1), it is an important member of the POU gene family. It has now been officially named "POUIF1" internationally. It is encoded by the Pit-1 gene and is a transcription factor with important functions that is specifically expressed in the anterior pituitary. It was first discovered by researchers such as MeCORMICK in 1991. The promoter of the Pit-1 gene contains a TATA region element, which is an element required for the 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 3 functional regions: the amino terminal region, the carboxyl terminal region, and the POU protein region.

[0003] The Pit1 gene is closely related to the development of the anterior pituitary and the expression of related genes in the embryonic period. Pit-1 affects the development of three types of pituitary cells: thyroid stimulating hormone (TSH), lactotrophin (PRL) and growth hormone (CH). It can specifically identify and bind to the regulation and activation of target gene transcription such as TSH, PRL, GH. PIT1 is of great value in the diagnosis and pathological classification of pituitary adenomas. For example: growth hormone cell adenoma, thyrotropin cell adenoma, etc. Summary of the invention

[0004] The inventors provide an anti-Pit-1 protein monoclonal antibody, 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; 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 also provided a method for preparing an anti-Pit-1 protein monoclonal antibody, which selected the amino acid sequence (positions 372-448) shown in SEQID NO.3 in the Pit-1 protein, added a cysteine ​​to its carboxyl terminus, and then coupled it with the carrier protein KLH as an immunogen.

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

[0009] Furthermore, the immunoassay includes immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

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

[0011] Different from the prior art, the beneficial technical effects produced by the present invention are as follows: the above technical scheme selects the 372-448 amino acids of the Pit-1 protein as the antigen peptide, and the immunogen obtained after coupling with KLH is used to immunize mice, and through cell fusion, screening and subcloning, a mouse hybridoma cell line 4G5 that efficiently secretes anti-Pit-1 protein monoclonal antibodies, and the anti-Pit-1 protein monoclonal antibodies secreted by the cell line are obtained. The antibodies obtained in this scheme have high specificity and sensitivity, can specifically identify cells expressing Pit-1 protein, and are suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The figure is a comparison of the immunohistochemical staining results of a pituitary tumor, the left one is the Pit-1 of the present invention, and the right one is the commercially available Pit-1. DETAILED DESCRIPTION

[0013] In order to explain the technical content, structural features, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.

[0014] Example 1 Preparation of immunogen

[0015] 1. Preparation of Immunogen

[0016] According to the sequence and secondary structure analysis of the protein sequence with accession number NP_005140.1 in the NCBI database, the molecular weight of the Pit-1 protein with a total length of 448 amino acids is about 74 kDa. According to the secondary structure and surface accessibility parameters of the protein predicted by the online server http: / / www.cbs.dtu.dk / services / NetSurfP / , and the analysis results of its antigenicity index, the amino acid sequence of positions 372-448 (SEQ ID NO.3) was selected as the antigen for chemical synthesis. For the convenience of coupling, a cysteine ​​was added to the carboxyl end of the polypeptide to provide thiol coupling.

[0017] 2. Peptide coupling and purification

[0018] Select the maleimide activated keyhole limpet hemocyanin kit from Thermo Scientific (Cat. No. 77653) and follow the procedure provided in the kit. For the peptide to be coupled, first use Ellman's reagent (ThermoScientific, Cat. No. 22582) to detect the free thiol group in the peptide: add 100 μL of Ellman's reagent stock solution to a 96-well plate, then add 10 μL of peptide solution, and use a Nano Drop spectrophotometer to measure its ultraviolet absorption value at λ = 412nm. If the OD value is >0.15, proceed to the next step; if the OD value is <0.15 or >0.05, add additional peptide until the requirement is met; if the OD value is <0.05, return to the peptide synthesis step for re-quality control. At the beginning of coupling, add 200 μL of deionized water to each mcKLH package to prepare a 10 mg / mL KLH solution, dissolve 2 mg of hapten in 500 μL of Imject EDC coupling buffer, add 500 μL of polypeptide solution to 200 μL of carrier protein solution, add 1 mL of deionized water to a package of EDC (10 mg), shake slowly until completely dissolved, take 50 μL and add it to the mcKLH polypeptide solution. After reacting for 2 hours, remove the uncoupled cross-linker and salts through a desalting column to obtain Pit-1-KLH.

[0019] Example 2 Establishment of hybridoma cell lines

[0020] 1. Immunity

[0021] The cross-linked polypeptide in Example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881) and used to immunize ICR mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) and injected subcutaneously at 6 points in the abdomen of each mouse at a dose of 60 μg / mouse. The booster immunization was performed once every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 30 μg / mouse. Seven days after the third booster immunization, the polyantibody titer against the immunogen in the mouse serum was detected by indirect ELISA (wavelength 450nm). The mouse with the highest titer was immunized by tail vein injection, and the antigen was mixed with physiological saline at a dose of 50 μg / mouse.

[0022] 2. Cell Fusion

[0023] Prepare aseptically the spleen cell suspension of immune-qualified mice, mix it with mouse myeloma cells sp2 / 0 (ATCC Number CRL-8287) at a ratio of 5:1, and centrifuge at 1500rpm for 5min. After discarding the supernatant, place the centrifuge tube in a 37℃ water bath, slowly add 1mL of PEG1500 (Roche) within 1 minute, and stir the cells. After standing in warm water for 1min, add 10mL of serum-free IMDM (Sigma), mix well, and centrifuge at 1000rpm for 5min. After discarding the supernatant, add 10mL of serum (PAA) and carefully blow up the cells, and add 5mL of thymocytes mixed with 10×HAT (Sigma) and mix well. Then add 25mL of semi-solid culture medium containing 2.1% nitrocellulose (Sigma) and mix well, and then pour it evenly into 20 cell culture dishes. Place the cell culture dish in a humidified box and culture it in a 37℃ 5% CO2 incubator.

[0024] 3. Cloning and ELISA screening of positive hybridoma cells

[0025] 7 days after fusion, the size and density of cloned cell clusters are moderate. Under the dissecting microscope, round, solid, and large clone clusters are taken and placed in a 96-well culture plate with culture medium prepared in advance, and cultured in a 37°C 5% CO2 incubator. After 3 days, the cell volume accounts for about 2 / 3 of the bottom area. Take 100 μL of supernatant and use immunogen and synthetic peptide for ELISA screening respectively. The positive clones are completely replaced with 200 μL of complete culture medium containing feeder cells and 1% HT (Sigma). Two days later, the second ELISA screening is carried out, and the positive clones are transferred to a 24-well plate with culture medium (containing feeder cells and HT) prepared in advance for culture. Five days later, take 100 μL of supernatant for the third ELISA screening, and the positive clones are successively transferred to 6-well plates and cell culture bottles for expansion and cryopreservation.

[0026] Example 3 Preparation of monoclonal antibodies by ascites induction method

[0027] 1. Ascites Preparation

[0028] The cells in the logarithmic growth phase were washed with serum-free medium and suspended, and approximately 5 × 10 5 , 1mL. The suspended cells were intraperitoneally injected into mice sensitized with paraffin oil in advance. Ascites was collected 7 days later. The ascites was centrifuged at 4000rpm for 10min at 4℃. The ascites in the middle was carefully aspirated and collected in a centrifuge tube and stored at 4℃ or -20℃.

[0029] 2. Purification of Monoclonal Antibodies

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

[0031] Example 4 Monoclonal Antibody Characterization

[0032] 1. Subclass Identification

[0033] Dilute the coated goat anti-mouse IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) to 0.5μg / mL with 100mM PBS (pH7.4), add 100μL to each well, and incubate at 4℃ overnight. Pour out the liquid, wash 3 times with PBS containing 0.05% Tween (PBS-T), add 200μL blocking solution (PBS containing 2% BSA and 3% sucrose) to each well, and incubate at 37℃ for 1h. Pour out the liquid, wash 3 times with PBS-T. Add 0.1mL hybridoma supernatant to each well, and incubate at 37℃ for 1h. Pour out the liquid and wash 3 times with PBS-T. 0.1 mL of HRP-labeled goat anti-mouse (κ, λ) antibody diluted 1:1000 or HRP-labeled goat anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech) diluted 1:2000 with blocking solution was added to the appropriate wells and incubated at 37°C for 1 hour. The liquid was emptied and washed 3 times with PBS-T. 50 μL of citric acid buffer (PH4.0) containing 0.15% ABTS (Southern Biotech) and 0.03% H2O2 was added to each well for color development, and the OD value at 405 nm wavelength was measured within 10-20 minutes.

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

[0035] 2. Determination of affinity constant

[0036] Take the Pit-1 polypeptide prepared in Example 1, the coating concentration is 2μg / mL, 100μL / well, coat overnight at 4℃, and wash 3 times with PBS-T. Add 200μL of blocking solution to each well and block at 37℃ for 2h, wash 3 times with PBS-T. Dilute the monoclonal antibody purified in Example 3 by 2 times starting from 1:200, leave a blank control in the last well, incubate at 37℃ for 1h, and wash 3 times with PBS-T. Dilute the HRP-labeled goat anti-mouse secondary antibody 1:20000, 100μL per well, incubate at 37℃ for 1h, and wash 3 times with PBS-T. Add 100μL of citric acid-phosphate buffer containing 0.1% TMB (Sigma) and 0.03% H2O2 to each well for color development for 10min, and add 50μL of 0.5M sulfuric acid solution to terminate the reaction. Use an enzyme reader to measure the absorbance value at a wavelength of 450nm. Draw a curve of OD value versus antibody dilution multiple, find the dilution multiple A corresponding to half of the maximum binding OD value, and use the following formula to calculate the affinity constant of the antibody to be 9.6×10 8 .

[0037]

[0038] 3. Monoclonal antibody reaction specificity and application effect

[0039] Take the Pit-1 polypeptide prepared in Example 1, use the immunoblotting method to detect the recognition specificity of the monoclonal antibody of the present invention, and perform 12% polyacrylamide gel electrophoresis. Transfer the gel protein band to a PVDF membrane (Millipore) in a Bio-Rad electrotransfer system according to conventional methods. Place the membrane in a TBS-T blocking solution containing 5% skim milk powder at 4°C overnight. Add the Pit-1 monoclonal antibody purified in Example 3 (1:1000 dilution) and incubate at 4°C overnight. After washing the membrane with TBS-T, add a 1:5000 diluted goat anti-mouse secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) and incubate at room temperature for 1 hour. Wash the membrane again with TBST, add ECL ultrasensitive colorimetric solution (Beijing Pulilai Gene Technology Co., Ltd.), and use the ChemiDocMP multicolor fluorescence imaging system (Bio-Rad) to collect chemiluminescent image data.

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

[0041] Take freshly cultured hybridoma cells and take the supernatant to verify the antigen binding characteristics to confirm that the cell line used for cloning can indeed secrete the required antibodies. After the results are confirmed, centrifuge and collect for 10 6The above hybridoma cells. Trizol method was used to extract total RNA of hybridoma cells. 9 μL of total RNA was taken, 2.5 μL of oligo(dT)12–18primer (10 mM) and 5 μL of dNTPs were added, mixed evenly, kept at 70°C for 5 minutes and then placed on ice for 5 minutes, or denatured according to the reverse transcriptase used. Then 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 obtained was subjected to PCR amplification, and 25 pmol of each primer was added to the 50 μL reaction system. The sequences of the primers for amplification of the heavy chain variable region and the light chain variable region were designed and synthesized according to the mouse monoclonal antibody primer sequences in the book "Recombinant Antibodies" edited by Shen Beifen (Science Press, published in 2005).

[0042] The remaining dNTPs and buffer were added as usual, and finally 1 μL of cDNA template and 1U hot start Taq DNA polymerase were added. The PCR amplification program was set to 94°C for 40 seconds, 52°C for 40 seconds, and 72°C for 40 seconds for 20 to 25 cycles, and finally extended at 72°C for 3 minutes. The product can be placed at 4°C for standby or directly electrophoresed. Take 20 μL of PCR product for electrophoresis analysis and separate on 1.5% agarose gel. The length of the light chain (κ light chain) is between 320-340 bp, and the length of the heavy chain is between 340-370 bp. When there is a specific product in this region, the gel is cut and recovered, cloned into a T vector or expression vector for sequencing.

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

[0044] 1. Chip preparation process

[0045] Each sample was first stained with HE slices to determine the tumor site. The tissue chip was made using the fully automatic tissue chip instrument of 3DHISTECH. The prepared tissue chip wax block was placed in the wax block mold, placed in a 68°C oven for 10 minutes, so that the tissue core and the wax of the receptor wax block were integrated, and then the mold was gently removed from the oven, and the semi-melted paraffin was allowed to cool at room temperature for about 30 minutes, and then placed in a -20°C refrigerator for 6 minutes. After the tissue chip wax block was removed from the mold, sliced ​​or placed in a 4°C refrigerator for later use. After trimming, continuous sections were made with a thickness of 3μm. The continuous sections were floated in 40% alcohol and allowed to unfold naturally. The separated sections were then transferred to 50°C warm water for 30 seconds, and the sections were mounted on slides treated with polylysine. The prepared tissue chip was placed in a 68°C oven for 2 hours, taken out, cooled at room temperature, and stored in a -4°C refrigerator.

[0046] II. IHC staining and analysis

[0047] Dewax in conventional xylene for 3 times, 6 minutes each time, hydrate in 100%, 100%, 95%, 85% gradient ethanol, 3 minutes each time, and finally rinse with tap water. Perform antigen retrieval, then place the slices in a wet box and rinse with PBS for 3×3 minutes. Add 3% H2O2 for 10 minutes, and rinse with PBS for 3×3 minutes. Shake dry the slices, add the primary antibody diluted in an appropriate proportion (the first dilution is designed according to the antibody concentration) and incubate at room temperature (25°C) for 1 hour, rinse with PBS for 3×3 minutes, add the secondary antibody and incubate at room temperature for 15-30 minutes, rinse with PBS for 3×3 minutes, shake off PBS, and use freshly prepared DAB color development solution for 3-10 minutes. Counterstain with hematoxylin for 25 seconds, and return to blue with PBS for 30 seconds. Dehydrate in a gradient of 85% (3 minutes)-95% (3 minutes)-100% (3 minutes)-100% (3 minutes) alcohol, and finally transparentize with xylene for 3 minutes and seal with neutral gum.

[0048] Immunohistochemical staining results are divided into: positive and negative. Positive expression must be in specific antigenic sites of cells and tissues to be considered positive. In the case of clear tissue staining distribution and accurate cell localization, the staining results are further divided according to the difference in staining intensity, as follows:

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

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

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

[0052] 4. The sample is negative and marked as “-”.

[0053] 3. Data Statistics

[0054] 1. Tumor tissue chip test results:

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

[0056]

[0057] The results showed that the anti-Pit-1 protein monoclonal antibody prepared by the present invention had accurate staining positioning, clear staining without nonspecific staining, and clean background; its positive rate was comparable to that of commercially available antibodies, but its positive intensity was higher than that of commercially available antibodies. Among them, the staining intensity of the Pit-1 protein monoclonal antibody secreted by 1 case of 4G5 cell line was significantly higher than that of the commercially available Pit-1, indicating that it has higher sensitivity and effectively avoids false negative results.

[0058] Figure 1 The figure is a comparison of the immunohistochemical staining results of a pituitary tumor (the left one is the Pit-1 of the present invention, and the right one is the commercially available Pit-1).

[0059] 2. Normal tissue chip test results:

[0060] The normal tissue chip includes 30 normal tissue samples, which are mainly selected from fresh and timely fixed surgical specimens; each tissue includes 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectum, parathyroid gland, liver, pituitary gland, salivary gland, testis, kidney, thyroid gland, prostate gland, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infants), skin, bone marrow, peripheral nerve, lung, mesothelial cells. The antibody (4G5) and the commercially available antibody were synchronously detected on the normal tissue chip, and the positive and negative test results were consistent, indicating that the specificity of the antibody in normal tissues is equivalent to that of the commercially available antibody.

[0061] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that the process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "include..." or "comprise..." do not exclude the existence of other elements in the process, method, article or terminal device including the elements. In addition, in this article, "greater than", "less than", "exceed" and the like are understood to exclude the number itself; "above", "below", "within" and the like are understood to include the number itself.

[0062] It should be noted that, although the above embodiments have been described in this article, the patent protection scope of the present invention is not limited thereby. Therefore, based on the innovative concept of the present invention, changes and modifications made to the embodiments described herein, or equivalent structures or equivalent process changes made using the contents of the present invention specification and drawings, directly or indirectly applying the above technical solutions to other related technical fields, are all included in the patent protection scope of the present invention.

Claims

1. An anti-Pit-1 protein monoclonal antibody, 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 clone number of the monoclonal antibody is 4G5.

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

4. A method for preparing an anti-Pit-1 protein monoclonal antibody, characterized in that: The amino acid sequence shown in SEQ ID NO.3 in the Pit-1 protein was selected, a cysteine ​​was added to its carboxyl terminus, and then coupled with the carrier protein KLH as an immunogen.

5. A Pit-1 protein immunoassay reagent, characterized in that: The immunoassay reagent contains the anti-Pit-1 protein monoclonal antibody according to claim 1 as its effective ingredient.

6. The immunoassay reagent according to claim 5, characterized in that: The immunoassay includes immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay.

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