Preparation And Characterization Of The Monoclonal Antibody Against Human Soluble Mesothelin-Related Proteins

Objective: Mesothelin (MSLN) is a differentiation antigen recognized by Mab K1 present on normal mesothelial cells and overexpressed in several human tumors, including mesothelioma and ovarian and pancreatic adenocarcinoma.The mesothelin cDNA is 2138-bp long ,contains an open reading frame of 1884bp, and encodes a 69-kDa protein. The precursor protein contains 628 amino acids and has four potential N-linked glycosylation sites. After glycosylation at one or more of its four putative glycosylation sites, it is cleaved by furin to yield the 40-kDa fragment ,mesothelin, attached to the cell membrane by a glycosyphosphatidy inositol linkage and a smaller 32-kDa fragment named megakaryocyte-potentiating factor, that is released from the cell. Two minor spliced forms of the major mesothelin transcript have been detected that encode two slightly altered proteins termed variant 1 and variant 2. Variant 2 retains the intron between exons 16 and 17 and probable leads to premature termination of the protein, resulting in its release from the cell, named soluble mesothelin-related protein (SMR). SMR is a soluble 42-45 kDa protein with an NH2-terminal amino acid sequence, identical to that of the membrane-bound portion of mesothelin. The antigen was also detected in the cell-free culture supernatant from antigen positive carcinoma and in the cell-free malignant effusions of patients with mesothelin-positive carcinoma, to be valuable for diagnosis or follow-up of cancer patients.At present, no Mab reacted to SMR can be obtained in China.To prepare Mab binds with SMR, compound MSLN polypeptide was synthesized selectively. The specificity of antibody was evaluated by immuocytochemistry and Western blot respectively.Methods: 1 Examining the amino acid sequence fragment of human MSLN from NCBI with GOLDKEY analysis software , the fragment was used to predict the mono-parameter of Hopp&Woods hydrophilicity, antigenicity, accessibility,β-turns of MSLN respectively and then used Wu's method to synthetically analyse the veracity of prediction.2 Prepare anti-SMR antibody BALB/c mice were immunized with compound SMR polypeptide. The splenic cell of the mice were fused with SP2/0 mouse myeloma cells. The positive clone was screened and the fragment was subcloned. A stable hybridoma cell lines which can excrete antibody was expand cultivation. The titer of antibody was detected with ELISA. BALB/c mice were immunized with some hybridoma cells by hypoderm injection. Ascites was taken after two weeks. High titer antibody of anti-SMR were obtained. The specificity of antibody were evaluaeted with immuocytochemistry and Western blot. 3 Identification of titer,subtype and specificity of the antibody.3.1 Titration of the antibody The titer of the antibody was detected with ELISA using the compound SMR polypeptide coating on the microwell plate at the dilution of 1: 1000 (1μg /mL ).3.2 Isotyping identification of antibody Dilute the monoclone antibody with phosphatebuffer saline, Ph7.2-7.6. 150μl of diluted sample was added to the development tube, incubating at room temperature for 30 minutes .Place one isotyping strip with the black end at the bottom in development tube for 5 minutes.3.3 Specificity identification of antibody (1) Using immuocytochemistry to detect the reactivity of monoclone antibody to native protein. After pickling,washing and autoclaving, coverslips were put into six-hole microwell plate. MSLN positive ovarian cell--OVCAR-3,SKOV3,3AO were added into the hole respectively to make cell slices. For staining, cell slices endogenous peroxidase activity was removed using 3%H202 at room temperature and nonspecific antigens were blocked with normal calf serum. The cell slices were then incubated overnight at 4℃with primary antibody in a humidity chamber. Staining was achieved using a biotinylated anti-mouse secondary antibody and antibody binding was amplified using biotin. The complex was visualized using diaminovenzidine (DAB). (2) Using Western blot analysis to detect the reactivity of monoclone antibody to native protein. Total protein was extracted from the ovarian cell mentioned above and a measure of 50μg of protein was electophoresed in sodium dodecyl sulfate (SDS)-polyacrylamide at 80V for 30 min and then at 120 V for 90 min. Then transfer the protein onto a PVDF membrane at 250 mA, 4h. Sequential incubation with 5% nonfat milk powder to block nonspecific antigens for 1h. Then incubated overnight at 4℃with primary antibody. And add the horseradish peroxidase-conjugated goat anti-mouse IgG for 1h. The complex was visualized using diaminovenzidine (DAB).Results: 1 The epitopes prediction results using the multi-parameter prediction of B-cell epitopes were different, but the predicted values of 153-162,282-292and 471-481 epitope domains were identical. The epitope domains are adjacent toβ-turns. Therefore, B-cell epitopes were likely at or adjacent to the three domains. According to the synthesis results, the SMR compound peptides with four branch strutures were synthesized with 471-481 epitope domains.2 The titer of anti-SMR mmonoclone antibody was 1 :102400(ELISA).3 The specificity identification of the antibody (1) immuocytochemistry confirmed that the monoclone antibody—2H10 reacted to the cell membrane of three ovarian cell mentioned above specifically. (2) Results of Western blot confirmed that antibody–2H10 reacted with native protein at Mr 40 000.Conclusions: Using B-cell epitopes prediction method, we selected epitopes of amino acid residues in MSLN. The anti-SMR antibody were obtained with the compound peptide. The antibody could react with native MSLN and had high specificity.