Marker for diagnosing liver cancer containing anti-ATIC autoantibody, and composition for diagnosing liver cancer containing antigen thereof
An autoimmune antibody and composition technology, applied in the field of cleotide formyltransfera, can solve the problems of limited implementation, low practicability, and little diagnostic effect, and achieve effective development results
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[0082] Example 1: Obtain XC154 autoimmune antibodies derived from HBx mice
[0083] In order to obtain the autoimmune antibodies that are produced when cancer occurs, HBx transformed mice are used. It is known that the liver cancer that occurs is similar to human liver cancer. From HBx-transformed mice in rearing, spleen cells of individuals confirmed to have liver cancer were taken and used as B cells, and cell fusion with mouse myeloma cells Sp2 / 0 was carried out to prepare B cell hybridomas cell. The cell fusion is based on the general preparation method of B-cell hybridoma cells. The fused cells were screened once using hypoxanthine-aminopt erin-thymidine medium (HAT medium), and only the cells that formed clones were cultured. Re-select cells that can detect cancer cell-reactive antibodies in the cell culture medium and continue to culture.
[0084] The reactivity of autoimmune antibodies to cancer cells is confirmed by intracellular staining of fixed and permeabilized canc...
Example Embodiment
[0086] Example 2: Analysis of Complementary Determining Region (CDR) of XC154 antibody and purification of antibody
[0087] After confirming that the XC154 antibody can specifically recognize the antigen expressed in cancer cells, in order to obtain information about the antigen recognition specificity of the XC154 antibody, the complementarity determining region sequence of the antibody was analyzed. The detailed method is as follows:
[0088] Collect 10 6 About two XC154 antibody producing cells, use RNA extraction kit (Q iagen) to extract total RNA. CDNA was synthesized from 5μg of total RNA using the complementary DNA (cDNA synthesis) kit (Invitrogen), and the synthesized 1μg cDNA and mouse heavy chain constant region primers (Mou se Heavy chain constant region primer) F5'-CTT CCG GAA TTC S AR GTN MAG CTG SAG SAG TCW GG-3' (SEQ NO.21), R5'-GGA AGA TCT GAC ATT TGG GAA GGA CTG ACT CTC-3' (S EQ NO .22) and Mouse light chain constant reg ion primer F5'-GGG AGC TCG AYAT TGT GMT S...
Example Embodiment
[0090] Example 3: Confirmation of the expression of XC154 antibody specific reactive antigen in various cancer cells
[0091] In order to confirm the reactivity of the XC154 antibody to a variety of cancer cell lines, various cancer cells were stained intracellularly using the method of Example 1 above, and then analyzed by flow cytometry ( image 3 ). From the results, it was confirmed that the XC154 antibody-reactive protein was overexpressed in liver cancer cell lines such as HepG2, SK-Hep-1, and various types of cancer cell lines such as HeLa, LNcap-LN3, and A549.
[0092] In addition, in order to confirm the expression position in the cell, intracellular staining is performed and then observed with a confocal fluorescence microscope. A relatively strong staining can be confirmed at the position of the cytoplasm or the position of the cell membrane ( image 3 ).
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