Generation of a Polyclonal Mouse Anti-canine MAGE-A Antibody

• Main Authors: Yu-Chiang Feng, 馮郁青
• Other Authors: Shih-Chieh Chang
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/e28xyw

碩士 === 國立中興大學 === 獸醫學系暨研究所 === 100 === Melanoma antigen-A (MAGE-A) which comprises 12 members named MAGE-A1 to MAGE-A12 was first discovered in 1991. These antigens are predominantly expressed in a wide variety of tumors but only limited expressed in normal human testes and placentas. The aim of this study was to generate a polyclonal mouse anti-canine MAGE-A antibody and to establish the immunohistochemical (IHC) staining protocol of this in-house antibody against canine tissues. First, a pair of primers was designed based on conserved region of canine MAGE-A. Given the size of this product amplified by polymerase chain reaction (PCR), the amplicon was named MAGE-A237. A plasmid with two repeats of MAGE-A237 sequence was constructed, and then cloned and expressed in bacteria host, E.coli BL21. The purified recombinant MAGE-A protein as the antigen was mixed with adjuvant for immunizing mice by intramuscular injection every other week. To test the performance of serum antibody, sera that collected at day 11, 25 and 39 were served as the first antibody against recombinant MAGE-A protein by western blot analysis, and against normal canine tissues by IHC staining. Results showed that all the generated serum antibodies are able to detect recombinant MAGE-A protein by western blot, while the most suitable dilution ratio were 1:20000 for sera on day 11, 25 and 1:500000 for sera on day 39. The optimization of IHC protocol was: use of serum on day 25 at 1:2000 dilution as the first antibody and incubated for 10 minutes. Among all the tested normal canine tissues, the IHC staining results showed negative immunoreactivity except testicular spermatogonia. MAGE-A may be a promising tumor marker for further clinical application on benign and malignant tumors.