[1734] The Isoelectric Point (pI) of Epitope May Be the One of the Determinant Factor for the Effective Heat-Induced Epitope Retrieval

H Kajiya, S Takekoshi, M Takei, RY Osamura. Tokai University School of Medicine, Isehara, Kanagawa, Japan; Nippon Medical School, Sendagi, Tokyo, Japan

Background: The localization of nuclear proteins has been benefitted by heat induced epitope retrieval(HIER). This technical study is aimed at to elucidate the relationship between the pH of retrieval buffer used in HIER and the isoelectric point (pI) of epitopes for various antibodies against human pituitary transcription factors, i.e. NeuroD1, TBX19, GATA2, DAX1, PROP1, PITX1, MSX1, in order to clarify the background mechanisms in HIER method for the particular antibodies using FFPE pituitary adenomas.
Design: Tissues of pituitary adenomas were obtained by surgery and immediately formalin-fixed and paraffin-embedded(FFPE). Deparaffinized sections were treated for HIER method (autoclave heating; 121C, 5min) using universal buffer (Teorell-Stenhagen citrate-phosphate-borate buffer), which has a buffering capacity over a wide range pH from 2.0 to 12.0. For immunostainig, biotin avidin peroxidase complex (ABC) method was used.The pI of each transcription factor and corresponding adenomas cases are summarized in the Table.
Results: In our study, all pituitary transcription factors were strongly immunostained in the nuclei after HIER in universal buffer, simply depending on buffer pH value.

pH-dependent antigen retrieval for human pituitary transcription factors
AntigensPituitary adenomapINon heatingpH 3.0pH 6.0pH 9.0
Neuro D1ACTHoma4.53---+
Ptx1GHoma4.83-+/--+++
GATA2TSHoma7.40-++++/-++
DAX1Non functioning8.49-++++++
TBX19ACTHoma9.31-++-+/-
Prop1GHoma12.70-+++-+/-
immunostaining was scored as follows: +++, strong; ++, moderate; +, weak; faint; -, negative

It should be particularly emphasized that, in order to give the most appropriate staining by the efficient HIER, antigen epitopes with acidic pI(negatively charged) required basic pH buffer conditions, while antigen epitopes with alkaline pI(positively charged) required acidic pH buffer conditions, This suggests that electrostatic charges of epitopes are essential to achieve the best results by HIER for individual antibodies.
Conclusions: From our studies, it should be pointed out that the pH of the buffer and pI of the epitopes are of great importance for electrostatic charges of the individual transcription factors for retrieval. The pI of epitopes may be the one of the determinant factor for the mechanism of HIER to achieve the best results on immunohistochemical staining.
Category: Techniques

Wednesday, March 11, 2009 1:00 PM

Poster Session VI # 223, Wednesday Afternoon

 

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