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MAP dendrimer elicits antibodies for detecting rat and mouse GH‐binding proteins
Author(s) -
Aguilar Roberto M.,
Talamantes Frank J.,
Bustamante Juan J.,
Muñoz Jesus,
Treviño Lisa R.,
Martinez Andrew O.,
Haro Luis S.
Publication year - 2009
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.1096
Subject(s) - dendrimer , antibody , chemistry , computational biology , microbiology and biotechnology , biology , biochemistry , genetics
The membrane‐bound rat GH‐R and an alternatively spliced isoform, the soluble rat GH‐BP, are comprised of identical N ‐terminal GH‐binding domains; however, their C ‐terminal sequences differ. Immunological reagents are needed to distinguish between the two isoforms in order to understand their respective roles in mediating the actions of GH. Accordingly, a tetravalent MAP dendrimer with four identical branches of a C ‐terminal peptide sequence of the rat GH‐BP (GH‐BP 263–279 ) was synthesized and used as an immunogen in rabbits. Solid‐phase peptide synthesis of four GH‐BP 263–279 segments onto a tetravalent Lys 2 ‐Lys‐β‐Ala‐OH core peptide was carried out using Fmoc chemistry. The mass of the RP‐HPLC‐purified synthetic product, 8398 Da, determined by ESI‐MS, was identical to expected mass. Three anti‐rat GH‐BP 263–279 MAP antisera, BETO‐8039, BETO‐8040, and BETO‐8041, at dilutions of 10 −3 , recognized both the rat GH‐BP 263–279 MAP and recombinant mouse GH‐BP with ED 50 s within a range of 5–10 fmol, but did not cross‐react with BSA in dot blot analyses. BETO‐8041 antisera (10 −3 dilution) recognized GH‐BPs of rat serum and liver having M r s ranging from 35 to 130 kDa, but did not recognize full‐length rat GH‐Rs. The antisera also detected recombinant mouse GH‐BPs. In summary, the tetravalent rat GH‐BP 263–279 MAP dendrimer served as an effective immunogenic antigen in eliciting high titer antisera specific for the C ‐termini of both rat and mouse GH‐BPs. The antisera will facilitate studies aimed at improving our understanding of the biology of GH‐BPs. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.