MAP dendrimer elicits antibodies for detecting rat and mouse GH-binding proteins.
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-beta-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.
Aguilar
Roberto M
RM
Reeve-Irvine Research Center, University of California, Irvine, Irvine, CA 92697, USA.
Talamantes
Frank J
FJ
Bustamante
Juan J
JJ
Muñoz
Jesus
J
Treviño
Lisa R
LR
Martinez
Andrew O
AO
Haro
Luis S
LS
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002417:Cattle; D050091:Dendrimers / Q000138:chemical synthesis / Q000737:chemistry*; D005260:Female; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D011247:Pregnancy; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D011986:Receptors, Somatotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity
D000906:Antibodies; D050091:Dendrimers; D010455:Peptides; D020033:Protein Isoforms; D011986:Receptors, Somatotropin