TY - JOUR
T1 - A new antiserum with conformational specificity for LHRH
T2 - Usefulness for radioimmunoassay and immunocytochemistry
AU - Ellinwood, William E.
AU - Ronnekleiv, Oline K.
AU - Kelly, Martin J.
AU - Resko, John A.
N1 - Funding Information:
Synthetic LHRH was conjugated to bovine serum albumin (BSA) using the bis-diazotized benzidine (BDB) method described by Bassiri and Utiger for thryotropin-releasing hormone \[6\]. This method favors conjugation of LHRH to BSA at either the tyrosine or histidine residues \[21\]. The BDB was prepared by dissolving 0.23 g benzidine dihydrochloride (Grade II, Sigma Chemical Co., St. Louis) in 45 ml ice-cold 0.2 N HC1 and then adding 0.175 g NaNO2 in 5 ml distilled H20. The reaction was allowed to proceed for I hr at 4°C with stirring. The BDB was used fresh on the day of preparation. Thirty mg LHRH (NIH) was mixed with 100 mg BSA in 10 ml 0.16 M boric acid, 0.13 M NaCI (pH 8.3), and 1.4× 10 ~ dpm \[3,4-3H\]LHRH. (42.3 Ci/mM, New England Nuclear) in 100 p,l ethanol was added. A solution of 1.5 ml ~The work described in this article, Publication No 1378 of the Oregon Regional Primate Research Center, was supported by grants HD15888 (WEE), NSI8848, HD16793 {OKR), NS18989 (MJK), HD16022 (JAR) from the National Institutes of Health. 2Requests for reprints should be addressed to W. E. Ellinwood, Department of Physiology, Oregon Health Sciences University.
PY - 1985/1/2
Y1 - 1985/1/2
N2 - We have produced and characterized a new high titer, highly specific antiserum for luteinizing hormone-releasing hormone (LHRH), and demonstrated its usefulness for radioimmunoassay (RIA) and immunocytochemistry. The antiserum can be used at a final dilution of 1:500,000 to 1:600,000 for RIAs with a sensitivity of 0.2 pg/tube. Both the amino and carboxy terminal ends of the LHRH molecule are required for antibody recognition, and the antigenic determinant appears to be part of a three-dimensional structure of LHRH. Fragments of LHRH and other brain peptides are not recognized by the antiserum. Using immunocytochemical techniques, we have localized LHRH-containing neurons in the medial basal hypothalamus of the rhesus monkey, guinea pig, and rat. Staining of LHRH fibers and cell bodies was eliminated by preabsorbtion of the immune serum with synthetic LHRH. This antiserum should be useful in studies that require quantification of very low amounts of LHRH and in studies that require correlation between immunocytochemical localization and tissue content or secretion of LHRH.
AB - We have produced and characterized a new high titer, highly specific antiserum for luteinizing hormone-releasing hormone (LHRH), and demonstrated its usefulness for radioimmunoassay (RIA) and immunocytochemistry. The antiserum can be used at a final dilution of 1:500,000 to 1:600,000 for RIAs with a sensitivity of 0.2 pg/tube. Both the amino and carboxy terminal ends of the LHRH molecule are required for antibody recognition, and the antigenic determinant appears to be part of a three-dimensional structure of LHRH. Fragments of LHRH and other brain peptides are not recognized by the antiserum. Using immunocytochemical techniques, we have localized LHRH-containing neurons in the medial basal hypothalamus of the rhesus monkey, guinea pig, and rat. Staining of LHRH fibers and cell bodies was eliminated by preabsorbtion of the immune serum with synthetic LHRH. This antiserum should be useful in studies that require quantification of very low amounts of LHRH and in studies that require correlation between immunocytochemical localization and tissue content or secretion of LHRH.
KW - Immunoctyochemistry
KW - LHRH
KW - Radioimmunoassay
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U2 - 10.1016/0196-9781(85)90075-0
DO - 10.1016/0196-9781(85)90075-0
M3 - Article
C2 - 3887335
AN - SCOPUS:0021987660
SN - 0196-9781
VL - 6
SP - 45
EP - 52
JO - Peptides
JF - Peptides
IS - 1
ER -