University of Virginia Health System

Center for Research in Reproduction
Ligand Assay and Analysis Core

ASSAY METHODS PAGE

For % CV data, please refer to our 2005 CV% data page. If you are uncertain which method was used to generate your data, please contact the laboratory at (434) 982-3675 or email ligandcore@virginia.edu.

Mouse LH Sandwich Assay (MLHS):
LH was measured in serum by a modified supersensitive two-site sandwich immunoassay (1,2) using monoclonal antibodies MAB1 (no. 581B7) against bovine LH (@) and TMA (no. 5303: Medix Kauniainen, Finland) against the human LH-beta subunit as described previous (2). The tracer antibody, (no. 518B7 kindly provided by Dr. Janet Roser (3), Department of Animal Science, University of California, Davis) was iodinated by the chloramine T method and purified on Sephadex G-50 columns. The capture antibody (no. 5303) was biotinylated and immobilized on avidin-coated polystyrene beads (7mm; Nichols Institute, San Juan Capistrano, CA). Mouse LH reference prep. provided by Dr. A.F. Parlow and the National Hormone and Peptide program was used as standard. The assay has a sensitivity of 0.07 ng/ml, and the average intra-assay and inter-assay coefficients of variation for the Quality Controls are 3.6% and 9.2%, respectively.

(1) Fallest PC, et al. Regulation of Rat Luteinizing Hormone B Gene Expression in Transgenic Mice by Steroids and a Gonadotropin-Releasing Hormone               Antagonist. Biol of Reprod 1995 53:103-109.

(2) Haavisto AOM, et al. A supersensitive immunofluorometric assay for rat luteinizing hormone. Endocrinology 1993 132:1687-1691.

(3) Matteri RL et al. Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species. Domest Anim Endocrinol 1987 4:157-165.

Rat LH Sandwich Assay (RLHS):
LH was measured in serum by a modified supersensitive two-site sandwich immunoassay (1,2) using monoclonal antibodies MAB1 (no. 581B7) against bovine LH (@) and TMA (no. 5303: Medix Kauniainen, Finland) against the human LH-beta subunit as described previous (2). The tracer antibody, (no. 518B7 kindly provided by Dr. Janet Roser (3), Department of Animal Science, University of California, Davis) was iodinated by the chloramine T method and purified on Sephadex G-50 columns. The capture antibody (no. 5303) was biotinylated and immobilized on avidin-coated polystyrene beads (7mm; Nichols Institue, San Juan Capistrano, CA). Rat LH reference prep. provided by Dr. A.F. Parlow and the National Hormone and Peptide program was used as standard. The assay has a sensitivity of 0.07 ng/ml, and the intra-assay and inter-assay coefficients of variation for the Quality Controls were 2.6% and 8.4%, respectively.

(1) Fallest PC, et al. Regulation of Rat Luteinizing Hormone B Gene Expression in Transgenic Mice by Steroids and a Gonadotropin-Releasing Hormone               Antagonist. Biol of Reprod 1995 53:103-109.

(2) Haavisto AOM, et al. A supersensitive immunofluorometric assay for rat luteinizing hormone. Endocrinology 1993 132:1687-1691.

(3) Matteri RL et al. Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species. Domest Anim Endocrinol 1987 4:157-165.

RAT FSH RIA:
Rat FSH measurements were determined by RIA using reagents provided by Dr. A.F. Parlow and the National Hormone and Peptide Program and procedures validated earlier (1). Rat FSH reference prep RP-2 was used for assay standards and anti-rat FSH (S-11) diluted to a final concentration of 1:446,000 was used as primary antibody. Secondary antibody was purchased from the Richard Henninger Lab (phone: 801-224-2468) and was diluted to a final concentration of 1:40. The assay has a sensitivity of 1.5 ng/ml and less than 0.5% cross-reactivity with other pituitary hormones. The average intra-assay and inter-assay coefficients of variation are 5.7 and 9.4%, respectively.

(1) Gay VL, Midgley AR Jr., Niswender GD. Patterns of gonadotropin secretion associated with ovulation. Fed Proc 1970 29:1880-1887.

MOUSE FSH RIA:
Mouse FSH measurements were determined by RIA using reagents provided by Dr. A.F. Parlow and the National Hormone and Peptide Program and procedures validated earlier (1). Mouse FSH reference prep was used for assay standards and Mouse FSH antiserum (guinea pig) AFP-1760191 diluted to a final concentration of 1:400,000 was used as primary antibody. For assays performed prior to September 1,   2006, the secondary antibody was purchased from Antibodies, Inc. (cat #51-134) and was diluted to a final concentration of 1:60. For assays performed after September 1, 2006, the secondary antibody was purchased from Equitech-Bio, Inc. and was diluted to a final concentration of 1:25.  The assay has a sensitivity of 2.0 ng/ml and less than 0.5% cross-reactivity with other pituitary hormones. The intra-assay  and inter-assay coefficients of variation are 6.9% and 13.3%, respectively.

(1) Gay VL, Midgley AR Jr., Niswender GD. Patterns of gonadotropin secretion associated with ovulation. Fed Proc 1970 29:1880-1887.

COMMERCIALLY PREPARED KIT REFERENCES:

Click on the links below to see information about the commercially available kits from the vendors we use for our assays.

DSL

Ligand Assay and Analysis Core Laboratory Home Page