Assay Methods

Assay Methods

Center for Research in Reproduction
Ligand Assay and Analysis Core

Assay Methods

For % CV data, please refer to our 2013 % 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 is measured in serum by a sensitive two-site sandwich immunoassay (1,2) using monoclonal antibodies against bovine LH (no. 581B7) and against the human LH-beta subunit (no. 5303: Medix Kauniainen, Finland)  as described previous (2). The tracer antibody, (no. 518B7) is kindly provided by Dr. Janet Roser (3), (Department of Animal Science, University of California, Davis) and iodinated by the chloramine T method and purified on Sephadex G-50 columns. The capture antibody (no. 5303) is biotinylated and immobilized on avidin-coated polystyrene beads (7mm; Epitope Diagnostics, Inc., San Diego, CA). Mouse LH reference prep (AFP5306A; provided by Dr. A.F. Parlow and the National Hormone and Peptide program) is used as standard. The assay has a sensitivity of 0.04 ng/ml.

(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 is measured in serum by a sensitive two-site sandwich immunoassay (1,2) using monoclonal antibodies against bovine LH (no. 581B7) and against the human LH-beta subunit (no. 5303: Medix Kauniainen, Finland)  as described previous (2). The tracer antibody, (no. 518B7) is kindly provided by Dr. Janet Roser (3), (Department of Animal Science, University of California, Davis) and iodinated by the chloramine T method and purified on Sephadex G-50 columns. The capture antibody (no. 5303) is biotinylated and immobilized on avidin-coated polystyrene beads (7mm; Epitope Diagnostics, Inc., San Diego, CA). Rat LH reference prep (RP-3; provided by Dr. A.F. Parlow and the National Hormone and Peptide program) is used as standard. The assay has a sensitivity of 0.04 ng/ml.

(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 (RFSH):
Rat FSH is measured by RIA using reagents provided by Dr. A.F. Parlow and the National Hormone and Peptide Program, as previously described (1). Rat FSH reference prep RP-2 is used for assay standards and anti-rat FSH (S-11) diluted to a final concentration of 1:446,000 is used as primary antibody. Secondary antibody is purchased from the Richard Henninger Lab (phone: 801-224-2468) and diluted to a final concentration of 1:40. The assay has a sensitivity of 1.5 ng/ml, with less than 0.5% cross-reactivity with other pituitary hormones.

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

MOUSE FSH RIA (MFSH):
FSH is assayed by RIA using reagents provided by Dr. A.F. Parlow and the National Hormone and Peptide Program, as previously described (1). Mouse FSH reference prep AFP5308D is used for assay standards and Mouse FSH antiserum (guinea pig; AFP-1760191) diluted to a final concentration of 1:400,000 is used as primary antibody. The secondary antibody is purchased from Equitech-Bio, Inc. and 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.

(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:

For information about the commercially available kits we use for our assays, please see the websites of the vendors below: