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.

 

Multiplex Assays for Rat/Mouse LH and FSH

The development of protein microarrays have allowed for the measurement of several biomarkers within a single sample.  These Multiplex systems utilize combinations of antibody-coupled color-coated microspheres and fluorescent-tagged secondary antibodies within a single microtiter plate well.  The system uses a liquid suspension array of up to 100 sets of 5 um beads, each internally dyed with different ratios of two spectrally distinct fluorophores to assign it a unique spectral address.  Each set of beads can be conjugated with different capture antibodies; the beads are mixed and incubated with sample in a microplate well to allow binding to specific anlytes.  To detect and quantitate each analyte, a fluorescently labeled reporter molecule that specifically binds the analyte is added.  Following incubation, the contents of each microplate well are automically drawn into a flow-cytometer, and the beads are aligned in single file where 2 lasers excites the beads individually.  The red laser excites the dyes in each bead, identifying the spectral address for each analyte.  The green laser excites the reporter molecule associated with the bead, which allows quantification of each analyte.  System software records the fluorescent signals simultaneously for each bead, translating the signals into quantitative data, based on standard curves included within each microplate.  The Ligand Core uses Millipore Pituitary Panel Multiplex kits to measure LH and FSH in rat and mouse serum samples (25 ul per singlet determination).


COMMERCIALLY PREPARED KIT REFERENCES:

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