Assay Methods
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; RRID:AB_278503) as described previous (2). The tracer antibody, (no. 518B7; RRID:AB_2784498) 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.
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; RRID:AB_278501 ) diluted to a final concentration of 1:400,000 is used as primary antibody. The secondary antibody is purchased from Equitech-Bio, Inc. (RRID:AB_2784502) 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.
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 (20 ul per singlet determination).
The Ligand Core implemented an In-House Ultra-Sensitive Mouse and Rat FSH ELISA. The method is based on a recent manuscript published by Orgaro et al (1). This assay allows for a FSH measurement in 5ul of serum or plasma [2.5ul for rodent samples expected to have elevated FSH (e.g. gonadectomy, post-GnRH)]; see sample processing instructions below. Whole blood is not acceptable, as the method validation study showed assay interference using whole blood (1). The capture polyclonal antibody (guinea pig anti-mouse FSH; AFP-1760191; RRID: AB_2665512) is provided by the National Hormone and Peptide Program (NHPP). The detection polyclonal antibody (rabbit anti-rat FSH-S11; AFP-C0972881; RRID: AB_2687903) is also provided by the NHPP. The HRP-conjugated polyclonal antibody (donkey anti-rabbit, Cat #AP182P; RRID: AB_92591) is purchased from EMD Millipore, Temecula, CA. The Mouse FSH reference preparation (mFSH-RP, AFP5308D; NHPP) is used as the assay standard.
(1) Ongaro L, Alonso CAI, Zhou X, Brule E, Li Y, Schang G, Parlow AF, Steyn FJ, Bernard DJ. (2021) Development of a highly sensitive ELISA for measurement of FSH in serum, plasma and whole blood in mice. Endocrinology 162, PMC7894055.
Method Correlation
| MOUSE | RAT | |
| ELISA / RIA | 52% | 54% |
| ELISA / Multiplex | 49% | 40% |
Sample Preparation – FSH in Diluted Serum or Plasma
This method is designed to measure FSH in diluted serum or plasma (whole blood is not acceptable). Samples (serum, plasma) must be pre-diluted (1:10 or 1:20 – if FSH is expected to be elevated) in assay buffer (see recipe below) prior to shipment (for 1:10 dilution singlet measurement – 6ul serum or plasma plus 54ul assay buffer; for duplicate measurement – 12ul serum or plasma plus 108ul assay buffer; for 1:20 dilution singlet measurement – 3ul serum or plasma plus 57ul assay buffer; for duplicate measurement – 6ul serum or plasma plus 114ul assay buffer). Samples must be stored and shipped frozen.
Formula for Ultra-Sensitive Mouse & Rat FSH Assay Buffer (0.2% BSA – 0.05% Tween 20 – PBS; pH 7.5)
For 1 Liter PBS
- 2 grams Bovine Serum Albumin (BSA)
- 500ul Tween 20
Method Description for Publication
The Ultra-Sensitive Mouse & Rat FSH ELISA is an in-house method based on a manuscript published by Ongaro et al (1). This assay allows for a FSH measurement in 2.5 – 5ul of serum or plasma. The capture polyclonal antibody (guinea pig anti-mouse FSH; AFP-1760191; RRID: AB_2665512) is provided by the National Hormone and Peptide Program (NHPP). The detection polyclonal antibody (rabbit anti-rat FSH-S11; AFP-C0972881; RRID: AB_2687903) is also provided by the NHPP. The HRP-conjugated polyclonal antibody (donkey anti-rabbit, Cat #AP182P; RRID: AB_92591) is purchased from EMD Millipore, Temecula, CA. The Mouse FSH reference preparation (mFSH-RP, AFP5308D; NHPP) is used as the assay standard. The Limit of Quantitation (Functional Sensitivity) is defined as the lowest concentration that demonstrates accuracy within 20% of expected values and intra-assay coefficient of variation (%CV) <20%, and was determined by serial dilutions of a defined sample pool. Functional sensitivity is 0.016 ng/ml. Intra-assay %CV is 3.6%. Inter-assay %CVs are 5.1% (Low QC, 0.265 ng/ml), 7.8% (Medium QC, 0.693 ng/ml) and 4.2% (High QC, 1.958 ng/ml).
(1) Ongaro L, Alonso CAI, Zhou X, Brule E, Li Y, Schang G, Parlow AF, Steyn FJ, Bernard DJ. (2021) Development of a highly sensitive ELISA for measurement of FSH in serum, plasma and whole blood in mice. Endocrinology 162, PMC7894055.
The Ligand Core implemented an In-House Ultra-Sensitive Mouse and Rat LH ELISA. The method is based on a manuscript published by Steyn et al (1). This assay will allow for a LH measurement in 2.5 or 5ul of serum, plasma or whole blood (our standard LH assay requires 50ul of serum or plasma); see sample preparation instructions below. The capture monoclonal antibody (anti-bovine LH beta subunit, 518B7; RRID:AB_2784498) is provided by Janet Roser, University of California. The detection polyclonal antibody (rabbit LH antiserum, AFP240580Rb; RRID:AB_2784499) is provided by the National Hormone and Peptide Program (NHPP). HRP-conjugated polyclonal antibody (goat anti-rabbit) is purchased from DakoCytomation (D048701-2; RRID:AB_2784500). Mouse LH reference prep (AFP5306A; NHPP) is used as the assay standard.
Sample Preparation – LH in Diluted Serum, Plasma, or Whole Blood
This method is designed to measure LH in diluted serum, plasma or whole blood samples. Samples (serum, plasma, whole blood) must be pre-diluted (1:10) in assay buffer (see recipe below) prior to(1:10 or 1:20 – if FSH is expected to be elevated) in assay buffer (see recipe below) prior to shipment (for 1:10 dilution singlet measurement – 6ul serum or plasma plus 54ul assay buffer; for duplicate measurement – 12ul serum or plasma plus 108ul assay buffer; for 1:20 dilution singlet measurement – 3ul serum or plasma plus 57ul assay buffer; for duplicate measurement – 6ul serum or plasma plus 114ul assay buffer). Whole blood samples MUST be diluted in assay buffer, immediately following collection.
Formula for Ultra-Sensitive Mouse & Rat LH Assay Buffer (0.2% BSA – 0.05% Tween 20 – PBS, pH 7.5)
For 1 Liter PBS: 2 grams Bovine Serum Albumin (BSA)
500ul Tween 20
Mouse and Rat LH Assays: Method Correlation Data
ELISA/Multiplex
Multiplex/RIA
141%
153%
Method Description for Publication
The Ultra-Sensitive Mouse & Rat LH ELISA is an in-house method based on a manuscript published by Steyn et al (1). This assay allows for a LH measurement in 2.5 – 5ul of serum, plasma or whole blood. The capture monoclonal antibody (anti-bovine LH beta subunit, 518B7; RRID:AB_2784498) is provided by Janet Roser, University of California. The detection polyclonal antibody (rabbit LH antiserum, AFP240580Rb; RRID:AB_2784499) is provided by the National Hormone and Peptide Program (NHPP). HRP-conjugated polyclonal antibody (goat anti-rabbit) is purchased from DakoCytomation (Glostrup, Denmark; D048701-2; RRID:AB_2784500). Mouse LH reference prep (AFP5306A; NHPP) is used as the assay standard. The Limit of Quantitation (Functional Sensitivity) is defined as the lowest concentration that demonstrates accuracy within 20% of expected values and intra-assay coefficient of variation (%CV) <20%, and was determined by serial dilutions of a defined sample pool. Intra-assay %CV is 2.2%. Inter-assay %CVs are 7.3% (Low QC, 0.13 ng/ml), 5.0% (Medium QC, 0.8 ng/ml) and 6.5% (High QC, 2.3 ng/ml). Functional sensitivity is 0.016 ng/ml.
(1) Steyn FJ, Wan Y, Clarkson J, Veldhuis JD, Herbison AE, Chen C. (2013) Development of a methodology for and assessment of pulsatile LH secretion in juvenile and adult male mice. Endocrinology 154:4939-45.
For information about the commercially available kits we use for our assays, please click on the “View Available Kits” link below.
The inclusive listing of manufacturers below:
- American Laboratory Products Co (ALPCO)
- Analox Instruments USA
- ANSH Labs
- BMA Biomedicals
- EMD Millipore Corporation
- Immuno-Biological Laboratories Inc (IBL)
- MP Biomedicals LLC
- Siemens Healthcare Diagnostics – Immulite
For % CV data, please refer to our 2023 % 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.