Assay Methods

Center for Research in Reproduction
Ligand Assay and Analysis Core
Assay Methods

 

Validation of New Steroid Assay Methods: 

Please review the attached document for discontinuing and replacement methods as based on the recommendations of Endocrine Society’s Sex Steroid Assays Reporting Task Force.

Steroid Method Validation  (Click to access document)

Mouse and Rat LH Sandwich Assay (MRLHS):

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.

Mouse and Rat FSH RIA (MRFSH):

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).

Ultra-Sensitive Mouse and Rat LH ELISA:

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) is provided by Janet Roser, University of California. The detection polyclonal antibody (rabbit LH antiserum, AFP240580Rb) is provided by the National Hormone and Peptide Program (NHPP). HRP-conjugated polyclonal antibody (goat anti-rabbit) is purchased from DakoCytomation (D048701-2). Mouse LH reference prep (AFP5306A; NHPP) is used as the assay standard.

Mouse and Rat LH Assays: Method Correlation Data

ELISA/RIA

ELISA/Multiplex

Multiplex/RIA

216%

141%

153%


Sample Preparation
(1) Steyn FJ, et al. Development of a methodology for and assessment of pulsatile LH secretion in juvenile and adult male mice. Endocrinology (2013) 154:4939-45.

This method is designed to measure LH in diluted serum, plasma or whole blood samples. Samples (serum, plasma, whole blood) must be pre-diluted [standard protocol is 1:10; if LH is expected to run high (e.g. gonadectomized animals) dilute samples 1:20] in assay buffer (see recipe below) prior to shipment:

–       for X10 singlet measurement – 6ul serum, plasma or whole blood plus 54ul assay buffer; for duplicate measurement – 12ul serum, plasma or whole blood plus 108ul assay buffer.

–       for X20 singlet measurement – 3ul serum, plasma or whole blood plus 57ul assay buffer; for duplicate measurement – 6ul serum, plasma or whole blood plus 114ul assay buffer.

After diluting samples, store and ship frozen. 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)

For 1 Liter PBS

–       2 grams Bovine Serum Albumin (BSA)
–       500ul Tween 20

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) is provided by Janet Roser, University of California. The detection polyclonal antibody (rabbit LH antiserum, AFP240580Rb) 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). 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.

 

COMMERCIALLY AVAILABLE KITS

For information about the commercially available kits we use for our assays, please see the attached document.  The inclusive listing of manufacturers below:

  • American Laboratory Products Co (ALPCO)
  • Analox Instruments USA
  • ANSH Labs
  • Beckman Coulter Inc
  • Calbiotech Inc
  • Eagle Biosciences Inc
  • EMD Millipore Corporation
  • Immuno-Biological Laboratories Inc (IBL)
  • MP Biomedicals LLC
  • Peninsula Laboratories Inc
  • R & D Systems Inc
  • Siemens Healthcare Diagnostics – Immulite
  • Wako Life Sciences Inc
  • Wilburn Medical USA

For % CV data, please refer to our 2019 % 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.