Researchers have trusted Cayman’s collection of steroid hormone ELISAs for decades to study effects in metabolism, inflammation, immune function, osmoregulation, sexual development, and stress responses. Designed with competitive assay principles, each kit offers highly sensitive detection in the low pg/ml range, reliable and reproducible results, and flexible use with multiple sample types. These kits are backed by the guidance of Cayman’s knowledgeable technical support scientists and in-house assay development team. Inevitably during an experiment, questions come up about the appropriate use of an assay. Here are some answers to the most frequently asked questions about our steroid hormone assays.

Species Specificity

Will this assay work in ________ (insert species) samples?

Steroid hormones are organic molecules with the same structure regardless of the species. If the sample has high enough levels of the analyte (above the sensitivity limit listed in the kit booklet) and is free of interferents, analyte should be detected by the assay. Some instances of analyte detection may be state dependent. For example, estradiol levels in most rodent samples are extremely low, rising to detectable levels only during the pro-estrus period and in pregnant females.

Cayman’s steroid ELISA kits use antibodies (e.g., goat anti-mouse, mouse anti-rabbit, rabbit anti-sheep) immobilized on the plate, so samples containing IgG from the antigen species may interfere with these assays. For example, a rabbit sample containing IgG may interfere with an assay using a mouse anti-rabbit antibody on the plate. Removal of the IgG prior to performing the assay by precipitating the protein or by other extraction methods is recommended.

Total versus Free Steroid Hormone

Does this kit measure total or free __________ (insert steroid hormone)?

Steroids can be associated with protein or are glucuronidated in some samples. To ensure measurement of total non-glucuronidated steroid (or total parent steroid), we recommend following the extraction protocol in the kit booklet provided with the assay. Several different protocols are suggested, depending on the steroid to be analyzed. This includes performing extractions using methanol, chloroform, ethyl acetate, hexane, diethyl ether, acetone, and/or methylene chloride along with the option of using a tritium-labeled steroid that corresponds to your analyte of interest to trace sample recovery. You may use a different protocol based on your own requirements, sample type, and expertise.

A set of kits to measure certain glucuronidated steroids in urine is also available. Levels of these glucuronidated steroid hormones correlate highly with levels of the parent hormone measured in serum. Analysis of glucuronidated metabolites offers an advantageous, non-invasive alternative for indirect measurement of hormone levels and accordingly may be useful for biomarker detection in certain biological or pathological activities.

Glucuronidated Steroid Hormone ELISAs

Estrone-3-Glucuronide ELISA Kit

Pregnanediol-3-Glucuronide (PDG) ELISA Kit

Testosterone Glucuronide ELISA Kit

Sensitivity

What is the sensitivity of these assays?

Cayman’s steroid assays were developed to give the best sensitivity possible, while maintaining good signal-to-noise ratio. The assay range and sensitivity of each assay is listed in the kit booklet and can be viewed in the product tables below. Low concentrations of steroid hormones are often physiologically relevant, and variable concentration ranges of steroid hormones are typically seen depending on the sample in question. Cayman’s steroid assays report pg/ml or ng/ml levels of sensitivity and support a wide dynamic range to accommodate these factors. For example, serum levels of testosterone range from <70-200 pg/ml in women to <70-12,000 pg/ml in men, depending on age. Cayman’s Testosterone ELISA Kit offers an assay range of 3.9-500 pg/ml and a sensitivity of 6 pg/ml. In the infographic below, the range of concentrations of physiological steroid hormones in human serum reported by the Mayo Clinic is compared to the assay range and sensitivity of Cayman’s steroid hormone assay kits. Note that these ranges are offered as a guide only. Each laboratory should establish their own range for their experimental variables.

Cayman’s assay kits undergo rigorous quality testing to deliver the sensitivity and specificity needed to detect biologically significant analyte levels. Sensitivity is defined as the 80% B/B₀* point on the standard curve. In general, this corresponds to the Lower Limit of Quantitation (LLOQ). 80% binding is the minimum level where reliable, reproducible data can be obtained and represents the minimal functional sensitivity outside of the range of statistical bias. This point of quantitation can be trusted to give you reliable, reproducible data. Cayman also provides researchers the Lower Limit of Detection (LLOD) along with the assay’s high-low range and 50% B/B0 mid-point (the direct center of the most reliable portion of the curve) to further qualify the ELISA’s robustness. Several of our competitors define sensitivity by the LLOD, which is usually considered the mean zero plus two standard deviations. While this is the lowest level of analyte detected by the kit, this range is typically not reliable nor reproducible and may reside at a concentration below the linear range of the assay. This value typically lies below an assay’s functional sensitivity range.

*%B/B₀ - (%Bound/Maximum Bound): ratio of the absorbance of a particular sample or standard well to that of the maximum binding (B₀) well.

Sample Competitive ELISA Standard Curve

Plasma versus Serum

Which should I use, plasma or serum?

For most steroid hormones, their levels in plasma and serum should be roughly equivalent. The clotting process does not significantly influence steroid levels. However, there is a large body of literature reporting serum levels of these analytes, demonstrating serum is a commonly used measure.

Saliva Samples

Will this assay work for saliva samples?

In general, steroids are present at lower concentrations in saliva than in the blood. If the sample has high enough levels of the analyte (above the sensitivity limit listed in the kit booklet) and is free of interferents, analyte should be detected by the assay. Saliva samples are relatively clean samples but may require concentration and purification to eliminate interferents.

Fecal Samples

Will this assay work for fecal samples?

Though they can be measured in serum and other sample matrices, biologists often measure stress-induced glucocorticoids (i.e., cortisol, corticosterone) in fecal material of the species they are studying. This non-invasive sample collection procedure has the advantage of not causing stress and thereby interfering with experimental results. Though urine and saliva have also been explored as alternative non-invasive methods, fecal sampling is the most widely used. Cayman’s glucocorticoid assays have been validated for measurement in serum, feces, and other sample matrices.

Steroid Hormone Assay Kits Available from Cayman

Adrenal Androgens & Androgen Precursor

Estrogens