The Ultrasensitive Luteinizing Hormone (LH) ELISA Gets a New Lease on Life

In 2013, Dr. Derik Steyn and colleagues published, in Endocrinology, the development of an ultrasensitive luteinizing hormone (LH) enzyme-linked immunosorbent assay (ELISA) (1). Although many hormone assays have been reported in Endocrinology articles in the past, this one was different and had an immediate and significant impact on the field, especially for those working with mice. While the power and versatility of genetic manipulation has made the mouse an invaluable model system in neuroendocrinology and other disciplines, their small size can and does create obstacles. This was certainly the case for the accurate measurement of LH in murine circulation prior to the development of the Steyn assay. LH is secreted from pituitary gonadotrope cells in pulses. These pulses are driven by pulsatile gonadotropin-releasing hormone (GnRH) from the brain. Whereas GnRH pulses can be measured at the level of the median eminence, this is an invasive procedure, which has limited its broad implementation (2). As LH pulses faithfully recapitulate GnRH pulses, the former has become a surrogate for the latter (3). That is, in both the clinic and the research lab, LH pulses are used to indirectly measure GnRH pulses. In larger animals, including humans, it is trivial to take repeated blood samples (eg, every 5-15 minutes) over extended periods of time (eg, up to 24 hours), a prerequisite for precisely measuring episodic LH secretion. Whereas repeated blood sampling is possible in some rodents, such as rats, the method has proven more problematic in mice, in light of their substantially lower blood volumes. This all changed with the Steyn assay, which has remarkable sensitivity, enabling accurate measurement of LH in as few as 2 to 4 µL of whole blood in mice. This has made it possible to collect blood samples every 3 to 10 minutes for up to 6 hours, providing great precision on the nature of LH release in individual animals (4). The assay has also proven quite versatile in detecting LH in various sample preparations, including serum, plasma, whole blood, pituitary protein lysates, and media from cultured pituitaries or cell lines (4, 5). Since its publication, the Steyn LH assay has been implemented around the world. Its ease of use and low cost have enabled investigators to establish the assay in their own labs. The Ligand Assay Core at the University of Virginia also provides it as an affordable service. The Steyn et al paper has been cited more than 170 times, but the assay has undoubtedly been more broadly implemented. Some of the most impactful research in reproductive neuroendocrinology over the past 10 years has used the Steyn assay. This includes, but is not limited to, seminal studies demonstrating that kisspeptin neurons in the arcuate nucleus form a critical part of the GnRH pulse generator, if not the pulse generator itself (6). The assay has also been instrumental in determining how metabolic and stress signals regulate fertility (7). The Steyn assay is a sandwich ELISA, which relies on a mouse monoclonal anti-bovine LHβ subunit capture antibody (518B7) and a rabbit polyclonal anti-rat LH antiserum for detection (AFP240580Rb). The latter was provided by Dr. Al Parlow, the founder of the National Hormone Pituitary Program. Sadly, Dr. Parlow passed away in 2021. As a result, the detection antiserum (as well as other valued reagents from the National Hormone Pituitary Program) are no longer available or not easily acquired. This development threatened the viability of the Steyn assay and the important discoveries it enables. Fortunately, as recently reported in Endocrinology, a replacement LH assay has been developed and validated by Dr. Kellie Breen and colleagues (8) (hereafter Breen assay). The major innovation in the Breen assay is the substitution of the Parlow rabbit LH antiserum with a mouse monoclonal antibody directed against human LH (5303 SPRN-5). Therefore, the Breen assay uses distinct monoclonal antibodies for both capture and detection. This approach has both positive and negative features, although the latter are easily addressed. On the positive side, monoclonal antibodies are renewable, making it unlikely that the loss of a critical reagent will challenge this assay in the future (provided the hybridomas remain available for academic or commercial antibody production). A second positive is that the Breen assay is more sensitive than the Steyn assay, making analyses of low LH levels even more reliable. The Breen assay shows similar versatility to the Steyn assay, measuring LH from a variety of sample sources. Although LH was not measured in plasma, the ability of the assay to detect LH in whole blood and serum suggests that plasma will not be a challenge. Indeed, absolute LH values in whole blood and serum were more similar in the Breen assay than in the Steyn assay, and LH could be measured reliably from multiple routes, including from the submandibular vein, which has been problematic with the Steyn assay in our experience. Absolute LH values with the Breen assay are slightly lower than in the Steyn assay, although the results from the 2 are highly correlated. Still, some care should be taken if comparing LH values between the assays. Because the Breen assay relies on 2 mouse monoclonal antibodies, a horseradish peroxidase–conjugated secondary antibody cannot be used with the detection antibody as in the Steyn assay. Instead, the detection antibody is biotinylated, enabling its revelation using horseradish peroxidase–conjugated streptavidin. As a result, investigators should measure the efficiency of the antibody biotinylation to enable comparisons between their assays over time. Breen and colleagues employed their assay in a variety of experimental contexts, including comparing males and females, intact vs gonadectomized mice, LH pulsatility in response to restraint stress, and LH release following kisspeptin treatment. The assay stood up to each challenge. Therefore, investigators can confidently rely on this new ultrasensitive LH ELISA in their own mouse work, regardless of the nature of their experiments. The Steyn assay will no doubt remain in use for several years as investigators or core facilities employ the original Parlow antiserum they may still have on hand. Nevertheless, the field can be confident that as this resource is exhausted, a robust replacement assay will be waiting in the wings. Supported by Canadian Institutes of Health Research Project Grants PJT-162343 and -169184 to DJB. The authors have nothing to disclose. Data sharing is not applicable to this article as no data sets were generated or analyzed during the current study.

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