Hormone assays

Ideally, hormone assays should be evaluated in animal models where effects with test compounds or surgical removal have been described sufficiently well by other investigators. Alternatively, evidence from histopathology examinations can be correlated with hormone measurements in studies where there are marked effects on the target organ and relevant hormone. 

Antibodies developed for hormone assays can be monoclonal antibodies or polyclonal antibodies there is a greater degree of nonspecificity for these polyclonal 

As the molecular masses and amino acid sequences of the protein hormones increase, the cross-reactivity of antisera in the different species becomes more problematic, and homologous antisera are required (Miller and Valdes 1992 Valdes and Miller 1992). Several homologous reagents are commercially available (e.g., prolactin, thyrotrophin, luteotrophin, and follitrophin for rats, and canine thyrotrophin). Some very specialized centers are able to produce suitable antisera given purified hormones, but this is a time-consuming process and the majority of toxicology laboratories use antisera from external suppliers who often can provide information on cross-reactivity. 

Immunoassays are subject to some effects and interferences unique to immunoassay and some common to all chemical assays (Wu 2000). Establishment of an assay should include some assessment of antibody interference, signal interference, and matrix and hook effects. Interference may be caused by cross-reactivity or by an endogenous metabolite (e.g., in steroid hormone immunoassays) or a xenobi-otic (or metabolite) with a similar structure this problem occurs more frequently 

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It is sometimes necessary to suppress the production of ACTH to identify the source of a particular hormone or to establish whether its production is influenced by the secretion of ACTH. In these circumstances, it is advantageous to use a very potent substance such as dexamethasone because the use of small quantities reduces the possibility of confusion in the interpretation of hormone assays in blood or urine. For example, if complete suppression is achieved by the use of 50 mg of cortisol, the urinary 17-hydroxycorticosteroids will be 15-18 mg/24 h, since one third of the dose given will be recovered in urine as 17-hydroxycorticosteroid. If an equivalent dose of 1.5 mg of dexamethasone is used, the urinary excretion will be only 0.5 mg/24 h and blood levels will be low. 

Peas are a low-fat plant, and it is likely, therefore, that our results should not be freely extended to all other auxin- and gibberellin-induced systems. In fact, the equally classic oat coleoptile section hormone assay does not seem to respond to the fatty substances. Even the pea section fails to respond, unless the seedling has received some red light during the early part of its development. 

Nuclear Reprogramming Methods and Protocols, edited by Steve Pells, 2006 324 Hormone Assays in Biological Fluids, edited... 

Steroid hormones assay standardization Immunoassay vs. GC-MS and LC-MS/MS, validation of assay methodologies, establishing standard pools of steroid hormones in women and man, utilizing the pools for cross comparison of various methodologies. [18]... 

StanczykFZ, Lee JS, Santen RJ (2007) Standardization of steroid hormone assays why, how, and when Cancer Epidemiol Biomarkers Prev 16 1713-1719... 

Specific dissection is performed on the brain and pituitary gland. Hypothalamic fragments may be dissected and shock frozen immediately in liquid nitrogen, for later extraction and determination of hypothalamic peptides. The pituitary gland is dissected, the posterior pituitary is stored separately, and the anterior pituitary is halved by a median-sagittal cut to obtain separate tissue samples for histology (fixation), and for subsequent analysis of hormone contents (stored frozen at -20 °C until hormone assay). 

This section describes the measurement of pituitary hormones in the context of repeated dose studies of 1-4 weeks duration. When performing an endocrine safety pharmacology study in rats it is advisable to determine pituitary hormone concentrations at autopsy, because in this species specific pituitary hormone assays are readily available. The time point two hours after last dosing is in general suitable, when changes in pituitary hormone contents need to be assessed. 

For the steroid hormone assays, they are are many suppliers nowadays, an internet search for suitable reagents... 

DSL Diagnostic Systems Inc. (2005) Steroid hormone assays for clinical diagnostics and research applications. http //secure.dslabs.com/Products/ProdInfo.asp Lieberman BA (ed) (2001) Steroid receptor methods protocols and assays. Methods in molecular biology, vol 176. Humana Press, Totowa, NJ... 

In order to assess pituitary function, inclusion of the LHRH stimulation test for LH and FSH is recommended in male rats and female rats, and the mono-iodo-tyrosin (MIT) stimulation test for prolactin secretion is recommend at least for the female animals. Pituitary content of LH, FSH and prolactin should always be determined at autopsy (half of the pituitary for hormone assays and the other half for histology). 

Wheeler MJ, Morley Hutchinson JS (eds) (2005) Hormone assays in biological fluids, Methods in molecular biology, vol 324. Humana Press, Totowa, NJ Wittliff LJ, Raffelsberger W (1995) Mechanisms of signal transduction sex hormones, their receptors and clinical utility. J Clin Ligand Assay 18 211-235... 

Perry WF (1951) A method for measuring thyroid hormone secretion in the rat with its application to the bioassay of thyroid extracts. Endocrinology 48 643-650 Reineke EP, Turner CW (1950) Thyroidal substances. In Em-mens CW (ed) Hormone Assay, Chapter XIX. Academic Press Inc., New York, pp 489-511 Turner CW, Premachandra BN (1962) Thyroidal substances. In Dorfman RI (ed) Methods in Hormone Research, Vol II, Chapter 10. Academic Press, New York and London, pp 385-411... 

Fig. 1. Dose-response curve for amount of dextran-coated charcoal added versus percentage free and bound, adsorbed. These are theoretical curves drawn from generalizations derived from several assays. —, Curve showing binding of free ligand —, curve showing binding of antibody bound ligand. The exact amounts of charcoal giving a particular bound value vary with each assay (e.g., growth hormone assay differs from insulin assay). For significance of arrows A and B, see the section on selection of charcoal dose.

We consider here a hypothetical hormone assay, for which the manufacturer or a research laboratory wants to estimate the LoD, The default values a = P = 5% are used. It is supposed that the manufacturer has 10 samples available from patients lacking the hormone because of disease or pharmacological suppression. Ten measurements of each blank sample are performed on 10 different days to ensure that the total assay variation is reflected. Only nonnegative values are provided by the assay, and the distribution of the 100 blank measurements is skew (Figure 14-5). Thus LoB is estimated nonparametrically as the 95th percentile of the measurement distribution. The 95th percentile corresponds to the 95.5 ordered observation (= 100 x (95/100) + 0.5). The 95 and 96 observations have the values 0.0539 and 0.0548 U/L, respectively, Linear interpolation between the 95 and 96 observations yields an LoB estimate of 0.0544 U/L (= 0.0539 + 0.5 X (0.0548 - 0.0539)). 

Andress DL, Endres DB, Maloney NA. Comparison of parathyroid hormone assays with bone histomor-phometry in renal osteodystrophy. J Clin Endocrinol Metab 1986 63 1163-9. 

Malluche HH, Mawad H, Truba D, Monier-Faugere MC. Parathyroid hormone assays—evolution and revolutions in the care of dialysis patients. Chn Nephrol 2003 59 313-8. 

Loraine JA, BeU ET, eds. Hormone assays and their clinical application, 4th ed. Edinburgh Churchill Livingstone, 1976. 

Mikhail G. Hormone assays and the gynecologist. Fertil Steril 1976 27 229-37. 

Sealey J, Campbell G, Preibisz J. Hormone assays Renin, aldosterone, peripheral vein, and urinary assays. In Laragh J, Brenner B, eds. Hypertension pathophysiology, diagnosis, and management. New York Raven Press, 1990 1443-60,... 

FT4 assays based on direct equilibrium dialysis or ultrafiltration measure free hormone without the need for total hormone measurements. These methods are unaffected by either variations in serum binding proteins or thyroid hormone autoantibodies. However, IV heparin administration can cause spurious elevations m FT4 determined by these techniques as a consequence of in vitro generation of free fatty acids. Mean values obtained in euthyroid healthy subjects are reported to be slightly higher when using ultrafiltration methods than when using equilibrium dialysis. Analytical performance goals have been recommended for free thyroid hormone assays.When an FT4 assay... 

Gorden P, Weintraub B. Radioreceptor and other functional hormone assays. In Wilson JD, Foster DW,... 

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