This kit is a competitive enzyme immunoassay designed specifically for the quantitative measurement of testosterone in rat and mouse serum or EDTA plasma. It is intended strictly for Research Use Only (RUO) and is not for use in diagnostic procedures.

The procedure is optimized for preclinical models where sample volume is limited, requiring only 10µl of matrix (serum or EDTA plasma) per well.

The lowest analytical detectable level of testosterone that can be distinguished from the Zero Calibrator is 0.024 ng/ml at the 2SD confidence limit.

The kit shows high specificity for testosterone. The most significant cross-reactivity is with 5𝛼-Dihydrotestosterone (56.7%)5. It shows minimal cross-reactivity ($<0.1\%$) with other common steroids such as Estradiol, Cortisol, Progesterone, and Estrone.

Based on studies of healthy, untreated animals, expected ranges include: Male Rat Serum: 1.81 – 11.59 ng/mL Female Rat Serum: 0.57 – 1.42 ng/mL Male Mouse Serum: 0.44 – 21.36 ng/mL Female Mouse Serum: 0.09– 0.47 ng/mL

The total incubation time is 90 minutes, divided into two stages: 1) Conjugate Incubation: 60 minutes at room temperature on a microplate shaker (900 rpm) 2) Substrate Incubation: 30 minutes in the dark without shaking13.

If samples are expected to exceed the highest calibrator (25ng/mL), they should be diluted with Standard A (Calibrator 0) before being assayed. The dilution factor must be applied to the final result calculation.

Unopened reagents should be stored at 2 – 8°C. Once opened, reagents remain stable for 30 days if stored properly at the same temperature range.

Unlike general assays, this ELISA is specifically validated for the unique hormonal profiles of rodents, including considerations for brain sexual dimorphism and Leydig cell differentiation studies. The kit includes 96 break-apart wells, ready-to-use standards, and a 4-Parameter Logistics (4 PL) curve fit recommendation for the most accurate data reduction.