MEASUREMENT OF SERUM TESTOSTERONE IN KACANG GOAT BYUSING ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TECHNIQUE: THE IMPORTANCE OF KIT VALIDATION (Pengukuran Testosteron Serum Kambing Kacang dengan Teknik Enzyme-Linked Immunosorbent Assay (ELISA): Pentingnya Validasi Kit)

Abstract

This study was conducted to validate a commercial testosterone enzyme-linked immunosorbent assay (ELISA) kits (DRG EIA-1559) inanalytic and biological manner for measuring serum testosterone concentrations in kacang goats. This study used 18 healthy kacang goats, sixbucks (>2 years), six kids (<6 months), and six does (>2 years). Blood samples were collected from jugular vein and prepared as serum. Twovalidation tests were performed, an analytical validation comprises a parallelism, accuracy, precision and sensitivity and a biological validationby comparing testosterone concentration from bucks, kids, and does. Testosterone concentrations were measured using ELISA technique. Data ofanalytical validation were analyzed descriptively and test of equality of slope was performed to see the parallelism between samples and standardcurves. Analysis of variance (ANOVA) was used for biological validation data. Results of parallelism showed that sample curve was parallel tothe standard curve. Accuracy, precision (% CV of intra-and inter-assay) and sensitivity of the assay were 99.65±4.27%, <10%, <15% and 0.083ng/ml, respectively. Results of biological validation showed that the assay used were accurately measured testosterone which testosteroneconcentrations in bucks were significantly higher compared to kids and does (P<0.05). In conclusion, a commercial testosterone ELISA kits(DRG EIA-1559) is a reliable assay for measuring serum testosterone concentration in kacang goats. Key words: analytical and biological validations, ELISA, testosterone, kacang goat

The validation of a commercial enzyme-linked immunosorbent assay and the effect of freeze-thaw cycles of serum on the stability of cortisol and testosterone concentrations in Aceh cattle

“…EIA-1559) ELISA kits produced by DRG Instruments GmbH, Germany, were evaluated using analytical and biological validations. The procedure for assay validation was conducted as described by Rangel-Negrin et al 14 and Gholib et al 4 . Briefly, the analytical validation was comprised of parallelism (i.e.…”

“…Steroid hormones can be measured by an enzyme-linked immunosorbent assay (ELISA) technique using various samples (e.g., plasma or serum 4 , urine 5 , feces 6,7 , saliva 8,9 , and hair 10 ). This technique is now widely used, as it is simple, rapid, convenient, relatively inexpensive, requires a lower sample volume, and particularly, as it is free of radioisotope waste 11,12 .…”

“…In order to reliably measure C and T concentrations in cattle using a human commercial ELISA kit, the ELISA kit needs to be validated analytically, physiologically or biologically 4,14 . Analytical validation can be performed by examining the specificity (cross-reactions), sensitivity, precision, and parallelism (linearity) of the ELISA kit 1,4 .…”

“…In order to reliably measure C and T concentrations in cattle using a human commercial ELISA kit, the ELISA kit needs to be validated analytically, physiologically or biologically 4,14 . Analytical validation can be performed by examining the specificity (cross-reactions), sensitivity, precision, and parallelism (linearity) of the ELISA kit 1,4 . In addition, it is crucial to perform the biological validation of the assay to examine the ability of the assay to differentiate the variation of the hormone concentration based on the physiological conditions of the animals 14 .…”

“…In addition, it is crucial to perform the biological validation of the assay to examine the ability of the assay to differentiate the variation of the hormone concentration based on the physiological conditions of the animals 14 . The biological validation of T measurements can be achieved by comparing the concentrations of T from individuals of different age (juvenile versus adult), or sex (male versus female) classes 4 . For C measurements, the comparison of C concentrations of the same animal before and after some known stressful events (eg., capture, translocation, transportation, and agonistic interactions) can be used as a biological validation procedure 14 .…”