Parallelism data were analyzed by slope equality test, precision and accuracy calculated by % CV of intra-and inter-assay, and % recovery, respectively. Five treatments were performed to test delayed to centrifugation time: blood centrifuged at 1 h (control, P1), 6 h (P6), 12 h (P12), 18 h (P18), and 24 h (P24) after collection. Validation test of EIA-1887 kit was performed through: a) analytical (parallelism, accuracy, and precision tests), and b) biological validations (measuring cortisol concentrations before and after transportation). Blood was collected from eight kacang goats. This study aimed to evaluate the validity of human cortisol ELISA kit (EIA-1887, DRG Instruments GmbH, Germany) for cortisol measurement and effect of delayed to blood centrifugation time on cortisol concentrations in kacang goats. The use of human cortisol ELISA kit for measuring cortisol in animals and delayed to blood centrifugation time for hormone measurement need to be evaluated. (Evaluation the Validity of Human Cortisol Enzyme-Linked Immunosorbent Assay (ELISA) Kit and Centrifugation Time of Blood Sample for Measuring the Concentration of Cortisol in Kacang Goats)ĪBSTRACT. Keterlambatan waktu sentrifugasi selama 24 jam berpengaruh terhadap konsentrasi kortisol. Kesimpulan, human cortisol ELISA kit (EIA-1887) memiliki validitas yang baik secara analitik dan biologis untuk digunakan dalam pengukuran konsentrasi kortisol pada kambing kacang. Adanya penurunan secara nyata konsentrasi kortisol pada darah yang disentrifugasi 24 jam (P24) setelah koleksi (p0,05). Konsentrasi kortisol setelah transportasi secara signifikan lebih tinggi daripada sebelum transportasi (p0,05). Akurasi kit EIA-1887 adalah 103,43☗,85%, dan % CV intra-assay dan inter-assay adalah 10%. Hasil uji parallelism menunjukkan kurva sampel kambing kacang sejajar/parallel dengan kurva standar kortisol. Data uji parallelism dianalisis dengan uji persamaan kemiringan, uji presisi dihitung % CV (coefficient variation) intra-assay dan inter-assay, uji akurasi dihitung % recovery, uji T untuk validasi biologis, dan uji ragam (One Way Anova) untuk pengaruh waktu sentrifugasi. Uji keterlambatan waktu sentrifugasi terhadap stabilitas konsentrasi kortisol dilakukan dengan 5 perlakuan yaitu disentrifugasi kurang dari 1 jam (P1/kontrol), 6 jam (P6), 12 jam (P12), 18 jam (P18),dan 24 jam (P24) setelah darah dikoleksi. Uji validitas kit EIA-1887 dilakukan secara: a) analitik (uji parallelism, akurasi, dan presisi), dan b) biologis (pengukuran kortisol sebelum dan setelah transportasi). Sampel darah dikoleksi dari delapan ekor kambing kacang. Penelitian ini bertujuan untuk mengevaluasi validitas human cortisol ELISA kit (EIA-1887, DRG Instruments GmbH, Germany) untuk pengukuran konsentrasi kortisol dan menguji pengaruh keterlambatan waktu sentrifugasi sampel darah terhadap stabilitas konsentrasi kortisol pada kambing kacang. Penggunaan human cortisol enzyme-linked immunosorbent assay (ELISA) kit untuk pengukuran hormon kortisol pada hewan dan keterlambatan waktu sentrifugasi sampel darah untuk analisis hormon perlu dievaluasi.
Plasma cortisol concentrations in Aceh cows show a diurnal variation which is higher in the morning than afternoon, but the concentration of cortisol in the fecal sample did not.ĪBSTRAK. In conclusion, ELISA Kit (EIA-K003H5, Arbor Assays ® ) is a reliable assay for measuring cortisol in the feces of Aceh cows. The results from the biological validation test showed that the concentrations of fecal cortisol post-transportation were significantly higher compared to the pre-transportation (P0.05). The results of a parallelism test showed the diluted sample curve from the fecal extract was not significantly different (parallel) to the standard curve of EIA-K003-H5 kits (P>0.05), the accuracy of the assay: 99,76 ± 3.77%, and the % CV of intra-and inter-assay less than 10%.
EIA-K003-H5, Arbor Assays ® ) was validated using analytical (parallelism, accuracy, and precision/% CV of intraand inter-assay) and biological validations (by analyzing sample preand post-transportation).
Blood and fecal samples were collected in the morning and afternoon from 8 Aceh cows aged 2-3 years. This study aims to validate a commercial ELISA kit and evaluate the sampling time on the diurnal variations of cortisol concentration in blood and feces of Aceh cattle. The time of sample collection is also another important parameter needed to be evaluated due to a known diurnal variation of cortisol concentrations. The most important one is to test the validity of the ELISA kit itself since most of the commercially available ELISA kit is not designed for animal use. Some parameters should be evaluated before the analysis of cortisol hormone using enzyme-linked immunosorbent assay ( ELISA ).
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