Evaluasi Validitas Human Cortisol Enzyme-Linked Immunosorbent Assay (ELISA) Kit dan Waktu Sentrifugasi Sampel Darah untuk Pengukuran Konsentrasi Hormon Kortisol pada Kambing Kacang

Gholib Gholib, Sri Wahyuni, Rahma Melinda, Muslim Akmal

Abstract


ABSTRAK. 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. 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. Sampel darah dikoleksi dari delapan ekor kambing kacang. Uji validitas kit EIA-1887 dilakukan secara: a) analitik (uji parallelism, akurasi, dan presisi), dan b) biologis (pengukuran kortisol sebelum dan setelah transportasi). 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. 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. Hasil uji parallelism menunjukkan kurva sampel kambing kacang sejajar/parallel dengan kurva standar kortisol. Akurasi kit EIA-1887 adalah 103,43±7,85%, dan % CV intra-assay dan inter-assay adalah 10%. Konsentrasi kortisol setelah transportasi secara signifikan lebih tinggi daripada sebelum transportasi (p<0,05). Adanya penurunan secara nyata konsentrasi kortisol pada darah yang disentrifugasi 24 jam (P24) setelah koleksi (p<0,05). Kesimpulan, human cortisol ELISA kit (EIA-1887) memiliki validitas yang baik secara analitik dan biologis untuk digunakan dalam pengukuran konsentrasi kortisol pada kambing kacang. Keterlambatan waktu sentrifugasi selama 24 jam berpengaruh terhadap konsentrasi kortisol.

 

(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)

 

ABSTRACT. 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. 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. Blood was collected from eight kacang goats. 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). 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. Parallelism data were analyzed by slope equality test, precision and accuracy calculated by % CV of intra-and inter-assay, and % recovery, respectively. Data of biological validation and centrifugation time effects were analyzed by Student t-test, and one way ANOVA, respectively. Results of parallelism showed that serial dilution curve of kacang goat plasma was parallel with cortisol standard curves. Accuracy of EIA-1887 kit was 103.43±7.85%, and % CV of intra-and inter-assay were <10%. Concentration of cortisol after transportation was significantly higher than before transportation (p<0.05). Concentration of cortisol was significantly decreased when blood was centrifuged at 24 h after collection (P<0.05). In conclusion, human cortisol ELISA kit (EIA-1887) is a reliable assay for measuring cortisol in plasma of kacang goat. Delayed to blood centrifugation time affect cortisol concentrations.

Keywords


human cortisol ELISA kit; kambing kacang; kortisol; validasi; waktu sentrifugasi; Cortisol; kacang goats; time of centrifugation; validation

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References


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DOI: https://doi.org/10.17969/agripet.v21i1.18153

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