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

Background: To obtain accurate measurements of cortisol (C) and testosterone (T) in Aceh cattle, commercial enzyme-linked immunosorbent assay (ELISA) kits need to be carefully validated. Moreover, repeated freeze-thaw cycles during the storage of the samples may affect the stability of the hormones in the serum. Here, the reliability of C and T concentration measurements in the serum of Aceh cattle, was tested using commercial C and T ELISA kits designed to measure human C and T concentrations. Further, the effect of repeated freeze-thaw cycles on the stability of C and T concentrations in the serum was evaluated. Methods: Commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) ELISA kits from DRG Instruments GmbH were validated through an analytical validation test (i.e., parallelism, accuracy, and precision) and a biological validation test (for C: effect of transportation on the C excretion; for T: the concentrations of T between bulls and cows). To test the effects of freeze-thaw cycles, cattle serum was subjected to the following treatments: (i) remained frozen at -20OC (control group); (ii) exposed to freeze-thaw cycles for two, four, six, and eight times (test groups). Results: Parallelism, accuracy, and precision tests showed that both C and T ELISA kits adequately measured C and T in the serum of Aceh cattle. Concentrations of C post-transportation were significantly higher than pre-transportation (p<0.05). Concentrations of T in bulls were significantly higher than in cows (p<0.05). After four to eight freeze-thaw cycles, C concentrations were significantly lower compared to the control group (all p < 0.05). In contrast, T concentrations remained stable (all p>0.05). Conclusions: Commercial C (EIA-1887) and T (EIA-1559) ELISA kits are reliable assays for measuring serum C and T, respectively, in Aceh cattle. Repeated freeze-thaw cycles significantly affected the stability of serum C, but did not for T.

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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

F1000Research ◽

10.12688/f1000research.19804.3 ◽

2020 ◽

Vol 8 ◽

pp. 1220

Author(s):

Gholib Gholib ◽

Sri Wahyuni ◽

Muslim Akmal ◽

Muhammad Hasan ◽

Muhammad Agil ◽

...

Keyword(s):

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Analytical Validation ◽

Commercial Enzyme ◽

Freeze Thaw ◽

Validation Test ◽

Accuracy And Precision ◽

Cattle Serum ◽

The Stability

Background: To obtain accurate measurements of cortisol (C) and testosterone (T) in Aceh cattle, commercial enzyme-linked immunosorbent assay (ELISA) kits need to be carefully validated. Moreover, repeated freeze-thaw cycles during the storage of the samples may affect the stability of the hormones in the serum. Here, the reliability of C and T concentration measurements in the serum of Aceh cattle, was tested using commercial C and T ELISA kits designed to measure human C and T concentrations. Further, the effect of repeated freeze-thaw cycles on the stability of C and T concentrations in the serum was evaluated. Methods: Commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) ELISA kits from DRG Instruments GmbH were validated through an analytical validation test (i.e., parallelism, accuracy, and precision) and a biological validation test (for C: effect of transportation on the C secretion; for T: the concentrations of T between bulls and cows). To test the effects of freeze-thaw cycles, cattle serum was subjected to the following treatments: (i) remained frozen at -20OC (control group); (ii) exposed to freeze-thaw cycles for two, four, six, and eight times (test groups). Results: Parallelism, accuracy, and precision tests showed that both C and T ELISA kits adequately measured C and T in the serum of Aceh cattle. Concentrations of C post-transportation were significantly higher than pre-transportation (p<0.01). Concentrations of T in bulls were significantly higher than in cows (p<0.01). After four to eight freeze-thaw cycles, C concentrations were significantly lower compared to the control group (all p < 0.05). In contrast, T concentrations remained stable (all p>0.05). Conclusions: Commercial C (EIA-1887) and T (EIA-1559) ELISA kits are reliable assays for measuring serum C and T, respectively, in Aceh cattle. Repeated freeze-thaw cycles significantly affected the stability of serum C, but did not for T.

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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

F1000Research ◽

10.12688/f1000research.19804.1 ◽

2019 ◽

Vol 8 ◽

pp. 1220 ◽

Cited By ~ 4

Author(s):

Gholib Gholib ◽

Sri Wahyuni ◽

Muslim Akmal ◽

Muhammad Hasan ◽

Muhammad Agil ◽

...

Keyword(s):

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Analytical Validation ◽

Commercial Enzyme ◽

Freeze Thaw ◽

Validation Test ◽

Accuracy And Precision ◽

Cattle Serum ◽

The Stability

Background: To obtain accurate measurements of cortisol (C) and testosterone (T) in Aceh cattle, commercial enzyme-linked immunosorbent assay (ELISA) kits need to be carefully validated. Moreover, repeated freeze-thaw cycles during the storage of the samples may affect the stability of the hormones in the serum. Here, we test the reliability of C and T concentration measurements in the serum of Aceh cattle, obtained using commercial C and T ELISA kits designed to measure human C and T concentrations. Further, we evaluate the effect of repeated freeze-thaw cycles on the stability of C and T concentrations in the serum. Methods: Commercial C (Cat. no. EIA-1887) and T (Cat. no. EIA-1559) ELISA kits from DRG Instruments GmbH were validated through an analytical validation test (i.e., parallelism, accuracy, and precision) and a biological validation test (for C: effect of transportation on the C excretion; for T: the concentrations of T between bulls and cows). To test the effects of freeze-thaw cycles, cattle serum was subjected to the following treatments: (i) remained frozen at -20OC (control group); (ii) exposed to freeze-thaw cycles for two, four, six, and eight times (test groups). Results: Parallelism, accuracy, and precision tests showed that both C and T ELISA kits adequately measured C and T in the serum of Aceh cattle. Concentrations of C post-transportation were significantly higher than pre-transportation (p<0.05). Concentrations of T in bulls were significantly higher than in cows (p<0.05). After four to eight freeze-thaw cycles, C concentrations were significantly lower compared to the control group (all p < 0.05). In contrast, T concentrations remained stable (all p>0.05). Conclusions: Commercial C (EIA-1887) and T (EIA-1559) ELISA kits are reliable assays for measuring serum C and T, respectively, in Aceh cattle. Repeated freeze-thaw cycles significantly affected the stability of serum C, but did not for T.

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

Jurnal Agripet ◽

10.17969/agripet.v21i1.18153 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Gholib Gholib ◽

Sri Wahyuni ◽

Rahma Melinda ◽

Muslim Akmal

Keyword(s):

Immunosorbent Assay ◽

Enzyme Linked Immunosorbent Assay ◽

Time Effects ◽

Elisa Kit ◽

Validation Test ◽

Accuracy And Precision ◽

Before And After ◽

Equality Test ◽

One Way Anova ◽

Blood Centrifugation

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 (p0,05). Adanya penurunan secara nyata konsentrasi kortisol pada darah yang disentrifugasi 24 jam (P24) setelah koleksi (p0,05). 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 (p0.05). Concentration of cortisol was significantly decreased when blood was centrifuged at 24 h after collection (P0.05). 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.

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Can Osteopontin Be Considered a Biomarker for Endometriosis?

Revista de Chimie ◽

10.37358/rc.17.9.5840 ◽

2017 ◽

Vol 68 (9) ◽

pp. 2132-2134

Author(s):

Daniela Roxana Albu (Matasariu) ◽

Elena Mihalceanu ◽

Alina Pangal ◽

Carmen Vulpoi ◽

Mircea Onofriescu ◽

...

Keyword(s):

Serum Level ◽

Immunosorbent Assay ◽

Pelvic Pain ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Multifactorial Disease ◽

Elisa Kit ◽

Healthy Women ◽

Progesterone Treatment ◽

Large Dispersion

Endometriosis is a multifactorial disease that is manifested by infertility and pelvic pain. The purpose of the study was to evaluate the effect of progesterone treatment on the serum level of osteopontin, a multipotent cytokine, in patients with endometriosis. The study was prospective and we evaluated osteopontin levels that were measured in the serum of 40 patients with endometriosis and 12 healthy women using a standardized Enzyme-Linked Immunosorbent Assay (ELISA) kit. Osteopontin seric levels were lower in endometriosis patients and increased after progesterone treatment. Because of the large dispersion of data even in the control group, we find the association between osteopontin and endometriosis questionable.

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