Uncertainty analysis of trypsin inhibitor activity determination using microtiter plate method
2013
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Authors
Župunski, VesnaNikolić, Zorica
Zdjelar, Gordana
Jovičić, Dušica
Ignjatov, Maja
Milošević, Dragana
Tamindžić, Gordana
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Measurement of proteinase inhibitor activity of legume seeds is widely used since some of these
exert toxic and antinutritional effects. Standard method assay for trypsin inhibitor activity (TIA) determination is based on the hydrolysis of benzoyl-DL-arginine-p-nitroanilide (BAPA) by trypsin and includes spectrophotometric measurement of the reaction products. Assaying of trypsin inhibitor activity is usualy performed using reaction mixture volumes of 4-10 ml, however, it was shown that microtitration method can also be used for TIA determination. Although the results obtained by microtitration method were consistent with those obtained by French Association for Normalization reference method (AFNOR), it was noted that any experimental error in absorbance values was increasingly magnified as the sample size was diminshed. The aim of this study was to analyse measurement uncertanty of TIA determination using microtiter plate method. Assaying procedure was conducted at 37 °C on sample ...(soybean variety Vojvoñanka), positive and negative control, which were set in four replicats in each row of the microtiter plate, with preincubation time of 10 minutes and incubation time of 30 minutes. The trypsin inhibitor activity was expresed in number of trypsin units inhibited (TUI) per miligram of sample, taking into account the fact that one trypsin unit is defined as an increase of 0.01 absorbance units at 405 nm. Large number of experiments was conducted and uncertainty analysis was obtained using uncertainty budget. Values of calculated trypsin inhibitor activity were approximately 70 TUI/mg and estimated expanded measurement uncertainty (with coverage factor k=2) was aproxitemately 15%.
Keywords:
trypsin inhibitor activity / microtiter plate / analysisSource:
Book of Abstracts, 1st Legume Society Conference 2013: A Legume Odyssey, 9-11 May 2013, Novi Sad, 2013, 131-131Publisher:
- International Legume Society
- Novi Sad : Institute of Field and Vegetable Crops
Funding / projects:
- Interdisciplinary Approach to Development of New Soybean Varieties and Improvement of the Cultivation Practices and Seed Production (RS-MESTD-Technological Development (TD or TR)-31022)
- Increasing the market significance of forage crops by breeding and optimizing seed production technology (RS-MESTD-Technological Development (TD or TR)-31024)
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FiVeRTY - CONF AU - Župunski, Vesna AU - Nikolić, Zorica AU - Zdjelar, Gordana AU - Jovičić, Dušica AU - Ignjatov, Maja AU - Milošević, Dragana AU - Tamindžić, Gordana PY - 2013 UR - http://fiver.ifvcns.rs/handle/123456789/3013 AB - Measurement of proteinase inhibitor activity of legume seeds is widely used since some of these exert toxic and antinutritional effects. Standard method assay for trypsin inhibitor activity (TIA) determination is based on the hydrolysis of benzoyl-DL-arginine-p-nitroanilide (BAPA) by trypsin and includes spectrophotometric measurement of the reaction products. Assaying of trypsin inhibitor activity is usualy performed using reaction mixture volumes of 4-10 ml, however, it was shown that microtitration method can also be used for TIA determination. Although the results obtained by microtitration method were consistent with those obtained by French Association for Normalization reference method (AFNOR), it was noted that any experimental error in absorbance values was increasingly magnified as the sample size was diminshed. The aim of this study was to analyse measurement uncertanty of TIA determination using microtiter plate method. Assaying procedure was conducted at 37 °C on sample (soybean variety Vojvoñanka), positive and negative control, which were set in four replicats in each row of the microtiter plate, with preincubation time of 10 minutes and incubation time of 30 minutes. The trypsin inhibitor activity was expresed in number of trypsin units inhibited (TUI) per miligram of sample, taking into account the fact that one trypsin unit is defined as an increase of 0.01 absorbance units at 405 nm. Large number of experiments was conducted and uncertainty analysis was obtained using uncertainty budget. Values of calculated trypsin inhibitor activity were approximately 70 TUI/mg and estimated expanded measurement uncertainty (with coverage factor k=2) was aproxitemately 15%. PB - International Legume Society PB - Novi Sad : Institute of Field and Vegetable Crops C3 - Book of Abstracts, 1st Legume Society Conference 2013: A Legume Odyssey, 9-11 May 2013, Novi Sad T1 - Uncertainty analysis of trypsin inhibitor activity determination using microtiter plate method EP - 131 SP - 131 UR - https://hdl.handle.net/21.15107/rcub_fiver_3013 ER -
@conference{ author = "Župunski, Vesna and Nikolić, Zorica and Zdjelar, Gordana and Jovičić, Dušica and Ignjatov, Maja and Milošević, Dragana and Tamindžić, Gordana", year = "2013", abstract = "Measurement of proteinase inhibitor activity of legume seeds is widely used since some of these exert toxic and antinutritional effects. Standard method assay for trypsin inhibitor activity (TIA) determination is based on the hydrolysis of benzoyl-DL-arginine-p-nitroanilide (BAPA) by trypsin and includes spectrophotometric measurement of the reaction products. Assaying of trypsin inhibitor activity is usualy performed using reaction mixture volumes of 4-10 ml, however, it was shown that microtitration method can also be used for TIA determination. Although the results obtained by microtitration method were consistent with those obtained by French Association for Normalization reference method (AFNOR), it was noted that any experimental error in absorbance values was increasingly magnified as the sample size was diminshed. The aim of this study was to analyse measurement uncertanty of TIA determination using microtiter plate method. Assaying procedure was conducted at 37 °C on sample (soybean variety Vojvoñanka), positive and negative control, which were set in four replicats in each row of the microtiter plate, with preincubation time of 10 minutes and incubation time of 30 minutes. The trypsin inhibitor activity was expresed in number of trypsin units inhibited (TUI) per miligram of sample, taking into account the fact that one trypsin unit is defined as an increase of 0.01 absorbance units at 405 nm. Large number of experiments was conducted and uncertainty analysis was obtained using uncertainty budget. Values of calculated trypsin inhibitor activity were approximately 70 TUI/mg and estimated expanded measurement uncertainty (with coverage factor k=2) was aproxitemately 15%.", publisher = "International Legume Society, Novi Sad : Institute of Field and Vegetable Crops", journal = "Book of Abstracts, 1st Legume Society Conference 2013: A Legume Odyssey, 9-11 May 2013, Novi Sad", title = "Uncertainty analysis of trypsin inhibitor activity determination using microtiter plate method", pages = "131-131", url = "https://hdl.handle.net/21.15107/rcub_fiver_3013" }
Župunski, V., Nikolić, Z., Zdjelar, G., Jovičić, D., Ignjatov, M., Milošević, D.,& Tamindžić, G.. (2013). Uncertainty analysis of trypsin inhibitor activity determination using microtiter plate method. in Book of Abstracts, 1st Legume Society Conference 2013: A Legume Odyssey, 9-11 May 2013, Novi Sad International Legume Society., 131-131. https://hdl.handle.net/21.15107/rcub_fiver_3013
Župunski V, Nikolić Z, Zdjelar G, Jovičić D, Ignjatov M, Milošević D, Tamindžić G. Uncertainty analysis of trypsin inhibitor activity determination using microtiter plate method. in Book of Abstracts, 1st Legume Society Conference 2013: A Legume Odyssey, 9-11 May 2013, Novi Sad. 2013;:131-131. https://hdl.handle.net/21.15107/rcub_fiver_3013 .
Župunski, Vesna, Nikolić, Zorica, Zdjelar, Gordana, Jovičić, Dušica, Ignjatov, Maja, Milošević, Dragana, Tamindžić, Gordana, "Uncertainty analysis of trypsin inhibitor activity determination using microtiter plate method" in Book of Abstracts, 1st Legume Society Conference 2013: A Legume Odyssey, 9-11 May 2013, Novi Sad (2013):131-131, https://hdl.handle.net/21.15107/rcub_fiver_3013 .