Development of monoclonal antibodies against porcine blood for detection in fish-based products

Animal blood mainly from porcine and bovine have been used in human food as a binder, gelling agent, emulsifier, coloring agent and iron’s supplementary in most meat and fish-based products. However, certain communities including Muslims and Jews are strictly prohibited to consume animal blood in...

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Main Author:	Raja Nhari, Raja Mohd Hafidz
Format:	Thesis
Language:	English
Published:	2017
Subjects:	Monoclonal antibodies - Research
Online Access:	http://psasir.upm.edu.my/id/eprint/70288/1/FBSB%202017%2028%20IR.pdf
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spelling	my-upm-ir.702882019-08-28T04:01:42Z Development of monoclonal antibodies against porcine blood for detection in fish-based products 2017-06 Raja Nhari, Raja Mohd Hafidz Animal blood mainly from porcine and bovine have been used in human food as a binder, gelling agent, emulsifier, coloring agent and iron’s supplementary in most meat and fish-based products. However, certain communities including Muslims and Jews are strictly prohibited to consume animal blood in food due to prohibition assigned by each religious and related to health and personal matter. The addition of porcine plasma in fish surimi has been highlighted by the local authority in Malaysia. To date, a specific method to detect porcine plasma in food is unavailable. In this study, we have developed monoclonal antibodies (mAbs) against heated soluble proteins (HSPs) of porcine blood using fusion technology for detection of porcine plasma in fish surimi. Specificity of mAbs against blood, non-blood (meat and nonmeat) and commercial animal plasma proteins from different species were determined using indirect non-competitive ELISA. Antigenic components of porcine blood were determined using Western blot. The sensitivity of ELISA has been determined to analyze fish surimi that spiked with porcine plasma. After several screening of hybridoma was made, 27 hybridomas produced mAbs were selected. Based on that, fifteen mAbs are specific to raw and heated porcine blood, one mAb only specific to raw porcine blood and another 11 mAbs are cross-reacted with other animal blood. The fifteen mAbs specific to porcine blood are also not cross-reacted with meat and non-meat proteins. Based on specificity to animal plasma, twelve mAbs from 15 mAbs are specific to porcine plasma while other 3 mAbs are cross-reacted to chicken plasma. Western blot showed that 2 mAbs bind 60 kDa, 8 mAbs bind 85 kDa and 2 mAbs bind 250 kDa of the antigenic protein of porcine blood. The mAb labeled as B4E1 was selected to be used for detection of porcine plasma. The developed ELISA has a limit of detection (LOD) and limit of quantification (LOQ) of 0.2 μg/g and 1 μg/g, respectively for a standard solution of porcine plasma. The intra- and inter-assays of ELISA with coefficients of variation (CVs) less than 20% were able to detect at least 0.25% (w/w) porcine plasma in fish surimi. In conclusion, this study has successfully obtained the hybridoma-producing mAbs that are specific to porcine blood and porcine plasma. This study also has developed indirect non-competitive ELISA for detection of porcine plasma in laboratory model fish ball and fish surimi products using mAb B4E1 with LOD and LOQ of 0.2 μg/g and 1 μg/g, respectively with the sensitivity of the ELISA is 0.25% (w/w) porcine plasma in fish surimi. Monoclonal antibodies - Research 2017-06 Thesis http://psasir.upm.edu.my/id/eprint/70288/ http://psasir.upm.edu.my/id/eprint/70288/1/FBSB%202017%2028%20IR.pdf text en public masters Universiti Putra Malaysia Monoclonal antibodies - Research
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Monoclonal antibodies - Research
spellingShingle	Monoclonal antibodies - Research Raja Nhari, Raja Mohd Hafidz Development of monoclonal antibodies against porcine blood for detection in fish-based products
description	Animal blood mainly from porcine and bovine have been used in human food as a binder, gelling agent, emulsifier, coloring agent and iron’s supplementary in most meat and fish-based products. However, certain communities including Muslims and Jews are strictly prohibited to consume animal blood in food due to prohibition assigned by each religious and related to health and personal matter. The addition of porcine plasma in fish surimi has been highlighted by the local authority in Malaysia. To date, a specific method to detect porcine plasma in food is unavailable. In this study, we have developed monoclonal antibodies (mAbs) against heated soluble proteins (HSPs) of porcine blood using fusion technology for detection of porcine plasma in fish surimi. Specificity of mAbs against blood, non-blood (meat and nonmeat) and commercial animal plasma proteins from different species were determined using indirect non-competitive ELISA. Antigenic components of porcine blood were determined using Western blot. The sensitivity of ELISA has been determined to analyze fish surimi that spiked with porcine plasma. After several screening of hybridoma was made, 27 hybridomas produced mAbs were selected. Based on that, fifteen mAbs are specific to raw and heated porcine blood, one mAb only specific to raw porcine blood and another 11 mAbs are cross-reacted with other animal blood. The fifteen mAbs specific to porcine blood are also not cross-reacted with meat and non-meat proteins. Based on specificity to animal plasma, twelve mAbs from 15 mAbs are specific to porcine plasma while other 3 mAbs are cross-reacted to chicken plasma. Western blot showed that 2 mAbs bind 60 kDa, 8 mAbs bind 85 kDa and 2 mAbs bind 250 kDa of the antigenic protein of porcine blood. The mAb labeled as B4E1 was selected to be used for detection of porcine plasma. The developed ELISA has a limit of detection (LOD) and limit of quantification (LOQ) of 0.2 μg/g and 1 μg/g, respectively for a standard solution of porcine plasma. The intra- and inter-assays of ELISA with coefficients of variation (CVs) less than 20% were able to detect at least 0.25% (w/w) porcine plasma in fish surimi. In conclusion, this study has successfully obtained the hybridoma-producing mAbs that are specific to porcine blood and porcine plasma. This study also has developed indirect non-competitive ELISA for detection of porcine plasma in laboratory model fish ball and fish surimi products using mAb B4E1 with LOD and LOQ of 0.2 μg/g and 1 μg/g, respectively with the sensitivity of the ELISA is 0.25% (w/w) porcine plasma in fish surimi.
format	Thesis
qualification_level	Master's degree
author	Raja Nhari, Raja Mohd Hafidz
author_facet	Raja Nhari, Raja Mohd Hafidz
author_sort	Raja Nhari, Raja Mohd Hafidz
title	Development of monoclonal antibodies against porcine blood for detection in fish-based products
title_short	Development of monoclonal antibodies against porcine blood for detection in fish-based products
title_full	Development of monoclonal antibodies against porcine blood for detection in fish-based products
title_fullStr	Development of monoclonal antibodies against porcine blood for detection in fish-based products
title_full_unstemmed	Development of monoclonal antibodies against porcine blood for detection in fish-based products
title_sort	development of monoclonal antibodies against porcine blood for detection in fish-based products
granting_institution	Universiti Putra Malaysia
publishDate	2017
url	http://psasir.upm.edu.my/id/eprint/70288/1/FBSB%202017%2028%20IR.pdf
_version_	1747812803936256000

Development of monoclonal antibodies against porcine blood for detection in fish-based products

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