Citation
Raja Nhari, Raja Mohd Hafidz
(2017)
Development of monoclonal antibodies against porcine blood for detection in fish-based products.
Masters thesis, Universiti Putra Malaysia.
Abstract
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.
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