Adaptation of an enzyme-linked immunosorbent assay for determination of diminazene aceturate in goat serum and tissue residues of animals
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Date
2014-11-27
Authors
Karanja, Wycliff
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Abstract
The importance of food safety through the reduction of residues in our food supply
cannot be overemphasized. Food safety remains a major challenge confronting
contemporary society. Analytical methods are needed to generate the data on which
dietary exposure assessments are based and to enforce statutory maximum residue limits
(MRLs) that are set. Diminazene aceturate is one of the few drugs used for animal trypanosomosis. Because of it's wide use in livestock, the risk of unwanted residues in
edible products may exist. A competitive enzyme-linked immunosorbent assay (ELISA)
for determination of diminazene residues in edible animal tissues after extraction in 0.1
M borax at pH 9.7 was investigated. The assay used rabbit anti-diminazene polyclonal
antibody on the solid phase support. Horseradish peroxidase-labeled diminazene was
incubated with sample overnight at 4°C. After five washes with buffer enzyme activity
was determined by adding tetramethyl-benzidine and hydrogen peroxide as substrate.
The resulting blue colour whose intensity was inversely proportional to the drug
concentration changed to yellow when the reaction was stopped by addition of 0.1 M
orthophosphoric acid. The assay was optimized and validated for determination of
diminazene in tissues. The assay exhibited high specificity (99.997%) for diminazene
recognizing only isometamidium at 0.003% and this may be contributed by the
amidinophenyl that is common in both drugs. Recoveries from spiked tissues were above
77% while Dilutional parallelism experiments demonstrated a recovery of 96.0% ± 9.5%.
The limit of detection (LaD) for the assay was 2.4ng/g for muscle, 2.5ng/g for liver and
2.2ng/g for kidney while limits of quantification (LOQ) were 5.51ng/g, 4.11 ng/g and
3.74 ng/g respectively. The LaDs are 4.4x103 to 3.5x103 lower than the MRLs that are
500llg/kg, 12,000Ilg/kg and 6,000Ilg/kg of muscle liver and kidney respectively: Assay
precision was characterized by a within assay coefficient of variation (CV) of 2.4% and
between assays CV of 15.5%. When diminazene was administered intramuscularly at
3.5mg/kg to five goats that were sacrificed seven days later, the mean diminazene residue
levels were 0.75Ilg/g±0.14Ilg/g for skeletal muscle, 32.05Ilg/g±5.7Ilg/g for liver and
4.29Ilg/g±0.66Ilg/g for kidney. The analysis of tissue samples collected from
slaughterhouses around Nairobi showed that out of 35 muscle samples, only one was
positive and had a diminazene concentration of 0.039 ug/g. Four out of 32 kidney
samples were positive for diminazene with levels of 0.63, 1.66,2.61 and 3.96Ilg/g. From
ten liver samples two were positive with levels of 1.07 and 1.74 ug/g. From this analysis
none of the positive samples had levels above the MRL values. This study has
demonstrated that competitive ELISA can be employed for the determination of
diminazene residues. The results of this study are relevant to food scientists,
toxicologists and analysts working in the area of detection and safety assessment of food
residues, companies developing veterinary drugs, regulatory bodies involved in safety
assessment of veterinary drugs and residue monitoring and to regulatory bodies
responsible for veterinary drugs registration.
Description
Department of Biochemistry and Biotechnology,91pg. March 2007
QP 519.9.E48K3