| dc.description.abstract | Studies aimed at developing enzyme immunoassay and immunohistochemical
techniques for the determination of aflatoxins in animal tissues were carried
out.
Highly specific antibodies against aflatoxin B1 were produced in rabbits using
aflatoxin Bi-bovine serum albumin conjugate as immunogen. The antibodies
were employed in ultrasensitive enzyme immunoassay for aflatoxin B1 and in
the production of immunoaffinity columns with very high binding capacities for
most aflatoxin analogues. With average detection limits of 14.83±00.77 pgjml
for AFB1dissolved in methanol:PBS (10:90) and 16.81±1.97 pgjml for AFB1
dissolved in methanol:acetone:PBS (5:5:90), the ELISA established with these
antibodies is the most highly sensitive so far reported. The binding capacities
of lAC columns produced were 694ng for aflatoxin B1, 393ng for aflatoxin B2
and over 83 ng for aflatoxin G1, G2, M1,B2(1,G2(1,PI, Q1 and aflatoxicol.
To determine the extraction methods that were most appropriate for the
preparation of liver tissues for aflatoxin analysis by the established ELISA
method, different extraction methods were compared. The criteria for
comparison of these methods were the efficiency of extraction as determined by
AFB1recoveries in chicken liver tissues spiked at 1ngjg AFB1and ability to
eliminate tissue matrix effects as determined by the recoveries in tissues
spiked at Ongj g. A method involving prior protein denaturation with
methanol:acetone (50:50), followed by AFB1extraction by
methanol:acetone:PBS was found to be most suitable. Extracts obtained by use
of this method were defatted and applied to ELISAwith and without further
immunoaffinity chromatography (IAC)purification. The average percentage
recoveries in chicken, cattle, swine and dog liver tissues spiked at between 1to
5ng/g were 58.2, 44.2, 32.2 and 41.8 and the percentage coefficients of
variation were 27.1, 26.3, 25.0 and 20.2, respectively. The use ofIAC
purification reduced the coefficients of variation between recovery values of
replicates to less than 20%. In tissues spiked at Ongj g (n=24) and at l ng Zg
(n=24), the diagnostic sensitivity and specificity were 91.7% and 100%,
respectively. Both the diagnostic sensitivity and specificity were 100% when the
tissue extracts were processed with lAC. In liver tissues from chicken dosed
with the AFBl, recovery values obtained with this method were comparable to
those obtained with a reference thin layer chromatography method. The
method was applied to the analysis of AFBI in liver tissue from 63 suspected
natural cases of aflatoxicosis in chicken and dogs. Aflatoxins were
demonstrated in 34.9% and 44.4% of chicken and dog tissues, respectively.
Immunohistochemical studies were aimed at localization of aflatoxin BI-DNA
adducts in liver tissues. The immunoreactivity of three different anti-aflatoxin
antibodies with a synthetic aflatoxin BI-DNAadducts was investigated through
a competitive ELISA.An anti-aflatoxin B2awhich exhibited high
immunoreactivity with aflatoxin BI-DNAin ELISAand a monoclonal that is
specific for a persistent aflatoxin BI-DNAadduct were employed in
immunohistochemical studies. Different tissue fixing and processing
procedures were compared to determine the most suitable for aflatoxin
localization by the two antibodies. Specific immunostaining was obtained only
with the monoclonal antibody in tissue sections that were digested with
proteinase K. Positive immunostaining was observed in liver tissues from
chicken dosed with AFB! and from some field cases of suspected aflatoxicosis
in chicken and dogs. However, in the analysis of tissues from suspected cases
of aflatoxicosis in chicken and dogs, the immunohistochemical test localized
aflatoxin metabolites in fewer cases than was recorded with the ELISA test.
The ELISA and immunohistochemical tests described here may be useful in the
diagnosis of aflatoxicosis, in quality control of meat and in research on
aflatoxin poisoning | en |
