Note: Descriptions are shown in the official language in which they were submitted.
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ASSAY FOR AFLATOXINS
This invention relates to an assay for testing for the
presence of aflatoxins. The invention use immunoassay
techniques in simultaneously testing for aflatoxin Bl and
Gl.
Background Of The Invention
Aflatoxins are a group of toxic secondary metabolites
produced by Aspergillus flavu~ and A. ~ara~iticus.
Aflatoxin ~1 (AFB1), the most toxic compound in this series,
has been found to be one of the mo~t poten~ carcinogens
occurring naturally. Another ~revalent aflatoxin, aflatoxin
Gl ("AFGl"), is also carcinoqenic. Because of frequent
contamination of ~FBl and AFGl in agricultural commodities,
aflatoxins are a potential hazard to human and animal
health. There i~, therefore, a need to accurately identify
aflatoxin contaminated food supplie~.
Investigation~ in our laboratory have led to the produc-
tion of specific antibodies against some aflatoxins (e.g.
AFBl), and several immunoassay methods for the analysis of
aflatoxin in different commoditie~ have been developed which
u~e these antibodies. See F. Chu, 47 J. Fd. Prot. 562-569
(1984); F. Chu, in Mycotoxins And Phycotoxins 277-292
(1986); F. Chu, in Modern Method~ In The Analysis And
Structural Elucidation Of Mycotoxins 207-237 (1986).
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The degree of cross-reactivity of these antibodies with
various aflatoxins varies considerably. As a result, the
reliability of prior art immunoassays is adversely
affected. For example, if the antibodies used in the
immunoassay are 100% cross-reactive with AFB1, but only 33~
with AFG1 (see F.S. Chu et al., 33 Appl. Environ. Microbiol.
1125-28 (1977)) the presence of ~FG1 in the sample could
result in an inaccurately low "apparent total aflatoxin
level" by these immunoassay methods. Since current govern~
ment safety standards require total aflatoxin levels to be
below certain amounts, this could result in a false
negative. False positives could also result, depending on
the aflatoxin being tested for and the content of the
sample, thereby leading to a waste of food.
Prior art approaches for the preparation of immunogens
for the production of antibodies against aflatoxins can
generally be classified into two groups. In the first
group, aflatoxin is conjugated to a protein carrier through
a carbonyl group at the cyclopentane ring, by making a car-
boxymethyl oxime (CMO) derivative. In the second group,
conjugation is made through the dihydrofuran portion of
molecule by using a hemiacetal-type derivative. The prior
art for the antibodies which were generated by immunization
of animals with the first group of immunogens generally
recognized the dihydrofuran portion of the molecule, wherea~
those generated by the second group of immunogens had a
specificity directed toward the cyclopentane ring. This led
to differences in affinity and antibodies that generally
cannot recognize both AFBl and AFGl.
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Thus, there is a need to obtain an antibody which reacts
with AFGl with about the same affinity as with AFBl.
Summar~ Of The Invention
In one aspect, the invention provides an assay for the
presence of aflatoxin in a sample. One uses an antibody
capable of binding to an aflatoxin to test for the presence
of aflatoxin. The antibody is of a type that recognizes
both AFBl and AFGl, and does so with substantially equal
affinity for both. "Substantially equal affinity" is
intended to mean that the concentration of AFGl needed to
cause 50% inhibition ("I-50") of binding of either
tritiated-AFBl or enzyme-labelled-AFBl (or both) using the
antibody under the radioimmunoassay (RIA~ or enzyme-linked
immunosorbent assay (ELISA) conditions specified below is
lS between 50% to 200% of the concentration of AFBl needed to
do so. Preferably, the antibody also binds AFB3 and was
derived by immunizing a rabbit (or other animal) with a
conjugate of AFB3.
In another form, the invention comprises a kit with an
antibody of the above type (e.g. anti-A~B3) together with
either a radioactively labelled aflatoxin that the antibody
will al50 bind to (for RIA) or an enzyme - labelled
aflatoxin (for enzyme immunoassay)~
In yet another form, the invention provides an antibody
of the above type. An immunogen is created by converting an
OH group of AFB3 to a hemi-succislate, and then conjugating
AFB3-HS to BSA. The conjugate is then used to immunize a
rabbit (or other animals) so as to produce polyclonal anti-
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bodies of interest. Monoclonal (as opposed to polyclonal)
antibodies can also be ob~ained by immunization of mice with
the AFs3-HS-sSA conjugate.
An objective of the invention therefore includes
providing an immunoassay of the above kind.
Another object is to provide an assay of the above kind
which is simple, relatively inexpensive, and easy to
perform, and which reduces the risk of false positives and
false negatives.
Another objective is to provide antibodies and kits for
conducting assays of the above kind.
Still other objectives and advantages of the present
invention will be apparent from the description which
follows.
Description Of The Drawings
~ better understanding of the present invention will be
accomplished by reference to the drawings. It should be
understood, however, that the drawings and the description
of the preferred embodiments are merely examples of the
invention. They are not intended to represent the full
scope of the invention. Rather, the claims should be looked
to in order to determine the full scope of the invention.
Fig. 1 depicts the chemical structures of aflatoxin B
and Gl; and
Fig. 2 is a schematic depiction of the chemistry
involved in the development of an antigen for the antibodies
of the present invention.
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Description Of The Preferred Embodiments
Materials And Methods
toxins Bl, B2, Gl, G2 were produced by Aspergillus
parasiticus NRRL 2999, and were purified according to the
method of F. Chu, 54 J. Ass. Off. Analyt~ Chem. 1304-1406
(1971). Aflatoxin B3 was either prepared from A.
parasiticus culture according to the method of J. Heathcote
_ al., 25 Tetrahedron 1497-1500 (1969), or prepared from
AFGl according to the method of R. Cole et al., 19 Agri. &
Fd. Chem. 222-223 (1971).
An AFB3 standard was kindly supplied by the National
Peanut Laboratory, USDA. Tritiated AFBl (14 Ci/mmol) was
obtained from Moravek Biochemicals, City of Industry,
California. Bovine serum albumin (BSA, RIA grade) was pur-
chased from Siqma Chemical Co. (St. Louis, Missouri). Water
soluble carbodiimide, i.e. l-ethyl-3, 3-dimethylamino-
propyl-carbodiimide (EDPC) and succinic anhydride (SA) were
obtained from Aldrich Chemical Co. (Milwaukee, Wisconsin).
Complete Freund's adjuvant containing Mycobacterium
tuberculosis (H 37 Ra) and incomplete Freund's adjuvant were
obtained from Difco Laboratories (Detroit, Michigan).
Albino rabbits (female) of approximately 2 kg size were
purchased from Smith's Rabbitry (Seymour, Wisconsin). The
health status of the rabbits were analyzed by a swab test.
Rabbits demonstrated to be Pasteurella negative were
selected for immunization. All chemicals and organic
solvents were reagent grade or better.
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Preparation Of Antigen
The method of preparation of the immunogen is schemati-
cally summarized in Fig. 2. Aflatoxin B3 was first con-
verted to the AFB3-hemisuccinate (AFB3-HS) under conditions
partially adapted from those used for the conversion of T-2
toxin to its hemisuccinate in the presence of dimethylamino-
pyridine (DAMP), F. Chu et al., 37 Appl. Environ. Microbiol.
104-108 (1979). Conjugation of AFB3-HS to BSA (or the
enzyme horseradish peroxidase (HRP) which can be used in an
ELISA) was then carried out in the presence of a water
soluble coupling agent, carbodiimide (E~PC), by procedures
analoyous to those previously described for T-2 toxin in
that same article.
The molar ratio of hapten to carrier protein in the
reaction mixture was 26. After reaction and dialysis to
remove the free AFB3-HS, the molar ratio was found to be
around 10 as determined according to the method of A.
Habeeb, 14 Anal. Biochem. 328-336 (1966).
Production Of Polyclonal Antibody
Immunization schedule and methods of immunization were
essentially the same as those described for T-2 toxin in F.
Chu et al., 37 Appl. Environ. Microbiol. 104-108 (1979)
using the multiple injection technique in three rabbits.
The rabbits were each injected intradermally with 500 mg of
the immunogen in 1.0 ml of 0.1 M sodium phosphate buffer
(pH 7.4) containing 0.85% NaCl (PBS), (emulsified with
2.0 ml of complete Freund's adjuvant~ on the back of each
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rabbit (40 sites) using the multiple-injection technique, F.
Chu et al. 33 Appl. Environ. Microbiol. 1125-1128 (1977).
For booster injections 500 mg of antigen in 1.0 ml of
PBS and 2.0 ml incomplete Freund's adjuvant was used and the
injection was made in the thighs (4 sites). The collected
antisera were precipitated with (NH4)2SO4 to a final satura-
tion of 33.3% using a 100~ saturated (NH4)2SO4 solution.
Finally, the precipitates were reconstituted to half of the
original volume with distilled water, dialyzed against dis-
tilled water for 0.5 to one hour (membrane cut-off was
10,000 daltons), against 0.01 M PBS overnight at 6C, and
then lyophilized.
Production of Monoclonal Antibody
Monoclonal antibodies were produced by fusion of P3/NS-
1/1-AG4-1 myeloma cells with spleen cells isolated from a
Balb/c mouse that had been immunized with AF33-HS conjugated
to bovine serum albumin (AFB3-HS-BSA). Protorols for the
production and characterization of the monoclonal antibodies
were similar to the procedure our lab recently reported for
the production of monoclonal antibody for T-2 toxin. T. Fan
et al., 54 Appl. Environ. Microbiol. 2959-2963 (1989).
Applicant has deposited hybridoma H575G4H7, which is
capable of producing such antibody, with the American Type
Culture Collection, Rockville, Maryland, as ATCC No.
HB10106. Samples from the deposit will be made available in
accordance with U.S. patent law requirements upon issuance
of the patent and in accordance with the requirements of any
applicable foreign patent laws. No patent license is
intended by such availability.
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Radioimmunoassay ("RIA")
Protocols for RIA were partially adapted from those
described for AFBl, in which an ammonium sulfate
precipitation method was used to separate the free and bound
toxin. F. Chu et al., 37 Appl. Environ. Microbiol. 104-108
(1979). 50 ul of tritiated AFBl (10,000 to 12,000 dpm) (the
hot competitive compound) was incubated with 0.15 ml of
antiserum solution of various dilutions in PBS at room
temperature for 30 minutes and then in a cold room (6C) for
at least one hour, followed by separation of the bound and
free ligand with an ammonium sulfate precipitation method as
described in the prior article. Antibody titer was defined
as the reciprocal of the antiserum volume (in milliliters)
required to give 50% binding of tritiated toxin under the
conditions described.
Protocols for determination of antibody specificity were
essentially the same as for the titer determination, except
that various non-radioactive aflatoxins at concentratior.s
between 0.1-1,000 ng/mL, were added to the reaction mixture
to set up a standard curve. Different derivatives were
first dissolved in methanol and then diluted with Ool M PBS
(pH 7.4). The final volume of the reaction mixture was
0.2 ml, and the final methanol concentration was 5%.
It will be appreciated that in testing aflatoxin levels
in natural samples, the samples can be extracted using pre-
viously published procedures (e.g. El Nakib et al., 64 J.
Ass. Off. ~nal. 1077-1082 (1981)), the e~tract then being
used in place of the knowns, with the resulting radio-
activity being compared to a standard curve.
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Radioactivity was determined in a Beckman Instrument,
Inc. (Fullerton, California) model LS-5801 liquid scintil-
lation spectrometer using 4.5 ml of Aquasol ~New England
Nuclear Corp., Boston, Massachusetts).
Enzyme-linked_Immunosorbent (ELISA) Assay
ELISA assays (e.g. direct and indirect) have been used
for analysis of some mycotoxins. See generally F. Chu,
Mycotoxins And Phycotoxins (1986); F. Chu, 47 J. Fd. Prot.
562-569 (1984), F. Chu et al., 70 J. Assoc. Off. And. 854-
857 (1981) T. Fan et al., 47 J. Fd. Prot. 263-266 (1984).
Both types are competitive assays, and both can readily be
adapted to use this antibody. A direct competitive ELISA
such as the one described for the analysis of aflatoxin B
previously established by our laboratory in F. Chu et al.,
70 J. Assoc. Off. Analy. Chem. 854857 (1987j was adapted to
evaluate cross-reactlvity where AFB3-NS-HRP was used as the
marker enz~me. An ELISA can also be de~eloped using the
monoclonal antibody instead of polyclonal antibodies, and
indirect competitive ELISA (double antibody) and other
standard ELISA techniques can be applied.
General Discussion
A part of the invention is the realization that AFB3 has
structural features common to both Bl and Gl. Another part
of the invention is the realization that a conjugation point
; 25 on B3 can be obtained. Another part of the invention is the
realization that this~point leaves regions of similarity to
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1 31 5221
AFBl and AFGl available for antibody formation. Moreover,
results obtained from the present study indicate that the
antibodies produced by this method have substantially equal
affinity for both aflatoxins Bl and Gl.
The relative cross-reactivity (RCR, A~Bl AFB as 100%) of
the polyclonal antibody with AFBl, AFB2, AFGl, AFG2, and
AFB3, as determined by RIA, are found to be lO0, 7.5, 78.1,
4.6, and 47.2 respectively. The RCR of ~he polyclonal
antibody as determined by ELISA using AFB3-HS-HRP AFB, as
the marker, are found to be 100, 8, 100, 8, and 80,
respectively. Note that in this latter system, the affinity
for AFBl and AFGl is indentical!
With respect to the monoclonal antibodies, competitive
radioimmunoassay using tritiated AFBl as the marker ligand
revealed that two clones produced an antibody that cross-
reacted well with both AFBl and AFGl. The relative cross-
reactivities (RCR) of antibody produced by clone 575B8F12
for AFBl, AFB2, AFGl, and AFG2 were 100, <0.1, 150 and 120
respectively. The RCR of antibody produced by clone 575G4H7
for AFBl, AFB2, AFGl, and AFG2 were 100, 50, 150 and 103,
respectively.
It should be noted that the low cross-reactivity of the
antibodies with AFB2 and AFG2 does not limit the wide use of
these antibodies because AFB2 and AFG2 rarely occur in
agricultural commodities, and because the toxicity and
carcinogenicity of these two are relatively low.
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