CA1315221C - Assay for aflatoxins - Google Patents
Assay for aflatoxinsInfo
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- CA1315221C CA1315221C CA000613230A CA613230A CA1315221C CA 1315221 C CA1315221 C CA 1315221C CA 000613230 A CA000613230 A CA 000613230A CA 613230 A CA613230 A CA 613230A CA 1315221 C CA1315221 C CA 1315221C
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- aflatoxin
- antibody
- assay
- recognizes
- binding
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Abstract
Abstract An immunoassay for aflatoxin is disclosed. It relies on developing antibodies which recognize both aflatoxin G1 and B1 with substantially equal affinity. The antibodies are preferably derived using an aflatoxin B3-HS-BSA conjugate as an antigen.
Description
`- ~31522~
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).
~- a t315221 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.
131522t 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--` 1 3 1 5 22 1 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.
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.
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 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.
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.
~ 31 ~221 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 _9_ 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.
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).
~- a t315221 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.
131522t 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--` 1 3 1 5 22 1 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.
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.
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 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.
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.
~ 31 ~221 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 _9_ 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.
Claims (10)
1. An assay for the presence of aflatoxin in a sample, comprising the step of:
using an antibody capable of binding to the afla-toxin to test for the presence of aflatoxin;
wherein the antibody is of a type that recognizes both aflatoxin B1 and aflatoxin G1, and does so with sub-stantially equal affinities for both.
using an antibody capable of binding to the afla-toxin to test for the presence of aflatoxin;
wherein the antibody is of a type that recognizes both aflatoxin B1 and aflatoxin G1, and does so with sub-stantially equal affinities for both.
2. The assay of claim 1, wherein the antibody also recognizes aflatoxin B3.
3. An antibody capable of binding to an aflatoxin, the antibody recognizing both aflatoxin B1 and aflatoxin G1, and the antibody having substantially equal affinity for both.
4. A competitive assay kit comprising:
an antibody capable of binding to an aflatoxin, the antibody recognizing both aflatoxin B1 and aflatoxin G1, and the antibody having substantially equal affinity for both;
and a radioactively labelled aflatoxin that the anti-body will also bind to.
an antibody capable of binding to an aflatoxin, the antibody recognizing both aflatoxin B1 and aflatoxin G1, and the antibody having substantially equal affinity for both;
and a radioactively labelled aflatoxin that the anti-body will also bind to.
5. An ELISA test kit for aflatoxin comprising:
an antibody capable of binding to an aflatoxin, the antibody recognizing both aflatoxin B1 and aflatoxin G1, and the antibody having substantially equal affinity for both;
and an enzyme linked aflatoxin that the antibody will bind to.
an antibody capable of binding to an aflatoxin, the antibody recognizing both aflatoxin B1 and aflatoxin G1, and the antibody having substantially equal affinity for both;
and an enzyme linked aflatoxin that the antibody will bind to.
6. A hybridoma capable of producing antibody that binds to both aflatoxin B1 and aflatoxin G1, said antibody having substantially equal affinity for both.
7. An assay for the presence of aflatoxin in a sample, comprising the steps of:
exposing the sample to an antibody capable of bind-ing to the aflatoxin to test for the presence of aflatoxin;
and determining the presence or absence of aflatoxin in the sample;
wherein the antibody is of a type that recognizes both aflatoxin B1 and aflatoxin G1, and does so with sub-stantially equal affinity for both;
wherein the antibody also recognizes aflatoxin B3 and is of a type that had been produced as a result of an aflatoxin B3 conjugate with a polypeptide having been used as an antigen where the polypeptide was linked to an aflatoxin B3 moiety at the carbon that normally bears the OH group in aflatoxin B3.
exposing the sample to an antibody capable of bind-ing to the aflatoxin to test for the presence of aflatoxin;
and determining the presence or absence of aflatoxin in the sample;
wherein the antibody is of a type that recognizes both aflatoxin B1 and aflatoxin G1, and does so with sub-stantially equal affinity for both;
wherein the antibody also recognizes aflatoxin B3 and is of a type that had been produced as a result of an aflatoxin B3 conjugate with a polypeptide having been used as an antigen where the polypeptide was linked to an aflatoxin B3 moiety at the carbon that normally bears the OH group in aflatoxin B3.
8. The assay of claim 7, wherein the antibody had been derived from a cell line bearing ATCC designation HB 10106.
9. An antibody capable of binding to an aflatoxin, wherein:
the antibody is of a type that recognizes both afla-toxin B1 and aflatoxin G1, and does so with substantially equal affinity for both;
wherein the antibody also recognizes aflatoxin B3 and is of a type that has been produced as a result of an aflatoxin B3 conjugate with a polypeptide having been used as an antigen where the polypeptide was linked to an aflatoxin B3 moiety at the carbon that normally bears the OH group in aflatoxin B3.
the antibody is of a type that recognizes both afla-toxin B1 and aflatoxin G1, and does so with substantially equal affinity for both;
wherein the antibody also recognizes aflatoxin B3 and is of a type that has been produced as a result of an aflatoxin B3 conjugate with a polypeptide having been used as an antigen where the polypeptide was linked to an aflatoxin B3 moiety at the carbon that normally bears the OH group in aflatoxin B3.
10. The antibody of claim 9, wherein the antibody had been derived from a cell line bearing ATCC designation HB
10106.
10106.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US35051789A | 1989-05-11 | 1989-05-11 | |
| US07/350,517 | 1989-05-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1315221C true CA1315221C (en) | 1993-03-30 |
Family
ID=23377064
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000613230A Expired - Fee Related CA1315221C (en) | 1989-05-11 | 1989-09-26 | Assay for aflatoxins |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1315221C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2090590A1 (en) * | 2006-11-17 | 2009-08-19 | Horiba, Ltd. | Antibody against aflatoxins, support using the antibody, method of immunologically detecting aflatoxins and method of concentrating and purifying aflatoxins |
| CN101270146B (en) * | 2008-05-08 | 2010-09-29 | 中国农业科学院油料作物研究所 | Method for preparing aflatoxin G1 artificial antigen |
| CN102617589A (en) * | 2012-02-24 | 2012-08-01 | 中国农业大学 | Artificial antigen for aflatoxin M1 and antibody prepared by same |
| CN103792359A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Preparation and detection method of aflatoxin G1 enzyme-linked immunosorbent assay kit |
| CN103792358A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Preparation and detection method of aflatoxin G1 colloidal gold immunochromatograohic assay detection board |
| CN110386980A (en) * | 2019-07-16 | 2019-10-29 | 河南检通生物技术有限公司 | A kind of aspergillus flavus-resistance mycin G1(AFG1) method for preparing monoclonal antibody |
-
1989
- 1989-09-26 CA CA000613230A patent/CA1315221C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2090590A1 (en) * | 2006-11-17 | 2009-08-19 | Horiba, Ltd. | Antibody against aflatoxins, support using the antibody, method of immunologically detecting aflatoxins and method of concentrating and purifying aflatoxins |
| EP2090590A4 (en) * | 2006-11-17 | 2010-07-28 | Horiba Ltd | Antibody against aflatoxins, support using the antibody, method of immunologically detecting aflatoxins and method of concentrating and purifying aflatoxins |
| US8153767B2 (en) | 2006-11-17 | 2012-04-10 | Horiba, Ltd. | Antibody against aflatoxins, support using the antibody, method of immunologically detecting aflatoxins and method of concentrating and purifying aflatoxins |
| CN101270146B (en) * | 2008-05-08 | 2010-09-29 | 中国农业科学院油料作物研究所 | Method for preparing aflatoxin G1 artificial antigen |
| CN102617589A (en) * | 2012-02-24 | 2012-08-01 | 中国农业大学 | Artificial antigen for aflatoxin M1 and antibody prepared by same |
| CN103792359A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Preparation and detection method of aflatoxin G1 enzyme-linked immunosorbent assay kit |
| CN103792358A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Preparation and detection method of aflatoxin G1 colloidal gold immunochromatograohic assay detection board |
| CN110386980A (en) * | 2019-07-16 | 2019-10-29 | 河南检通生物技术有限公司 | A kind of aspergillus flavus-resistance mycin G1(AFG1) method for preparing monoclonal antibody |
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