Vol. 34, No. 1 Printed in U.S.A.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1977, p. 94-96 Copyright ©D 1977 American Society for Microbiology
Enzyme-Linked Immunosorbent Analysis for Aflatoxin B1 D. W. LAWELLIN,* D. W. GRANT, AND B. K. JOYCE Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523
Received for publication 9 March 1977
An enzyme-linked immunosorbent analysis (ELISA) permitted the detection of less than 10 pg of aflatoxin B1 per ml. The antitoxin was most specific for aflatoxins B1 and B.,, and least specific for aflatoxin G1.
ml. Using antiserum AT-9, the assay reliably measured between 100 to 1 pg per ml of AFBI, within ±1 standard deviation of the concentration derived from the stand curve, in 92% (n = 18) of the test samples for each concentration tested.
Current analyses for aflatoxin B, (AFB,) lack both the sensitivity and specificity for quantifying trace amounts of AFBI found in biological fluids from animals subjected to experimentally induced aspergillosis (1; D. W. Lawellin, M.S. thesis, Colorado State University, Fort Collins, 1974). An enzyme-linked immunosorbent analysis (ELISA) has been developed to permit analysis of such samples. The immunogen was prepared according to the procedure of DeCarvalho et al. (2) using tetrazobenzidine (3.98 mmol) to couple aflatoxin B2a (AFB.2,; 1.2 mmol) to bovine serum albumin (0.12 mmol) in a total volume of 320 ml. AFB, was converted to the acetal form AFB.,a by acid catalysis before coupling. AFB2a is a metabolic end product of AFB, in several animal species. The immune amplifier peroxidase (Sigma Chemical Co., type VI, 400 U/mg) was conjugated to AFB.,a in the same manner, maintaining the same molar ratio for the reac-
TABLE 1. Inhibition of AFB, by AFB20, AFM, and AFG, in the ELISA procedure a concn Hapten inhibitor Hapten (,ug/ml)
AFB,
10-4 10-4 10-4 10-4
Inhibition (range) 100
100 (88-100) 50 (60-25) 7.5 (1-10) a Sensitization: 1:5 x 104 dilution of antiserum AT-9 in borate saline buffer (BSB), pH 8.2, 56°C for 90 min. Test: 10-4 Ug of aflatoxin per ml in BSB (pH 8.2)-0.025% Tween 80, 22°C for 4 h; peroxidase-AFB, (0.4 U) in BSB (pH 8.2)-0.025% Tween 80, 22°C for 16 h.
AFB2. AFM, AFG,
tants.
Antiserum was produced in 3-month-old female New Zealand white rabbits. Multiple-site subcutaneous injections of 1 mg of the immunogen per animal were given dorsally, first in Freund complete adjuvant and thereafter in Freund incomplete adjuvant. The injections were given twice monthly for 3 months and then weekly during the 4th month. The animals were bled by cardiac puncture 7 days after the last injection. Individual serum collections were stored at -27°C. The AFB1 assay protocol, modified after Engvall et al. (3-5), is outlined in Fig. 1. Antiserum titration curves from two animals (Fig. 2) indicated that the optimal serum dilution ranged from 103 to 104. An antibody dilution that gives 50% of the maximal colorimetric absorption in the region of antigen excess should be used to reduce antibody competition for adsorption sites on the polystyrene surface. Standard hapten competition curves for the ELISA (Fig. 3) demonstrated that the procedure could detect less than 10 pg of AFB, per
TABLE 2. Effect of interfering materials in biological fluids on the aflatoxin ELISA a
Bilogical fluids
Biological
Initial AFB, concn ml)
(pAg/
AFB, recovered (/Ag/ml) Untreated XAD-treated
1 0.71 0.75 Urine lb (0.5) 0.05 0.001 Plasma lb (0.5) 0.25 Serum 0.005 a See Table 1. b By thin-layer chromatography analysis, the measured AFB, contents of the serum and plasma were one-half the actual amounts added.
The ability of various aflatoxins to inhibit the binding of AFB, was determined (Table 1). AFBu produced complete inhibition. AFM1 and AFG, caused 50 and 7.5% inhibition, respectively. The results of analyses for AFB, in biological fluids are presented in Table 2. The urine contained no materials that interfered substantially with the analyses. Blood fractions, how94
VOL. 34, 1977
NOTES
95
Place 1.0 ml of appropriate dilution of antitoxin (U) in 0.05 M borate saline buffer, pH 8.2 (BSB; 0.034 M NaCl) in polystyrene tubes (Corning tissue culture, 16 by 125 mm). Incubate tubes in an upright position at 560C for 90 min.
Ku) Wash tubes three times with 1 ml of BSB (0.15 M NaCl)-0.025% Tween 80.
Add to the sensitized tubes 0.4 ml of appropriate dilution of AFBI (B) in BSB (0.15 M NaCl)-0.025% Tween 80 for 4 h at 22°C. Place tubes in a culture tube roller set at an angle so the entire antibody-sensitized area is contacted.
Wash tubes three times with 1 ml of BSB (0.15 M NaCl)-0.025% Tween 80. Add 0.5 ml of peroxidase-AFB, (0.4 peroxidase units, BE) in BSB (0.15 M NaCl)-0.025% Tween 80 for 8 h at 22°C. Place in culture tube roller as above.
i13eu
>"LV Wash tubes three times with 1 ml of BSB (0.15 M NaCl)-0.025% Tween 80.
Add 1 ml of 0.01 M phosphate buffer, pH 6.0, containing 19 mmol of o-tolidine and 10 mmol of H202 for 90 min at 3700 (S). Stop reaction by addition of 0.1 ml of 1 N NaOH. Read colorimetric response at 425 nm.
FIG. 1. Protocol for AFB, ELISA .
,
s
96
APPL. ENVIRON. MICROBIOL.
NOTES 0.22r
z
£
z i
zI 0.14j H
I0.1 4.
£
0.10F 0.06 2
3
4
- LOG PG 2
3
4
5
6
LOG RECIPROCAL AMIBODY DILUTION
FIG. 2. Antibody titering curves for anti-aflatoxin sera. Sensitization: Appropriate dilution of antiserum in BSB, pH 8.2, 56°C for 90 min. Test: Peroxi-
dase-AFB, (0.4 U) in 0.4 ml of BSB (pH 8.2)-0.05% Tween 80; 16 h incubation at 22°C; 1 ml of o-tolidine substrate for 90 min. Antiserum AT-9, 6 mg of immunoglobulin per ml (A, + standard deviation). Antiserum AT-4, 1 mg of immunoglobulin per ml (U, + standard deviation). ever, required pretreatment with XAD-2 resin columns (6) to remove interfering materials. The enzyme-linked immunosorbent analysis for AFB, described here is of particular value for studying in vivo aflatoxin formation during experimental aspergillosis and as a diagnostic aid in cases of suspected aflatoxicosis.
LITERATURE CITED 1. Brewster, T. C., and D. W. Grant. 1972. Excretion of aflatoxin by frogs after implantation with Aspergillus flavus. J. Infect. Dis. 125:66-71. 2. DeCarvalho, S., H. J. Rand, and A. Lewis. 1964. Cou-
S
6
7
8
9
FREE AFLATOXIN B1 / ML
FIG. 3. Representative hapten competition curves for free AFB, in the ELISA procedure. Test: Appropriate dilution AFB, in BSB (pH 8.2)-0.025% Tween 80, 22°C for 4 h; peroxidase-AFB, (0.4 U) in BSB (pH 8.2)-0.025% Tween 80, 220C for 16 h; o-tolidine for 90 min. Antiserum AT-4, 1:5 x 10: dilution (U, + standard deviation). Antiserum AT-9, 1:5 x 104 dilution (A, ± standard deviation).
3. 4.
5.
6.
pling of cyclic chemotherapeutic compounds to immune gamma-globulins. Nature (London) 202:255258. Engvall, E., and P. Perlmann. 1971. Enzyme-linked immunosorbent assay (ELISA); quantitative assay of immunoglobulin G. Immunochem. 8:871-872. Engvall, E., K. Jonsson, and P. Perlmann. 1971. Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of labeled antigen and antibody coated tubes. Biochem. Biophys. Acta 251:427-431. Engvall, E., and P. Perlmann. 1972. Enzyme-linked immunosorbent assay. III. Quantitation of specific antibody by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109:129-134. Rohm-Haas. 1972. XAD-2 resin bibliography. RohmHaas technical bulletin, Ion Exchange Dept., Philadelphia.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1977, p. 94-96 Copyright ©D 1977 American Society for Microbiology
Enzyme-Linked Immunosorbent Analysis for Aflatoxin B1 D. W. LAWELLIN,* D. W. GRANT, AND B. K. JOYCE Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523
Received for publication 9 March 1977
An enzyme-linked immunosorbent analysis (ELISA) permitted the detection of less than 10 pg of aflatoxin B1 per ml. The antitoxin was most specific for aflatoxins B1 and B.,, and least specific for aflatoxin G1.
ml. Using antiserum AT-9, the assay reliably measured between 100 to 1 pg per ml of AFBI, within ±1 standard deviation of the concentration derived from the stand curve, in 92% (n = 18) of the test samples for each concentration tested.
Current analyses for aflatoxin B, (AFB,) lack both the sensitivity and specificity for quantifying trace amounts of AFBI found in biological fluids from animals subjected to experimentally induced aspergillosis (1; D. W. Lawellin, M.S. thesis, Colorado State University, Fort Collins, 1974). An enzyme-linked immunosorbent analysis (ELISA) has been developed to permit analysis of such samples. The immunogen was prepared according to the procedure of DeCarvalho et al. (2) using tetrazobenzidine (3.98 mmol) to couple aflatoxin B2a (AFB.2,; 1.2 mmol) to bovine serum albumin (0.12 mmol) in a total volume of 320 ml. AFB, was converted to the acetal form AFB.,a by acid catalysis before coupling. AFB2a is a metabolic end product of AFB, in several animal species. The immune amplifier peroxidase (Sigma Chemical Co., type VI, 400 U/mg) was conjugated to AFB.,a in the same manner, maintaining the same molar ratio for the reac-
TABLE 1. Inhibition of AFB, by AFB20, AFM, and AFG, in the ELISA procedure a concn Hapten inhibitor Hapten (,ug/ml)
AFB,
10-4 10-4 10-4 10-4
Inhibition (range) 100
100 (88-100) 50 (60-25) 7.5 (1-10) a Sensitization: 1:5 x 104 dilution of antiserum AT-9 in borate saline buffer (BSB), pH 8.2, 56°C for 90 min. Test: 10-4 Ug of aflatoxin per ml in BSB (pH 8.2)-0.025% Tween 80, 22°C for 4 h; peroxidase-AFB, (0.4 U) in BSB (pH 8.2)-0.025% Tween 80, 22°C for 16 h.
AFB2. AFM, AFG,
tants.
Antiserum was produced in 3-month-old female New Zealand white rabbits. Multiple-site subcutaneous injections of 1 mg of the immunogen per animal were given dorsally, first in Freund complete adjuvant and thereafter in Freund incomplete adjuvant. The injections were given twice monthly for 3 months and then weekly during the 4th month. The animals were bled by cardiac puncture 7 days after the last injection. Individual serum collections were stored at -27°C. The AFB1 assay protocol, modified after Engvall et al. (3-5), is outlined in Fig. 1. Antiserum titration curves from two animals (Fig. 2) indicated that the optimal serum dilution ranged from 103 to 104. An antibody dilution that gives 50% of the maximal colorimetric absorption in the region of antigen excess should be used to reduce antibody competition for adsorption sites on the polystyrene surface. Standard hapten competition curves for the ELISA (Fig. 3) demonstrated that the procedure could detect less than 10 pg of AFB, per
TABLE 2. Effect of interfering materials in biological fluids on the aflatoxin ELISA a
Bilogical fluids
Biological
Initial AFB, concn ml)
(pAg/
AFB, recovered (/Ag/ml) Untreated XAD-treated
1 0.71 0.75 Urine lb (0.5) 0.05 0.001 Plasma lb (0.5) 0.25 Serum 0.005 a See Table 1. b By thin-layer chromatography analysis, the measured AFB, contents of the serum and plasma were one-half the actual amounts added.
The ability of various aflatoxins to inhibit the binding of AFB, was determined (Table 1). AFBu produced complete inhibition. AFM1 and AFG, caused 50 and 7.5% inhibition, respectively. The results of analyses for AFB, in biological fluids are presented in Table 2. The urine contained no materials that interfered substantially with the analyses. Blood fractions, how94
VOL. 34, 1977
NOTES
95
Place 1.0 ml of appropriate dilution of antitoxin (U) in 0.05 M borate saline buffer, pH 8.2 (BSB; 0.034 M NaCl) in polystyrene tubes (Corning tissue culture, 16 by 125 mm). Incubate tubes in an upright position at 560C for 90 min.
Ku) Wash tubes three times with 1 ml of BSB (0.15 M NaCl)-0.025% Tween 80.
Add to the sensitized tubes 0.4 ml of appropriate dilution of AFBI (B) in BSB (0.15 M NaCl)-0.025% Tween 80 for 4 h at 22°C. Place tubes in a culture tube roller set at an angle so the entire antibody-sensitized area is contacted.
Wash tubes three times with 1 ml of BSB (0.15 M NaCl)-0.025% Tween 80. Add 0.5 ml of peroxidase-AFB, (0.4 peroxidase units, BE) in BSB (0.15 M NaCl)-0.025% Tween 80 for 8 h at 22°C. Place in culture tube roller as above.
i13eu
>"LV Wash tubes three times with 1 ml of BSB (0.15 M NaCl)-0.025% Tween 80.
Add 1 ml of 0.01 M phosphate buffer, pH 6.0, containing 19 mmol of o-tolidine and 10 mmol of H202 for 90 min at 3700 (S). Stop reaction by addition of 0.1 ml of 1 N NaOH. Read colorimetric response at 425 nm.
FIG. 1. Protocol for AFB, ELISA .
,
s
96
APPL. ENVIRON. MICROBIOL.
NOTES 0.22r
z
£
z i
zI 0.14j H
I0.1 4.
£
0.10F 0.06 2
3
4
- LOG PG 2
3
4
5
6
LOG RECIPROCAL AMIBODY DILUTION
FIG. 2. Antibody titering curves for anti-aflatoxin sera. Sensitization: Appropriate dilution of antiserum in BSB, pH 8.2, 56°C for 90 min. Test: Peroxi-
dase-AFB, (0.4 U) in 0.4 ml of BSB (pH 8.2)-0.05% Tween 80; 16 h incubation at 22°C; 1 ml of o-tolidine substrate for 90 min. Antiserum AT-9, 6 mg of immunoglobulin per ml (A, + standard deviation). Antiserum AT-4, 1 mg of immunoglobulin per ml (U, + standard deviation). ever, required pretreatment with XAD-2 resin columns (6) to remove interfering materials. The enzyme-linked immunosorbent analysis for AFB, described here is of particular value for studying in vivo aflatoxin formation during experimental aspergillosis and as a diagnostic aid in cases of suspected aflatoxicosis.
LITERATURE CITED 1. Brewster, T. C., and D. W. Grant. 1972. Excretion of aflatoxin by frogs after implantation with Aspergillus flavus. J. Infect. Dis. 125:66-71. 2. DeCarvalho, S., H. J. Rand, and A. Lewis. 1964. Cou-
S
6
7
8
9
FREE AFLATOXIN B1 / ML
FIG. 3. Representative hapten competition curves for free AFB, in the ELISA procedure. Test: Appropriate dilution AFB, in BSB (pH 8.2)-0.025% Tween 80, 22°C for 4 h; peroxidase-AFB, (0.4 U) in BSB (pH 8.2)-0.025% Tween 80, 220C for 16 h; o-tolidine for 90 min. Antiserum AT-4, 1:5 x 10: dilution (U, + standard deviation). Antiserum AT-9, 1:5 x 104 dilution (A, ± standard deviation).
3. 4.
5.
6.
pling of cyclic chemotherapeutic compounds to immune gamma-globulins. Nature (London) 202:255258. Engvall, E., and P. Perlmann. 1971. Enzyme-linked immunosorbent assay (ELISA); quantitative assay of immunoglobulin G. Immunochem. 8:871-872. Engvall, E., K. Jonsson, and P. Perlmann. 1971. Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of labeled antigen and antibody coated tubes. Biochem. Biophys. Acta 251:427-431. Engvall, E., and P. Perlmann. 1972. Enzyme-linked immunosorbent assay. III. Quantitation of specific antibody by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J. Immunol. 109:129-134. Rohm-Haas. 1972. XAD-2 resin bibliography. RohmHaas technical bulletin, Ion Exchange Dept., Philadelphia.
