Aflatoxin B1 : Specific Antibodies and Their Use in Radioimmunoassay 1,2, S John J. Langone 4 and Helen Van Vunakis

4, 5

SUMMARY-New Zealand White rabbits immunized with covalent conjugates prepared from polylysine and the O-carboxymethyloxime derivatives of either aflatoxin Bl (AFBl) or an analogue, 5, 7-dimethoxycyclopentenon (2,3-c}coumarin, produced antibodies that bind 3H-AFBl. The specificities of the antisera with respect to aflatoxins B2, B2a, GI, G2, QI, PI, and some other structurally related compounds were determined. Radioimmunoassays that can detect levels as low as 0.27 pmoles (0.06 ng) of AFBI were used to analyze serum, urine, and crude extracts of corn and peanut butter supplemented with aflatoxin. In the foodstuffs, as little as 1 p,g AFBdkg was measured. The immunoassay was at least as sensitive and specific as other available analytic methods, but did not require the purification of samples by chromatography before analysis. The technique may be particularly useful in epidemiologic studies designed to study the possible relationship between chronic anatoxin ingestion and cancer.-J Natl Cancer Inst 56:

591-595, 1976.

Aflatoxin Bl (AFBl; text-fig. I) is the most toxic and carcinogenic member of a group of compounds produced by the mold Aspergillus flavus. It can undergo several metabolic processes including the hydration of the 2,3double bond (AFB2a), the reduction of the keto gr~up (aflatoxicol). Q-dimethylation (AFPl)' and hydroxylatIOn





(AFMl and AFQl) (1~ 2). AFBI also can bind covalently to cell constituents including DNA and RNA in vitro and in vivo, possibly through the intermediary of the 2,3 epoxide (3-7). An alternative mechanism of carcinogenesis may involve the competition of aflatoxin for steroid hormone binding sites on the endoplasmic reticulum, which results in impaired polysome binding (8). Aflatoxins can contaminate many foods, especially grain and peanut products that are not stored properly (1~ 9). In some countries, the ingestion of food contaminated with aflatoxin could cause the deaths of several hundred persons a year (9, 10). Chronic effects resulting in carcinogenesis cannot be estimated as reliably (9). The aflatoxins (and several other mycotoxins) may present a health hazard that is far more extensive and serious than has been realized. Growing concern with aflatoxin poisoning and the potentially hazardous effects of these compounds acting as carcinogens at doses insufficient to manifest the symptoms of aflatoxicosis have led to a concerted effort to devise analytic methods for the accurate determination of aflatoxin in foods, bedding, physiologic fluids, and tissues. Procedures have been developed to extract aflatoxin from several sources including corn and peanut products (11). The crude extracts are chromatographed on a column of silica gel and the purified mixture of aflatoxins is resolved by thin-layer chromatography (TLC) (11) or high-pressure liquid chromatography (12). The accurate determination of aflatoxin levels in contaminated foods and physiologic fluids is still a formidable problem. The available chromatographic techniques are time consuming; they usually require highly purified samples, and their application to many samples is cumbersome. Radioimmunoassay (RIA) has been applied to the quantitative determination of several classes of pharmacologically important compounds (13-15). The high specificity of antibodies and the sensitivity afforded by radiolabeled tracers often permit the use of RIA's to measure picomole levels of the target compound in unprocessed physiologic fluids or other samples. Here we report the production of antibodies to AFBI and the development of sensitive RIA's that have been used to analyze crude extracts of corn and peanut butter contaminated with aflatoxin. Serum and urine supplemented with AFBI also can be analyzed by this technique. MATERIALS AND METHODS

Aflatoxins BlJ B 2 , G 1, and G 2 were purchased from Aldrich Chemical Co., Milwaukee, Wisconsin, or Calbiochern, Los Angeles, California. Aflatoxins B 2a, PI' and Ql were obtained from Dr. G. N. Wogan, Department of Received July 15, 1975; accepted October 6, 1975. Supported by Public Health Service contract CP23243 from the Division of Cancer Cause and Prevention, National Cancer Institute. S Publication #1044 from the Department of Biochemistry, Brandeis University. 4 Department of Biochemistry, Brandeis University, Waltham, Mass. 02154. 5 Recipient of Public Health Service Research Career Award 5K6-AI2372 from the National Institute of Allergy and Infectious Diseases. 1


Aflatoxin Analogu& I.-Structures of aflatoxins Bl, B2, G 1 , G 2 and the aflatoxin analogue 5,7 -demethoxycyclopentenon(2,3-c)coumarin. Asterisk. denotes positions of oxime formation for coupling to polylysine.



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Nutrition and Food Science, Massachusetts Institute of Technology, Boston, l\1assachusetts, and samples of corn and peanut butter conta~inated with aflatoxin w~re obtained from Dr. J. Rodncks, Food and Drug AdmInistration, \Vashington, D.C. Chemicals used in ~ynth~ses were reagent grade and used without punficatIOn. 3H-AFB 1 (sp act, 4.0 Ci/mmole) was. pu~chased from Nuclear Dynamics, Inc., El lVIonte, Cahfor~lla. . . For the preparation of conjugates for ImmUnIZatIon, O-carboxymethyloxime derivatives of AFBI and the aflatoxin analogue 5,7 -dimethoxycyc1opentenon(2,3-c)coumarin were covalently bound to the free amino groups of polylysine. AFB 1 O-carboxymethyloxime.-A solution of AFBI (17 mg; 0.054 mmole) and O-carboxymethylhy~roxyla­ mine hemi hydrochloride (140 mg; 0.64 mmole) In 4 ml ethanol and 0.80 ml of 2 M aqueous NaOH (1.6 mmole) was refluxed for 3 hours. After standing at 25° C overnight the brown reaction mixture was concentrated to I mI.' Water (5 ml) was added, the pH adjusted to 9.5 with INN aOH, and the solution extracted with two 10-ml portions of ethyl acetate. The aqueous layer was acidified to pH 2 with 6 N HCI and store~ a.t 0° C overnight to yield th~ oxi~e as a yel~ow. preCIpItate. It was collected by centnfugatIOn and dned In vacuo over anhydrous CaS0 4 • By TLC (silica gel G) in ethyl acetatemethanol-NH 4 0H (65: 35: 11), the oxime (;:::;10 mg) was homogeneous with a retardation factor (R f ) of 0.60. The product was stored in 1.0 ml dimethyl sulfoxide (DMSO) frozen at -10° C. 5,7 -Dimethoxycyclopentenon(2,3-c )coumarin O-car.b~xy­ methyloxime.-This derivative was prepared by a SImIlar procedure from 5,7 -dimethoxycyclopentenon(2,3-c)coumarin (70 mg; 0.27 mmole) (16) and O-carboxymethylhydroxylamine hemi hydrochloride (294 mg; 1.35 mmole) in the presence of NaOH (6.0 mmole; 1.25 ml of 5 M solution). This product (40 mg) also showed one spot by TLC (R f =0.62) in the same solvent system. Conjugates for immunization.-The O-carboxymethyloximes of AFBl and the aflatoxin analogue were coupled to polY-L-lysine (mol w.t, 90,000). In a ty'pi~al reaction, 10 mg 3-(3-dimethylammopropyl)-carbodllmIde·HCI was added to 5 mg AFBI O-carboxymethyloxime dissolved in 0.5 ml DlVlS0. After 5 minutes, 10 mg polylysine (0.1 ml of 100 mg/ml solution, pH 8.0) ~as added to gi,:e a clear brown solution. The pH was adjusted to 7.0 with 0.1 N NaOH and the reaction mixture stirred at 25° C for 18 hours. Buffer (0.4 ml; 0.005 M phosphate, 0.15 M NaCI; pH 7.4) was added, and the solution chromatographed on a 35 X 1.5-cm column of Sephadex G-50 M eluted with buffer. The fractions corresponding to standard blue dextran 2000 were dialyzed against six changes of buffer (I liter each) before being used for immunization. Based on the UV-visible spectrum of a standard sample of A~Bl (text-fig. 2), the incorporation was 6 moles of mycotoxin/ mole polylysine. . To couple the oxime derivative of .the aflatOXIn a?alogue, we replaced DMSO with dl.methylforma.m~de (DMF). After chromatography, the conjugate, contaInIng 10 moles hapten per mole polylysine, was used directly. Immunization procedure.-Electrostatic complexes between each hapten-polylysine conjugate and succin.ylbovine serum albumin were prepared and used to Immunize New Zealand White rabbits according to a schedule like that described in (17) for preparation of antibodies to morphine. Downloaded from by University of Durham user on 05 January 2018


















Wavelength (nm)

2.-Spectra in water at pH 6.0 of polylysine ( .. : . ), 100 JLg/ml; AFBl ( - - - ) , 7.5 JLgjml; and the polylysmeAFBl conjugate ( - - ) .


RIA procedures.-The diluent for all reagents was buffer [0.14 M NaCI:O.OI M Tris (pH 7.5)] containing 10% horse serum. To 0.1 ml buffer were added a O.I-ml solution of sample to be analyzed, 3~-AFBl [~7,000 counts per minute (cpm)], and 0.1 ml dIluted antibody. The mixture was incubated at 37° C for 1 hour, treated with goat antiserum to rabbit y-globulin (0.05 ml), and stored at 4° C overnight. After centrifugation at 2,000 rpm for 30 minutes, the supernatants were decanted and the walls of the tubes wiped dry. The precipitates were dissolved in 0.2 ml of I N NaOH, and transferred to counting vials. Scintillation flui? [2.5 ml toluene so~ution of 5 g 2,5-diphenyloxazole/hter, 0.33 g 1,4-b!s[2-(5phenyloxazolyl)]benzene/liter, and 12% Beckman BIO-Solv Solubilizer (formula # 3)] was added an~ the samples counted in a Hewlett-Packard model 3320 Instrument. In the absence of inhibitor, I ILl antiserum to AFBI (0.1 ml of a 1/100 dilution) precipitated 5,300 cpm, whereas 2 ILl antiserum to the aflatoxin analogue (0.1 ml of a I / 5~ dilution) precipitated 4,800 cpm. When no~~al rabbIt serum replaced antiserum, 300 cpm were preCIpItate~. Mutagenicity tests.-Samples of Salmonella typhtmurium strains T A 1535, T A 1536, T A 1537, and T A 1538 were supplied by Dr. B. N. Ames, University of California at Berkeley. The tests were done accordIng to the procedures in (18, 19), except that the homogenate was prepared from the liver of an adult male New Zealand ''''hite rabbit rather than from a rat. RESULTS

The O-carboxymethyloxime derivatives of AFBI and the aflatoxin analogue 5,7 -dimethoxycyclopentenon(2,3-c)coumarin were prepared and covalen.tly b?und t? the f~ee amino groups of polylysine. RabbIts gIven eIther Immunogen produced antibodies that bound 3H-AFB 1 • The homologous compound and several othe~ afl~toxins could inhibit the binding of 3H-AFB 1 by antIbodIes produce~ against either of the two haptens (text-fig. 3). ~ot~ antIsera were highly sensitive and showed speCIfiCIty for AFB 1 • In the anti-AFB 1 system (text-fig. 3a), only 1.5 pmoles (0.45 ng) of homologous antigen were required for 50% inhibition and as little as 0.27 pmoles (0.060 ng)





Aflatoxin added to corn or peanut butter

80 c:::



1.-Recovery of aflatoxin from corn and peanut butter a





~ 40










lGl G2

Picomoles Inhibi10r


Bl Bl Bl t JB B2

Cone en tra tion (J.&gjkg)

J.&g AFBI equivalents/kg by RIAb In corn

In peanut butter

0 1 10 50

0 O.4±O.O 7.9±O.1 49.5±2.5


3.4±O.1 27.3±1.6

1O} ~ 20



a Samples of corn (100 g) and two 50-g samples of peanut butter supplemented with aflatoxin by the Food and Drug Administration were extracted according to procedures 26.042 and 26.023, respectively, of the Association of Official Analytical Chemists (11). The final organic extracts were dried in a vacuum oven at a temperature which did not excecd 35° C, and the residues dissolved in 0.025-0.1 ml DMF made up to 0.5 ml with buffer. Further dilutions as required were made in buffer. Control samples of solvents used in the extractions or as diluents were carried through the assay procedure and used to check for nonspecific effects. b Two samples were analyzed in duplicate. Values are meanS±8D.


80 60









Picomoles Inhibitor

3.-Inhibition of A) 3H-AFB 1-anti-AFB 1 and B) a H - AI-B l - anti -5,7 -dimethoxycyclopentenon (2 ,3-c) coumarin immune systems by AFBI (e), AFB2 (~), AFB 2a (V), AFG 1 (0), AFG2 (..), AFQl (0), and AFP 1 (.).


could be detected. In the other system (text-fig. 3b), 5.6 pmoles (1.5 ng) AFBI inhibited 50%, and the lower limit of detection was 1.5 pmoles (0.50 ng). In the homologous system, the levels of the available aflatoxins and structurally related compounds required to give 50% inhibition varied over a wide range. AFB2 with a saturated C-2,3 double bond inhibi ted 2.3 times less effectively than AFBl" AFG l and AFG z inhibited 6.6 and 21 times less effectively. The quantities of AFP I, AFQl' and AFB2a required to give 50% inhibition were 447-, 12-, and 87-fold greater, respectively, than for AFB 1 • Sterigmatocystin inhibited 28% when used 1,500 times in excess, whereas coumarin and warfarin did not inhibit even at the 3-nmole level. Compared to this antiserum, the antiserum to the aflatoxin analogue was three times less sensitive for AFB l , but was more specific (text-fig. 3b). The amounts (pmoles) of the other aflatoxins relative to AFBI required to give 50% inhibition were: B z, 5.3; G l , 61; G 2 , 309; Bza, 127; QlJ 69; PI' 611. Sterigmatocystin, coumarin, and warfarin were ineffective inhibitors in this system also. Ames and his co-workers (18, 19) reported that AFBl was mutagenic in their Salmonella-mammalian microsome assay. We used rabbit liver homogenates in similar test systems to determine that the aflatoxin analogue was not mutagenic in any of the four test systems even at 100 times the level at which AFBl was strongly active in strain TA 1538 and mildly active in strain TA 1537. For the aflatoxins, the order of mutagenicity is B l > G l > B2 > G 2 and is the same as the relative toxicity found in a similar bacterial system (20). Downloaded from by University of Durham user on 05 January 2018

Serum and urine to which known amounts of AFBl had been added were also analyzed by RIA. The standard curves in buffer, serum, or urine differed less than 15% at the point of 50% inhibition, and each was used with appropriate samples to convert the inhibition data to the concentration of AFBl or AFBl equivalents. When serum was supplemented with 50 ng AFBl/ml, 10-,al samples were assayed directly and gave 37% inhibition. This value corresponded to 0.5 ng AFBd sample or 50 ng AFBl/ml. Quantitative recovery indicated that there was no significant interference by binding of the AFBI to serum components. Similarly, 100 :,al urine supplemented with 10 ng AFBdml gave 47'% inhibition. In this instance, the recovery was 90% of the input. The RIA for AFBl was used to analyze extracts of corn and peanut butter samples contaminated with different levels of AFBI or with a mixture of AFBv B2 , G 1, and G 2 (table 1). Each sample was extracted in duplicate and each extract was analyzed without further purification. The estimated levels of aflatoxin compared to the actual input were within the limits of recoveries expected from the extraction procedures. Because the RIA was sensitive, most extracts (dissolved in DMF buffer) could be diluted before analysis. For example, extracts of corn samples contaminated with 10 or 50 .,ag AFBl/kg were diluted 1/30 and 1/300, respectively. Even when samples contained only l,ag AFB1/kg, a 1/3 dilution could be used. DISCUSSION

The RIA's for aflatoxin are at least as sensitive as the available spectral and chromat~graphic techniques (11, 20-22) and show specificity for AFB 1 • The relative abilities of the available aflatoxins to inhibit the immune reactions indicate that the methoxyl group is immunodominant. AFP l , the demethylated product of AFB 1, is 450-600 times less effective than AFB l , and is the poorest inhibitor in both systems. These results probably reflect the difference in polarity between the nonpolar ether moiety and the polar phenolic hydroxyl group. The polarity of ligands is important in determining the effectiveness of immune binding (23), and results like these were observed in other systems (24). The ability of the antibodies to discriminate between AFBI and AFP 1 may be of practical significance. AFP l' a major urinary metabolite in monkeys, is excreted mainly as the glucuronide



conjugate and, to a lesser extent, as the sulfate (25). These derivatives were not available to be tested as inhibitors, but they would be expected to inhibit even less effectively than AFP l' Although AFP 1 has not yet been found in human urine, its production by human liver in vitro has been demonstrated (26). Saturation of the C-2,3 double bond (to give Hz from Bl> or G z from G 1 ), hydration of the double bond (Bza), hydroxylation at the carbon f3 to the keto group (Ql)' and modification of the cyclopentenone ring to a sixmembered ring lactone (B to G series) all produce compounds that inhibit less effectively than AFBl' However, the differences are not as great as those observed between PI and B 1 • Use of the analogue to develop an RIA for AFBI offers some practical advantages. It is safe to handle, it can be synthesized readily on a large scale, and good antibody specificity can be achieved. Experiments have shown that at least 100 f-tl serum or urine (at pH 7) can be assayed for aflatoxin without unacceptable nonspecific inhibition that can occur in some immune systems; thus levels of aflatoxin as low as 750 pg/ml fluid can be assayed directly. However, lower levels of aflatoxin can be determined after larger volumes of fluid are extracted to concentrate the mycotoxins. As little as 75 pg AFBdO.l ml concentrate could then be measured. Unfortunately, data on levels in blood are lacking, and data on urine levels are sparse. The available information (9) indicates that such levels range between 0.02 and 0.05 p.g/24-hour sample in urine from normal and cirrhotic children exposed to aflatoxin. Thus under. these conditions, urine would have to be extracted and concentrated before analysis. In a recently reported outbreak of hepatitis due to aflatoxicosis, it was discovered that several hundred people had consumed contaminated maize containing 6-16 mg aflatoxin/kg/day for I month (10). This level of contamination is unusually high. However, even when the concentration of aflatoxin is within the more commonly observed range (0.11.0 mg/kg) (9), the RIA may be useful in epidemiologic studies to determine if a causal relationship exists between aflatoxin consumption and the incidence of chronic liver disease. The RIA's have been used to analyze extracts of corn or peanut butter contaminated with aflatoxin at levels of I or 10 p.g/kg, respectively, without the additional column chromatography step required by other techniques. Due to nonspecific interference, accurate results could not be obtained on samples of peanut butter that contained 1 p.g AFBI/kg, though analyses probably could be performed after further purification. The agreement between experimental and input values was best at higher concentrations of aflatoxin, possibly because more DMF could be used to dissolve the extracted aflatoxin before analyses by RIA. The assay has not been used to analyze other types of foods, but the extended applications clearly are possible. Samples of diverse foods from various countries have shown that aflatoxin contamination at greater than 30 p.g/kg is common, and that concentrations exceeding I mg/kg are possible (9). Hence use of the assay to measure meaningful aflatoxin concentrations has been demonstrated. Since AFBI usually is the major food contaminant, the more specific RIA based on the aflatoxin analogue could be used to estimate levels of this toxin in the presence of AFG 1 , G 2 , or low levels of B2 that can be found in some Downloaded from by University of Durham user on 05 January 2018

products. Results obtained with the less specific but more sensitive anti-AFB 1 RIA may be considered AFBI equivalents. Limited specificity among the aflatoxins may not be a requisite for a practical assay, since the detection of any aflatoxin, whether it is only AFBI or a mixture of compounds, is the ultimate goal in a program designed to screen foods or physiologic fluids of persons who may have ingested contaminated products. For metabolic studies or determination of levels of individual compounds derived from AFB 1 , antibodies with high specificity for each metabolite will be required. In conclusion, the sensitivity, specificity, and simplicity of this RIA technique should make the assay a useful tool in epidemiologic studies and other research when accurate levels of this potent, toxic, and carcinogenic agent must be determined. REFERENCES (1) CIEGLER A: Micotoxins: Occurrence, chemistry, biological activity. Lloydia 38:21-35, 1975 (2) PATTERSON DS: Metabolism as a factor in determining the toxic action of the aflatoxins in different animal species. Food Cosmet Toxicol 1l:287-294, 1973 (J) ALEXANDROV K, FRAYSSINET C: Microsome-dependent binding of benzo(a)pyrene and aflatoxin Bl to DNA, and benzo(a)pyrene binding to aflatoxin-conjugated DNA. Cancer Res 34:3289-3295, 1974 (4) SWENSON DH, MILLER EC, MILLER J: Aflatoxin B1"2,3-oxide: Evidence for its formation in rat liver in vivo and by human liver microsomes in' vitro. Biochem Biophys Res Commun 60:1036-1043, 1974 (5) GURTOO HL, DAHMS R: On the nature of the binding of aflatoxin B 2a to rat hepatic microsomes. Res Commun Chern Pathol Pharmacol 9:107-113, 1974 (6) GARNER RC: Chemical evidence for the formation of a reactive aflatoxin Bl metabolite by hamster liver microsomes. Fed Eur Biochem Soc Lett 36:261-264, 1973 (7) GURTOO HL, DAVE CV: In vitro metabolic conversion of aflatoxins and benzo(a)pyrene to nucleic acid-binding metabolites. Cancer Res 35:382-389, 1975 (8) WILLIAMS DJ, RABIN BR: Disruption by carcinogens of the hormone dependent association of membranes with polysomes. Nature 232:102-105, 1971 (9) CAMPBELL TC, S'IOLOFF L: Implication of mycotoxins for human health. J Agr Food Chern 22: 1006-1014, 1974 (10) KIRSHNAMACHARI KA, NAGARAJAN V, BHAT RV, et al: Hepatitis due to aflatoxicosis. Lancet I: 1061-1063, 1975 (11) Natural Poisons: In Official Methods of Analysis of the Association of Official Analytical Chemists (Horwitz W, ed.), chapt 26. 1975, pp 1-24 (12) SEITZ LM: Comparison of methods of aflatoxin analysis by high-pressure liquid chromatography. J Chromatogr 104:8191, 1975 (IJ) BERSON SA, YALOW R: Radioimmunoassay: A status report. In Immunobiology (Good RA, Fisher PW, eds.). Stamford, Conn., Sinauer Assoc, 1971, pp 287-293 (14) BUTLER VP, JR, BEISER SM: Antibodies to small molecules: Biological and clinical applications. Adv Immunol 17:255310, 1973 VAN VUNAKIS H, LEVINE L: Antibodies: Ana(15) LANGONE lytical tools to study pharmacologically active compoundS. Accounts Chemical Research 8:335-342, 1975 (16) ASAO T, BikHI G, ABDEL-KADER MM, et al: The structures of aflatoxins Band G 1 . J Am Chern Soc 87:882-886, 1965 (17) VAN VUNAKIS H, WASSERMAN E, LEVINE L: Specifications of antibodies to morphine. J Pharmacol Exp Ther 180:514521, 1972 (18) AMES BN, LEE FD, DURSTON WE: An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc Natl Acad Sci USA 70:782-786, 1973 (19) AMES BN, DURSTON WE, YAMASAKI E, et al: Carcinogens are mutagens: A simple test system combining liver homagenates for activation and bacteria for detection. Proc Nat! Acad Sci USA 70:2281-2285, 1973



(20) GARNER RC, MILLER EC, MILLER JA: Liver microsomal metabolism of aflatoxin BI to a reactive derivative toxic to Salmonella typhirnurium T A 1530. Cancer Res 32:20582066, 1972 (21) PURCHASE IF: Aflatoxin residues in food of animal origin. Food Cosmet Toxicol 10:531-544, 1972 (22) GARNER RC: Aflatoxin separation by high-pressure liquid chromatography. J Chromatogr 103:186-189, 1975

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(23) LANDSTEINER K: The Specificity of Serological Reactions. Cambridge, Raryard Univ Press, 1945 (24) LEVINE L, RICEBERG LJ: Radioimmunoassay for melatonin. Res Commun Chern Pathol Pharmacol 10:693-702, 1975 (25) DALEZIOS J, Wogan GN: Aflatoxin PI: A new aflatoxin metabolite in monkeys. Science 171:584-585, 1971 (26) BucHI GR, MUELLER PM, ROEBUCK BD, et al: Aflatoxin QI major metaholite of aflatoxin BI produced in human liver. Res Commun Chern Pathol Pharmacol 8:585-592, 1974

Aflatoxin B; specific antibodies and their use in radioimmunoassay.

New Zealand White rabbits immunized with covalent conjugates prepared from polylysine and the O-carboxy-methyloxime derivatives of either aflatoxin B1...
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