Production of Antibody Against Ochratoxin A

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1976, p Copyright D 1976 American Society for Microbiology Vol. 31, No. 6 Printed in U.SA. Production of Antibody Against Ochratoxin A F. S. CHU,* FRED CHI C. CHANG, AND RONALD D. HINSDILL Food Research Institute and Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, Wisconsin Received for publication 13 February 1976 Antibody against ochratoxin A was obtained after repeated injection of different protein-ochratoxin A conjugates to rabbits. Among many protein-ochratoxin conjugates tested, bovine serum albumin-ochratoxin A was found to be the best antigen. The antibody is specific for ochratoxins A, C, and T, but is not specific for ochratoxins B, a, and other coumarin derivatives. The sensitivity for ochratoxin A detection using a binding assay is in the range of 0.5 to 10 ng/0.5- ml sample. Detailed methods for the preparation of protein-ochratoxin conjugates, preparation of immune serum, and methods for antibody titers are described. Ochratoxins are a group of toxic metabolites produced by a number of fungi in the Aspergillus and Penicillium genera (1, 4, 7, 15). Ochratoxin A (OA), the most potent toxin in this group, has been considered primarily as a nephrotoxin. Because of the widespread occurrence of the toxin-producing fungi and of the occurrence of the toxin in several agricultural commodities (9, 11-14), the ochratoxin problem has received considerable attention in recent years and has been the topic of several reviews (1, 4, 7, 15). Like many other mycotoxins, methods for ochratoxin determination generally depend on chemical analysis, biological assay, or a combination of both methods (4, 7, 10). These methods are either lacking in specificity or limited by their sensitivity. To overcome some of these difficulties, attempts were made to develop an immunochemical method for ochratoxins. In a preliminary report (F. S. Chu, C. C. Chang, and R. D. Hinsdill, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, Q32, p. 210), we compared the binding of ochratoxins with bovine serum albumin and with antibodies against OA and discussed the possible use of the binding assay methods for ochratoxin determination. Details on the preparation of ochratoxin-protein conjugates, methods for antisera production, the specificity of the antibody against ochratoxins, as well as other immunochemical properties are presented in this paper. MATERIALS AND METHODS Materials. Ochratoxins A, B, C, and a (OA, OB, OC, Ga; Fig. 1) were prepared by the method of Chu and Butz (5) and of Chu (3). For some experiments, crystalline OA was kindly supplied by Alex Ciegler of the National Regional Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture. Ochratoxin Tc (OTc) was prepared according to Wei and Chu (16). Tritiated OA (:1Hlabeled OA) was prepared by New England Nuclear Corp. (Boston, Mass.) using the method of Chang and Chu (2). Water-soluble carbodiimide 1-ethyl- 3,3-dimethylamino-propyl-carbodiimide (EDPC) was obtained from Story Chemical Corp., Otta Chemical Division (Muskegon, Mich.). Bovine serum albumin (BSA), lysozyme, polylysine, and phenylalanine were products of Sigma Chemicals (St. Louis, Mo.). Scintisol-complete was obtained from Isolab Inc. (Akron, Ohio). 4-Hydroxycoumarin was kindly provided by Collin Schroeder of WARF Institute, Inc. (Madison, Wis.). Coumarin was obtained from ICN-K and K Laboratories, Inc. (Cleveland, Ohio). Complete Freund adjuvant was purchased from Difco Laboratories (Detroit, Mich.). Diethylaminoethyl-cellulose was obtained from Schleicher and Schuell, Inc. (Keene, N.H.). Rabbits were obtained from Roger Guetschow Rabbit Farm 831 (Arlington, Wis.). All other chemicals were either reagent grade or chemically pure. Preparation of OA-protein conjugates. Antigens were prepared by conjugation of OA with proteins in the presence of EDPC. In general, 40 mg of protein in 4 ml of 0.1 M NaCl was mixed with 20 mg of OA in 0.5 ml of ethanol and 3 ml of 0.1 M sodium phosphate buffer (NaPB), ph 7.0, and 150 mg of EDPC. The solution was adjusted to ph 7.0 and stirred in the dark at room temperature for 24 h. After reaction, the solution was dialyzed against 0.1 M NaPB, ph 7.0, for 72 h and kept frozen until ready for injection or lyophilized to dryness. The amount of OA conjugated to the proteins was determined by a spectrophotometric method (4) after the conjugates had been either exhaustively dialyzed against an acid solution (0.01 M HCI) or extracted with chloroform. Data obtained from both the unreacted OA or the OA conjugated to protein were used for final calculation. In some cases 3Hlabeled OA was used to facilitate the quantitation. Immunization schedule. The immunization

2 832 CHU, CHANG, AND HINSDILL OR O CH2CH (COOR). NHCO 0 Ochrotoxins I R R' x A H H CI B H H H C CH2CH3 H CI FIG. 1. Structures of ochratoxins. schedule consisted of an initial injection of 5 mg of conjugate into each rabbit via footpads and a booster of 2.5 mg of conjugate through the thigh 9 days later. In general, 1 ml of antigen was mixed with 1 ml of complete adjuvant, and 1 ml of this mixture was injected into each rabbit. Trial bleedings were made using the marginal ear vein 10 to 14 days after the booster. Subsequent booster injections were made 7 days after the bleeding. Preparation of immunoglobulin G. Immunoglobulin G (IgG) was prepared from the antisera by two precipitations with (NH4)2S04 at a final saturation of 33.3%. The precipitate was redissolved in 0.02 M NaPB, ph 8.0, and further purified by chromatography on a diethylaminoethyl-cellulose column which was equilibrated with 0.02 M NaPB at ph 8.0 (6). The column was then eluted with 0.02 M NaPB at ph 8.0. IgG was eluted from the column with the initial buffer and was either lyophilized to dryness or stored in the freezer for subsequent tests. Determination of antibody titers against OA. The antibody titers of antisera obtained from different bleedings were determined by an equilibrium dialysis in cells with a volume of either 0.5 or 1.0 ml. Because of the nonspecific interaction of ochratoxins with albumin (3), purified IgG was used throughout all the experiments. IgG at different concentrations in 0.1 M NaPB (ph 7.2) was dialyzed against appropriate amounts of 3H-labeled OA also in 0.1 M buffer (ph 7.2) at 6 C in the dark overnight. The amount of OA bound to the IgG was determined from the radioactivity data in both sides of the cell. Immunoglobulin concentration was determined spectrophotometrically at 278 nm using absorptivity (El % ) of 15.5 (8) Ḋetermination of specificity of antibody against ochratoxins. The equilibrium dialysis method was also used for the determination of specificity of antibody obtained from the immunized abbits. A 0.5- ml portion of an IgG-3H-labeled OA solution (4 to 6 4g of IgG and 2.3 ng of 3H-labeled OA; with 75% OA bound to IgG) was dialyzed against 0.5 ml of unlabeled test ochratoxin or compound in 0.1 M NaPB at ph 7.2 over a wide concentration range. At least three controls in which the IgG-3H-labeled OA complex was dialyzed against buffer (0.1 M NaPB, ph 7.2) were included for each experiment. The degree of displacement of radioactivity from the IgG-3H- x APPL. ENVIRON. MICROBIOL. labeled OA complex was then determined. A similar experiment was performed using BSA as the binding protein. Determination of radioactivity. An appropriate amount (in general less than 0.5 ml) of solution was mixed with 10 ml of Scintisol-complete and counted in a Beckman model LS-330 liquid scintillation spectrometer for a certain time (preset at standard deviation of 0.5%). RESULTS Determination of antibody titers. Since it has been previously reported that OA strongly binds with serum albumin, all the antisera were purified by (NH4)2SO4 precipitation and diethylaminoethyl-cellulose chromatography for subsequent binding studies. An appropriate amount of the purified IgG was dialyzed against different amounts of 3H-labeled OA, and the amount of OA bound by IgG at the saturation was then determined. A typical binding curve which shows the concentrationdependent binding ofoa by IgG is given in Fig. 2. The extrapolated value obtained from the curve indicates that 0.59 ng of OA is bound by 1 jig of IgG for this typical bleeding. Production of antisera against OA. The effect of different protein conjugates on the antibody production is shown in Table 1. The degree of conjugation of OA to different proteins and polypeptides was different, with more OA conjugating to polylysine of molecular weight 85,000 on the molar basis. Table 1 clearly shows OA USED, ng FIG. 2. Concentration dependent on binding of OA with IgG. IgG (25 pg/ml) obtained from the third bleeding of a rabbit immunized with BSA-OA was used. The extrapolate value (i.e., 0.59 ng/pg) was used for the calculation ofantibody titer in the subsequent studies.

3 VOL. 31, 1976 TABLE 1. Production of antibody against OA by different protein-oa conjugatesa Mol of OA/ OA bound Conjugates mol of pro- Rabbit no. (ng) per ml tein of antiserum PLL-30-OA PLL-85-OA" LYS-OA BSA-OA Purified IgG obtained from the second bleeding was used in the binding assay. The degree of binding was calculated as described in Fig. 1. PLL-30-OA, PLL-85-OA, LYS-OA and BSA-OA represent ochratoxin A conjugating with polylysine 30,000 (molecular weight) polylysine 85,000, lysozyme, and bovine serum albumin, respectively. ' First bleeding serum was used because the rabbits were infected with Pasteurella. that the antibody production varied considerably with the conjugate used and also varied with individual rabbits. BSA-OA was found to be the best antigen among all the conjugates tested. Antibodies against carrier proteins were demonstrated in the sera against all the conjugates even as in the first bleedings by immunodiffusion tests. However, antibody against OA increased appreciably only after the second and subsequent booster injections. Figure 3 shows the production of antibody against OA by rabbit no. 19 after five injections of BSA-OA conjugate over a period of 3 months. Whereas the binding capacity per unit of IgG increased after the booster, the IgG concentration also increased. Similar results were obtained from other rabbits which had been injected with BSA-OA. Antibody titers did not increase appreciably in rabbits which had been injected with lysozyme-oa conjugate, even after eight injections over a period of 1 year. Since the rabbits which had been injected with polylysine-oa conjugates subsequently became infected with Pasteurella, those rabbits were sacrificed 2 months after the initial injection of antigen. Tests of the IgG obtained from all preimmunization sera revealed no binding between the yg fraction and OA. Specificity of antisera against ochratoxins. The specificity of the antisera against ochratoxins produced by rabbits after repeated injections of BSA-OA conjugate was determined by a competitive binding assay in which the IgG- 3H-labeled OA complex was dialyzed against ANTI-OCHRATOXIN A ANTIBODY 833 different unlabeled ochratoxin analogs or structurally related substances over a wide concentration range, and the results are shown in Fig. 4. The concentrations to give 50% inhibition of IMMUNIZATION TIME, DAYS FIG. 3. Production of antibody against OA. The rabbit was immunized and given booster injections with BSA-OA at times indicated by arrows and bled at times indicated by circles. The antibody titers were expressed as OA bound per milliliter of antisera (a) and OA bound per microgram of IgG (0). 100 (5 z B \08 QA'~~~~~ CONCENTRATION, ng / SYSTEM FIG. 4. Effect of structural analogs of OA on the binding of 3H-labeled OA with IgG. A mixture of 3Hlabeled OA (2.3 ng) and IgG (4.6 pg) in 0.5 ml of 0.1 M NaPB was dialyzed against 0.5 ml of different unlabeled analogs at different concentrations. The extent of binding of 3H-labeled OA with IgG in the absence of unlabeled toxin, i.e., by dialyzing against 0.5 ml of buffer alone, was considered as 100% of binding. OA, OB, OC, OTc, and Oa represent ochratoxins A, B, C, Tc, and a, respectively. Phe, OH-C, and C represent phenylalanine, 4-hydroxycoumarin, and coumarin, respectively. HE

4 834 CHU, CHANG, AND HINSDILL binding of 3H-labeled OA with IgG by unlabeled OA, OTc, OC, and OB were found to be 3, 5, 8, and 400 ng, respectively. However, the concentrations to give 50% inhibition ofbinding of 3H-labeled OA with IgG by other structurally related compounds such as phenylalanine Oa, 4-hydroxycoumarin, and coumarin were greater than 5,000 ng in the present assay. Thus, the results indicate that the IgG is specific for OA, OTc, and OC, is less specific for OB, but is not specific to phenylalanine, Oa, 4- hydroxycoumarin, and coumarin. In the system described, a total of 7,000 counts/min of radioactive OA was used. From the inhibition-binding curve of unlabeled OA, it is readily seen that the sensitivity for OA determination falls in the range of 0.5 to 10 ng. Since OA also strongly binds with albumin, an experiment was carried out to determine the concentrations to give 50% inhibition by different ochratoxin derivatives when bovine serum albumin was substituted for the antibody. The results as shown in Fig. 5 show that the concentrations for OA and OB were 23 and 330 ng, respectively. For OC, Oa, 4-OH coumarin, and coumarin, the concentrations were greater than 1,000 ng. DISCUSSION OA, like most other mycotoxins, is a lowmolecular-weight organic compound and thus is devoid of any antigenicity. Nevertheless, through the conjugation of the toxin to a protein or polypeptide carrier, an antibody against the haptenic group can be obtained from rabbits immunized with the toxin-protein conjugate. Although the best antisera in the current test series were obtained from rabbits immu- CONCENTRATION, FIG. 5. Effect of structural analogs of OA on the binding of3h-labeled OA with BSA. A mixture of3hlabeled OA (2.5 ng) and BSA (6.25 pg) in 0.1 M NaPB was dialyzed against 0.5 ml of unlabeled analogs at different concentrations. Calculations ofbindings was the same as in Fig. 3. ng APPL. ENVIRON. MICROBIOL. nized with BSA-OA conjugate, polylysine-oa cannot be ruled out as a good antigen, since the immunization was interrupted by infection. Pronounced increase in antibody production by rabbits in the BSA-OA group occurred only after 40 days and three boostering injections. One of the most difficult problems in determining antibody titers against OA was the interference of the binding of OA with albumin (3) when the whole antisera were used. In our earlier analyses, no clear-cut results were obtained when we compared the binding of OA with the preimmunization sera and the binding of OA with the whole antisera. Most meaningful quantitative results were obtained, however, only after the use of purified IgG for binding assays. Consequently, all the antisera were purified for different experiments reported in this paper. The specificity of binding of OA by IgG obtained from rabbits after immunization with BSA-OA was established from the fact that unlabeled OA was the only compound which gave best inhibition for the binding of radioactive OA by IgG. Since the chemical structure of OC closely resembles that of OA and OTc differs from OC only with an additional hydroxyl group in the phenylalanine residues, it is not surprising that the binding of both toxins with IgG appears to be in the same magnitude as OA. On the other hand, OB, which differs from OA only in that it lacks a chlorine atom in the dehydroisocoumarin ring residues, binds considerably less strongly than OA. The failure of both Oa and phenylalanine to compete in the binding of OA with IgG indicates that several of the features of the OA molecule, including the amide bond, chlorinated dehydroisocoumarin residues, and perhaps the conformation of intact OA molecule, are important for the binding with antibody. The binding properties between OA and IgG antibody or albumin differ considerably. Interaction of OA with IgG appears more specific and strong than with albumin (Fig. 4 and 5). With the availability of antisera, radioimmunoassay for OA can be readily developed. The sensitivity of radioimmunoassay, however, will depend upon the specific activity of radioactive OA available. In the present system (:H-labeled OA specificity was 3 Ci/mmol), the standard curve for OA analysis appears in the range of 0.5 to 10 ng/0.5 ml. This method is comparable or slightly more sensitive than any presently available assay for OA and yet has the advantage of specificity. The finding that OTc can completely displace the binding of :Hlabeled OA from IgG suggests the possibility of using iodinated OTc as the radioactive species;

5 VOL. 31, 1976 thus the sensitivity of the assay system will be even higher. This and possibly the use of solidstate techniques for radioimmunoassay of OA are presently under investigation in our laboratory. ACKNOWLEDGMENTS We wish to express our thanks to Alex Ciegler of the National Regional Research Laboratory, U.S. Department of Agriculture, for providing ochratoxin A in some experiments. This work was supported by the College of Agricultural and Life Sciences, the University of Wisconsin, and by Public Health Service Research grant no. CA from the National Cancer Institute. LITERATURE CITED 1. Applegate, K. L., and J. R. Chipley Ochratoxins. Adv. Appl. Microbiol. 16: Chang, F. C. C., and F. S. Chu Preparation of 3Hochratoxins. J. Labelled Compd. Radiopharm., in press. 3. Chu, F. S A comparative study of the interaction of ochratoxins with bovine serum albumin. Biochem. Pharmacol. 23: Chu, F. S Studies on ochratoxins. Crit. Rev. Toxicol. 2: Chu, F. S., and M. E. Butz Spectrophotofluorodensitometric measurement of ochratoxin A in cereal products. J. Assoc. Off. Anal. Chem. 53: Fahey, J. L Chromatographic separation of immunoglobulins, p In C. A. Williams and M. W. Chase (ed.), Methods in immunology and immunochemistry, vol 2. Academic Press Inc., New York. 7. Harwig, J Ochratoxin A and related metabolites, p In I. F. Purchase (ed.) Mycotoxins. Elsevier Scientific Publishing Co., Amsterdam, The Netherlands. ANTI-OCHRATOXIN A ANTIBODY Karush, F., S. S. Karush, H. N. Eisen, and J. E. Mc- Guigan Hapten reaction and kinetics of interaction with antibodies. B. Equilibrium dialysis, and C. Quenching of antibody fluorescence by hapten and antigens, p In C. A. Williams and M. W. Chase (ed.), Methods in immunology and immunochemistry. Academic Press Inc., New York. 9. Krogh, P., B. Hald, P. Englund, L. Rutqvist, and 0. Swahn Contamination of Swedish cereals with ochratoxin A. Acta Pathol. Microbiol. Scand. 32: Nesheim, S., N. F. Hardin, 0. J. Francis, Jr., and W. S. Langham Analysis of ochratoxin A and B and their esters in barley, using partition and thin layer chromatography. I. Development of method. J. Assoc. Off. Anal. Chem. 56: Scott, P. M., W. van Walbeek, B. Kennedy, and D. Anyeti Mycotoxins (ochratoxins A, citrinin, and sterigmatocystin) and toxigenic fungi in grains and other agricultural products. Agric. Food Chem. 20: Shotwell, 0. L., C. W. Hesseltine, and M. L. Goulden Ochratoxin A: occurence as natural contaminant of a corn sample. Appl. Microbiol. 17: Shotwell, 0. L., C. W. Hesseltine, M. L. Goulden, and E. E. Vandegraft Survey of corn for aflatoxin, zearalenone and ochratoxin. Cereal Chem. 47: Shotwell, 0. L., C. W. Hesseltine, E. E. Vandegraft, and M. L. Goulden Survey of corn from different regions for aflatoxin, ochratoxin and zearalenone. Cereal Sci. Today 16: Steyn, P. S Ochratoxin and other dihydroisocoumarins, p In A. Ciegler, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, vol. 6. Academic Press Inc., New York. 16. Wei, R. D., and F. S. Chu Synthesis of ochratoxins Ta and Tc, analogs of ochratoxin A and C. Experientia 30: