New Horizon of Mycotoxicology for Assuring Food Safety (Proceedings of ISMYCO Kagawa'03) Edited by Takumi Yoshizawa (C)2004 Japanese Association of Mycotoxicology 249 Immunoaffinity column for mycotoxins, its problems and solutions Masahiro NAKAJIMA Food Department, Nagoya City Public Health Research Institute (1-11, Hagiyama-cho, Mizuho-ku, Nagoya 467-8615, Japan) Summary The immunoaffnity column (IAC) methods using specific antibodies against mycotoxins have been developed. The use of IACs in the cleanup step of mycotoxin analysis provides a number of advantages over the conventional chemical methods, such as highly specific, simple and rapid, and saving toxic solvents. Recently, with the availability of commercial IACs for several mycotoxins, the IACs have become the powerful tools in the cleanup stage of mycotoxin analysis. However, there are some disadvantages of commercial IACs also. One of the major disadvantages is cost of IAC. To solve this problem, methods of regenerating IACs for reuse have been investigated. Other disadvantages are low and varied recoveries because of low mycotoxin affinity of IACs. In general, aflatoxins (AFs) are eluted with methanol from IAC. However, AFs are decomposed during evaporation of methanol eluate. To avoid this decomposition, acetonitrile elution from IAC was very effective. In the case of IAC for ochratoxin A (OTA), low recoveries result from low OTA affinity of antibody at ph condition other than neutral ph. By introducing 10 mm ammonium acetate buffer (ph 6.6) into the washing steps of IAC, high and stable recoveries of OTA could be achieved. Because of low toxin affinity of antibody against deoxynivalenol, only a small portion of sample solution can be applied onto IAC. To solve this problem, the combination of solid phase extraction column and IAC was effective. Key words: immunoaffinity column, aflatoxins, ochratoxin A, deoxynivalenol, cost Introduction The conventional chemical cleanup methods for mycotoxins consume large amount of time and toxic solvents such as chlorinated solvents, and sometimes require practical experience. Furthermore, from a viewpoint of the earth environment and human health protection, we analysts are asked for reduction in the use of toxic solvents. Under these circumstances, the Immunoaffinity column (IAC) methods using specific antibodies against mycotoxins have been developed. The use of IACs in the purification step
250 provides a number of advantages over the conventional methods, such as clean extracts due to the high specificity of the antibodies for mycotoxins, rapidity of the purification step, and reduction in the use of toxic solvents. The first IAC for aflatoxins (AFs) was developed by Groopman et al. in 1984, and we first developed the IAC for ochratoxin A (OTA) in 1990. Recently, with the availability of commercial IACs for AFs, OTA, zearalenone, fumonisins (FUMs), deoxynivalenol (DON) and T-2 toxin, these IACs have become the powerful tools in the cleanup stage of mycotoxin analysis. Moreover, because of the accuracy and precision of IAC method at low ƒêg/kg level of mycotoxin contamination, the Official Methods of Analysis of AOAC International has adopted eight IAC methods for AFs, AFM,, OTA and FUMs until now. In spite of the above advantages of the IACs, there are some disadvantages of commercial IACs, such as low and varied recoveries of mycotoxins, and high cost. In some cases, low recoveries result from low mycotoxin affinity of antibody or insufficient antibodies bounded onto the gel of IACs. As a result, some kinds of IACs are easy to affect by loading solutions such as sample solution and washing solution. In this paper, some problems of commercial IACs and theer solutions are mentioned. Cost One of the major disadvantages is cost of the commercial IACs. Because, IACs require use of larger amounts of antibodies than other immunochemical methods, cost per assay is too high. In Japan, costs of commercial IACs are 3 to 10 times higher than solid phase extraction column. To reduce costs, methods of regenerating IACs have been investigated. I have reported the reuse of AflaTest column (VICAM, Watertown, MA) in 1999. By washing used column with phosphate buffered saline (ph 7.4, PBS) and 48 hours regeneration time in the refrigerator, AflaTest was usable at least 6 times. Santos and Vargas reported that OchraTest column (VICAM) could be reused only one more time by regeneration with PBS washing and keeping at 7 to 8 Ž for 1 month. By washing the used column with water and allowing the column to stand for 24 hours at room temperature, the ZearalaTest column (VICAM) could be regenerated at least 3 times without altering their performance and without affecting the results of repeated determinations. The DONTest column (VICAM) could be reused up to 2 times (personal communication). To reduce costs of IACs, further research is needed on techniques for the recombinant technology, which will provide a uniform supply of large amount of antibodies needed for IACs preparation. Aflatoxins In Japan, IAC method for AFs has not been so popular, because most researches
251 have reported that recoveries of AFs are low. Most of the Japanese researchers have adopted the pre-column derivatization method, in which AFB1 and G1 is treated with trifluoroacetic acid (TFA) to increase their fluorescence intensity in reversed phase HPLC-florescence detection. In general, the instruction of manufacturer recommend the use of methanol to elute AFs bound to IAC. To treat with TFA, methanol eluate from IAC for AFs has to be evaporated to dryness. AFs could be decomposed during evaporation of methanol eluate. However, by introducing acetonitrile elution instead of methanol, high recoveries of AFs can be achieved. As shown in Table 1, methanol elution revealed large variation and low recoveries of AFs, although acetonitrile elution had good recoveries of AFs. Table 1. Recoveries of aflatoxins (AFs) from immunoaffinity column by methanol or acetonitrile elution. Pistachio nut sample was spiked with 10 ng each with aflatoxins/g. Twenty-five g of sample was extracted with 125 ml of methanol-water (7:3, v/v). After filtration thorough Whatman No. 4, 15 ml filtrate was diluted with 30 ml of water or PBS. After re-filtration thorough Whatman 934 AH, 15 ml filtrate was applied onto AflaTest P column, followed by washing with 10 ml of water. Toxins were eluted with 3 ml of methanol or acetonitrile into 4 ml Silanized amber screw top vial. Eluate was evaporated to dryness under gentle stream of nitrogen gas at 40 Ž. One-hundred ƒêl of trifluoroacetic acid was added into vial, capped, mixed well on Vortex mixer for 30 s. Nine-hundred ƒêl of acetonitrile-water (1:9, v/v) was added into vial and mixed well. One-hundred ƒêl of sample solution was injected into HPLC-fluorescence detector. Average of triple measurements were represented. Ochratoxin A I have been using the commercial IAC for OTA for more than 6 years however, I have had experience that the recovery of OTA is sometimes low. To solve this problem, I checked the column performance of commercial IACs. In Japan, 4 kinds of IACs for OTA are available viz., OchraTest (VCAM), Ochraprep (R-Biopharm Rhone Ltd,
252 Glasgow), RIDA Ochratoxin column (R-Biopharm AG, Darmstadt), and OchraStar (proto type was IMSORB, Romer Labs, Herzogenburg). The method for performance test was as follows. OTA solution containing 5 ng of OTA in methanol-pbs (1:10, v/v) was applied onto IACs. After washing the column with 3 ml of water, OTA was eluted with 3 ml of methanol. Methanol eluate was evaporated to dryness under gentle stream of N2 gas at 40 Ž, and residue was dissolved in 1 ml acetonitrile-water-acetic acid (30:70:1, v/v/v). One-hundred ƒêl aliquot of sample solution was injected into HPLCfluorescence detector. As shown in Table 2, OchraTest column revealed the variation of recovery between the lots, and the other IAC showed too low recoveries. Instructions of IACs other than OchraTest recommend PBS washing after sample application. PBS washing revealed that all recoveries of OTA from each TAG were near to 100%. However, the salts from PBS origin in the final solution affected the retention time of OTA on HPLC chromatogram, that is, faster retention time than that of OTA standard was observed. To solve this problem, a volatile 10 mm ammonium acetate buffer (ph 6.6) was used as washing solution instead of PBS. This washing solution showed more than 95 % recovery of OTA from IACs with no change of retention time of on chromatogram. Table 2. Performance of commercial immunoaffinity columns. OTA solution including 5 ng of OTA in methanol-pbs (1:10, v/v) was applied onto immunoaffinity column, followed by washing with 3 ml of water. OTA was eluted with 3 ml of methanol into Silanized amber screw top vial. Eluate was evaporated to dryness under gentle stream of nitrogen gas at 40 Ž. Residue was dissolved in 1 ml of acetonitrile-water-acetic acid (30:70:1, v/v/v). One-hundred ml of sample solution was injected into HPLC-fluorescence detector. Average of duplicate measurements were represented. Deoxynivalenol Typical analytical procedure of IAC for DON is as follows. Sample (wheat or barley) was extracted with water in the presence of Polyethylene glycol 8000. After filtration, only 1 ml of filtrate was applied onto IAC, followed by water washing. DON bound on IAC were eluted with methanol, and detected by HPLC-ultraviolet detector. If
253 large volume of sample solution can be loaded onto the IAC, lower detection limit could be achieved. However, more than 2 ml of sample solution could not be applied onto DONTest (VICAM) as shown in Fig. 1, although binding capacity of DONTest was found to be 1.5 pg per column (Fig. 2). These phenomenons might be observed because Fig. 1. Effect of loading volume of sample solution onto DONTest column. Fifty g of unpolished wheat (fortified 0.5 mg/kg of DON) were extracted with 200 ml of water in the presence of 10 g of polyethylene glycol 8000. After filtration through Whatman No.4 and Whatman 934 AH, 1 ml (equivalent to 0.25 g sample) to 10 ml filtrate was applied onto DONTest, followed by washing with 5 ml of water. DON was eluted with 3 ml of methanol into Silanized amber screw top vial. Eluate was evaporated to dryness under gentle stream of nitrogen gas at 40 Ž. Residue was dissolved in 0.5 ml of mobile phase (acetonitrile-methanol-water = 5:5:90, v/v/v). Ten ƒêl of sample solution was injected into HPLC-UV detector. Averages of duplicate measurements were represented. Fig. 2. Binding capacity of DONTest column. Two ml of each concentration of DON in PBS was applied onto DONTest. After washing with water, DON was eluted with 3 ml of methanol. Eluate was evaporated to dryness under gentle stream of nitrogen gas at 40 Ž. Residue was dissolved in appropriate volume of mobile phase (acetonitrilemethanol-water = 5:5:90, v/v/v). Ten ƒêl of sample solution was injected into HPLC-UV detector. Averages of duplicate measurements were represented.
254 of low toxin affinity of antibody against DON, and ingredients from sample origin may affect antibodies of DONTest easily. To remove the ingredients of sample, pre-column cleanup should be done. By introducing the multifunctional cleanup before IAC, up to 10 ml of wheat sample solution (2.5 g equivalent to sample) could be applied without affecting the recoveries (Fig. 3). Fig. 3. Effect of combination of multifunctional column and DONTest. Fifty g of unpolished wheat (naturally contaminated with DON at average concentration of 0.45 mg/kg) were extracted with 200m1 of acetonitrile-water (85:15, v/v). After filtration through Whatman No.4, appropriate volume of filtrate was applied onto MultiSep #227 (Romer Labs). After discarding the first 3 ml of eluate, eluate was collected into flask. After evaporation, residues were dissolved in PBS (0.25 g sample/ml). Two to 10 ml of PBS solution was applied onto DONTest. Averages of duplicate measurements were represented. In conclusion, I would like to ask the manufacturers the following things; set up the cost of IACs suitable for routine analysis of mycotoxins, temperature management under transportation to prevent from denature of antibodies of IACs, and development of more stable antibodies. References 1) Groopman, J.D., Trudel, L.J., Donahue, P.R. Marshak-Rothstein, A., Worgan, G.N.: Proc. Nat. Acad. Sci. USA, 81, 7728-7731 (1984) 2) Nakajima, M., Terada, H., Hisada, K., Tsubouchi, H., Yamamoto, K., Uda, T., Itoh, Y., Kawamura, O., Ueno, Y.: Food Agric. Immunol., 2, 189-195 (1990) 3) Nakajima, M.: "Proceeding of International Symposium of Mycotoxicology '99, Mycotoxin Contamination: Health Risk and Prevention Project" (eds, Kumagai, S., Goto, T., Kawai, K., Takahashi, H., Yabe, K., Yoshizawa, T., Koga, H., Kamimura, H., Akao, M.), pp.172-179 (1999), Keibundo Matsmoto Printing Co., Inc., Tokyo 4) Santos, E.A., Vargas, E.A.: Food Addit. Contam.,19, 447-458 (2002) 5) Fazekas, B., Tar, A.: J. AOAC Int., 84, 1453-1459 (2001)