Immunoassays for Residue Analysis - ACS Publications - American

Chapter 18

Detection and Quantification of Glycoalkaloids

Downloaded by UNIV OF SOUTHERN CALIFORNIA on August 30, 2013 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch018

Comparison of Enzyme-Linked Immunosorbent Assay and High-Performance Liquid Chromatography Methods 1

1

1

Larry H. Stanker , Carol Kamps-Holtzapple , Ross C. Beier , Carol E . Levin , and Mendel Friedman 2

2

1

Food Animal Protection Research Laboratory, Agricultural Research

Service, U.S. Department of Agriculture, 2881 F & Β Road, College Station, TX 77845-9594 Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710 2

Glycoalkaloids (GA's) represent an important group of naturally occurring plant toxins. They are found in a wide variety of plants including potato and tomato. Analysis of G A ' s is complicated, particularly in tomato because these compounds do not readily absorb in the U V . Thus, extensive sample cleanup is usually required followed by HPLC. In this chapter we summarize our efforts to develop simple, rapid immunoassays for the major GA's found in potato and in tomato. The assay we developed is a monoclonal antibody (Mab) based competition enzyme-linked immunosorbent assay (cELISA). Hapten synthesis and antibody production is discussed. G A levels measured in identical samples using an HPLC method and the cELISA showed a high degree of correlation (correlation coefficient = 0.998). The cELISA clearly has widespread application and is able to rapidly and accurately measure G A levels. Glycoalkaloids (GA's) are naturally occurring, potentially toxic, lûtrogen-containing secondary plant metabolites that are found in a number of agriculturally important species including potatoes, tomatoes, and eggplant (1). In commercial potatoes (Solatium tuberosum) there are two major glycoalkaloids, α-chaconine and a-solanine, both glycosylated forms (trisaccharides) of the aglycon solanidine (Figure 1). They are thought to function as a defense against insects and other pests (2). These two G A ' s , along with a number of other natural chemicals in potatoes, are phytoalexins (3). Phytoalexins are low-molecular-weight antimicrobial compounds that are both synthesized by and accumulated in plants as a result of exposure to microorganisms (4). Recent studies suggest that the potato G A ' s provide protection to the Colorado potato beetle and to the potato leafhopper (2, 5-7). Wild potatoes (Solarium chacoense) and eggplants (Solarium melongena) contain the glycoalkaloid solasonine (Figure 1). Because wild potatoes often contain higher This chapter not subject to U.S. copyright Published 1996 American Chemical Society In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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IMMUNOASSAYS FOR RESIDUE ANALYSIS

IMMUNOGEN

Figure 1. Chemical structure of the potato glycoalkaloids, α-chaconine, ccsolanine, solasonine, the aglycon solanidine, and the immunogen used to produce monoclonal anti-GA antibodies.

In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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ELISA and HPLC Detection of Glycoalkaloids

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G A levels than commercial varieties, they have been used by plant breeders who are attempting to generate improved cultivars. Such cultivars, however, can have G A levels above 20 mg/100 g of tuber, the generally accepted cutoff level between safe and unsafe potatoes {8,9). This guideline limiting the glycoalkaloid content of new potato cultivars has been recommended (10,11) because of the potential human toxicity of these compounds, including reported deaths. One mode of G A toxicity might be their ability to inhibit cholinesterases (12-14). Three cholinesterases can be differentiated which are inhibited by a-solanine (12), and α-chaconine is a stronger inhibitor than a-solanine (14). Teratogenic effects were produced in mice by a-chaconine (15). Neural-tube defects were reported in Syrian hamsters by α-chaconine and a-solanine (16,17). Keeler et al. (18) reported that the new sprouts of the seven potato varieties they tested were teratogenic in hamsters. Likewise, sprouts of the British potato, Arran Pilot, were reported by Renwick et al. (17) to cause cranial bleb, encephaly, exencephaly, and spina bifida in Syrian hamsters. α-Chaconine and α-solanine have been shown to be embryotoxic in a frog embryo assay (19,20). This effect may be due to the ability of the steroidal glycoalkaloids to alter ion fluxes across cell membranes (21,22). In tomatoes (Lycopersicon esculentum), the major glycoalkaloid is a-tomatine, which is a glycosylated (tetrasaccharide) derivative of the aglycon tomatidine (Figure 2). As with the Sclanum glycoalkaloids, α-tomatine is reported to be potentially toxic (19,23-24). It has been found to possess antifungal activity (25), to inhibit fruitworm and spiny bollworm larval growth (26,27), and to interfere with moth eggs (28). Figures 1 and 2 illustrate the structures of these potato and tomato glycoalkaloids. Current methods for analysis of GA's include gas chromatography (GC) (29-31), and high-performance liquid chromatography (HPLC) (11,32-37). While these methods are accurate and sensitive, they are time consiirning, require complex instrumentation and are not easily adapted to rapid screening programs. Part of the difficulty associated with analysis, especially for the tomato G A , α-tomatine, is its lack of absorbency except at low U V wavelengths (e.g., at 205 nm). Thus, extensive cleanup is necessary prior to HPLC in order to remove confounding substances (38,39). This has been partially solved by Friedman et al. (37), who developed an H P L C method for α-tomatine that utilized pulsed amperometric detection. This method was found to be useful for both tomatoes and processed tomato products such as juice, ketchup, sauce and soup (40). The difficulties associated with the analysis of G A ' s have resulted in the development of alternative immunochemical methods. Enzyme immunoassays, radio immunoassays, and fluorescence polarization immunoassays (FPA) have been reported for the potato GA's by a number of different groups (41-46). Immunoassays for the tomato G A α-tomatine also have been reported (47). The above studies all have used the G A itself, or a modified G A as immunogen, linking it to the carrier protein via a modification in the sugar. In contrast, our studies have utilized a protein conjugate of the aglycon of α-solanine as the immunogen. Thus, our immunogen does not contain any structural information in the sugar component of the G A . Using this immunogen, we report here on the ability of one of our monoclonal antibodies, Sol-129, to bind both tomato and potato G A ' s with a high degree of correlation between the competition ELISA we developed and HPLC methods.




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Figure 2. Chemical structure of the tomato glycoalkaloid α-tomatine, its aglycon tomatidine, and the glycoside digitonin.


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Materials and Methods The anti-glycoalkaloid monoclonal antibody Sol-129 and the competition (c)ELISA used in these studies were reported earlier (48). Briefly, 96-well microtiter wells were coated with a solanidine-bovine serum albumin (Sol-BSA) conjugate (Figure 1). Standards and/or unknowns were then added, followed by Mab Sol-129. The Mab was then allowed to partition between the G A bound to the bottom of the microtiter well and the G A present in solution. The plates were washed to remove unreacted reagents. The amount of bound Mab was detected by addition of an enzymeconjugated anti-mouse inununoglobulin sera (Sigma, St. Louis, MO) and substrate. Thus, the color developed in the assay is inversely proportional to the amount of analyte in the sample. The data is expressed as a percent inhibition of control (IC) using the following expression % IC = (1-B/Bo) X 100 where Bo is the value obtained when no G A is present in the sample (buffer is added instead of sample or standard). Sample extraction and HPLC analysis was as previously described (36,37). Tomato samples were extracted, an aliquot of the extract was then injected for H P L C analysis, and a second aliquot was used in the cELISA. The aliquot used in the cELISA was dried under a stream of nitrogen gas, resuspended in D M F (approximately 1 mL), diluted in assay buffer (usually a 1/1000 dilution was made), and then further diluted in a 1:2 fashion in assay buffer. Unknown concentrations were determined by comparison to a percent B/Bo standard curve near the I C point (B/Bo between 40-60%). 50

Results and Discussion Monoclonal Antibodies. We have previously described a set of eleven Mabs that bound different potato and tomato glycoalkaloids (48). The most sensitive of these Mabs, referred to as Sol-129, had 50% of its binding activity inhibited (IC ) when 100 pL of a 2.5 ppb solution of α-solanine was added to the reaction. A typical standard curve for α-tomatine is shown in Figure 3. Extensive cross reactivity studies, using various potato and tomato glycoalkaloids, their aglycons, and nonnitrogen-containing glycosides as competitors have been reported (48) for each of the 11 Mabs described. A subset of these data is shown in Table 1. Cross reactivity was calculated by comparison of the I C ^ values and assigning 100% activity to that for solanidine (the immunizing compound). These data clearly showed that only one of the 11 Mabs isolated, Sol-129, recognized the tomato glycoalkaloid, α-tomatine, in addition to the potato G A ' s . Furthermore, of the 11 Mabs isolated, Sol-129 was observed to have the greatest relative affinity for all of the antigens recognized. A possible interpretation of this data is that a greater number of contacts between residues in the combining site of the antibody and the analyte exists for Mab Sol-129, than there is for the other ten Mabs. 50



248


0

H

1

1

1—ι—ι

ι ι ι 11

1

10

1

1—I

I I 1II

1

1

1—I I I I I 1

100

1000

Competitor (ppb) Figure 3. A typical cELISA curve for Mab Sol-129 using α-tomatine as competitor.



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The ability of Mab Sol-129 to bind tomato G A ' s and solasonine in addition to the potato G A ' s is more difficult to explain since the animals were immunized with a protein conjugate of the aglycon of α-solanine (Figure 1). The orientation of the Fring of solanidine and the commercial potato glycolalkaloids α-solanine and achaconine is fixed (Figure 1). In contrast, the F-ring in solasonine, α-tomatine and tomatidine (Figures 1 and 2) is not fixed and can adopt various conformations. Since conjugation to the carrier protein was achieved through the hydroxyl group on the number 3 carbon of solanidine, conventional wisdom suggests that the resultant antibodies should be most sensitive to that portion of the molecule most distal to the attachment site, which would be the F-ring. This is in fact what we observed (Table 1). Ten of the 11 Mabs bound only a-solanine, α-chaconine or solanidine. Using solanidine as the immunogen, we would not have predicted that an antibody capable of binding the tomato GA's would be isolated. Table 1. % Crossreactivity*observed with different anti-glvcoalkaloid Mabs. Monoclonal Antibody Compound Tested

8

solanidine a-solanine a-chaconine solasonine α-tomatine tomatidine

100 18 16 0 0 0

48

100 4 5 0 0 0

54

55

59

67

68

100 61 80 0 0 0

100 51 29 0 0 0

100 46 64 0 0 0

100 56 77 0 0 0

100 0b 0 0 0 0

71

100 0 0 0 0 0

91

100 35 44 0 0 0

106

129

100 29 26 0 0 0

100 96 89 7 46 24

Original data adapted from Stanker et al. (48) Cross reactivity was calculated by comparing the I C ^ value obtained with that from solanidine. No competition was observed at the highest level of competitor tested. a

A number of explanations can be put forth to explain our observations. The Mab Sol-129 may not form any contacts with the F-ring of the immunogen but instead bind exclusively via contacts on the A-E rings; these regions of the potato and tomato aglycons are similar. This mterpretation is not favored since binding to nonnitrogen containing G A ' s such as digitonin (Figure 2) was not observed. Conversely, the antibody may have contacts with the F-ring, but enough contacts with other regions of the aglycon to provide sufficient binding energy to allow binding (albeit with a lower relative affinity) with the tomato GA's. This latter explanation would require that the orientation of the F-ring in the tomato GA's not inhibit binding because of steric or electronic differences. A third possibility is that the higher affinity of Sol-129 forces the F-ring of the tomato GA's into an orientation preferred by the antibody. We have not yet determined which, if any, of these hypotheses most accurately describes the situation with Sol-129 binding. Studies aimed at cloning the genes and expressing




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functional single-chain variable fragments (scFv) (Kamps-Holtzapple et al. this volume) will provide us with the tools (e.g., the amino acid sequences of the antibody heavy- and light-chains) necessary to test these hypotheses. Regardless of the exact nature of Sol-129 bmding, it represents a specific interaction that probably would not be observed in a polyclonal antiserum obtained from animals immunized as above, because most of the resulting B-cell clones appeared to be producing potato-GA-specific antibodies. Barbour et al. (47) were able to produce polyclonal antibodies to tomato GA's. Rather than the hapten we used in our studies, they used a B S A conjugate of α-tomatine for immunization. Linkage was achieved via the sugar molecules using the sodium periodate cleavage reaction. Thus their immunogen included the aglycon (tomatidine) as well as sugar (albeit modified by the periodate cleavage reaction). The resulting antisera bound α-tomatine and tomatidine with equal affinity, as well as having a 41% and 24% cross reactivity with a-solanine and demissine, respectively. They also noted that their antisera had a 30% cross reactivity with digitonin. Following an analysis of extracts of tomato foliage, Barbour et al (47) conclude that their polyclonal antisera suffered from significant interference from "other compounds" resulting in a "non-linear" and nonproportional response in their ELISA. While monoclonal antibody Sol-129 described here cross-reacted with potato and tomato GA's, we did not observe cross reactivity to digitonin, cholesterol, stigmasterol, or β-sitosterol. The difference in binding specificity is not surprising. A l l of the antibodies in a given Mab preparation bind the same epitope compared to a collection of antibodies in a sera that may all bind the immunogen but with different affinities and to slightly different epitopes. Thus, Mabs often display a more restricted specificity than observed with antisera. Furthermore, the immunogen we used did not contain any of the sugar component of the alkaloid. Table 2.

Glycoalkaloid level (mg/g) measured in five freeze-dried potato samples by HPLC and cELISA

Sample

q-Chaconine Klamath tuber flesh (no peel) Russet whole tuber 3194 Whole tuber Ζ whole tuber Lenape tuber peel

ELISA

HPLC

trace 0.1 0.5 0.8 2.1

a-Solanine 0 0.1 0.3 0.5 0.8

0.004 0.13 0.97 1.63 >2.5

Data from Stanker et al (48). Analysis of Potato and Tomato Extracts. Monoclonal antibody Sol-129 bound the potato GA's α-solanine and α-chaconine with virtually identical affinity. Therefore, it was used in initial experiments to quantify the total G A level in a series of potato



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samples. The data from these experiments with potato samples was reported previously and is summarized in Table 2. The potato samples analyzed by H P L C were first extensively extracted using a multistep process previously described (36). Briefly, the samples were extracted with a 2% acetic acid solution, basified, reextracted with butanol, concentrated, filtered, and analyzed by HPLC. In contrast, duplicate samples analyzed by the cELISA were simply extracted in an acetic acid solution, neutralized and analyzed. The cELISA results are expressed as glycoalkaloid equivalents since Mab Sol-129 has a comparable binding affinity for α-chaconine and α-solanine, and they are the major GA's in commercial potatoes. These data clearly indicate that the cELISA analysis gave results comparable to that obtained using HPLC. However, because of the simplified sample preparation used with the cELISA, it is a much faster assay and could be used to rapidly screen large numbers of potato samples. The above data obtained with potato samples encouraged us to apply the immunoassay for measurement of α-tomatine in tomatoes. α-Tomatine is an ideal candidate for immunochemical analysis. It is readily water soluble, and since it contains no chromophore, it is not easily measured by spectrophotometric detection using traditional methods. Furthermore, since there is only one major G A in tomato, atomatine, the cELISA results should more closely match the HPLC results than was seen when potatoes were analyzed. A number of samples representing different tomato cultivars and maturation stages, processed tomato products, and tomato plant root samples were analyzed using both the cELISA method and an HPLC method described by Friedman et al. (37). Specifically, tomatoes were extracted by stirring 1 g in 20 mL of 1% acetic acid for 2 h. The suspension was then centrifuged for 10 min at 13,000 relative centrifugal force, and the supernatant was filtered through a Whatman GF/C filter. The pellet was resuspended in 10 mL of 1% acetic acid, centrifuged, and filtered, and the two extracts were combined. This extract was further purified using solid phase extraction (SPE). A C SPE tube, equipped with 60 mL reservoir (Supelco) was conditioned with 5 mL of methanol followed by 5 mL of water. The aqueous extract (now about 30 mL) was applied and allowed to gravity drip. When the sample was fully absorbed onto the packing, the tube was washed with about 10 mL of water, followed by 5 mL of 30:70 acetonitrile/1% N H O H and then 5 mL of water. The atomatine was eluted with 10 mL of 70:30 acetonitrile/pH 3 citric acid/disodium phosphate buffer. The organic solvent was then evaporated off. The aqueous residue was basified with ammonia water and extracted twice into water-saturated butanol, using a separatory funnel. This sample was then dried on a rotovapor. The residue was taken up in 1 mL of 50% methanol/0.1% acetic acid and filtered through a 0.45 μπι H V membrane obtained from Millipore (Bedford, MA). This filtrate was ready for HPLC injection. The extracts were then split and analyzed by ELISA and H P L C . We felt it important to demonstrate that regardless of the detection method, H P L C or ELISA, the same G A value was observed if the samples were prepared using the same preparation scheme, (thus eliminating differences of analyte recovery associated with different extraction methods). These data are summarized in Table 3 and Figure 4. The values obtained by the cELISA and HPLC were highly correlated having a correlation coefficient of 0.998 (N = 20). In addition, we observed good correlations regardless of the matrix (Table 3). 1 8

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Conclusions The protein conjugate we developed based on the aglycon of α-solanine, solanidine, was a highly effective immunogen. We were able to isolate a large number of monoclonal anti-GA antibodies. Cross reactivity studies (48) suggested that this collection of Mabs could be divided into four epitope groups, each group presumably interacting differently with the aglycon. A l l of the Mabs except one, Sol-129, reacted only with potato GA's, and some of these bound only the aglycon, solanidine. In contrast, Sol-129 was capable of binding the largest number of GA's including the tomato G A α-tomatine and its aglycon. Mab Sol-129 also demonstrated the greatest relative affinity for each of the GA's it bound. We have yet to determine the molecular details of the binding site for Sol-129. However, studies aimed at cloning the heavyand light-chain genes (Kamps-Holtzapple and Stanker, this volume) will aid in such studies. The results from analysis of a variety of potato and tomato samples by HPLC methods and using the cELISA method are highly correlated. Little if any matrix effect was observed in the tomato samples. Future studies are aimed at determining whether a Qig/g)

Table 3· Analysis of α-Tomatine in Freeze Dried Tomatoes

SAMPLE Control Red Tomato Mature Green Control Tomato Manteca Red Tomato Manteca Green Tomato Precipitated Control Red Tomato Immature Green Tomato Immature Green Tomato Replicate Mature Green Tomato Mature Green Tomato Replicate Breaker Tomato Breaker Tomato Replicate Large Immature Tomato Large Immature Tomato Replicate Tomato Plant Roots Tomato Plant Roots Replicate Tomatillos Tomatillos Replicate Canned Tomato Sauce Pickled Tomatoes Commercial Mature Green Tomatoes

HPLC 60 122 10 308 19 168 192 39 38 61 92 409 385 320 353 6 6 64 121 144

ELISA 67 115 11.3 312 15 173 204 39 37 59 90 400 371 308 347 6.2 6.1 57 114 135

±4 ±9 ±0.9 ±31 ±2 ±13 ±4.4 ±2.2 ±2 ± 1.5 ± 1.5 ±27.5 ±9.6 ± 14.5 ±7.8 ±0.1 ±0.2 ± 1.8 ±2.5 ±2.3

more simplified sample extraction and preparation method can be used for analysis of GA's in plant material and body fluids and tissues. In addition, studies are underway to adapt the cELISA method for GA's in potato and tomato leaves. Preliminary studies



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20

HPLC

30

253

40

α-tomatine)

Figure 4. Analysis of α-tomatine in tomatoes using the cELISA and an H P L C method. The line represents the linear regression of this data, with an R o f 0.998. 2


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suggest that the cELISA accurately measures α-tomatine in tomato leaves. The ability to measure G A ' s in individual leaves would be useful for early screening of newly developed cultivars. Literature Cited


1.

Friedman, M. In Evaluation of Food Safety: Finley, J.W., Robinson, S.F., Armstrong, Α., eds.; ACS Symposium Series 484; American Chemical Society: Washington DC, 1992, pp 429-462. 2. Norris, D. M. In Chemistry of Plant Protection; Haug, H., Hoffman, H. eds.; Springer Verlag: Berlin, Germany, 1986; pp 97-146. 3. Beier, R. C.; Nigg, Η. N. In Foodborn Disease Handbook, Diseases Caused by Hazardous Substances; Hau, Y. H.; Gorham, J. R.; Murrell, K. D.; Oliver, D. O., eds.; Marcel Dekker, Inc.: New York, NY, 1994, Vol. 3; pp 68-70. 5. Flanders, K. L.; Hawkes, J. G.; Radcliffe, Ε. E.; Florian, I. L. Euphytica 1992, 61, 83-111. 6. Gaffield, W.; Keeler, R. F.; Baker, D. C. In Handbook of Natural Toxins, Toxicology of Plant and Fungal Compounds; Keeler, R. F., Tu, A. T., eds.; Marcel Dekker, Inc.: New York, NY, 1991, Vol. 6; pp 135-158. 7. Roddick, J. G.; Rijnenberg, A. L.; Osman, S. F. J. Chem Ecol. 1988, 14, 889-902. 8. Slalina, P. Food Chem. 1990, 28, 759-761. 9. Van Gelder, W. J. M. In Handbook of Natural Toxins, Toxicology of Plant and Fungal Compounds; Keeler, R. F.; Tu, A. T., eds.; Marcel Dekker Inc.: New York, NY, 1991, Vol 6; pp 101-134. 10. McMillian M.; Thompson J. C. Q. J. Med. 1979, 48, 227-243. 11. Morris S. C.; Lee T. H. Food Technol. Aust. 1984, 36, 118-124. 12. Harris, H.; Whittaker, M. Ann. Hum. Genet. Lond. 1962, 26, 73-76. 13. Patil, B. C.; Sharma, R. P.; Salunkhe, D. K.; Salunkhe, Κ. Food Cosmet. Toxicol. 1972, 10, 395-398. 14. Alozie, S. O.; Sharma, R. P.; Salunkhe, D. K. J. Food Biochem. 1979, 2, 259-276. 15. Pierro, L. J.; Haines, J. S.; Osman, S. F. Teratology 1977, 15, 31A. 16. Renwick, J. H. Lancet 1982, 1, 748. 17. Renwick, J. H.; Claringbold, D. B.; Earthy, M. E.; Few, J. D.; McLean, A. C. S. Teratology 1984, 30, 371-381. 18. Keeler, R. F.; Young, S.; Brown, D.; Stallknecht, G. F.; Douglas, D. Teratology 1978, 17, 327-334. 19. Friedman, M.; Rayburn, J. R.; Bantle, J. A. Food. Chem. Toxicol. 1991, 29, 537-547. 20. Friedman, M.; Rayburn, J. R.; Bantle, J. A. J. Agric. Food Chem. 1992, 40, 1617-1624. 21. Blankemeyer, J. T.; Stringer, Β. K.; Rayburn, J. R.; Bantle, J. Α.; Friedman, M. J. Agric. Food Chem. 1992, 40, 2022-2026. 22. Blankemeyer, J. T.; Atherton, R.; Friedman, M. J. Agric. Food Chem. 1995, 43, 636-639.


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ELISA and HPLC Detection of Glycoalkaloids 255

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