A Pyrrolizidine Alkaloid Enzyme-Linked Immunosorbent Assay

Chapter 16

A Pyrrolizidine Alkaloid Enzyme-Linked Immunosorbent Assay Detection Strategy 1

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Mary A. Bober , Mark J. Kurth , Larry A. Milco , David M. Roseman , R. Bryan Miller , and Henry J. Segal 1

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Downloaded by PEPPERDINE UNIV on August 23, 2017 | http://pubs.acs.org Publication Date: December 26, 1990 | doi: 10.1021/bk-1990-0451.ch016

Department of Chemistry and Department of Veterinary Medicine, Pharmacology, and Toxicology, University of California, Davis, CA 95616

A two-pronged class-specific and compound-specific immunochemical approach targeting naturally occurring macrocyclic pyrrolizidine alkaloids was developed. Antibodies have been developed against a retronamine-BSA conjugate and then used to detect the necine base retronecine, a common substructural feature of many naturally occurring macrocylcic pyrrolizidine alkaloids. In addition, three macrocyclic alkaloids isolatedfromthe plant Senecio vulgaris (retrorsine, senecionine, and seneciphylliine) were detected using antibodies to retronamine. A comparison of antibodies produced against the retronamine conjugate in both rabbits and mice showed that the mouse antibodies possessed a slightly higher affinity (I50 = 1.1 ppm) for the retronecine analyte than antibodies raised in rabbits (I50 = 1.5 ppm). Antibodies produced to this retronamine conjugate may prove useful in assaying the toxic macrocyclic pyrrolizidine alkaloids in plant extracts. Antibodies were also developed to monocrotaline and can be used to detect quaternarized monocrotaline (I50 = 0.25 ppm at pH 7.6), N-methylated monocrotaline (I50 = 5.3 ppm at pH 7.6), and protonated monocrotaline (I50 = 6.0 ppm at pH 6.0). Antibodies to monocrotaline do not cross-react with retrorsine, retrorsine N-oxide, ridelliine or retronecine.

The pyrrolizidine alkaloids (PAs) constitute a class of secondary plant metabolites of notably wide geographical and botanical distribution, occurring in numerous plant families, (including Boraginaceae, Compositae, Gramineae, Leguminosae, Orchidaceae, Rhizophoraceae, Santalaceae, and Saptoaceae) which are indigenous to various environments throughout the world [1]. Indeed, the number of PA-producing plant species may approach 6,000, accounting for 3% of all known flowering plants [2] — many of which produce more than one PA. Frequently these compounds coexist in the plant with varying amounts of their corresponding N-oxide derivatives. Most of the more than 200 known PAs are toxic to mammals [3,4], exhibiting a broad range of cytotoxic and pathological actions including hepatotoxic, pneumotoxic, embryotoxic, mutagenic, carcinogenic, and teratogenic effects [5,6,7], Chronic gastrointestinal [8], cardiopulmonary [9], and central nervous system [10] disorders are further manifestations of PA poisoning. The toxicity plus the ubiquitous nature of these alkaloids makes them a world-wide health concern. 0097-6156/91/0451-0176$06.00/0 © 1991 American Chemical Society

Vanderlaan et al.; Immunoassays or Trace Chemical Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1990.


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A Pyrrolizidine Alkaliod ELISA Detection Strategy 177

While non-competitive enzyme-linked immunosorbent assays (ELIS As) have been used to detect snake venom protein in whole blood in experimental [11] and clinical settings [12], competitive ELISAs have been recently developed against toxic alkaloids. The ability to detect ergotamine in spiked grain samples in concentrations of 10 ng/g [13] and tropane alkaloids as a class (with atropine in concentrations as small as 10 n^mL) have been reported [14]. Thus the proven clinical, field, and research applications of ELISAs in both the detection and quantification of toxins prompted us to develop immunoassay techniques for the rapid, reliable screening of biological samples for PA contamination. As presented below, our strategy is to develop a twopronged class-specific and compound-specific immunochemical approach to PA detection. The suitability of the ELISA in PA detection encounters certain limitations which must be addressed and which have posed significant problems in the development of a broad spectrum immunoassay. Two important issues which must be considered in developing an immunoassay for the detection of PAs are: (i) selection of a hapten and linker arm that constitute a suitable immunogen, and (ii) use of an amplified immunoassay versus an indirect non-amplified system. While these issues are important in any immunoassay, we have found them to be critical in establishing a sensitive PA immunoassay. Class-specific Immunoassay: In reviewing the structural features of the known macrocyclic PAs, one common similarity becomes obvious: retronecine, the necine base, is found in alkaloids from six botanical families and 26 genera [15]. Thus, one aspect of our approach to hapten design was based on the premise that antibodies produced in response to PA analogues possessing the retronecine moiety would deliver a class-specific immunoassay capable of detecting most macrocyclic PAs: for example, retrorsine, senecionine, and seneciphylliine are all PAs from the plant Senecio vulgaris which contain the retronecine substructural unit (Figure 1). In previous work from our laboratory [16], we coupled retronecine to bovine serum albumin (BSA) and produced retronecine antibodies that competed for retronecine and monocrotaline. More recently, we have developed anti-retronamine to a retronamine hapten, an antigen with improved in vitro (and presumably in vivo) stability, which also target the retronecine moiety. Figure 2 presents a comparison in the ability of anti-retronecine and anti-retronamine to detect the analyte retronecine in a competitive ELISA. Compound-specific Immunoassay: A second aspect of our PA detection strategy targeted developing a compound-specific immunoassay - a strategy which required presenting the necic acid moiety as well as the necine base moiety to the immune system. Monocrotaline, which possesses the necine base retronecine and, by bisesterification (i.e., macrolactonization), the necic acid monocrotalic acid, is present in numerous plants making it an ideal substrate for compound-specific ELISA development. Further, monocrotaline occurs in a sufficiently high concentration in some plants (over 9% by weight in the seeds of Crotalaria retusa, for example [17]) to make ELISA detection useful for some samples without the need for concentrating extracts before obtaining a sufficient mass of alkaloid to provide a working I50, the alkaloid concentration at which 50% antibody inhibition is seen. Materials and Methods Antibodies were developed against three different hapten-BSA conjugates in rabbits: retronecine-BSA, retronamine-BSA, and monocrotaline-BSA. Antibodies to each of the hapten conjugates were assayed using the antibody detection ELISA. While each hapten was capable of stimulating an immune response, the ability of


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IMMUNOASSAYS F O RT R A C E C H E M I C A L ANALYSIS

HQi

£5=·


to retroresine

bS

senecionine seneciphyliine HO

OH

/—OR

X-N: monocrotaline X«N CH CB-(^C0 H monocrotaline immunogen HO r-NHR +

2

2

CD

R-H retronecine R-C(0)(CH )2C02H retronecine immunogen

R-H retronamine R-C(0)(CH2) C02H retronamine immunogen

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4

Figure 1. Immunogen strategy based on the premise that antibodies produced in response to the retronecine moiety would deliver a class-specific immunoassay capable of detecting retrorsine, senecionine, seneciphylliine, and monocrotaline. 100 anti-retronamine

80-

g

anti-retronecine

60-

3 40H

20 H

-n-r^

10

100

1000

r

10000

retronecine [ppm]

Figure 2. Rabbit antibodies to retronecine-BSA (1 jigfaell retronecine-OVA coating antigen) and retronamine-BSA (10 μg/vίell retronamine-OVA coating antigen).


100000


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SOBER ET AL.

179 A Pyrrolidine Alkaliod ELISA Detection Strategy

antibodies to detect retronecine and/or monocrotaline [Trans World Chemicals] in a competitive ELISA varied considerably. The amplified assay system for retronecine was based upon the work of Laurent et al [18] who were able to detect desmosine, a cross-linked amino acid present in minute quantities in rat connectivetissueand urine using the avidin-biotin amplified ELISA. While it is possible to use a non-amplified indirect ELISA for antibody detection, such a strategy has the disadvantage of requiring increased substrate incubation time and generally results in lower overall absorbance values. As discussed below, the amplified ELISA has also proven superior in assays using antisera developed against retronamine and monocrotaline haptens. Retronamine and monocrotaline antibodies were used to detect retronecine and monocrotaline. In an attempt to improve on the relatively high I50 value of our antiretronecine (1.68 ppt), we conjugated retronamine, a synthetic derivative of retronecine, to BS A by acylation of the retronamine primary amine with hexanedioic anhydride giving a hapten intermediate which was then coupled to BSA by watersoluble l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). The tertiary nitrogen of monocrotaline was alkylated with the N-hydroxysuccinimide ester of 4bromocrotonic acid giving a hapten intermediate which was then coupled to BSA via N-acylation of the lysine primary amines. Microtiter plates were coated with 1 or 10 μ^ΛνβΙΙ of hapten-chicken egg ovalbumin conjugates (hapten-OVA). The retronamine conjugate provided a more sensitive assay with a more concentrated coating solution (10 μg/well) than the retronecine conjugate, which was used at a 1 μg/well concentration. Both concentrations were tested with each assay. The concentration which provided the more sensitive assay was selected for later experiments. Standard concentrations of the hapten were assayed on each microtiter plate. The standards (retronecine and monocrotaline) were incubated with various dilutions of antisera for one hour at 37°C in culture tubes. Retrorsine [Aldrich], senecionine, and seneciphylliine were tested at a single concentration for cross-reactivity against the retronamine antiserum. A 100 aliquot of the standard solution was added to each well on the microtiter plate. Following a one hour incubation the plates were washed and biotinylated goatantirabbit antibody [Sigma] was added. This step was followed by the addition of avidin labelled horseradish peroxidase. After the final washing the enzyme substrate 0-phenylenediamine was added. The development of colored product was measured using a Molecular Devices UVmax microtiter plate reader. Results and Discussion Resultsfromthe assay using anti-retronecine are provided in our prior publication [16]. Under neutral conditions (pH 7.6) the I50 for retronecine detection using antiretronamine was 1.5 ppm; a significant improvement when compared to our previous results using anti-retronecine (I50 = 1.68 ppt). For comparative purposes, it is useful to mention here that anti-retronecine was not as sensitive for detecting retronecine as the homologous system which uses anti-retronamine. In both cases, the antisera did not cross-react with monocrotaline. However, anti-monocrotaline was found to detect unconjugated monocrotaline to a far greater extent than retronecine. This would be expected since the necic acid portion of the molecule is most likely important to antibody-antigen interaction with anti-monocrotaline. Anti-retronamine was used in a competitive ELISA for the detection of purified retronecine and for the detection of retronecine-containing PAs (i.e., retrorsine, senecionine, and seneciphylline) from S. vulgaris. Indeed, these studies have established that anti-retronamine is superior to anti-retronecine in detecting not only purified PAs, but also the mixture of PAs found in chloroform extracts of 5. vulgaris.



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A complete description o f the hapten conjugation, antibody production, and assay development has been published [16]. It is worthwhile to reiterate that although antiserum to retronecine can detect the homologous hapten (retronecine) as w e l l as a heterologous hapten (i.e., monocrotaline Iso's o f 1.68 vs. 1.21 μ g / μ L , respectively), we consistently find that the homologous system gives a significantly higher maximum inhibitory response than the heterologous system. W h i l e this retronecine assay indicates that the E L I S A technique might prove useful i n screening samples for the presence o f the necine base, our initial results also indicated that assay sensitivity needed to be enhanced. Since the presentation o f the retronecine moiety to the immune system is paramount to obtaining a sensitive E L I S A , we investigated different sites for hapten-protein conjugation. A n important question considered i n our immunochemical study was whether significant benefit w o u l d be derived from an amplified immunoassay. Indeed, previous work from our laboratory has shown that, when using retronecine as an immunizing hapten, an avidin-biotin amplified system is far superior in the competitive immunoassay [16]. This amplified E L I S A , which takes advantage o f the high binding affinity o f avidin labelled horseradish-peroxidase for biotinylated second antibody (avidin/biotin K D = 1 0 ~ ) > provides a useful means for detecting antigen-antibody interactions of both the homologous and the heterologous competitive assays. A s presented here (see Figure 2), rabbit anti-retronamine can detect retronecine with over a thousand-fold greater sensitivity than rabbit anti-retronecine. This is an intriguing result, especially i n light of the structural similarity between the two haptens. W h i l e conclusive evidence is not available, the decreased sensitivity with anti-retronecine is consistent with partial hydrolysisof the retronecine-BS A ester bond and concomitant loss o f the retronecine moiety — an unwanted difficulty which is circumvented by the amide linkage of retronamine-BSA. G i v e n the sensitivity o f rabbit anti-retronamine, we decided to immunize mice with a r e t r o n a m i n e - B S A conjugate and were pleased to find the mice d i d produce polyclonal 15

Table I. Mouse Competitive E L I S A Using Anti-retronamine Retronecine

Anti-retronamine*

^g/rhD 0.0 0.5 1.0 10.0 15.0 20.0

(O.D. at 490 nm) 1.84 0.66 0.27 0.19 0.12 0.08

Pyrrolizidine Alkaloids from

Anti-retronamine*

Senecio vulgaris (5-7 retrorsine senecionine seneciphylline

(O.D. at 490 nm) 0.23 0.01 0.01

Antibodies to retronamine were raise i n mice against retronamine-BSA and then used in the competitive E L I S A at a 1:1,335 dilution. Microtiter plates were coated with ^ g / w e l l o f retronamine-OVA and a standard curve of retronecine was assayed with each microtiter plate.


SOBER ETAL

A Pyrrolidine Alkaliod ELISA Detection Strategy


retonamine-OVA .025ug/well .05ug/well l.Oug/well 10ug/well

1/500

1/1000

1/5000

Antibody Dilutions

Figure 3. D i l u t i o n s o f mouse antibodies t o r e t r o n a m i n e - B S A

(4a) Coating Antigen: Retronamine-OVA (10 μg/well)

Retronecine [ppm] (4b) Coating Antigen: Retronamine-OVA (1 μg/well)

Retronecine [ppm]

Figure 4. Percent inhibition of polyclonal antisera against retronamine versus retronecine (analyte). A 50% inhibition was observed at retronecine concentrations of 1 to 2 ppm.



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IMMUNOASSAYS FOR TRACE CHEMICAL ANALYSIS

antibodies (Figure 3) which could be detected at serum dilutions up to 1:10,000. In competition studies, we found that mouse anti-retronamine provides a slightly better I 5 0 (1-1 \ig/mL) than the corresponding rabbit anti-retronamine (1.5 μ g / m L ) . In addition, mouse polyclonal anti-retronamine detects the macrocyclic P A s (senecionine, seneciphylline and retrorsine) isolated from Senecio vulgaris i n a range o f 5 - 7 μ g / μ L (Table I). Figure 4 shows a comparison between rabbit and mouse antisera developed against the retronamine-OVA immunogen. In addition, Figure 4 illustrates that assay sensitivity increases slightly when the coating antigen is decreased from 10 μ § ^ β 1 1 to 1 |ig/well. These promising results suggested the possibility o f raising monoclonal antibodies to the retronamine-BSA conjugate. The antibody detection assay indicated that the monocrotaline-BSA immunogen had elicited a specific immune response in rabbits and that, at serum dilutions as high as 1:150,000, antibodies to monocrotaline c o u l d be detected. Moreover, these antibodies to monocrotaline are capable o f detecting monocrotaline at a dilution o f 1:50,000. These initial results were exciting since they demonstrated the first immunogenic response to a naturally occurring macrocyclic P A . The next steps undertaken were (i) screening available macrocyclic P A s (i.e., monocrotaline, ridelliine, retrorsine, and retrorsine N-oxide) i n competitive assays, and (ii) assaying for the detection o f retronecine i n a competitive assay. Our results establish that antimonocrotaline is specific for monocrotaline and does not compete with the other macrocyclic alkaloids tested. Antibodies to monocrotaline d i d not compete for retronecine. Hapten design and selection o f a hapten that provides the desired sensitivity and immunogenicity i s critical to establishing a useful P A immunoassay. Since protonation o f the tertiary amine o f monocrotaline i n neutral buffer appears important i n antibody-antigen recognition, our next strategy was to investigate the use o f quaternarized monocrotaline (i.e., N-alkylated monocrotaline) as a competitor. N (Methyl)- and N-(crotonic acid) monocrotaline were analyzed under neutral conditions ( p H 7.6) providing Iso's o f 0.53 and 0.25 ppm, respectively, while monocrotaline ( p H 6.0) gave an I 5 0 o f 6.0 ppm. The increased sensitivity obtained with N-(crotonic acid)monocrotaline over N-(methyl)monocrotaline reflects linker-arm recognition o f the crotonic acid moiety. ÇonclusiQns The competitive, amplified E L I S A can be used to detect monocrotaline and retronecine and within the range o f 5-7 μ g / μ L o f retrorsine, seneciphylline and senecionine. Assay sensitivity is dependent on the immunizing hapten and the type o f E L I S A selected. Retronamine and monocrotaline are currently our haptens o f choice for detecting a broad spectrum o f naturally occurring P A s , although, many more alkaloids need to be screened for cross-reactivity against these antisera. Acknowledgments W e thank Professor H . J . Segall o f the U . C . D a v i s Department o f Veterinary Pharmacology and Toxicology for the retrorsine, senecionine, and seneciphylliine standards, and are pleased to acknowledge support o f this work by the U . S. Department o f A g r i c u l t u r e ( 8 6 - C R S R - 2 - 2 8 5 2 ) and the N a t i o n a l Institutes o f Environmental Health Sciences by ES04274, ES04699, and ES00182. M J K is a Sloan Foundation Fellow, 1987-1991.


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Literature Cited 1. Smith, L. W.; Culvenor, C. C. J. J. Nat. Prod 1981, 44, 129-152. 2. Cullvenor, C. C. J. In: Toxicology in the Tropics; Smith, R. L., Bababunmi, Ε. Α., Eds. Taylor and Francis: London 1980. 3. Kumana, C. R.; Ng, M.; Lin, H. J.; Ko, W.; Wu, P. C.; Todd, D. Gut 1985, 26, 101-104. 4. Tandon, Β. N.; Tandon, H. D.; Tandon, R. K.; Narndranathan, M.; Joshi, Y. K. The Lancet 1976, August, 271-272. 5. Bull, L. B.; Culvenor, C. C. J.; Dick, A. T. In: The Pyrrolizidine Alkaloids; pp. 133-225. North-Holland: Amsterdam 1968. 6. McLean, Ε. K. Pharmocol. Rev. 1970, 22, 429-483. 7. Peterson, J. E.; Culvenor, C.C.J. In: Handbook of Natural Toxins; R.F. Keeler and A.T. Tu, Eds.; Vol. 1, pp 637-671. Dekker: New York 1983. 8. Hooper, P. T. J. Comp. Path. 1975, 85, 341-349. 9. Chesney, C. F.; Allen, J. R. Am. J. Pathol. 1973, 70, 489-492. 10. Hooper, P.T. Vet. Rec. 1972, 90, 37-38. 11. Labrousse, H.; Nishikawa, A. K.; Bon, C.; Avrameas, S. Toxicon 1988, 26, 1157-1167. 12. Chandler, H. M.; Hurrell, J. G. Clinica Chimica Acta 1982, 121, 225-230. 13. Shelby, Richard Al.; Kelley, Virginia C. J. Agric Food Chem. 1990, 38, 1130-4. 14. Fliniaux, Μ. Α.; Jacquin-Dubreuil, A. Planta Med. 1987.53, 87-90. 15. Mattocks, A. R. Toxicity of pyrrolizidine alkaloids. In: Chemistry and Toxicology of Pyrrolizidine Alkaloids; pp 1-14. Academic Press: San Diego 1986. 16. Bober, Μ. Α.; Milco, L. Α.; Miller, B. R.; Mount, M.; Wicks, B.; Kurth, M. J. Toxicon 1989, 27, 1059-1064. 18. Laurent, P.; Magne, L.; DePamas, J.; Bignon, J.; Jaurance, M. C.J.Immun. Meth. 1988, 107, 1-11. 17. Kumari, S.; Kapur, K. K.; Atal, C. C. Curr. Sci. 1967, 35, 546-47. RECEIVED August 30, 1990


A Pyrrolizidine Alkaloid Enzyme-Linked Immunosorbent Assay

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