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Affinity of anti-lysergol and anti-ergonovine monoclonal antibodies to ergot alkaloid derivatives.

Immunology provides an important analytical tool in modern plant science. Antibodies have been used to assay plant growth regulators (Weiler, 1986), determine the commercial value and physiological significance of secondary plant products (Robbins, 1986), identify plant products possessing medicinal value (Hacker et al., 1984), and assay toxic chemicals of plant origin (Smith et al., 1970). Immunoassays offer numerous advantages to conventional chromatography, enzymology, or bioassays. Conventional analytical methods usually require extensive sample preparation and purification prior to analysis and may require concentration to reach detectable levels (Robbins, 1986). Conventional methods often require expensive detection equipment and rarely acomodate large numbers of samples. Conversely, immunoassays are extremely sensitive and specific, requiring little sample preparation and permit simultaneous analysis of hundreds to thousands of samples. Reagents for immunoassays are environmentally benign and are needed in microliter quantities, whereas those for some conventional methods may be caustic or generate volumes of hazardous waste.

Monoclonal antibodies are products of single lymphocytic cells that are specific to a chemically defined antigenic determinant (epitope) found on an immunogenic agent (Goding, 1986). A monoclonal antibody was previously generated to lysergol, the alcohol form of the lysergic moiety common to the ergot alkaloids. The role of ergot alkaloids in fescue toxicosis symptomology has been investigated by means of this antibody (Hill et al., 1994), and an immunoassay was developed to quantify ergot alkaloids in tall fescue tissue (Hill and Agee, 1994). The antibody from cell line 15F3 cross reacted with compounds containing intact lysergic moieties but had a higher affinity to simpler lysergic acid derivatives than to the ergopeptine alkaloids (Hill et al., 1994). Endophyte-infected tall fescue (Festuca pratensis Shreb.) contains both lysergic acid amide and ergopeptine alkaloids (Yates et al., 1985). Hence, antibody assay data from cell line 15F3 may provide misleading information on the role of each ergot alkaloid class in fescue toxicosis and quantities in tall fescue forage because of an immunological bias to the simple lysergic acid derivatives. By having antibodies with more uniform affinity to the lysergic acid-containing compounds, studies can be conducted without an immunological bias of one alkaloid class over another. The objective of this research was to develop monoclonal antibodies with uniform affinity to the ergot alkaloid derivatives.

MATERIALS AND METHODS

All ergot alkaloids and reagents used in this study were purchased from Sigma Chemical Co. (St. Louis). An ergonovine-glutaryl-keyhole limpet haemocyanin (EG-KLH) conjugate was manufactured for the purpose of eliciting an immune response in Balb/C mice. Ergonovine-glutarate (EG) was manufactured by reacting 100 mg free base ergonovine and 100 mg glutaric anhydride in 3 mL pyridine under [N.sub.2] for 72 h (refer to Hill et al., 1994, for reaction conditions and purification procedures). The conjugate was manufactured by mixing 100 mg EG with 100 mg keyhole limpet haemocyanin (KLH) in 20 mL 0.03 M phosphate buffer solution (pH = 7.6) (PBS). Fifty milligrams of 1-ethyl3(dimethylaminopropyl) carbodi-imidehydrochloride (Sigma Chemical Co., St. Louis) were dissolved in I mL of PBS and added dropwise to the KLH/EG mixture. Fifteen milligrams of dry N-hydroxy sulfosuccinimide (Sigma Chemical Co., St. Louis) was added, the final pH adjusted to 7.6 and the mixture stirred for 12 h at room temperature in the dark. The conjugate was dialyzed using 14 000 MW exclusionary tubing in 3-L distilled water for 48 h. Water was changed every 12 h. Ergonovine-glutarate was also conjugated to human serum albumin (HSA) by the KLH protocol. Protein conjugates were frozen at - 70 [degrees] C and lyophilized.

Three Balb/C mice were each immunized with 50 mg of EG-KLH in 150 mL PBS at the University of Georgia Monclonal Antibody Facility. Primary vaccinations were administered subcutaneously using 150 mL Freund's complete adjuvant (Sigma Chemical Co., St. Louis). Secondary vaccinations were administered subcutaneously 28 d later using 150 mL Freunds incomplete adjuvant (Sigma Chemical Co., St. Louis). Mice were serologically tested for antibody activity and vaccinated once again 14 d later using Freunds incomplete adjuvant. Three days after final vaccination, the mouse with the highest antibody titer was euthanized by cervical dislocation, the spleen removed, and suspended lymphocites hybridized with myeloma cells (see Hill et al., 1994, for details of methods used for hybridization, cell culture, and cell line screening).

Cell lines expressing antibody titer to EG were tested for cross reactivity to various ergot alkaloids. One-half of 96 well Immulon 4 microtiter plates (Dynatech Co., Chantilly, VA) were coated with EG-HSA to test anti-ergonovine antibodies, and the other half with lysergol-glutaryl-HSA (LYS-HSA) to test the antilysergol antibody. The wells were coated with 188 pg of conjugate diluted in 50 L borate saline solution (6.19 g boric acid, 9.5 g [Na.sub.2][B.sub.2][O.sub.7][H.sub.2]O, 4.9 g NaCl, 1 L water, pH 8.5) by incubating overnight at 4 [degrees] C. The plates were rinsed three times with ELISA wash (1.21 g Tris, 500 L Tween 20, 0.2 g Na[N.sub.3], 1 L distilled water, pH 8.0) using a Denley Wellwash 5000 (Denley Instruments Ltd., Billingshurst, England) to remove excess conjugate. The microtiter plate wells were blocked with 125 L of bovine serum albumin blocking solution (10 g bovine serum albumin, 1. 17 g [Na.sub.2][HPO.sub.4], 0.244 g Na[H.sub.2][PO.sub.4], 8.2 g NaCl, 1 L distilled water) for 30 min at room temperature, and washed three times with ELISA wash. Antibodies from cell lines produced from EG-KLH were tested using the EG-HSA coated wells while the 15F3 antibody was tested with LYS-HSA.

Lysergol, lysergic acid, ergonovine, ergonovine maleate, ergovaline, ergotamine tartrate, ergocryptine, and 2-bromo-ergocryptine were tested for cross reaction in a competitive ELISA assay. One milligram of each was dissolved in 1 mL methanol and serial diluted (1: 10) from [10.sup.-4] to [10.sup.-18] g [mL.sup.-1]. All dilutions had final concentrations of 10% (v/v) methanol in borate saline solution. Molar concentrations of each dilution were calculated for each alkaloid. Fifty-microliter aliquots of each concentration of the serial dilutions were added to three microtiter wells (repeated measures) for each antibody tested. Hybridoma supernatant was diluted 1:100 in borate saline solution and 50 L of the diluted antibody was added to microtiter wells containing the alkaloids. Antibodies were permitted to bind competitively with the ergot alkaloids and the lysergic moiety on the protein conjugates for 2 h at 25 [degrees] C. Plates were washed three times with ELISA wash. Bound antibody was measured with 50 L of rabbit anti-mouse antibody alkaline phosphatase conjugate (Sigma Chem. Co., St. Louis, Cat. #A1902) diluted 1:500 in ELISA diluent (1.17 g [Na.sub.2][HPO.sub.4], 0.243 g Na[H.sub.2][PO.sub.4], 8.175 g NaCl, 500 mL Tween 20, 0.2 g Na[N.sub.3], 10 g bovine serum albumin, 1 L distilled water). After incubating at 25 [degrees] C for 2 h, the plates were washed three times with ELISA wash. Fifty microliters of chromogen solution containing 0.83 mg p-nitrophenyl phosphate (Sigma ChemCo., St. Louis) [mL.sup.-1] substrate buffer (0. 10 9 Mg[Cl.sub.2], 96 mL diethanolamine, 1 L distilled water, pH 9-8) was added to each well. Plates were incubated at 25 [degrees] C and color development was stopped after 30 min by adding 50 [micro]L of 3 M NaOH. Optical density of each well was measured at 405 run with a Bio-Tek EL311 (Winooski, VT) microplate reader. Affinity of the antibodies to the ergot alkaloids was expressed as the molar concentration at which the absorbance value was 50% of the maximum (Hill et al., 1994).

RESULTS

Three cell lines were identified that expressed affinity to EG-HSA. One cell line lost its activity during culture and was dicarded. Two cell lines, 9A12 and 9D1, maintained their activity and were characterized for cross reactivity.

Cross reactivity of the anti-lysergol antibody from cell line 15F3 was similar to previous studies and had greater affinity to lysergol and lysergic acid than to the ergopeptine alkaloids (Hill et al., 1994) (Table 1). Antibodies from 15F3 had no activity on brome-ergocryptine, suggesting that intact lysergic ring structures were necessary for affinity. The anti-ergonovine antibody from cell line 9D1 had higher affinity to lysergol and lysergic acid than the anti-ergonovine antibody 9A12. Both had [10.sup.-2] to 10-1 less affinity to lysergic acid and lysergol than antibodies from 15F3. All antibodies had similar affinity to ergonovine, ergonovine maleate, ergovaline, and ergotamine tartrate. Antibodies from cell line 9D1 had [10.sup.3] greater affinity to ergocryptine than those from 15F3 or 9A12, but had similar affinity to brome-ergocryptine as 9A12.

Table 1. Molar concentration at which 50% of the maximum absorbance values occurred when monoclonal antibodies were competitively tested for cross reaction to ergot alkaloid derivatives.
                                  Cell line
Ergot alkaloid
derivative                  15F3                9A12
                        -- Concentration at 50% max. absorbance --
Lysergol              3.93 x [10.sup.15]      3.93 x [10.sup.-7]
Lysergic acid         3.72 x [10.sup.10]      3.72 x [10.sup.-7]
Ergonovine            3.07 x [10.sup.-9]      3.07 x [10.sup.-8]
Ergonovine maleate    2.27 x [10.sup.-9]      2.27 x [10.sup.-9]
Ergovaline            2.40 x [10.sup.-9]      1.32 x [10.sup.-10]
Ergotamine tartrate   7.61 x [10.sup.-9]      7.61 x [10.sup.-9]
Ergocryptine          1.74 x [10.sup.-9]      9.57 x [10.sup.-8]
Bromo-ergocryptine    N.D.([dagger])          1.54 x [10.sup.-8]

                                  Cell line
Ergot alkaloid
derivative                            9D1
                    -- Concentration at 50% max. absorbance --

Lysergol                           3.93 x [10.sup.-9]
Lysergic acid                      3.72 x [10.sup.-8]
Ergonovine                         1.72 x [10.sup.-9]
Ergonovine maleate                 1.25 x [10.sup.-10]
Ergovaline                         2.40 x [10.sup.-9]
Ergotamine tartrate                7.61 x [10.sup.-10]
Ergocryptine                       1.74 x [10.sup.-11]
Bromo-ergocryptine                 1.54 x [10.sup.-8]


([dagger]) Cross reaction not detected.

Cell lines 15F3, 9A12, and 9D1 produced unique antibodies with varying affinities to lysergic acid derivatives. Anti-ergonovine antibodies from cell lines 9A12 and 9D1 each varied by as much [10.sup.-3] among the alkaloid derivatives tested. The antibody from cell line 15F3 varied by as much as [10.sup.-7] among the alkaloids tested. Therefore 9A12 and 9D1 produced antibodies with more uniform affinity to the alkaloid derivatives than 15F3. All antibodies tested may provide immunological tools to ascertain contributions of the ergot alkaloids to fescue toxicosis symptomology and/or ascertain toxicity of plant tissue. These antibodies can be obtained for academic research through a Materials Transfer Agreement from the University of Georgia Research Foundation, Inc. Contact the author to arrange for an agreement.

Abbreviations: EG, ergonovine glutarate; HSA, human serum albumin: EG-HSA, ergonovine-glutaryl-human serum albumin; KLH, keyhole limpet haemocyanin; EG-KLH, ergonovine-glutaryl-keyhole limpet haemocyanin; LYS-HSA, lysergol-glutaryl- human serum albumin-. PBS. phosphate buffer solution.

REFERENCES

Goding, J.W. 1986. Monoclonal antibodies: Principles and practice. Academic Press, New York.

Hacker, M.P., J.R. Dank, and W.B. Ershler. 1984. Vinblastine pharmokinetics measured by a sensitive enzyme-linked immunosorbent assay. Cancer Res. 44:478-481.

Hill, N.S., and C.S. Agee. 1994. Detection of ergoline alkaloids in endophyte-infected tall fescue by immunoassay. Crop Sci. 34:530-534.

Hill, N.S., F.N. Thompson, D.L. Dawe, and J.A. Stuedemann. 1994. Antibody binding of circulating ergot alkaloids in cattle grazing tall fescue. Am. J. Vet. Res. 55:419.

Robbins, R.J. 1986. The measurement of low-molecular-weight. nonimmunogenic compounds by immunoassay. p. 86-136. In H.F. Linskens and J.F. Jackson (ed.) Immunology in plant science. Springer-Verlag, New York.

Smith, T.W., V.P. Butler, and E. Haber. 1970. Characterization of antibodies of high affinity and specificity for the Digitalis glycoside digoxin. Biochemistry 9:331-337

Weiler, E.W., 1986. Plant hormone immunoassays based on monoclonal and polyclonal antibodies. p. 1-17. In H.F. Linskens and J.F. Jackson (ed.) Immunology in plant science. Springer-Verlag, New York.

Yates, S. G.. R.D. Plattner, and G.B. Garner. 1995. Detection of ergopeptine alkaloids in endophyte-infected toxic KY-31 tall fescue by mass spectrometry/mass spectrometry. J. Agric. Food Chem. 33:719-722.

N. S. Hill(*)

Crop and Soil Sciences Dep., Univ. of Georgia, Athens, GA 30602. Received 3 June 1996. (*)Corresponding author (E-mail: nhill@uga.cc.uga.edu).
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Author:Hill, N.S.
Publication:Crop Science
Date:Mar 1, 1997
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