Journal of Immunological Methods, 131 (1990) 77-82 Elsevier


JIM 05621

Chromatographic enzyme immunoassay for T-2 toxin Beverly A. W a r d e n *, A b d e l l a h Sentissi * *, M a r k u s E h r a t * * *, D o u g l a s J. Cecchini * *, K a r i m a n A l a m a n d R o g e r W. G i e s e Department of Medicinal Chemistry in the College of Pharmacy and Allied Health Professions, and Barnett Institute of Chemical Analysis and Materials Science, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, U.S.A.

(Received 5 January 1990, revised received 26 March 1990, accepted 26 March 1990)

Both the active ester and maleimide moieties of the cross-linking reagent, N-[(v-maleimid0butyryl) oxy]succinimide (GMBS), were found to react with the primary amino groups on ribonuclease (RNase). This largely inactivated RNase towards a polymeric (but not monomeric) substrate. Citraconylating the RNase first, so that essentially only a single primary amino group remained to react with GMBS, overcame this problem. The subsequent maleimido-citraconyl-RNase was used to prepare a 1:1.1 M conjugate of anti-T-2 toxin Fab' and RNase (Fab'-RNase) in a 76% yield. The conjugate was used to detect as little as 0.1/~g of T-2 toxin based on the ability of T-2 toxin to specifically displace Fab'-RNase complexed to a T-2 agarose affinity gel. Key words: Enzyme immunoassay; Ribonuclease; T-2 toxin; Citraconylation; Chromatography

Introduction H ~

T-2 toxin, the structure of which is shown below, belongs to the tricothecene family of fungal byproducts (Betina, 1984). This poisonous compound can be found on molds when they develop on stored grains such as wheat. Animals which then eat the grain tend to become sick. Thus there is an interest in developing convenient assays for this toxin that can be used in-the-field.

Correspondence to: R.W. Giese, Department of Medicinal Chemistry in the College of Pharmacy and Allied Health Professions, and Barnett Institute of Chemical Analysis and Materials Science, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, U.S.A. * Present address: Florida International University, Miami, FL, U.S.A. ** Present address: State Laboratory Institute, Jamaica Plain, MA, U.S.A. *** Present address: Ciba Geigy, Basel, Switzerland.

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Starting with an antibody that recognizes T-2 toxin, we previously prepared an anti T-2 toxin Fab'-fluorescein conjugate and used it in a chromatographic 'hit-and-run' fluoroimmunoassay to quantitate T-2 (Warden et al., 1987). In this assay, T-2 toxin was detected by its ability to elute Fab'-fluorescein complexed to a T-2 agarose affinity gel. As little as 1 ng of T-2 was detected in 11 rain, and the assay device was used repetitively to detect successive doses of T-2 with only periodic recharging with Fab'-fluorescein. Potentially the usefulness of a 'hit-and-run' immunoassay can be broadened, especially for inthe-field applications, if this technique incorpo-

0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

78 rates a molecular label other than fluorescein. Because of our interest in using ribonuclease A (RNase) as a label for ligand binding assays (Cecchini et al., 1986; Ehrat et al., 1986), we chose to substitute the fluorescein label with this enzyme in this T-2 assay. It was first necessary to prepare a Fab'-RNase conjugate. We selected the cross-linking reagent N-[('t-maleimidobutyryl)oxy]succinimide(GMBS), which has been used to couple proteins (Kitagawa and Aikawa, 1976; Ghose et al., 1983; Nishida et al., 1985; Tsuruta et al., 1985). This reagent is intended to first react via its active ester moiety with a primary amino group on one protein, leaving its maleimide group to react with a sulfhydryl group on a second protein. The use of GMBS seemed to be attractive for our purposes since RNase contains many amino but no sulfhydryl groups, while Fab' contains a sulfhydryl group.

Materials and methods

Bovine pancreatic ribonuclease A (RNase), polycytidylic acid (5'), cytidine-2',3'-cyclic monophosphate, uridine 2',3' monophosphate, N-ethylmaleimide, 2-mercaptoethylamine, 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB), trinitrobenzene sulfonic acid (TNBS) and tris-(hydroxymethyl) aminomethane (Tris) were obtained from Sigma Chemical Co. Citraconic anhydride and mercaptoethanol were purchased from Eastman Organic Chemicals. Dimethylformamide, dimethylsulfoxide, N-methylmorpholine, and dimethylaminopyridine were from Pierce Chemical Co. N-[(7maleimidobutyryl)oxy]succinimide (GMBS), and Hepes buffer were from Calbiochem-Behring. Lanthanum acetate was obtained from Alfa products. The [3H]T-2 toxin (11 Ci/mmol) was from Amersham. Mouse ascites fluid containing an IgG1 anti T-2 antibody was provided by Dr. Kenneth Hunter. [14C]succinic anhydride (116 Ci/mmol) was purchased from New England Nuclear. Polyadenylic acid was from Boehringer Mannheim Biochemicals. Centricon-30 Microconcentrators were from Amicon. All other buffers and salts were purchased in the highest purity grade available from Fisher Scientific.

Maleimido-RNase 50 mg (3.7 ~mol) of RNase dissolved in 5 ml of 0.1 M potassium phosphate buffer, pH 7.6, was treated with 61 mg (165 #mol) of uridine 2',3' monophosphate. After stirring for 5 min at room temperature, 300 /~1 of a solution containing 14 mg (50 /~mol, 13.5 M excess over RNase) of GMBS in dimethylformamide was added and the mixture was stirred for 15 rain at room temperature. The reaction was stopped by adjusting the pH to 6 with glacial acetic acid, and the precipitate which formed was removed by centrifugation. The supernatant was desalted by gel filtration on a Bio-Gel P-2 column (1.6 x 50 cm) using 0.01 M ammonium bicarbonate pH 7.0 as eluent. The fractions containing RNase, determined by absorbance, were pooled and lyophilized. This product was characterized by assays for its enzymatic activity (both monomeric and polymeric substrates, see below), and by TNBS and mercaptoethanol/Ellman tests (see below). Maleimido-citraconyl-RNase 120 mg (8.8 #mol) of RNase were dissolved in 6 ml of 0.12 M Hepes pH 7.5 and 23.7/~1 (264 /tmol) of citraconic anhydride was added. This solution was magnetically stirred for 10 min at room temperature while the pH was maintained at 7.0 with 1 N NaOH. Dimethylformamide (100 #1) containing 60 mg (215/xmol) of GMBS was added and mixing was continued for 30 min. The reaction mixture was desalted using 3 PD-10 columns (Pharmacia) using 0.05 M sodium acetate pH 6.0 as the eluent. The void volume fractions containing the protein (based on absorbance) were pooled and lyophilized. Reaction of maleimido-citraconyl-RNase with mercaptoethanol and then dilute acid 25/~1 (0.35/~mol) of mercaptoethanol in 0.1 M potassium phosphate pH 7.0 were added to 3.0 mg (0.22 /xmol) of maleimido-citraconyl-RNase (1.6 maleimide residues/RNase, determined as described below) in 1.0 ml of the same buffer. The reaction mixture was stirred for 10 min at room temperature and subjected to gel filtration on a PD-10 column with 0.12 M Hepes pH 7.5. The fractions containing the citraconylated RNase were pooled and the citraconyl groups were removed by

79 adjusting the pH to 2.5 with 0.1 M HC1. After mixing at 37 °C for 2 or 4 h the pH was raised to 7.0 with 0.1 N NaOH.

[ 14C]succinyl.RNas e 200 mg of RNase (14.7/~mol) were dissolved in 35 ml of dimethylsulfoxide containing N-methylmorphohne (1.63 retool) and dimethylaminopyridine (1.47 /~mol). Succinic anhydride (1.25 mg, 12.55/~mol) was dissolved in 1 ml of dry dimethylsulfoxide and added to 250 /L1 of a solution of [lac]succinic anhydride (2.15 /~mol) in dimethylsulfoxide. The subsequent solution was vortexed and added to the RNase solution along with a dimethylsulfoxide wash (0.1 ml). After mixing on a rocking plate overnight at room temperature, the mixture was dialyzed for 48 h vs. 5 × 4 hters of distilled water. The dialysate was lyophihzed and stored at - 2 0 o C. The specific radioactivity of the succinylated RNase was 1790 cpm//tg. Fab '-RNase Anti-T-2 toxin monoclonal antibody was digested and reduced to Fab' as described previously (Warden et al., 1987). Fab' (7.4 mg) in 25 ml of 0.1 M sodium phosphate, 5 mM EDTA, pH 6.5 was mixed with maleimido-citraconyl-RNase (45.6 mg). After stirring for 20 h at 4 ° C, the solution was concentrated over a 4 h interval to a volume of 10 ml by ultrafiltration on a Centricon-30 Microconcentrator. The product was purified by gel filtration on a 1.6 × 40 cm column of Sephadex G-75 using 50 mM Tris-HC1, 150 mM NaC1 pH 7.2. Fractions (from 26 to 39 ml) containing the Fab'-RNase conjugate were pooled and the volume was reduced to 10 ml using Centricon-30 membrane filters. The purity of the conjugate was determined on a Pharmacia Supersose 12/30 size exclusion column calibrated and eluted as described (Warden et al., 1987). Fab'-RNase was also prepared by an analogous procedure using [a4C]succinyl-RNase as an internal standard to determine the stoichiometry of the Fab'-RNase. The molar ratio of RNase to Fab' was calculated from the concentration of protein as determined by the bicinchoninic acid assay (Smith et al., 1985) and from the amount of RNase as determined by counting a4C, using M r values of 57,000 for Fab' (Warden et al., 1987) and 13,700 for RNase.

Characterization of derivatized RNases Maleimide groups. The number of maleimide groups was determined by a modification of the method of Sedlak and Lindsay (Sedlak and Lindsay, 1968). N-ethylmaleimide standards (5-100 nmol) and RNase samples (10-70 nmol) were separately prepared in 1 ml of 0.1 M potassium phosphate, 20 mM EDTA pH 7.2. 100 nmol (1/~1) of mercaptoethanol in freshly prepared buffer (1 ml) was added to all tubes. After incubation at 37°C for 1 h, 3 ml of 0.4 M Tris-HC1, 20 mM EDTA pH 8.9 and 0.1 ml of 10 mM DTNB (Ellman's reagent) in methanol were added. The tubes were incubated for 10 rain at room temperature after which the absorbance at 412 nm was measured against a blank containing buffer and DTNB. Amino groups. The number of amino groups modified in each derivative was determined by the TNBS test (Snyder and Sobocinski, 1975). Briefly, 100 /~1 of protein solution (0.5 mg/ml) including unmodified RNase as a standard was added to 900/~1 of 0.10 M sodium borate pH 9.3 followed by 100/~1 of 0.03 M TNBS in water. The samples were vortexed and incubated for 30 min at room temperature and the absorbance was measured at 420 nm against a blank containing TNBS but no protein. Enzymatic activity. Monomeric substrate: 1 mg of cytidine-2',3'-cyclic phosphate was dissolved in 10 ml of 0.5 M Tris-HC1, 5 mM EDTA pH 7.5. 800 /~1 of this solution were pipetted into 1 ml cuvettes, 100/~1 samples of standard RNase (10100 #g) or modified RNase (100/~g) were added, and the change in absorbance at 284 nm with time was measured (Crooke et al., 1960). Polymeric substrate: the precipitation assay introduced by Anfinsen et al. (Anfinsen et al., 1954) as modified by P. Blackburn (personal communication) was used. RNase standard (30-1000 pg/50 ~tl) and sample solutions were prepared in 0.5 M Tris-HCl, 5 mM EDTA, 0.1% bovine serum albumin, pH 7.5.50 ILl of each solution were added to 100/~1 of 0.5% polycytidylic acid (w/v) in water. The tubes were incubated at 37 °C for 30 min, placed in an ice bath, and 5 0 / d of ice cold 14 mM lanthanum acetate in 24% perchloric acid was added. After a 15 min incubation the tubes were centrifuged at 1700 × g for 20 min at 4 ° C. A 100/~1 aliquot was

80 withdrawn from the supernatant, diluted ten-fold with water and the absorbance was measured at 260 nm.

Measurement of T-2 toxin 200 /~1 of T-2 tresyl adipic acid hydrazide agarose prepared as previously described (Allam et al., 1987) were poured into a 1 ml Superclean (Supelco) column. The column was equilibrated with 10 ml of 10 m M Tris-HC1 p H 7.5 and 1 ml of 1 m g / m l anti T-2 Fab'-RNase was applied at the rate of 0.1 ml/min. The column was washed at a flow rate of 0.3 m l / m i n while monitoring absorbance at 280 nm. Washing was stopped when the absorbance returned to baseline. Samples of T-2 (0, 100, 250, 500, 1000, and 3000 ng) in 0.2 ml of the Tris-HC1 buffer were applied to the column at a flow of 0.68 ml/min. After each sample had totally entered the gel, the flow was stopped for 5 rain. At the end of this incubation, released T-2. Fab'-RNase plus any background Fab'-RNase were eluted with the same buffer and flow rate. Fractions (0.68 ml) were collected and assayed for RNase activity (polymeric substrate). Ten determinations of T-2 (five samples in duplicate) plus two buffer blanks (no T-2) were done before the column was reloaded with Fab'-RNase.

Results and discussion

Maleimido-RNase We reacted RNase with a 13.5 M excess of GMBS at p H 7.6. Uridine 2',3' monophosphate was included to protect the active site lysines of the enzyme. In the product, 8.4 of the 12 amino groups normally present in RNase were found to be modified (TNBS test). However, this same material only contained 2.2 mol of intact (thiol-reactive) maleimide g r o u p s / m o l protein. Apparently, both the maleimide and active ester moieties of GMBS were reacting with the primary amino groups on RNase. It has been shown that N-ethylmaleimide can react with a primary amino group (Smyth et al., 1964), even at p H 6 (Brewer and Riehm, 1967) where this side reaction should be minimized (Means and Feeney, 1971; Liu et al., 1979). We observed that RNase reacted essentially the same with GMBS at pH 6 and 7.

The maleimido-RNase was 100% active towards the monomeric substrate cytidine-2',3'cyclic phosphate. However, it retained only a few percent activity towards polycytidylic acid (poly C). The amino groups of RNase make extensive ionic contact with a polynucleotide substrate (McPherson et al., 1986). Thus, the excess modified amino groups in the maleimido-RNase could account for the nearly complete loss of enzymatic activity towards poly C.

Maleimido-citraconyl-RNase In order to obtain a more enzymatically-active maleimido-RNase intermediate, we citraconylated the enzyme prior to reacting it with GMBS. This gave, in a 78% yield, a maleimido-citraconylRNase which contained 1.4 GMBS-modified amino groups and 1.2 intact maleimide groups. The former value was determined by the TNBS test after quenching the maleimide groups with mercaptoethanol and removing the citraconyl groups by lowering the p H to 2.5. Thus, the tendency of maleimide to react with primary amino groups had been overcome. Further, after removal of the citraconyl groups the product retained 94% of its activity towards monomeric substrate and 67% towards polycytidylic acid. Fab '-RNase An Fab' for T-2 toxin (Allam et al., 1987; W a r d e n et al., 1987) was conjugated to maleimido-citraconyl-RNase and the citraconyl groups were removed as described above. We purified the resulting mixture of products by size exclusion chromatography on Sephadex G-75, giving the chromatogram shown in Fig. 1A. Collection of the appropriate fraction gave a purified Fab'-RNase that was essentially a single peak when evaluated on a Superose 12/30 size exclusion column (Fig. 1B). The yield of this product was 76%, based on the amount of starting Fab', the limiting reagent in the coupling reaction. The molar stoichiometry of the conjugate was estimated to be I Fab' : 1.1 RNase based on the use of [14C]succinyl-RNase as an internal standard. Neither the titer for binding T-2 toxin in solution, nor the corresponding dissociation half-life at 4 o C (Warden et al., 1987) were different within experimental error for Fab'-RNase vs. intact bivalent


nificantly interferes with the enzymatic activity of the RNase component. T-2 assay



We evaluated the performance of this Fab'RNase in a 'hit-and-run' immunoassay for T-2 toxin. The Fab'-RNase reagent was first loaded onto a T-2 agarose gel followed by removal of unbound reagent by washing with buffer. Subsequent addition of T-2 analyte eluted a pulse of Fab'-RNase, and the latter was quantified by its enzymatic activity. As shown in Fig. 2, T-2 was repetitively detected (ten samples and two blanks) in amounts ranging from 0.1 to 3.0/~g.


41 min






t00 Conclusion

ELUTION VOL.(mL) Fig. 1. Chromatography of Fab'-RNase. A: Sephadex G-75 size exclusion chromatography of the Fab'/maleimidocitraconyl-RNase coupling reaction mixture: 1, Fab'-RNase; 2, RNase; 3, salts. B: re-chromatography of peak 1 on a Superose 12/30 size exclusion column. Detection: UV at 280 nm for both chromatograms. Absorbance values for peak l are: 0.85 in A, and 0.014 in B.

antibody. However, the conjugate only retained 2.4% hydrolytic activity towards polycytidylic acid and 9.8% towards cytidine-2',3'-cyclic monophosphate. Thus the Fab' moiety of Fab'-RNase sig-

The coupling of an anti T-2 toxin Fab' to RNase with the cross-linking reagent GMBS was improved by citraconylating the enzyme prior to its reaction with GMBS. Citraconylation is thereby anticipated to be useful in general for improving the effectiveness of GMBS as a cross-linking agent. Using the Fab'-RNase conjugate, as little as 0.1 /~g of T-2 toxin could'be detected in a chromatographic immunoassay. While this is 100-fold less sensitive than our prior fluoroimmunoassay, it may be adequate for some in-the-field applications. The lower sensitivity is due, at least in part, to the retention of only a few percent of the activity of the enzyme label in the Fab'-RNase conjugate.


Acknowledgements >l--

Supported by CRDEC Army contract no. DAAL03-86-K-0119 administered by the Army Research Office. Contribution no. 421 from the Barnett Institute.


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T-2 (M-g) Fig. 2. Standard curve for the determination of T-2 toxin by a 'hit-and run' immunoassay using Fab'-RNase. The error bars represent + 1 SD for duplicate measurements. Also shown is the value for the blank sample: amount of RNase eluted from the column by a sample of buffer containing no T-2.

References Allam, K.I., Ehrat, M., Cecchini, D., Warden, B.A. and Giese, R.W. (1987) Tresyl activation of a hydroxyalkyl ligand for coupling to a hydrazide gel: stable immobilization of T-2 toxin for affinity purification of T-2 antibody. Anal. Biochem. 162, 171-177.

82 Anfinsen, C.B., Redfield, R.R., Choate, W.L., Page, J. and Carroll, W.R. (1954) Studies on the gross structure, crosslinkages, and terminal sequences in ribonuclease. J. Biol. Chem. 207, 201-210. Betina, V. (Ed.) (1984) Mycotoxins Production, Isolation, Separation and Purification. Elsevier, Amsterdam. Brewer, C.F. and Riehm, J.P. (1967) Evidence for possible nonspecific reactions between N-ethylmaleimide and proteins. Anal. Biochem. 18, 248-255. Cecchini, D.J., Ehrat, M. and Giese, R.W. (1986) Substrateleash amplification of ribonuclease activity. J. Am. Chem. Soc. 108, 7841-7843. Crook, E.M., Mathius, A.P. and Rabin, B.R. (1960) Spectrophotometric assay of bovine pancreatic ribonuclease by the use of cytidine 2',3'-phosphate. Biochem. J. 74, 234-238. Ehrat, M., Cecchini, D.J. and Giese, R.W. (1986) Substrateleash amplification with ribonuclease S-peptide and S-protein. Ciin. Chem. 32, 1622-1630. Ghose, T.I., Blair, A.H. and Kulkami, P.N. (1983) Preparation of antibody-linked cytotoxic agents. Methods Enzymol. 93, 280-333. Kitagawa, T. and Aikawa, T. (1976) Enzyme coupled immunoassay of insulin using a novel coupling reagent. J. Biochem. 79, 233-236. Liu, F., Zinnecker, M., Hamaoka, T. and Katz, D.H. (1979) New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates. Biochemistry 18, 690-697. McPherson, A., Brayer, G., Cascio, D. and Williams, R. (1986) The mechanism of binding of a polynucleotide chain to pancreatic ribonuclease. Science 232, 765-768.

Means, G.E. and Feeney, R.E. (1971) Alkylating and similar reagents. In: Chemical Modifications of Proteins. HoldenDay, San Fransisco, CA, p. 105. Nishida, Y., Kawai, H. and Hishino, H. (1985) A sensitive sandwich enzyme immunoassay for human myoglobin using Fab'-horseradish peroxidase conjugate: methods and results in normal subjects and patients with various diseases. Clin. Chim. Acta 153, 93-104. Sedlak, J. and Lindsay, R.H. (1968) Estimation of total, protein bound, and nonprotein bound sulfhydryl groups in tissue with Ellman's reagent. Anal. Biochem. 25, 192-205. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 7685. Smyth, D.G., Blumenfeld, O.O. and Konigsberg, W. (1964) Reactions of N-ethylmaleimide with peptides and amino acids. Biochem. J. 91,589-595. Snyder, S.L. and Sobocinski, P.Z. (1975) An improved 2,4,6trinitrobenzenesulfonic acid method for the determination of amines. Anal. Biochem. 64, 284-288. Tsuruta, J., Yamamoto, T., Kozono, K. and Kambara, T. (1985) Application of a new method of antibody-enzyme conjugation with maleimide derivative for immunohistochemistry: HepatoceUular production, interstitial tissue distribution, and renal cell reabsorption of plasma albumin in guinea pig. J. Histochem. Cytochem. 33, 767-777. Warden, B.A., Allam, K., Sentissi, A., Cecchini, D.J. and Giese, R.W. (1987) Repetitive hit-and-run fluoroimmunoassay for T-2 toxin. Anal. Biochem. 162, 363-369.

Chromatographic enzyme immunoassay for T-2 toxin.

Both the active ester and maleimide moieties of the cross-linking reagent, N-[(gamma-maleimidobutyryl)oxy]succinimide (GMBS), were found to react with...
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