Quantification of diphtheria toxin-mediated ADP-ribosylation in a solid-phase assay.
The primary infection with corynebacteria takes place in the respiratory tract and affects mainly the pharyngeal tonsil. Death may result from obstruction of the respiratory tract and myocarditis. Because of the danger of diphtheria infections, diagnostic testing must be performed immediately. Although a number of methods are available to detect the gene and the protein, the only method to identify the active conformation of DT is the infection of guinea pigs with phase-infected corynebacteria [beta], 4).
DT causes cell death by inhibiting protein biosynthesis. It is composed of a catalytic-, a transmembrane-, and a receptor-binding domain. Binding to eukaryotic cells is mediated by the receptor-binding domain, which interacts with the heparin-binding epidermal growth factor (EGF)-like precursor on the cell surface (5). DT is internalized by receptor-mediated endocytosis to endosomes and subsequently cleaved by a protease of the furin family within a protease-sensitive loop between the catalytic and transmembrane domains (5). The catalytic domain is referred to as the A-chain of DT (DTA); the remainder is referred to as the B-chain. The A- and B-chain remain linked by a disulfide bridge. A cytosolic translocation factor complex of the cell seems to be involved in transfer of the A-chain and subsequent cleavage of the intramolecular disulfide bridge, releasing DTA into the cytosol (6).
DT is an ADP-ribosylatiog enzyme. The catalytic domain transfers ADP-ribose from nicotinamide adenine dinucleotide ([NAD.sup.+]) to eukaryotic elongation factor 2 (eEF2) and releases nicotinamide (7). eEF2 mediates the translocation step in peptide chain elongation by promoting transfer of peptidyl tRNA from the A- to the P-site of the ribosome (8). In contrast to bacterial EF2, eEF2 and EF2 from archaea contain a unique and strictly conserved posttranslationally modified histidine referred to as diphthamide (9). The ADP-ribosylation takes place on this diphthamide, resulting in inactivated eEF2 and blocked protein biosynthesis (10). eEF2-inactivation by DT is very effective, and 1 molecule modifies a vast number of eEF2 molecules. Therefore, a few toxin molecules are sufficient to inhibit cellular protein synthesis and induce apoptosis (11) with median lethal dose values in humans <100 ng/kg (12). Pseudomonas exotoxin A (PE) is a further bacterial toxin that ADP-ribosylates eEF2 at the diphthamide (13). Like DT, PE consists of separate catalytic and binding domains and has high toxicity, with a median lethal dose of approximately 3 [micro]g/kg in mice (14). The catalytic activity of PE is located at the C-terminus within domain III (15). Both bacterial toxins mediate cell death. When coupled to target-specific ligands, these chimeric toxins can act as specific cytotoxic agents. In modern tumor therapy, these chimeric toxins are a promising tool to overcome the low efficacy and higher incidence of side effects with conventional chemotherapeutics. Chimeric toxins bind specifically to their receptor on the tumor cell, are internalized, and kill the cell by inactivation of protein biosynthesis (16,17).
We developed an ADP-ribosylation assay for enzymatically active DT. Measuring the toxin activity may improve the current identification of DT by PCR or antibody detection, which fail to discriminate between active and inactive forms of the toxin.
Material and Methods
PURIFICATION OF YEAST eEF2
Endogenous eEF2 from Saccharomyces cerevisiae strain TKY675 was replaced by a genomically integrated histagged analog (kindly provided by Terri Goss Kinzy, Piscataway, NJ) (18). Yeasts were grown at 30 [degrees]C in 1 L yeast extract peptone dextrose to an [A.sub.600] of 2, harvested by centrifugation, resuspended in 15 mL buffer A-10 (50 mmol/L potassium phosphate, pH 7.6, 300 mmol/L potassium chloride, 10 mmol/L imidazole), and lysed by glass beads (0.5 mm diameter). After removal of the glass beads and subsequent centrifugation [beta]0 min, 60 000g, 4 [degrees]C) the supernatant was applied to a 2-mL nickel-nitrilotriacetic acid agarose (Qiagen) column, washed with 5 column volumes of A-10 and 4 column volumes of A-20 (A-10 with 20 mmol/L imidazole), and eluted with 3 column volumes of A-250 (A-10 with 250 mmol/L imidazole). The eluate was dialyzed twice against 0.5 L storage buffer [beta]00 mmol/L potassium phosphate, pH 7.0, 50 mmol/L potassium chloride, 50 mL/L glycerol), concentrated with Amicon Ultra-30 columns (Millipore), and stored at -80 [degrees]C. The yield was approximately 400 [micro]g eEF2 per liter yeast culture.
EXPRESSION AND PURIFICATION OF CHIMERIC TOXINS
The 2 chimeric toxins DTA-EGF (DTA coupled to EGF) (19) and scFv-PE (catalytic domain of PE coupled to an anti-interleukin-2 receptor single chain Fv antibody, kindly provided by Stefan Barth, Aachen, Germany) (20) were transformed into Escherichia coli Rosetta (DE3) pLysS (Merck), expressed in 200 mL Luria broth, and cells were harvested by centrifugation (15 min, 4 [degrees]C, 5000g). We purified DTA-EGF by nickel-nitrilotriacetic acid agarose chromatography (Qiagen) and scFv-PE using Protino nickel Tris(carboxymethyl)ethylene diamine columns (Macherey-Nagel). Both proteins were eluted with buffer containing 250 mmol/L imidazole, renatured by dialysis at 4 [degrees]C against 2 mol/L urea/10 mmol/L Tris (pH 7.4)/1 mmol/L reduced glutathione, twice against 20 mmol/L Tris (pH 7.4)/1 mmol/L EDTA, and twice against PBS (150 mM NaCl, 8.3 mM [Na.sub.2]HP[O.sub.4], 1.7 mM K[H.sub.2]P[O.sub.4], pH 7.4), concentrated as described above for eEF2 purification, and stored at -20 [degrees]C.
ADP-RIBOSYLATION OF EEF2 IN A WESTERN BLOT
We performed the ADP-ribosylation assay with 600 ng of yeast eEF2, 0.5 [micro]L biotinylated [NAD.sup.+] (250 [micro]mol/L; BD Biosciences), and 1 [micro]L DT (final concentration 420 [micro]g/L; Sigma) in a total volume of 24 [micro]L reaction buffer (50 mmol/L Tris, pH 7.6, 1 mmol/L EDTA, 1 mmol/L dithiothreitol) for 1 h at 37 [degrees]C. We separated the samples by electrophoresis (19) and detected biotinylated eEF2 by Western blotting with goat anti-biotin antibody (Sigma) as described (21). For negative controls, DT was heat-inactivated at 95 [degrees]C for 30 min.
ADP-RIBOSYLATION OF EEF2 IN A COLORIMETRIC SOLID-PHASE ASSAY
We precoated U16 MaxiSorp Immuno Modules (Nunc) with rabbit antimouse antibody (RAM) (Dako) (1:100 in 100 [micro]L PBS for 2 h) and incubated them overnight with mouse anti- his-tag antibody (Qiagen) (5 mg/L in 100 [micro]L PBS). We incubated his-tagged yeast eEF2 (1.2 [micro]g) with 2 [micro]L biotinylated [NAD.sup.+], 10 [micro]L BSA (2 g/L), 10 [micro]L L-arginine (100 mmol/L), 1 [micro]L ADP-ribosylating compound [DT, PE, and cholera toxin (CT); all obtained from Sigma], chimeric toxins (DTA-EGF or scFv-PE; 10 to 1000 ng/L), and reaction buffer in a total volume of 100 [micro]L at 37 [degrees]C for 2 h, transferred it into the precoated wells, and incubated it for an additional 2 h at room temperature. Imprecision of the method was analyzed by using different batches of eEF2. The wells were washed 3 times with PBS containing 0.05 g/L Tween 20 ([PBST.sub.0.05]), incubated for 5 min, and washed again. Wells were blocked with 200 [micro]L blocking solution I (50 g/L dry milk powder in PBS) for 30 min at room temperature followed by 30 min at 37[degrees]C and washed again as above. After incubation with horseradish peroxidase-labeled streptavidin (1 mg/L; Jackson ImmunoResearch Laboratories) in 100 [micro]L blocking solution II (100 mL/L fetal calf serum, 30 g/L BSA in PBS) for 30 min, wells were washed with [PBST.sub.0.05] for 30 min and finally incubated with 3,3',5,5'; tetramethylbenzidine (Merck) (0.2 g/L in 100 [micro]L 40 mmol/L citrate, pH 3.95) for color development. We stopped the reaction after 30 min by adding 50 [micro]L 2 mol/L [H.sub.2]S[O.sub.4] and measured the signal as difference of the absorbance at 450 run and 490 run by use of a microplate reader (Spectra Max 340PC).
To compare ADP-ribosylation of soluble eEF2, as described above, with immobilized eEF2,1.2 [micro]g eEF2 in PBS was incubated for 2 h directly after precoating with anti-his antibody and then washed to remove unbound eEF2. Thereafter, ADP-ribosylation and color development was performed in the same wells (without transfer) according to the protocol above.
To test the binding of soluble ADP-ribosylated eEF2 to different precoated antibodies, the wells were incubated with RAM followed by anti-his-tag antibody, as described above, or only directly with the anti-his-tag antibody, a commercially available polyclonal antibody (H118) against eEF2 (Santa Cruz Biotechnology), or a rabbit polyclonal peptide antibody against the yeast eEF2 peptide AVGGIYSVLNKKRGC (immunization by Bio Genes).
To find the association of DT enzymatic activity with absorbance, eEF2 was ADP-ribosylated with 1000 ng/L DT for 2 h as described above, bound to streptavidin-Sepharose within 2 h, and separated from unbound eEF2 by centrifugation (5 min, 4 [degrees]C, 5008). Bound and unbound eEF2 were quantified by use of the LAS-1000 System (Fujifilm) after Western blotting and detection with antihis-tag antibody (Qiagen) as described above.
[FIGURE 1 OMITTED]
We performed statistical analysis by use of SPSS version 11. We calculated the significance of increased signals by the paired t-test and estimated the significance of curve fits for linear regression curves. Statistical significance was assumed at P values <0.05.
DETECTION OF ADP-RIBOSYLATION AFTER WESTERN BLOTTING
His-tagged yeast eEF2 was successfully purified (>95% pure according to Coomassie staining) (Fig. 1A). Immunologic detection of the toxin-mediated transfer of ADPribose from [NAD.sup.+] to eEF2 is shown in Fig. 1B. Assay conditions gave a final DT concentration of 420 [micro]g/L; however, incubation of eEF2 without DT resulted in a weak but clear signal. Unspecific adsorption of the biotinylated [NAD.sup.+] was assumed to cause the signal. To minimize this nonspecific signal, the reaction was repeated in the presence of BSA, resulting in a modest reduction at 0.3 [micro]g BSA and a complete inhibition of the nonspecific signal after addition of 3 [micro]g BSA, without affecting DT activity. This optimized protocol was executed for DT and heat-inactivated DT, both at a concentration of 420 [micro]g/L (Fig. 1C). Whereas DT was able to ADP-ribosylate eEF2, heat-inactivated DT was apparently denatured after 30-min incubation at 95 [degrees]C. The purified chimeric toxins scFv-PE (containing the catalytically active domain of PE) and DTA-EGF (containing the catalytically active A-chain of DT) were obtained at a purity >90% according to Coomassie staining (Fig. 2A). The enzymatic activity of both chimeric toxins was demonstrated by ADP-ribosylation with 420 [micro]g/L protein and detection by Western blotting (Fig. 2B).
ADP-RIBOSYLATION IN A COLORIMETRIC SOLID-PHASE ASSAY
ADP-ribosylation by both DT and PE was detected by color development in the solid-phase assay, whereas heat-inactivated DT and CT, which ADP-ribosylates Gproteins, did not ADP-ribosylate eEF2, and no increase in the signals was observed (Fig. 3A). The increase in signal intensity for DT is concentration dependent and linear ([R.sup.2] = 0.997; P <0.001) (see Fig. 3B for a double logarithmic plot). Fig. 3, A and B, present the results of ADP-ribosylation combining several different eEF2 batches. The lowest detectable DT concentration for specific eEF2 batches is 30 ng/L DT, measured either by analysis of the 95% CIs or the comparison of the signal with the background mean plus the 3-fold SD (Table 1). The imprecision of the method is indicated by varying detection limits (background mean plus 3-fold SD) of different eEF2 batches (batch 1 = 0.248, batch 2 = 0.177). Collier and Kandel (22) defined the enzymatic activity of DT (1 unit DT transfers 1 pmol ADP-ribose in 15 min), and we used this definition to calibrate the solid-phase assay. We measured the amount of ADP-ribosyaated eEF2 as 1.3 pmol (10.5% of the total amount of eEF2) after incubation with 1000 ng/L DT for 2 h, which is thus equivalent to 0.163 U/L (or 1 mU/L corresponds to 6 ng/L DT). The lowest detectable concentration of DT [beta]0 ng/L, see above) thus corresponds to 5 mU/L (Table 1).
[FIGURE 2 OMITTED]
The linear regression curves for CT and inactivated DT resulted in [R.sup.2] values of 0.00 and 0.04, with P values of 0.97 and 0.8, respectively, showing no correlation. CT was shown to be enzymatically active in a proliferation assay (data not shown). PE revealed a significant correlation between signal intensity and toxin concentration (R2 = 0.96; P = 0.019) (Fig. 3B); however, 1000 ng/L PE resulted in only a 3.7-fold signal increase. Although 10 and 30 ng/L PE did not result in a measurable increase of the signal in comparison to the toxin-free background, all analyzed concentrations >30 ng/L PE did. A linear signal increase was also observed for both chimeric toxins. At 1000 ng/L, DTA-EGF enhanced the toxin-free signal 7.0fold and scFv-PE 5.2-fold. The [R.sup.2] value for the linear regression curves of DTA-EGF and scFv-PE was 0.97 for both chimeric toxins, with P values of 0.016 and 0.014, respectively.
OPTIMIZATION OF THE ADP-RIBOSYLATION ASSAY
When testing 2 different blocking solutions, Rotiblock resulted in increased background, larger variations in the results, and an increased lowest detectable amount, with only 1000 ng/L DT showing activity (Fig. 4A). Blocking with 50 g/L dry milk yielded more reproducible results (small error bars) and a concentration-dependent increase in the signal. Performing the ADP-ribosylation either in a separate vial or directly in the wells on immobilized eEF2 resulted in clear detection of DT activity (Fig. 4B). The weaker signal with the immobilized eEF2 may result from decreased accessibility of DT to diphthamide. A comparison of 4 different antibodies for the binding of ADPribosylated eEF2 to the solid phase revealed that best results were obtained with a combination of RAM and subsequent coating with anti-his-tag antibody (Fig. 4C). Neither the his-tag antibody alone nor the polyclonal antibodies against eEF2 were effective in binding eEF2 on the solid phase, probably owing to superior exposition and accessibility of the his-tag antibody's paratope after binding to RAM. The prolonged incubation of the ADP-ribosylation reaction provided only a minimal increase in the signal for 30 ng/L DT (Fig. 4D). This result may be linked to the degradation of 6-biotin-17-[NAD.sup.+], which is unstable in aqueous solutions.
[FIGURE 3 OMITTED]
Collier and Kandel (22) developed a system for the detection of the toxin-mediated ADP-ribosylation of eEF2. Enriched eEF2 was incubated with the toxin and radioactive-labeled [NAD.sup.+], and ADP-ribosylation was detected autoradiographically. Zhang (23) showed that DT accepts 6-biotin-17-[NAD.sup.+] as substrate for ADP-ribosylation. Based on these studies, we established a system for the measurement of enzymatically active DT (24). Western blot analyses were only semiquantitative, and detection was limited to approximately 40 [micro]g/L DT. To circumvent these drawbacks, we developed the quantitative colorimetric solid-phase assay. We used yeast his-tagged eEF2, and the source for ADP-ribose was biotinylated instead of radioactive [NAD.sup.+]. Differences in the lethal doses of DT for several species may be attributed to structural differences of eEF2 or different binding properties of DT to its extracellular receptor. Because DT showed high activity on yeast eEF2, we chose it as substrate for this assay. A certain background signal was observed in Western blotting, even in samples without DTA, likely attributable to endogenous ADP-ribosylation activity (25) or unspecific adsorption of the biotinylated [NAD.sup.+] or ADP-ribose moiety to eEF2. The addition of a 10- to 100-fold excess of BSA to the reaction successfully blocked this undesired side reaction. Specificity of the solid-phase assay was demonstrated with DT, PE, heat-inactivated DT, and CT (Fig. 3A). Heat-inactivation of DT was furthermore depicted in Western blotting, and the enzymatic activity of CT was measured in a cell culture experiment (data not shown). Because CT ADP-ribosylates G-proteins but not eEF2, it highlights the specificity of the solid-phase assay for ADP-ribosylation of eEF2. The assay is also suitable for PE, because PE shares substrate specificity with DT.
Analysis of ADP-ribosylation by DT and PE has previously used radioactively labeled [NAD.sup.+] (26). Armstrong and Merrill (27) used etheno-[beta]-[NAD.sup.+] to analyze the enzymatic activity of PE and the kinetic parameters of ADP-ribosylation in a fluorometric assay. This assay resulted in a specific signal at a concentration of 300 [micro]g/L. Other methods with lower detection limits have been based on the indirect measurement of toxin action, as shown by the detection of inhibited luciferase expression in cell culture experiments (28). In our solid-phase assay, the activity of the toxins appears not to be altered by the exchange of the binding domains by an antibody scFv or natural ligands such as EGF. Our results demonstrated that the catalytic domain of both enzymes is sufficient for detection. The solid-phase assay is thus a reliable method for the in vitro detection and quantification of therapeutic toxins that contain the catalytic domains of DT or PE.
[FIGURE 4 OMITTED]
A rapid and unquestionable identification of corynebacteria that are tox-positive and express active DT is necessary for an appropriate diagnosis of a diphtheria infection. All techniques known to date rely on 3 basic principles: (a) the immunologic detection of DT such as the common Elek test (29) and a number of derivatives such as immunoblots, ELISAs [beta]0, 31), immunochromatographic strip assays [beta]2), assays based on fluorescence nanocrystal quantum-dots [beta]3), hydrogel-based protein microchips [beta]4), and antibody-based microarrays [beta]5); (b) the molecular biological detection of the tox-gene by PCR assays [beta]6); and (c) the detection of the enzymatic activity of DT by infecting guinea pigs (4, 31), in vitro translation assays, and ADP-ribosylation assays (22).
These procedures have general disadvantages such as poor reliability, long analysis time, or a requirement for expensive and technically advanced equipment, as well as special reagents and experienced personnel [beta]1). The methods in categories a and b above do not enable differentiation between harmful strains and a number of less dangerous strains containing the tox-gene but producing enzymatically inactive DT [beta]0, 37). These bacteria are less toxic and have been found in North America. Nontoxigenic strains have also been reported with increasing frequency in Russia and the Ukraine [beta]1, 38,39). Although the 3rd analytical principle described earlier (c) provides the most immediate proof for active DT, the only existing assay for diagnostic purposes is guinea pig infection. In vitro translation assays are too indirect for sufficient reliability, and Western blot-based ADP-ribosylation assays do not allow quantification of DT activity.
Our assay overcomes a number of the problems with other methods. The imprecision of the assay reflects differences between the results obtained for individual eEF2 batches. To avoid batch-specific variations in a diagnostic assay, huge batches of eEF2 with acceptable results in DT detection have to be produced. Because of the low detection limit, this assay could detect enzymatically active DT directly from eluted patients' throat swabs. However, it is possible that bacterial cells and other cellular impurities may influence the assay. Removal of these cellular contaminants can be achieved by centrifugation, while the secreted DT remains in the supernatant. According to the studies of Engler and Efstratiou (40), different strains of C. diphtheriae produce different quantities of DT, with a mean of approximately 1 [micro]g/L DT produced by approximately 1 x [10.sup.7] colony-forming units of C. diphtheriae. Thus, we believe that 1 x [10.sup.7] DT-producing bacteria should be sufficient for the detection of active toxin in our assay. If, after final diagnostic assay development, the amount of DT in a positive throat swab is not sufficient for the detection of enzyme activity, the development of selective cultures as described by Engler et al. [beta]2) must be performed in all samples. Although it will not be possible to distinguish low DT concentrations from less active DT, by combining the antibody-based assay of Engler and Efstratiou (40) and our activity assay, it should be possible to characterize those C. diphtheriae strains that produce less active DT. Nevertheless, most important for diagnostic purposes is the ability to determine whether DT activity exists at all.
Grant/funding support: We acknowledge the generous financial support of the Stiftung fur Pathobiochemie and Molekulare Diagnostik der DGKL, the Deutsche Krebshilfe (grants 10-1670-Fu 1 and 10-1998-Fu 2), the Sonnenfeld-Stiftung, and the Wilhelm Sander-Stiftung (grants 2001.078.1 and 2001.078.2).
Financial disclosures: None declared.
Acknowledgments: We are grateful to Prof. Terri Goss Kinzy (UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ) who provided us with the yeast strain TKY675. Furthermore, we thank Prof. Stefan Barth (Universitaitsklinikum der Rheinisch-Westfdlischen Technischen Hochschule Aachen and Fraunhofer Institute for Molecular Biology and Applied Ecology, Aachen, Germany) for the valuable cooperation between our groups and for providing us with the plasmid RFT5(scFv)-ETA'pET27b.
Received January 3, 2007; accepted June 25, 2007. Previously published online at DOI: 10.1373/clinchem.2007.085365
(1.) Freeman VJ. Studies on the virulence of bacteriophage-infected strains of Corynebacterium diphtheriae. J Bacteriol 1951;61:675-88.
(2.) Impfkommission. Impfpraventable Krankheiten in Deutschland bis zum Jahr 2000. Epidemiologisches Bulletin, Robert-Koch-Institut 2002;15.02.2002:49-57.
(3.) Roux E, Yersin A. Contribution a l'etude de la diphtherie. Ann Inst Pasteur 1888;2:629-61.
(4.) Kohler W. Zentralblatt fur Bakteriologie-100 years ago: the controversy diphtheria and pseudodiphtheria bacilli versus virulent and avirulent diphtheria bacilli. Int J Med Microbiol 2004;294: 355-61.
(5.) Naglich JG, Metherall JE, Russell DW, Eidels L. Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor. Cell 1992;69:1051-61.
(6.) Falnes P0, Choe S, Madshus IH, Wilson BA, Olsnes S. Inhibition of membrane translocation of diphtheria toxin A-fragment by internal disulfide bridges. J Biol Chem 1994;269:8402-7.
(7.) Oppenheimer NJ, Bodley JW. Diphtheria toxin: site and configuration of ADP-ribosylation of diphthamide in elongation factor 2. J Biol Chem 1981;256:8579-81.
(8.) Moldave K. Eukaryotic protein synthesis. Annu Rev Biochem 1985;54:1109-49.
(9.) Van Ness BG, Howard JB, Bodley JW. ADP-ribosylation of elongation factor 2 by diphtheria toxin: NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products. J Biol Chem 1980;255:10710-6.
(10.) Honjo T, Nishizuka Y, Kato I, Hayaishi 0. Adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis by diphtheria toxin. J Biol Chem 1971;246:4251-60.
(11.) Kochi SK, Collier RJ. DNA fragmentation and cytolysis in U937 cells treated with diphtheria toxin or other inhibitors of protein synthesis. Exp Cell Res 1993;208:296-302.
(12.) Gill DM. Bacterial toxins: a table of lethal amounts. Microbiol Rev 1982;46:86-94.
(13.) Pavlovskis OR, Iglewski BH, Pollack M. Mechanism of action of Pseudomonas aeruginosa exotoxin A in experimental mouse infections: adenosine diphosphate ribosylation of elongation factor 2. Infect Immun 1978;19:29-33.
(14.) Callahan LT 3rd. Pseudomonas aeruginosa exotoxin: purification by preparative polyacrylamide gel electrophoresis and the development of a highly specific antitoxin serum. Infect Immun 1976; 14:55-61.
(15.) Carroll SF, Collier RJ. Active site of Pseudomonas aeruginosa exotoxin A: glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin. J Biol Chem 1987;262:8707-11.
(16.) Kreitman RJ, Squires DR, Stetler-Stevenson M, Noel P, FitzGerald DJ, Wilson WH, et al. Phase I trial of recombinant immunotoxin RFB4(dsFv)-PE38 (BL22) in patients with B-cell malignancies. J Clin Oncol 2005;23:6719-29.
(17.) Vallera DA, Todhunter DA, Kuroki DW, Shu Y, Sicheneder A, Chen H. A bispecific recombinant immunotoxin, DT2219, targeting human CD19 and CD22 receptors in a mouse xenograft model of B-cell leukemia/lymphoma. Clin Cancer Res 2005;11:3879-88.
(18.) Jorgensen R, Carr-Schmid A, Ortiz PA, Kinzy TG, Andersen GR. Purification and crystallization of the yeast elongation factor eEF2. Acta Crystallogr D Biol Crystallogr 2002;58:712-5.
(19.) Bachran C, Heisler I, Fuchs H, Sutherland M. Influence of protein transduction domains on target-specific chimeric proteins. Biochem Biophys Res Commun 2005;337:602-9.
(20.) Barth S, Huhn M, Matthey B, Schnell R, Tawadros S, Schinkothe T, et al. Recombinant anti-CD25 immunotoxin RFT5(SCFV)-ETA' demonstrates successful elimination of disseminated human Hodgkin lymphoma in SCID mice. Int J Cancer 2000;86:718-24.
(21.) Kaup M, Dassler K, Weise C, Fuchs H. Shedding of the transferrin receptor is mediated constitutively by an integral membrane metalloprotease sensitive to tumor necrosis factor alpha protease inhibitor-2. J Biol Chem 2002;277:38494-502.
(22.) Collier RJ, Kandel J. Structure and activity of diphtheria toxin. I. Thiol-dependent dissociation of a fraction of toxin into enzymatically active and inactive fragments. J Biol Chem 1971;246:1496-503.
(23.) Zhang J. Use of biotinylated NAD to label and purify ADP-ribosylated proteins. Methods Enzymol 1997;280:255-65.
(24.) Sutherland M, Bachran C, Heisler I, Fuchs H. Techniques for the analyses of immunotoxin activity. Kerala, India: Transworld Research Network, 2003;3:1-20.
(25.) Bektas M, Nurten R, Ergen K, Bermek E. Endogenous ADP-ribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions. Cell Biochem Funct 2006;24:369-80.
(26.) Lee H, Iglewski WJ. Cellular ADP-ribosyltransferase with the same mechanism of action as diphtheria toxin and Pseudomonas toxin A. Proc Natl Acad Sci U S A 1984;81:2703-7.
(27.) Armstrong S, Merrill AR. Application of a fluorometric assay for characterization of the catalytic competency of a domain III fragment of Pseudomonas aeruginosa exotoxin A. Anal Biochem 2001;292:26-33.
(28.) Zhao L, Haslam DB. A quantitative and highly sensitive luciferase-based assay for bacterial toxins that inhibit protein synthesis. J Med Microbiol 2005;54:1023-30.
(29.) Elek SD. The serological analysis of mixed flocculating systems by means of diffusion gradients. Br J Exp Pathol 1949;30:484-500.
(30.) Efstratiou A, Engler KH, Dawes CS, Sesardic D. Comparison of phenotypic and genotypic methods for detection of diphtheria toxin among isolates of pathogenic corynebacteria. J Clin Microbiol 1998;36:3173-7.
(31.) Efstratiou A, Engler KH, Mazurova IK, Glushkevich T, VuopioVarkila J, Popovic T. Current approaches to the laboratory diagnosis of diphtheria. J Infect Dis 2000;181(Suppl 1):S138-45.
(32.) Engler KH, Efstratiou A, Nom D, Kozlov RS, Selga I, Glushkevich TG, et al. Immunochromatographic strip test for rapid detection of diphtheria toxin: description and multicenter evaluation in areas of low and high prevalence of diphtheria. J Clin Microbiol 2002; 40:80-3.
(33.) Hoshino A, Fujioka K, Manabe N, Yamaya S, Goto Y, Yasuhara M, et al. Simultaneous multicolor detection system of the single-molecular microbial antigen with total internal reflection fluorescence microscopy. Microbiol Immunol 2005;49:461-70.
(34.) Rubina AY, Dyukova VI, Dementieva EI, Stomakhin AA, Nesmeyanov VA, Grishin EV, et al. Quantitative immunoassay of biotoxins on hydrogel-based protein microchips. Anal Biochem 2005;340: 317-29.
(35.) Rucker VC, Havenstrite KL, Herr AE. Antibody microarrays for native toxin detection. Anal Biochem 2005;339:262-70.
(36.) Mothershed EA, Cassiday PK, Pierson K, Mayer LW, Popovic T. Development of a real-time fluorescence PCR assay for rapid detection of the diphtheria toxin gene. J Clin Microbiol 2002;40: 4713-9.
(37.) Pallen MJ. Rapid screening for toxigenic Corynebacterium diphtheriae by the polymerase chain reaction. J Clin Pathol 1991;44: 1025-6.
(38.) Kolodkina V, Titov L, Sharapa T, Grimont F, Grimont PA, Efstratiou A. Molecular epidemiology of C. diphtheriae strains during different phases of the diphtheria epidemic in Belarus. BMC Infect Dis 2006;6:129.
(39.) Pallen MJ, Hay AJ, Puckey LH, Efstratiou A. Polymerase chain reaction for screening clinical isolates of corynebacteria for the production of diphtheria toxin. J Clin Pathol 1994;47:353-6.
(40.) Engler KH, Efstratiou A. Rapid enzyme immunoassay for determination of toxigenicity among clinical isolates of corynebacteria. J Clin Microbiol 2000;38:1385-9.
 Nonstandard abbreviations: DT, diphtheria toxin; EGF, epidermal growth factor; DTA, DT A-chain; [NAD.sup.+], nicotinamide adenine dinucleotide; eEF2, eukaryotic elongation factor 2; PE, Pseudomonas exotoxin A; scFv, single chain Fv antibody fragment; RAM, rabbit anti-mouse antibody; CT, cholera toxin; [PBST.sub.0.05], PBS containing 0.05 g/L Tween 20.
CHRISTOPHER BACHRAN,  MARK SUTHERLAND, ([dagger]) DIANA BACHRAN,  and HENDRIK FUCHS  *
 Zentralinstitut fur Laboratoriumsmedizin and Pathobiochemie, Charite-Universitatsmedizin Berlin, Campus Benjamin Franklin, Berlin, Germany.
([dagger]) Current address: University of Bradford, The Institute of Cancer Therapeutics, Bradford, West Yorkshire, United Kingdom.
* Address correspondence to this author at: Hendrik Fuchs, Zentralinstitut fur Laboratoriumsmedizin and Pathobiochemie, Charite--Universititsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany. Fax 4930 8445 4152; e-mail firstname.lastname@example.org.
Table 1. Response signals of ADP-ribosylation by DT in the solid-phase assay at different toxin concentrations for 2 separate eEF2 batches. (a) 95% CI DT DT Mean lower upper SD concentration, activity, response, bound bound ng/L U/L absorbance eEF2 batch 1 0 0 0.131 0.103 0.159 0.039 10 0.002 0.148 -0.03 0.326 0.02 30 0.005 0.23 0.173 0.278 0.069 100 0.016 0.265 0.217 0.313 0.057 300 0.049 0.609 0.456 0.762 0.214 1000 0.163 1.534 1.308 1.761 0.142 eEF2 batch 2 0 0 0.101 0.061 0.141 0.025 10 0.002 0.166 0.081 0.251 0.054 30 0.005 0.298 0.03 0.566 0.168 100 0.016 0.193 0.147 0.24 0.029 300 0.049 0.351 0.044 0.746 0.159 1000 0.163 1.44 0.849 2.03 0.066 (a) Means for each batch and DT concentration were calculated from 4 to 10 different samples. The detection limit was determined by addition of the 3-fold SD to the mean of the background and is 0.248 for eEF2 batch 1 and 0.177 for eEF2 batch 2.
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||Automation and Analytical Techniques|
|Author:||Bachran, Christopher; Sutherland, Mark; Bachran, Diana; Fuchs, Hendrik|
|Date:||Sep 1, 2007|
|Previous Article:||Improving healthcare accessibility through point-of-care technologies.|
|Next Article:||Plasma free metanephrine measurement using automated online solid-phase extraction HPLC-tandem mass spectrometry.|