The use of inosine 5'-monophosphate dehydrogenase (IMPDH) in the development of a new liquid homogeneous enzyme immunoassay technology.
IMPDH (EC 188.8.131.52) (1) catalyzes the NAD-dependent oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). The enzyme follows an ordered Bi-Bi reaction sequence of substrate and cofactor binding and product release. In the first step, IMP binds to IMPDH, followed by the binding of the cofactor NAD. After IMP oxidation, the reduced cofactor, NADH, is released from IMPDH, followed by the release of XMP. To monitor the reaction, the rate of NADH formation is measured at 340 urn. Uncompetitive inhibition occurs when MPA combines with the IMPDH-XMP complex at the active site of the enzyme to form IMPDH-XMP-MPA complex, which is unable to release XMP. IMPDH inhibition depends only on the concentration of MPA because of the uncompetitive nature of inhibition by MPA. Thus, the greater the concentration of MPA inhibitor, the greater the inhibition of the enzyme. An uncompetitive inhibitor of IMPDH inhibits by binding at the active site of the enzyme and does not compete with IMP or NAD for inhibition of the enzyme. Increasing substrate concentration does not reverse this type of inhibition.
Two properties of MPA inhibition of IMPDH may facilitate the development of homogeneous enzyme immunoassays. The first property is that an uncompetitive inhibitor as a target conjugate is preferred over a competitive inhibitor because uncompetitive inhibitors are rare in nature and should be less susceptible to interferences from drugs and naturally occurring substances, which frequently are competitive inhibitors of enzymes. The second property is that the sensitivity of MPA inhibition ([K.sub.1] = 10 nmol/L) (2) favors its use in enzyme immunoassays.
We developed this homogeneous immunoassay by covalently attaching theophylline to a position on MPA that did not interfere with the uncompetitive inhibition of IMPDH. To assess the inhibition of IMPDH by the theophylline-MPA derivative, we measured the I[C.sub.50], and compared it with the I[C.sub.50] for MPA (3). Both compounds were diluted to eight different concentrations in 750 mL/L dimethyl sulfoxide (DMSO)-250 mL/L [H.sub.2]O (75% DMSO in [H.sub.2]O). IMPDH-II was diluted to 3 U/L in 100 mmol/L Tris-HCl, 100 mmol/L KCl, 3 mmol/L EDTA, 100 mg/L bovine serum albumin, 5 mmol/L tris(2-carboxyethyl)phosphine-HCl, pH 8.0. The reaction buffer was 125 mmol/L Tris-HCl, 125 mmol/L KCl, 3.75 mmol/L EDTA, 125 mg/L bovine serum albumin, 0.0625 mmol/L IMP, 0.125 mmol/L NAD, pH 8.0. The theophylline-MPA derivative was synthesized by Roche Diagnostics Corp. Human IMPDH-II was used in all assays and was produced by recombinant techniques. The enzyme was partially purified by ammonium sulfate precipitation. The I[C.sub.50] assay was performed on the Roche COBAS FARA II analyzer. The assay reaction temperature was 40[degrees]C. Before the assay, 40 [micro]L of sample (DMSO-[H.sub.2]O blank or eight concentrations of test compound) and 320 [micro]L of reaction buffer were pipetted together and incubated for 5 min to warm to reaction temperature. The reaction was started by pipetting 40 [micro]L of enzyme reagent into the cuvette. The absorbance at 340 nm was read every 30 s for 10.5 min.
[FIGURE 1 OMITTED]
Results were calculated as [DELTA][A.sub.340]/min with a read window of 0.5-10.5 min. Each concentration of MPA and theophylline-MPA was run three times in triplicate (total of nine tests). In each analytical run, triplicate values were averaged and entered into Sigma Plot Ver. 4.01, which was then used to calculate the I[C.sub.50] by a hyperbolic decay two-parameter regression: y = ab/(b + x). The average I[C.sub.50] (three analytical runs) for each compound was reported. The results were as follows: MPA, I[C.sub.50] = 34 nmol/L (CV = 6.0%; RZ = 0.995); MPA-5'-isoprenyltheophylline (racemic), I[C.sub.50] = 116 nmol/L (CV = 1.8%; [R.sup.2] = 0.984). Thus, the theophylline-MPA derivative inhibited IMPDH activity but was less inhibitory than MPA itself.
Immunoassays for theophylline were performed on the Hitachi 917 analyzer as follows: 3 [micro]L of sample was added to a cuvette, and 150 [micro]L of R1 reagent was added, mixed, and incubated at 37[degrees]C for 5 min. R2 reagent (150 [micro]L) was then added and mixed. The change in absorbance at 340 nm was monitored during the 3.5-5.0-min interval after the addition of the R2 reagent. In-house theophylline calibrators were used with the Hitachi 917 analyzer for the theophylline-MPA method. Roche Integra theophylline calibrators and a Roche Integra fluorescence polarization theophylline reagent cassette were used with the Roche Integra 700 analyzer for the method-comparison studies.
The R1 reagent formulation used was 100 mmol/L Tris, 100 mmol/L KCl, 80 mmol/L IMP, 4 mmol/L TCEP, 6 mmol/L EDTA, 1.47 /.mol/L theophylline-MPA, 4 mmol/L Suttocide A, 0.1 g/L (theophylline) monoclonal antibody, IMPDH-II (adjusted to rate), final pH 8.0. The R2 reagent formulation used was 1 mmol/L NAD, 4 mmol/L Suttocide A, 1.75 mL/L Nonidet P-40 (0.175%), final pH 6.0. The (theophylline) monoclonal antibody was a purified Roche Diagnostics monoclonal.
The principle of the assay is as follows: Theophylline-specific antibody binds theophylline-MPA in the absence of theophylline and thus prevents the inhibition of IMPDH by theophylline-MPA. The enzyme activity is greatest when theophylline is absent. Theophylline, when present, binds to its antibody, thus freeing up theophylline-MPA. Free theophylline-MPA binds to catalytically active IMPDH and inhibits the enzyme by preventing the release of XMP. The rate of formation of NADH is measured at 340 nm and is correlated to theophylline concentration. The rates observed with 0, 5, 10, 20, and 40 mg/L theophylline calibrators were, respectively: 105, 98, 93, 85, and 78 milliabsorbance units/min at 340 nm on the Hitachi 917 analyzer.
We used Passing-Bablok regression statistics to compare the theophylline-MPA method on the Hitachi 917 with the fluorescence polarization method on the Integra 700. Patient plasma samples were used in the method comparison. Regression statistics were as follows: y = 0.962x + 0.077; median distance (95) = 1.991; n = 51; R = 0.982; median, 11.3 (x), 10.8 (y); minimum, 8.3 (x), 8.8 (y); maximum, 29.5 (x), 31.0 (y).
We conclude that these results indicate the potential use of IMPDH as a homogeneous enzyme immunoassay technology as shown for theophylline.
(1.) Fleming MA, Chambers SP, Connelly PR, Nimmersgern E, Fox T, Bruzzese FJ, et al. Inhibition of IMPDH by mycophenolic acid: dissection of forward and reverse pathways using capillary electrophoresis. Biochemistry 1996;22: 6990-7.
(2.) Carr SF, Papp E, Wu JC, Natsumeda Y. Characterization of human type I and type II IMP dehydrogenases. J Biol Chem 1993;36:27286-90.
(3.) Nelson PH, Carr SF, Devens BH, Eugui EM, Franco F, Gonzalez C, et al. Structure-activity relationships for inhibition of inosine monophosphate dehydrogenase by nuclear variants of mycophenolic acid. J Med Chem 1996;21:4181-96.
Allan R. Dorn,* Larry D. Mountain, Mitali Ghoshal, Raymond A. Hui, Lisa K. Klinedinst, Janice E. Rugaber, Andrew F. Schamerloh, and Salvatore J. Salamone
Roche Diagnostics Corporation, Centralized Diagnostics Business Unit, 9115 Hague Rd., PO Box 50457, Indianapolis, IN 46250-0457; * author for correspondence: fax 317-521-3085, e-mail firstname.lastname@example.org
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|Title Annotation:||Abstract of Oak Ridge Posters|
|Author:||Dorn, Allan R.; Mountain, Larry D.; Ghoshal, Mitali; Hui, Raymond A.; Klinedinst, Lisa K.; Rugaber,|
|Date:||Oct 1, 2001|
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