Analytical Biochemistry 344 (2005) 135–137

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ANALYTICALBIOCHEMISTRY

Notes & Tips

An immunosorbent, nonradioactive p38 MAP kinase assay comparable to standard radioactive liquid-phase assays

Stefan Laufer a,¤, Sabine Thuma a, Christian Peifer a, Cornelia Greim a,b, Yvonne Herweh b, Angela Albrecht b, Frank Dehner a

a Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Eberhard-Karls-University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany

b Department of Drug Research, Merckle GmbH, Graf-Arco-Str. 3, 89079 Ulm, Germany

Received 16 February 2005Available online 28 June 2005

We describe an advanced nonradioactive immunosor-bent p38 MAP kinase activity assay that is applicable forroutine screening of small-molecule enzyme inhibitors.When initially adopting Forrer’s protocol [1] for the sub-strate-bound nonradioactive procedure [2], the deter-mined IC50 value in our lab for p38 MAPK standardreference inhibitor SB203580 Wtted to Forrer’s resultsbut was approximately 10-fold higher than thoseobtained with the radioactivity assays (see Table 1). Theobjective of the present study was to achieve sensitivitycomparable to that of radioactivity assays. The protocoldescribed herein represents a readily accessible androbust screening assay for p38 MAPK inhibitors.

Forrer et al. [1] determined 10 �g/mL ATF-2 as anoptimal amount of substrate and this concentrationturned out to be optimal in our lab also (data notshown). Since SB203580 and other small-molecule inhib-itors compete with ATP for the same binding pocket inp38 MAPK [3], ATP concentration is critical and wasreduced from 1 mM to 100 �M in the assay [2] to obtainvalid liquid-phase IC50 values.

Using p38 MAPK enzyme concentrations in therange of 6–24 ng/well ATF-2-phosphorylation was linearup to an incubation time of 80 min (data not shown).For optimal routine applications, 12 ng/well p38 MAPKand incubation time of 60 min were selected. However,by kinetic measurements to characterize Km constant forATP, we observed a nonlinear rate of ATF-2 phosphor-ylation. A similar result was reported by Waas et al. [4]

* Corresponding author. Fax: +49(0)7071 29 5037.E-mail address: stefan.laufer@uni-tuebingen.de (S. Laufer).

0003-2697/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2005.06.015

who investigated the kinetics of ATF-2 phosphorylationin more detail. This eVect may be due to the following;p38 MAPK phosphorylates ATF-2 on multiple posi-tions [5–8]. However, only Thr69 and Thr71 are relevantand suYcient for activation in a two-step distributivemechanism, where p38 MAPK dissociates from ATF-2after primary phosphorylation of either Thr69 or Thr71.When Thr71 serves as the Wrst phospho acceptor the sub-sequent phosphorylation rate of Thr69 is barely aVectedwhereas initial Thr69 phosphorylation dramaticallyreduces the speciWcity for p38 by approximately 40-fold(kcat/Km). Since the anti-(Thr71)-phospho-ATF-2 [1] anti-body recognizes mono (Thr71) and bis-(Thr69/71)-phos-pho-ATF-2, we employed a speciWc anti-bis-(Thr69/71)-phospho-ATF-2 antibody (1. AB) that detects only thefully activated ATF-2 [6]. By using both the new 1. ABand an optimized concentration of the secondary anti-body (2. AB), a linear determination of diphospho-ATF-2 was achieved. In the Wnal assay protocol dilutions for1. AB and 2. AB were adjusted to 1:500 and 1:4000,respectively. Our protocol represents optimized cost-value ratios, minimized nonspeciWc binding of the 2. AB,and an IC50 value for SB203580 that approximates liter-ature data. The test system will be complementary toexisting assays and can be used to screen and developcompounds for activity against p38 MAPK.

All substances were purchased from Sigma, Germanyunless otherwise stated. ATF-2 (amino acids 19–96),anti-phospho-ATF-2 antibody (Thr69/71), and AP-conju-gated goat anti-rabbit IgG antibody were from UpstateCell Signaling Solutions, New England Biolabs, andSanta Cruz Biotechnology, respectively. Activated p38

136 Notes & Tips / Anal. Biochem. 344 (2005) 135–137

alpha MAP kinase was bought from Prof. Dr. J. Schultz,University of Tübingen. Immulon HBX microtiter plateswere purchased from ThermoLabsystems, Franklin,Massachusetts, USA. SB203580 was synthesized in ourlab whereas SB202190 and SB239063 were purchasedfrom Sigma, Deisenhofen, Germany and staurosporinewas bought from Calbiochem, Darmstadt, Germany.

Microtiter plates were coated with 50�L/well of thep38 MAPK substrate ATF-2 (10�g/mL in TBS)1 for1.5 h at 37 °C. After washing three times with bidistilledwater, remaining open binding sites were blocked withblocking buVer (BB; 0,05% Tween 20 (Bio-Rad), 0.25%BSA, 0,02% NaN3 in TBS) for 30 min at room tempera-ture. Plates were washed again, 50�L of the respectivetest solution was Wlled into the wells, and the plates wereincubated for 1 h at 37 °C. Test solutions containing12 ng/well p38 MAPK were diluted in kinase buVer(50 mM Tris, pH 7.5, 10 mM MgCl2, 10 mM �-glycero-phosphate, 100�g/mL BSA, 1 mM dithiothrectol,0.1 mM Na3VO4, 100�M rATP) with or without testsubstance (10¡4–10¡8 M). Test substances were dissolvedin dimethyl sulfoxide to form stock solutions of 10¡2 M,all further dilution steps were carried out in kinasebuVer. After subsequent washing, plates were blockedagain with BB for 15 min followed by a fourth washingstep. Wells were Wlled with 50 �L of the 1. AB (1:500 inBB) and incubated for 1 h at 37 °C followed by washingand consecutive incubation with 50 �L of the 2. AB(alkaline phosphatase conjugated) (1:4000 in BB). Then100�L of 4-NPP was pipetted in each well after a Wnalwashing step and color development was measured 1.5–2 h later with an enzyme-linked immunosorbent assayreader equipped with the SOFTmax PRO software at405 nm. After adapting all solution volumes, this assaywas performed automatically with the robotic liquidhandling system MultiPROBE II EX.

Based on the data provided by Forrer ey al. [1], weimproved the sensitivity of the solid-phase assay by opti-mizing several parameters. We have shown that IC50 val-ues for SB203580 [16], SB202190, and SB239063

1 Abbreviations used: TBS, Tris-buVered saline; BB, blocking buVer;BSA, bovine serum albumin.

Table 1Inhibition of p38� MAPK by diVerent inhibitors

Compilation of published IC50 values (�M) in comparison to theresults of this study (with asterisks). All cited values are derived fromradioactive solution-phase assays, except [1,2] which are nonradioac-tive enzyme-linked immunosorbent assay based.

¤ Mean of 22 values (SD: 0.019).¤¤ Means of at least two repeats in triple determination.

SB203580 Staurosporine SB202190 SB239063

0.03–0.05 [3,9–12] >100 [1] 0.05 [12] 0.044 [11]0.075–0.6 [13–16] 0.088 [2] 0.044 [17]0.15, 0.34 [1,2]0.056¤ >100¤¤ 0.04¤¤ 0.05¤¤

obtained from the immunosorbent p38 MAPK assaydescribed herein resemble the vast majority of resultsobtained by established radioactive solution-phaseassays. We assume that the results presented in this studyare supportive to a broadened application of nonradio-active, immunosorbent assays for routine and auto-mated testing of potential kinase inhibitors.

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