ANALYTICAL

BIOCHEMISTRY

192,

257-261

(1991)

Description of an Enzyme-Linked lmmunosorbent for the Detection of Protein Tyrosine Kinase’ Iciar

LBzaro,

Miguel

Servicio de Inmunologia,

Received

July

Gonzalez, Hospital

Garbiiie

Roy,

Luisa

Ramdn y Cajal, Carretera

de Colmenar

Press,

and Pedro

Gonzalez-Porqu&’

Km. 9,100, 28034 Madrid,

Spain

11, 1990

A solid immunoassay for the detection of protein tyrosine kinases has been developed. It is based on the binding of the synthetic polypeptide poly(Glu*Na,Tyr) 4:l to microELISA wells, where the phosphorylation reaction takes place in the presence of ATP and enzyme. The phosphorylated tyrosine residues produced in the reaction are finally detected, in the same well, by means of an ELISA using monoclonal antiphosphotyrosine antibody, peroxidase-labeled goat anti-mouse IgG antibody, and substrate. The amount of protein tyrosine kinase activity present in the sample is proportional to o 1991 the color at 492 nm developed in each well. Academic

M. Villar,

Assay

Inc.

The substitution of isotopic methods with safer and cheaper nonisotopic methods has improved the assays of many different systems. However, the use of radioactive assays based on the incorporation of the phosphate group from [T-~‘P]ATP by various proteins (1,2) has been preferred for the detection of protein tyrosine kinase (PTK)3 activity by most authors. Recently, a nonradioactive dot-blot assay for PTK activity employing the antiphosphotyrosine monoclonal antibody lG2 (3), followed by an immunogold staining procedure in polyvinylidene difluoride (PVDF) membranes, has been published (4). Here, we report the development and standardization of an ELISA for the detection of PTK activity as an alternative to the previous methods. The method is

1 Supported by a research grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social. ’ To whom correspondence should be addressed at Servicio de Inmunologia, Hospital Ramdn y Cajal, 28034 Madrid, Spain. 3 Abbreviations used: PTK, protein tyrosine kinase; ELISA, enzyme-linked immunosorbent assay; EGTA, ethylene glycol bis(& aminoethyl ether) N,N’-tetraacetic acid; TCA, trichloroacetic acid, PBS, phosphate-buffered saline; BSA, bovine serum albumin. 0003-2697191 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

based on the immobilization of the artificial substrate poly(Glu . Na,Tyr) 4:l on a microELISA plate where the phosphorylation reaction takes place in the presence of PTK and ATP. The phosphorylated substrate is then recognized by the antiphosphotyrosine monoclonal antibody lG2, which in turn is recognized by a peroxidaselabeled goat anti-mouse IgG antibody. Finally, upon the addition of the substrate for peroxidase (H,O, and ophenylenediamine), a color proportional to the amount of phosphotyrosine present in the well is developed. MATERIALS

AND

METHODS

Materials MicroELISA polystyrene plates (NUNC, The Netherlands) Cat. Ref. 449824, poly(Glu+ Na,Ala,Tyr) l:l:l, poly(Glu * Na,Ala,Tyr) 6:3:1, poly(Glu 0Na,Lys,Tyr) 6:3:1, and poly(Glu * Na,Tyr) 4:l were from Sigma (U.S.A.); ATP was from Pharmacia (Sweden); and [y32P]ATP (sp act: 5000 Ci/mmol) was from Amersham (England). Other substances were as follows: antiphosphotyrosine monoclonal antibody lG2 (4) (ATCCSD 990, U.S.A.); peroxidase-labeled goat anti-mouse IgG (TAGO, U.S.A.); DEAE-cellulose (DE-52, Whatman, U.S.A.); Sephacryl S-300 (Pharmacia, Sweden); and phenyl-Sepharose (Pharmacia, Sweden). Other chemicals were of the highest quality available and were purchased from Sigma and Merck. Protein Tyrosine Kinase Source Twenty grams of human spleen was homogenized with 100 ml of 0.01 M Tris . HCl, pH 7.7, 0.05 M NaCl and centrifuged for 1 h at 100,OOOg.The supernatant was absorbed onto a column of DEAE-cellulose (5 X 8 cm) and PTK was then eluted with a batch of 0.25 M NaCl, 0.01 M Tris * HCl, pH 7.7, adjusted to 0.2 M (NH,),SO, and absorbed onto a column of phenylSepharose 4B (2 X 8 cm). After the column was washed with 3 vol of 0.1 M (NH,),SO,, 0.01 M Tris . HCl, pH 7.7, 257

258

LAZARO OD

492

ET

AL.

nm

OD

492nm

l-

08

0.8

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0

2

20

40

60

pg/well OD

80

100

120

140

160

180

200

ng lgG/well

492nm

OD

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492nm

4 D

0.8

0.6

0.4

0.2

0

I

0

I

I

l/2000

I I

I I

I

I,

1/1000

I I

I,,

/

l/750

I,

l/500

dilution

0

10

20

30

40

Incubation

50

60

70

80

QO

time (min)

FIG. 1. Standard assay conditions. (A) Saturation curve for substrate binding to microtiter plates. Wells were coated with different amounts of the substrates and the phosphorylation reaction and ELISA were performed under the standard conditions described under Materials and Methods. *, poly(Glu * Na,Ala,Tyr) 1:l:l; 0, poly(Glu * Na,Tyr) 4:l; +, poly(Glu * Na,Ala,Tyr) 6:3:1; X, poly(Glu * Na,Lys,Tyr) 6:3:1. (B) Saturation curve for antiphosphotyrosine monoclonal antibody. Increasing amounts of the antiphosphotyrosine monoclonal antibody lG2 (ng IgG) were added to each well after the phosphorylation reaction had taken place. The ELISA was then performed as described under Materials and Methods. (C) Saturation curve for peroxidase-labeled goat anti-mouse IgG antibody. Serial dilutions of the antibody were used to detect the antiphosphotyrosine antibody bound to the phosphorylated substrate. The phosphorylation reaction and ELISA were performed under standard conditions. See Materials and Methods. (D) Kinetics of the phosphorylation reaction. At the times indicated on the abscissa, the phosphorylation reaction was stopped by withdrawing the reaction mixture and thoroughly washing the corresponding wells with PBS. The ELISA was performed under standard conditions as described under Materials and Methods.

PTK was then eluted with 0.005 M Tris 0HCl, pH 7.7, concentrated by ultrafiltration on an Amicon PM10 membrane to about 10 ml, and applied to a column of

Sephacryl S-300 (5 X 80 cm) equilibrated in 0.1 M NaCl, 0.01 M Tris - HCl, pH 7.7. The active fractions (40-60 kDa) were pooled and concentrated by ultrafiltration.

IMMUNOASSAY OD

FOR

PROTEIN

TYROSINE OD

492nm

259

KINASES

492nm

2

‘;

f

1.5

1

05-

OQ 0

200

100

300

400

500

/ 0

01

FIG. 2. ATP dependence of the phosphorylation activity was evaluated with variable ATP concentrations oping the reaction under standard conditions.

reaction. PTK and by devel-

The specific activity of PTK at this stage was 50 times higher than that in the crude extract, and was the material used throughout this work. Details on the further

pmol

PI Incorporated

30

25

/ 20

i

- 600

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,

- 500

=/a

i 10 I

,’

0

05

1

1’

- 400 q

- 300

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1 04

I 05

I 06

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FIG. 4. Standard curve for PTK: ELISA assay. The phosphorylation reaction and the ELBA were performed under standard conditions described under Materials and Methods. Increasing amounts of PTK extract expressed in pg of protein are plotted against optical density at 492 nm.

purification and characterization published elsewhere. Protein

thousands)

0.3

ug PTK extract

ATP IJ.M

cpm(

0.2

25

3

ug PTK extract FIG. 3. Standard curve for PTK: Radioactive assay. The phosphorylation reaction was performed under standard conditions as described under Materials and Methods. Results are expressed as pmol Pi incorporated on the substrate and as cpm incorporated with increasing amounts of PTK extract.

Tyrosine

of this enzyme will be

Kinase Assays

A. Radioactive assay. The phosphorylation reaction, according to (5), with minor modifications, was performed in a final volume of 50 ~1 containing the kinase extract and 50 PM poly(Glu. Na,Tyr) 4:1, 50 mM Hepes, pH 7.4,20 mM MgCl,, 5 mM NaF, 30 PM Na,VO,, 0.8 mM ethylene glycol bis(B-amino ethyl ether)iV,N’tetraacetic acid (EGTA), 0.2 mM EDTA, and 50 PM [y32P]ATP (sp act 40 mCi/mmol). It was incubated for 60 min at 37°C and, subsequently, 25 ~1 per assay was spotted on phosphocellulose paper (P81, Whatman), precipitated, and washed with 10% trichloroacetic acid (TCA). The papers were finally counted in a Packard scintillation counter. To each well of a microELISA plate, 100 B. ELISA. ~1 of poly(Glu * Na,Tyr) 4:l (0.01 mg/ml in phosphatebuffered saline (PBS)) was added and the plates were incubated overnight at 4°C. Prior to use, the plates were washed with PBS and blocked with 200 ~1 of 10 mg/ml bovine serum albumin (BSA) in PBS for 30 min at room temperature. The plates were then washed again with PBS, and 50 ~1 of the assay mix (50 mM Hepes, pH 7.4, 20 mM MgCl,, 5 mM NaF, 30 PM Na,VO,, 0.8 mM EGTA, 0.2 mM EDTA, and 300 PM ATP) and 50 ~1 of the enzyme at the appropriate dilution were added to each

260

LAZARO

well. After incubation for 60 min at 37°C the plate was washed with PBS, 0.1% Tween 20. Then, 100 ~1 of the antiphosphotyrosine monoclonal antibody at a concentration of 500 rig/ml in PBS, 0.1% Tween 20,5 mg/ml BSA was added, incubated at room temperature for 30 min, and washed as before with PBS, 0.1% Tween 20. Then, 100 ~1 of goat anti-mouse IgG antibody labeled with peroxidase at a l/500 dilution in PBS, 0.1% Tween 20, 10 mg/ml BSA was added, incubated for 30 min as before, and washed again with PBS, 0.1% Tween 20. Finally, 100 ~1 of a solution (3 mM H,O,, 10 mM o-phenylenediamine in citrate buffer 0.1 M, pH 5.5) was added and incubated at room temperature until color developed (usually less than 10 min). The reaction was stopped by the addition of 100 ~1 of 1 N H,SO, and the color was read at 492 nm on an &channel spectrophotometer (Titertek, multiscan). RESULTS

AND

DISCUSSION

Standard

Assay

Conditions

The setting of the conditions for the assay of PTK activity is shown in Fig. 1. Figure 1A shows the saturation curve for the binding of different substrates to the microELISA plate in the presence of an excess of all the other reagents. While poly(Glu. Na,Tyr) 4:l and poly(Glu * Na,Ala,Tyr) 1:l:l behaved as good substrates and saturated the well at about 1 pg per well, poly(Glu. Na,Lys,Tyr) 6:3:1 could not be used as substrate for this enzyme in neither the radioactive assay nor the ELISA. Paradoxically, poly(Glu . Na,Ala,Tyr) 1:l:l behaved as an adequate substrate when bound to the solid phase but did not incorporate 32P when assayed by the radioactive method. The opposite effect was found for poly(Glu l Na,Ala,Tyr) 6:3:1. Accordingly, all the studies that follow were performed with poly(Glu * Na,Tyr) 4:1, which was a suitable substrate in both assays and is a common artificial substrate for PTK (6,7). The binding of the substrate to the plate was stable to washes with PBS, 0.1% Tween 20, and incubations up to 1 M NaCl could be employed without interfering with the results. A convenient way to prepare the plates is, as described under Materials and Methods, to let the substrate bind overnight; however, several plates can be loaded with substrate and stored at 4°C for a week’s work. Longer periods of storage for “ready-to-use” plates are under study. Figure 1B shows the saturation curve for the antiphosphotyrosine monoclonal antibody. As can be observed, the assay is saturated for this antibody for concentrations above 50 rig/well. As described under Materials and Methods, this antibody is used in the standard assay in the presence of 5 mg/ml of BSA in PBS, 0.1% Tween 20. We have tested several concen-

ET

AL.

trations of BSA from 0.1 to 10 mg/ml and have found 5 mg/ml the most adequate. Although higher sensitivity can be achieved when the amount of BSA present in the incubation is decreased, the blanks obtained are also higher. Whenever higher BSA concentrations are used, longer incubation times are needed to obtain similar results. Figure 1C shows the saturation curve for the second antibody of the ELISA assay (peroxidase-labeled goat anti-mouse IgG). In the figure, the amount of antibody added is given as a dilution of the antibody solution provided by the manufacturer. A dilution of l/500 in PBS, 0.1% Tween 20,lO mg/ml BSA was found the most appropriate and as such was employed in the standard assay. Figure 1D shows the kinetics of the phosphorylation reaction. Under standard conditions the reaction was linear for the amount of enzyme added (0.7 pg of PTK extract) up to 90 min.

ATP Dependence

of the Phosphor-y&ion

Reaction

The phosphorylation reaction was performed under standard conditions with increasing ATP concentrations. As shown in Fig. 2, the reaction is saturated for ATP concentrations above 200 pM. From the results expressed in Fig. 2, a K, for ATP of about 20 FM could be calculated for the human spleen PTK used in this study.

Standard Curve for PTK Figure 3 shows the results obtained when increasing amounts of PTK extract are added and incubated under the standard conditions of the radioactive assay as described under Materials and Methods. For comparison, in Fig. 4, a similar curve was obtained by assaying the enzyme under standard conditions using the ELISA methodology. As can be observed, the ELISA method under these conditions is more sensitive than the radioactive assay by a factor of about 5. It can be argued that, by increasing the amount of [T-~~P]ATP in the assay, the sensitivity of the radioactive assay can be considerably improved, but this can also be accomplished in the ELISA method by allowing longer time for color development (which in this study varies between 3 and 10 min) to as much as 30 min without blank interference.

CONCLUDING REMARKS The assay of PTK activity by the enzyme immunoassay described has, in our opinion, several advantages over other methods already published. The assay is rapid, in a period of 3 h nearly 100 samples can be han-

IMMUNOASSAY

FOR

PROTEIN

dled (96 per plate). When columns are being monitored, this time can be further reduced by a factor of 3, decreasing the incubation times in the different steps. The method is inexpensive, has high sensitivity, is reproducible, is not interfered with by endogenous protein substrates, avoids the use of radioactive materials, and has no special requirements. A drawback of the method described is that although it can be used as a semiquantitative method for comparing the amount of PTK activity present in different samples, it does not give information on the amount of phosphate incorporated on the substrate. This information can be obtained if [T-~‘P]ATP is used to construct a standard curve in the microELISA plate, cutting the wells, counting the radioactivity incorporated, and comparing it with the optical density at 492 nm obtained under similar conditions.

TYROSINE

261

KINASES

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L., and Geahlen,

3. Huhn, R. D., Posner, M. R., Rayter, Frackelton, A. R. (1987) Proc. Natl. 4412. 4. Rijksen, Biochem.

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J. E., Harrison, 5. Casnellie, E. G. (1983) J. Biol. Chem. 6. Braun, S., Raymond, 259,2051-2054.

R. L. (1986)

S. I., Foulkes, Acad. Sci. USA

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G., Dalla Libera, L., and Moret, 7. Clari, Biophys. Res. Commun. 166, 1378-1383.

J. G., and 84, 440%

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