24
ASSAYS
[3]
[3] E n z y m e I m m u n o a s s a y s o f E i c o s a n o i d s Using Acetylcholinesterase B y PHILIPPE PRADELLES, JACQUES GRASSI, and JACQUES MACLOUF
The quality of measurement in immunoassays depends primarily on the distinct qualities of two molecules: a specific and high-affinity antibody and the corresponding labeled molecule with high specific activity. In recent years, nonisotopic labeling techniques ~ have been developed to overcome certain problems associated with the use of radioactive tracers. Among these methods, enzyme immunoassays (EIA) appear to represent an important alternative to radioimmunoassay. This approach has been widely used for all compounds that can be measured by radioimmunoassays; more recently such assays have emerged for eicosanoids. 2 We have developed such systems for these compounds and will describe our procedure using acetylcholinesterase (ACHE, EC 3.1.1.7) from Electrophorus electricus as a label with high specific activity. 3 The same approach has been used in developing these reagents for leukotrienes and the strategy that we have used for obtaining polyclonal antisera as well as for the preparation of corresponding tracers is described later in this volume (see [10]).
Reagents Polyclonal Antisera. All antisera were developed using eicosanoids coupled by their carboxylic group to bovine serum albumin as an antigenic carrier. These low molecular weight substances (around 350) need this covalent attachment to a macromolecule to elicit antibody production. Since this chapter is concerned with the preparation and use of a nonisotopic label, the reader is referred to other procedures for antibody production. 4 Preparation of Enzymatic Tracers. AChE is present in both the central and peripheral nervous systems of vertebrates and is found at even higher concentrations in the electric organ of electric fishes such as Electrophorus I j. Grassi, J. Maclouf, and P. Pradelles, in "Radioimmunoassay in Basic and Clinical Pharmacology" (C. Patrono and B. A. Peskar, eds.), p. 91. Springer-Verlag, New York, 1987. 2 y . Hayashi, T. Yano, and S. Yamamoto, Biochim. Biophys. Acta 663, 661 (1981). 3 p. Pradelles, J. Grassi, and J. Maclouf, Anal. Chem. 57, 1170 (1985). 4 j. Maclouf, this series, Vol. 86, p. 273.
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[3]
ENZYME IMMUNOASSAY OF EICOSANOIDS
25
electricus. This enzyme exists naturally in multiple molecular forms categorized as "globular" and "asymmetric." Globular forms (G1, G2, G4) consist of one (molecular weight 80,000), two, or four catalytic subunits, while asymmetric forms (A4, A8, A12) contain one, two, or three tetramers (G4) attached to a rodlike tail segment (molecular weight approximately 100,000) partly collagenous in nature (for a complete review and purification, see Massouli6 and Bon. 5'6 The G4 form of AChE obtained following trypsinization of the naturally abundant asymmetric forms A8 and A12 is covalently attached to the eicosanoid. 7 Briefly, the carboxylic function of a prostanoid or thromboxane is transformed into an activated ester using stoichiometric amounts of N-hydroxysuccinimide and N,N'dicyclohexylcarbodiimide, in anhydrous dimethylformamide (3-6/zmol/ ml). 3 Over 90% of the eicosanoid can be converted into the corresponding active ester. The success of the preparation of the esters can be verified by thin-layer chromatographic analysis on silica gel (solvent, ethyl acetate/ hexane, 8:2, v/v) using 3H-labeled eicosanoid as a tracer. When stored anhydrous at - 2 0 °, the active esters are stable for 4 to 5 months. The ester (100-200 nmol) is subsequently added to the globular G4 forms of acetylcholinesterase (0.4-0.6 nmol) in 500/~1 of 0.1 M borate buffer, pH 9 (except for prostaglandin E2 which is treated in a pH 7 buffer to avoid its dehydration into PGA2 or PGB2). After 1 hr at 22°, 1 ml of EIA buffer (see following section) is added to stop the reaction and to avoid adsorption of the enzyme during subsequent handling. Purification of the conjugates is performed with a BioGel A 15-m column (90 × 1.5 cm) eluted with 10-2 M Tris buffer, pH 7.4, containing ! M NaCI, 10-z M MgCI2, and 0.01% sodium azide. 3
Materials and Reagents for Enzyme Immunoassay Materials
Microtitration plates with 96 flat-bottomed wells (Microwell Immuno NUNC 96 F with certificate Cat. No 4 394 54, Nunc, Roskilde, Denmark) Microtiter plate plastic film (Flow Lab, Cat No 77 400 05, Helsinki, Finland) Microplate reader able to read at 412-414 nm Microplate shaker Polypropylene, polystyrene or glass test tubes, 5 ml 5 j. Massouli6 and S. Bon, Annu. Rev. Neuro Sci. 5, 57 (1982). 6 j. Massouli6 and S. Bon, Eur. J. Biochem. 68, 531 (1976). 7 L. Mc Laughlin, Y. Wei, P. T. Stockmann, K. M. Leahy, P. Needleman, J. Grassi, and P. Pradelles, Biochem. Biophys. Res. Commun. 144, 469 (1987).
26
ASSAYS
[3]
Reagents Affinity-purified anti-rabbit IgG (H + L) polyclonal (e.g., Jackson Immuno Research, Baltimore, MD; Zymed, San Francisco, CA) or mouse monoclonal (Cayman Chemicals, Ann Arbor, MI). Enzyme tracers and corresponding specific antisera. The tracers are commercially available (Cayman Chemicals) and the antisera can be purchased from the same or from others sources. Stock potassium phosphate buffer 1 M, pH 7.4. This buffer can be kept for several weeks at 4 ° . EIA buffer (0.1 M phosphate), pH 7.4, containing 0. I g of sodium azide, 23.4 g sodium chloride, 1 g tetrasodium EDTA, and 1 g bovine serum albumin in 1 liter. After addition of all salts, the pH should be readjusted to 7.4. This buffer is fairly stable for more than 4-8 weeks. Washing buffer (0.01 M phosphate), pH 7.4, containing Tween 20 (0.05%, v/v). Enzyme substrate (Ellman's reagent). Concentrated Ellman's reagent (enough for five plates) is prepared by dissolving in 2 ml potassium phosphate (0.5 M) buffer, pH 7.4, 0.175 g NaCI, 21.5 mg 5,5'dithiobis(2-nitrobenzoic acid) (Sigma, St. Louis, MO); when dissolved, 20 mg acetylthiocholine iodide (Sigma) are added. Immediately after this last addition, the mixture is rapidly frozen to avoid the hydrolysis of acetylthiocholine which may occur in the concentrated buffer. This concentrated reagent can be lyophilized and stored for several months at 4 °, protected from light. Just before use, the contents of a vial is dissolved in 100 ml distilled water and eventually kept up to 1 week at 4 ° in the dark. Standard solutions of precisely known concentrations of eicosanoids (e.g., Cayman Chemicals, Biomol Research Laboratories, Philadelphia, PA).
Preparation of Plates Coating with Mouse Monoclonal Anti-Rabbit IgG Antibodies. Add 20 ml potassium phosphate buffer 5 x 10-z M to 200 p~g of monoclonal IgG, and distribute 200 /xl of this solution into each well using the automatic dispenser. (Note: Considerable time can be saved by using an automatic dispensing system and a microplate washer. If not available, these can be substituted by an 8-tip microwell pipette.) Cover with plastic film. Allow this to incubate at least 18 hr at room temperature prior to the saturation step. Saturation of Plates. Add 100/zl of EIA buffer containing 3g/liter of bovine serum albumin and 0.3 g/liter sodium azide. Cover with microtiter
[3]
ENZYME IMMUNOASSAY OF EICOSANOIDS
27
plate, plastic film, and leave for at least 18 hr at 4 ° before use. [Note: For some affinity-purified polyclonal anti-rabbit IgG antibodies, the use of glutaraldehyde (2%) added to the coating buffer solution usually increases the yield of the coating reaction)] It is critical that plates are saturated with BSA prior to use in order to minimize nonspecific adsorption. In this condition (i.e., tightly sealed to avoid erratic evaporation from well to well and containing EIA buffer), the plates may be stored for several weeks (4-6 months) at 4 °. We recommend that the following volumes be employed: 200/zl for coating, 300/~1 for saturation, 150/zl for competitive binding, and 200/zl for the enzyme reaction. Competitive Binding Step
Determination of Antiserum Titer/Assay Dilution Doubling dilutions of antiserum (starting from 1/100, 1/200, 1/400 . . . . . 1/204,800) are prepared in EIA buffer. The protocol used on the plate is essentially similar to the one described below except that all wells are treated as if they were B0 (see below for definition of these terms). Figure 1 typifies titration curves. These curves differ from classical antibody dilution curves in two ways: first, there is a reduction of bound enzyme activity at high concentrations of antisera; second, the solid-phase bound-enzyme activity (B0) never exceeds a few percentage of the total enzyme activity (T) introduced [it may be as low as 3% of total enzyme activity (see Table I) without influencing the quality of the assay]. For a discussion of the likely explanation for this phenomenon, see Ref. 8. The relative value of Bo/T is unimportant in this procedure and the selection of the antiserum assay dilution is the one that gives between 0.2 and 1 absorbance unit (A) for given conditions of immunoreaction (time and temperature) and for a predetermined period of enzymatic reaction (usually between 30 min and 2 hr).
Preparation of Standards Dissolve a precisely weighed eicosanoid standard of known purity in acetonitrile/water (50:50, v/v) for 6-keto-PGF~ or in methanol/water (50:50, v/v) for other eicosanoids at a concentration of 100 /~g/ml. Store this stock solution at - 7 0 °. From this stock solution, prepare further dilutions in EIA buffer down to the "working highest concentration" s p. Pradelles, J. Grassi, D. Chabardes, and N. Guiso, Anal. Chem. 61, 447 (1989).
28
ASSAYS
[3]
70 6O
,--, ~9.
50
o 67 hr
40
-
47hr
30
-
16 hr
2O 10 .
0
.
.
.
.
.
.
.
.
.
.
.
.
.
1/1.000
.
.
.
°
.
1/10,000
Antiserum
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1/100,000
dilution
FIG. I. Titration of TxB2 antiserum. Titration was performed at room temperature as described in the text. The time for the immunological reaction are indicated in the box. Results are expressed in Bo (percentage of bound tracer in the absence of competitor) as a function of the final dilution of antiserum.
(usually 0.5-2 ng/ml depending on the eicosanoid and on the antiserum). See considerations on stability in Ref. 9. Prepare serial dilutiofis from 1 ml of the standard solution according the following scheme. Place 500/zl of EIA buffer in tubes 2-8. Transfer 500/zl of standard solution from tube 1 to tube 2 and vortex. Continue the same procedure from tube 2 to 3, then 3 to 4, etc. In order to improve the reproducibility of the assay, we recommend preparation of several tubes of the "highest working concentration" in EIA buffer and storage at - 7 0 ° for 4-6 months. For each assay, the standard solution will be thawed and the serial dilutions performed from this tube as described above.
Enzyme Immunoassay Procedure Definitions of abbreviations are as follows: BK, blank of instrument (empty wells) with Ellman's reagent at the last step; B, solid-phase bound enzymatic activity after competition binding; B0, solid-phase bound enzymatic activity (no competition with eicosanoid); T, total activity of enzy9 R. G. Stehle, this series, 86, p. 436.
[3]
ENZYME IMMUNOASSAY OF EICOSANOIDS
29
TABLE I BINDING PARAMETERS OF ENZYME IMMUNOASSAYSOF EICOSANOIDS USING ACETYLCHOLINESTERASE AS LABEL Antiserum a Compound
IC~ob
Bo(%)
Final dilution
TxB2 6-Keto-PGFI~ PGE2 PGD2-MO PGF2~ Bicyclo-PGE2 11-dehydro-TxB2 dinor-TxB2
4 1 1 2 2 13 2 2.5
40 3 5 25 11 6 3 7
1/60,000 1/300,000 1/30,000 1/600,000 1/300,000 1/ 60,000 1/ 1,500,000 1/60,000
Final working dilution to obtain 40% of maximal binding. b Picogram of eicosanoids corresponding to 50% of initial tracer binding.
matic tracer added (a fraction of the tracer is added to designated wells after all washing steps); NSB, nonspecific binding, i.e., binding of tracer to wells without specific antibodies present; and S1-8, standard solutions.
Incubating the Standards and the Samples Because this method can be automated, we suggest a constant arrangement of the samples on the 96-well plate (Fig. 2) as shown in the tabulation below. With the exception of the B0, all points are run in duplicate.
Row/plate number
Preparation
1A to 1H 2A and 2B
Used for the blank of the Ellman's reagent (see below) Usually kept to have an evaluation of the total enzymatic activity; 5/~1 of tracer are added after washing the plate and before addition of the Ellman's reagent (NSB, nonspecific binding) 100/~1 of EIA buffer (Bo) 50/~1 of EIA buffer 50/zl of the least concentrated standard (S 8) 50/~1 of the next concentration ($7) 50/~i of the other 6 points of the standard curve (S 1-6) 50/zl of each sample (*) to be measured at the appropriate dilution
2C to 2D 2E to 2H 4G, 4H 4E, 4F 3A to 4D 5A to 12H
30
ASSAYS
1
2
3
4
5
6
[3l
7
8
9
10 11 12
A®O@®®®®®®®®C B®©@@®®®®®®®C e®@@®®®®®®®®¢
o®@@®®®®®®®®® E®@@@®®®®®®®® F®@@@®®®®®®®® G®@@®®®®®®®®®
"®@@®®®®®®®®® FIG. 2. Plate setup.
Add 50/zl of the enzyme tracer to each well from column 2 to 12 with the exception of row 2A,B. Then, add 50/zl of the diluted specific antieicosanoid antiserum from row 2E-H and in all subsequent rows. For the addition of tracer and antiserum, we recommend the use of a repeating dispenser (Multipet, Eppendorf, Hamburg, FRG) that allows good precision in the pipetting.
Measurement of the Solid-Phase Bound AChE (B) After completion of the competitive binding step, the plate is washed as described above in "preparation of the plates"; 5/zl of enzymatic tracer are added in wells 2A,2B (for total activity assessment) and 200/.d of the Ellman's reagent are added to all wells. The plate is agitated gently using a 96-well plate shaker. At this stage, the enzymatic reaction should be carried Out in the absence of uv light, including fluorescent tubes or direct sunlight. In order to achieve sufficient accuracy with the optical density reading, the absorption of the B0 should have reached at least 0.2 A at 412-414 nm (Fig. 3). This step usually takes approximately from 30 min to 2 hr depending on conditions. The blank of the reader (BK) is set to the absorbance of the wells in the first row (1A to 1H). Care should be taken to keep the optical surface (the bottom) of the microtiter plates clean. If the absorbance is too high, the plate can be washed and the enzymatic reaction restarted. Calculation of the Results. This is done following the classical protocol of competitive binding studies using a linear log-logit transformation.I° The mean of the nonspecific binding (NSB) is subtracted from all sample 10 D. W. Rodbard, W. Bridson, and P. Rayford, J. Lab. Clin. Med. 74, 770 (1969).
[3]
31
ENZYME IMMUNOASSAY OF EICOSANOIDS
40
N 30
.~
-o- Bo =0.215A ~
-4"- B0 = 1.338 Z
C g
(7o)
~
B° = 1.644A
20
10
0
"
|
0.1
!
. . . .
|
I
. . . . . . .
1
|
|
.
.
.
10
| | . 1
100
pg FIG. 3. Precision profile of the TxB2 assay at different stages of enzymatic reaction. The values of absorbance for Bo of colorimetric reaction are indicated in the box.
values, but should not exceed more than 0.1% of the total enzymatic activity. The mean of the enzymatic activity (A) of each replicate is calculated and the percentage of the bound fraction is calculated from the following equation: B/Bo (%) =
(A of sample) - (A NSB) × 100 (ABo) - (A NSB)
Fitting of the curves is done following Ref. 10. Assay Assessment Titer and Sensitivity o f Different Antisera Using Enzyme Tracer As we can see from Table I, the use of acetylcholinesterase-eicosanoid as a label for different eicosanoids permits the use of a high dilution of all antisera that have been tested. In addition, the sensitivity obtained with the various eicosanoids is in the low picogram range. In all cases when comparisons are made, sensitivity obtained using the AChE-eicosanoid tracer is equal to or exceeds that obtained using the high specific radioactivity ~zsI-labeled eicosanoids. H In addition, in most cases the sensitivity i~ j. Maclouf, M. Pradel, P. Pradelles, and F. Dray, Biochem. Biophys. Acta 431, 139 (1976).
32
ASSAYS
[3]
TABLE II E F F E C T O F A N T I S E R U M C O N C E N T R A T I O N ON S E N S I T I V I T Y OF
Antiserum dflution 1/60,000 1/120,000 1/240,000
TxB~A S S A Y a
Bo(%) 40.6 26.5 19.6
B/Bo,50%(pg) 7.8 5.4 3.6
a Immunological reaction was performed for 16 hr at room temperature. of these a s s a y s follow the f u n d a m e n t a l principles of competitive immunoassay which m a y be affected either b y diluting the specific antibody (Table II) or b y changes in the t e m p e r a t u r e o f the immunological reaction (Fig. 4).
Inter- and Intraassay Reproducibility Table I I I shows the reproducibility o f m e a s u r e m e n t of different eicosanoids at low, m e d i u m , and high concentration and a s s a y e d on different days. As can be seen, the coefficient of variation (CV) was less than 10% for m o s t s y s t e m s at m o s t concentrations.
100 90 80
70 60
~
÷
+5°
-~ +11 °
5o
-it- + 2 2 °
40
-A-
+37 °
30
20 tO 0
0.01
" ° "
0.I
1
10
100
pg FIG. 4. Effect of the temperature on the sensitivity of the TxB2assay. The antiserum was used at 1/150,000 and the immunological reaction performed for 18 hr.
[3]
ENZYME IMMUNOASSAY OF EICOSANOIDS
33
TABLE III INTERASSAY VARIATION OF ENZYME IMMUNOASSAYS AT L O W , MEDIUM, AND H I G H CONCENTRATION EICOSANOIDS
Eicosanoid TxB2 (n = 63)
6-Keto-PGFI,~ (n --- 8)
Bicyclo-PGE2 (n --- 8)
Concentration (pg)
Coefficient of variation (%)
12.5 - 0.5 5 +- 0.25 1.2+-0.1 4.8 +- 0.45 1.1 -+ 0.1 0.45 - 0.05 51 +- 2.5 13 -+ 0.75 2.6 - 0.15
8 5 4 13 9 9 5 6 5
Specificity of Assay: Validation with Gas ChromatographyMass Spectrometry The optimal way of establishing the specificity of an immunoassay should be performed by an independent assay method in the relevant biological fluid. Comparison with gas chromatography-mass spectrometry represents an excellent validation criterion. The results obtained after measuring the same rat lung extracts by these two methods is shown
300 0
1r
200
~
100
III I
r
0
O. 99
I00 200 6-keto-PGFta ng/Iun 9 GC/MS
300
FIG. 5. Correlation between EIA and GC-MS for the analysis of 6-keto-PGFt~ from rat lung samples (from Ref. 12 with permission).
34
ASSAYS
[4]
in Figure 5. As can be observed, there is a very good correlation between the measurements obtained by enzyme immunoassay and GC-MS. 12 Concluding Remarks The protocol presented here is a reliable nonisotopic alternative to radioimmunoassays. In situations where we have made a direct comparison with radioimmunoassays using I25I-labeled eicosanoids, a enzymatic tracers have provided sensitivity equal to or better than with radioactivity. With growing concerns of the use of radioactivity in the laboratory and its inherent costs, an enzyme label is an interesting option. Furthermore, the techniques described are technically straightforward and have a potential for automation, representing a major advantage over radioimmunoassay. Although the acetylcholinesterase tracers provide very high sensitivity, all guidelines of validation for immunoassays in a particular biological fluid should be strictly applied before one can rely on the quantitative information provided by this technique. 13 Acknowledgments These studies were made possible by financial support from Commissariat ~ l'Energie Atomique and grants from CNAMTS/INSERM and CNRS. 12 j. y . Wescott, S. Chang, M. Balazy, D. O. Stene, P. Pradeiles, J. Maclouf, N. F. Voelkel, and R. C. Murphy, Prostaglandins 32, 857 (1986). 13 C. Patrono, in "Radioimmunoassay in Basic and Clinical Pharmacology" (C. Patrono and B. A. Peskar, eds.), p. 213. Springer-Verlag, New York, 1987.
[4] Radioimmunoassay of 11-Dehydrothromboxane B2 B y G I O V A N N I C I A B A T T O N I , PAOLA PATRIGNANI, and CARLO PATRONO
Introduction 11-Dehydrothromboxane (TxB2) is a major enzymatic derivative of TxB2 identified in the circulating plasma and/or urine of several animal species, including the rat, rabbit, nonhuman primates, and humans. The rationale for measurement of 11-dehydro-TxB2 as a reflection of changes in TxA2 production in humans, as well as the experimental evidence supporting this contention, are reviewed elsewhere (Patrono et al. 1). In this chapi C. Patrono, G. Ciabattoni, and P. Patrignani, in "Platelets and Vascular Occlusion" (G. A. FitzGerald and C. Patrono, eds.), p. 193. Raven Press, New York, 1989.
METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
ASSAYS
[3]
[3] E n z y m e I m m u n o a s s a y s o f E i c o s a n o i d s Using Acetylcholinesterase B y PHILIPPE PRADELLES, JACQUES GRASSI, and JACQUES MACLOUF
The quality of measurement in immunoassays depends primarily on the distinct qualities of two molecules: a specific and high-affinity antibody and the corresponding labeled molecule with high specific activity. In recent years, nonisotopic labeling techniques ~ have been developed to overcome certain problems associated with the use of radioactive tracers. Among these methods, enzyme immunoassays (EIA) appear to represent an important alternative to radioimmunoassay. This approach has been widely used for all compounds that can be measured by radioimmunoassays; more recently such assays have emerged for eicosanoids. 2 We have developed such systems for these compounds and will describe our procedure using acetylcholinesterase (ACHE, EC 3.1.1.7) from Electrophorus electricus as a label with high specific activity. 3 The same approach has been used in developing these reagents for leukotrienes and the strategy that we have used for obtaining polyclonal antisera as well as for the preparation of corresponding tracers is described later in this volume (see [10]).
Reagents Polyclonal Antisera. All antisera were developed using eicosanoids coupled by their carboxylic group to bovine serum albumin as an antigenic carrier. These low molecular weight substances (around 350) need this covalent attachment to a macromolecule to elicit antibody production. Since this chapter is concerned with the preparation and use of a nonisotopic label, the reader is referred to other procedures for antibody production. 4 Preparation of Enzymatic Tracers. AChE is present in both the central and peripheral nervous systems of vertebrates and is found at even higher concentrations in the electric organ of electric fishes such as Electrophorus I j. Grassi, J. Maclouf, and P. Pradelles, in "Radioimmunoassay in Basic and Clinical Pharmacology" (C. Patrono and B. A. Peskar, eds.), p. 91. Springer-Verlag, New York, 1987. 2 y . Hayashi, T. Yano, and S. Yamamoto, Biochim. Biophys. Acta 663, 661 (1981). 3 p. Pradelles, J. Grassi, and J. Maclouf, Anal. Chem. 57, 1170 (1985). 4 j. Maclouf, this series, Vol. 86, p. 273.
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[3]
ENZYME IMMUNOASSAY OF EICOSANOIDS
25
electricus. This enzyme exists naturally in multiple molecular forms categorized as "globular" and "asymmetric." Globular forms (G1, G2, G4) consist of one (molecular weight 80,000), two, or four catalytic subunits, while asymmetric forms (A4, A8, A12) contain one, two, or three tetramers (G4) attached to a rodlike tail segment (molecular weight approximately 100,000) partly collagenous in nature (for a complete review and purification, see Massouli6 and Bon. 5'6 The G4 form of AChE obtained following trypsinization of the naturally abundant asymmetric forms A8 and A12 is covalently attached to the eicosanoid. 7 Briefly, the carboxylic function of a prostanoid or thromboxane is transformed into an activated ester using stoichiometric amounts of N-hydroxysuccinimide and N,N'dicyclohexylcarbodiimide, in anhydrous dimethylformamide (3-6/zmol/ ml). 3 Over 90% of the eicosanoid can be converted into the corresponding active ester. The success of the preparation of the esters can be verified by thin-layer chromatographic analysis on silica gel (solvent, ethyl acetate/ hexane, 8:2, v/v) using 3H-labeled eicosanoid as a tracer. When stored anhydrous at - 2 0 °, the active esters are stable for 4 to 5 months. The ester (100-200 nmol) is subsequently added to the globular G4 forms of acetylcholinesterase (0.4-0.6 nmol) in 500/~1 of 0.1 M borate buffer, pH 9 (except for prostaglandin E2 which is treated in a pH 7 buffer to avoid its dehydration into PGA2 or PGB2). After 1 hr at 22°, 1 ml of EIA buffer (see following section) is added to stop the reaction and to avoid adsorption of the enzyme during subsequent handling. Purification of the conjugates is performed with a BioGel A 15-m column (90 × 1.5 cm) eluted with 10-2 M Tris buffer, pH 7.4, containing ! M NaCI, 10-z M MgCI2, and 0.01% sodium azide. 3
Materials and Reagents for Enzyme Immunoassay Materials
Microtitration plates with 96 flat-bottomed wells (Microwell Immuno NUNC 96 F with certificate Cat. No 4 394 54, Nunc, Roskilde, Denmark) Microtiter plate plastic film (Flow Lab, Cat No 77 400 05, Helsinki, Finland) Microplate reader able to read at 412-414 nm Microplate shaker Polypropylene, polystyrene or glass test tubes, 5 ml 5 j. Massouli6 and S. Bon, Annu. Rev. Neuro Sci. 5, 57 (1982). 6 j. Massouli6 and S. Bon, Eur. J. Biochem. 68, 531 (1976). 7 L. Mc Laughlin, Y. Wei, P. T. Stockmann, K. M. Leahy, P. Needleman, J. Grassi, and P. Pradelles, Biochem. Biophys. Res. Commun. 144, 469 (1987).
26
ASSAYS
[3]
Reagents Affinity-purified anti-rabbit IgG (H + L) polyclonal (e.g., Jackson Immuno Research, Baltimore, MD; Zymed, San Francisco, CA) or mouse monoclonal (Cayman Chemicals, Ann Arbor, MI). Enzyme tracers and corresponding specific antisera. The tracers are commercially available (Cayman Chemicals) and the antisera can be purchased from the same or from others sources. Stock potassium phosphate buffer 1 M, pH 7.4. This buffer can be kept for several weeks at 4 ° . EIA buffer (0.1 M phosphate), pH 7.4, containing 0. I g of sodium azide, 23.4 g sodium chloride, 1 g tetrasodium EDTA, and 1 g bovine serum albumin in 1 liter. After addition of all salts, the pH should be readjusted to 7.4. This buffer is fairly stable for more than 4-8 weeks. Washing buffer (0.01 M phosphate), pH 7.4, containing Tween 20 (0.05%, v/v). Enzyme substrate (Ellman's reagent). Concentrated Ellman's reagent (enough for five plates) is prepared by dissolving in 2 ml potassium phosphate (0.5 M) buffer, pH 7.4, 0.175 g NaCI, 21.5 mg 5,5'dithiobis(2-nitrobenzoic acid) (Sigma, St. Louis, MO); when dissolved, 20 mg acetylthiocholine iodide (Sigma) are added. Immediately after this last addition, the mixture is rapidly frozen to avoid the hydrolysis of acetylthiocholine which may occur in the concentrated buffer. This concentrated reagent can be lyophilized and stored for several months at 4 °, protected from light. Just before use, the contents of a vial is dissolved in 100 ml distilled water and eventually kept up to 1 week at 4 ° in the dark. Standard solutions of precisely known concentrations of eicosanoids (e.g., Cayman Chemicals, Biomol Research Laboratories, Philadelphia, PA).
Preparation of Plates Coating with Mouse Monoclonal Anti-Rabbit IgG Antibodies. Add 20 ml potassium phosphate buffer 5 x 10-z M to 200 p~g of monoclonal IgG, and distribute 200 /xl of this solution into each well using the automatic dispenser. (Note: Considerable time can be saved by using an automatic dispensing system and a microplate washer. If not available, these can be substituted by an 8-tip microwell pipette.) Cover with plastic film. Allow this to incubate at least 18 hr at room temperature prior to the saturation step. Saturation of Plates. Add 100/zl of EIA buffer containing 3g/liter of bovine serum albumin and 0.3 g/liter sodium azide. Cover with microtiter
[3]
ENZYME IMMUNOASSAY OF EICOSANOIDS
27
plate, plastic film, and leave for at least 18 hr at 4 ° before use. [Note: For some affinity-purified polyclonal anti-rabbit IgG antibodies, the use of glutaraldehyde (2%) added to the coating buffer solution usually increases the yield of the coating reaction)] It is critical that plates are saturated with BSA prior to use in order to minimize nonspecific adsorption. In this condition (i.e., tightly sealed to avoid erratic evaporation from well to well and containing EIA buffer), the plates may be stored for several weeks (4-6 months) at 4 °. We recommend that the following volumes be employed: 200/zl for coating, 300/~1 for saturation, 150/zl for competitive binding, and 200/zl for the enzyme reaction. Competitive Binding Step
Determination of Antiserum Titer/Assay Dilution Doubling dilutions of antiserum (starting from 1/100, 1/200, 1/400 . . . . . 1/204,800) are prepared in EIA buffer. The protocol used on the plate is essentially similar to the one described below except that all wells are treated as if they were B0 (see below for definition of these terms). Figure 1 typifies titration curves. These curves differ from classical antibody dilution curves in two ways: first, there is a reduction of bound enzyme activity at high concentrations of antisera; second, the solid-phase bound-enzyme activity (B0) never exceeds a few percentage of the total enzyme activity (T) introduced [it may be as low as 3% of total enzyme activity (see Table I) without influencing the quality of the assay]. For a discussion of the likely explanation for this phenomenon, see Ref. 8. The relative value of Bo/T is unimportant in this procedure and the selection of the antiserum assay dilution is the one that gives between 0.2 and 1 absorbance unit (A) for given conditions of immunoreaction (time and temperature) and for a predetermined period of enzymatic reaction (usually between 30 min and 2 hr).
Preparation of Standards Dissolve a precisely weighed eicosanoid standard of known purity in acetonitrile/water (50:50, v/v) for 6-keto-PGF~ or in methanol/water (50:50, v/v) for other eicosanoids at a concentration of 100 /~g/ml. Store this stock solution at - 7 0 °. From this stock solution, prepare further dilutions in EIA buffer down to the "working highest concentration" s p. Pradelles, J. Grassi, D. Chabardes, and N. Guiso, Anal. Chem. 61, 447 (1989).
28
ASSAYS
[3]
70 6O
,--, ~9.
50
o 67 hr
40
-
47hr
30
-
16 hr
2O 10 .
0
.
.
.
.
.
.
.
.
.
.
.
.
.
1/1.000
.
.
.
°
.
1/10,000
Antiserum
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1/100,000
dilution
FIG. I. Titration of TxB2 antiserum. Titration was performed at room temperature as described in the text. The time for the immunological reaction are indicated in the box. Results are expressed in Bo (percentage of bound tracer in the absence of competitor) as a function of the final dilution of antiserum.
(usually 0.5-2 ng/ml depending on the eicosanoid and on the antiserum). See considerations on stability in Ref. 9. Prepare serial dilutiofis from 1 ml of the standard solution according the following scheme. Place 500/zl of EIA buffer in tubes 2-8. Transfer 500/zl of standard solution from tube 1 to tube 2 and vortex. Continue the same procedure from tube 2 to 3, then 3 to 4, etc. In order to improve the reproducibility of the assay, we recommend preparation of several tubes of the "highest working concentration" in EIA buffer and storage at - 7 0 ° for 4-6 months. For each assay, the standard solution will be thawed and the serial dilutions performed from this tube as described above.
Enzyme Immunoassay Procedure Definitions of abbreviations are as follows: BK, blank of instrument (empty wells) with Ellman's reagent at the last step; B, solid-phase bound enzymatic activity after competition binding; B0, solid-phase bound enzymatic activity (no competition with eicosanoid); T, total activity of enzy9 R. G. Stehle, this series, 86, p. 436.
[3]
ENZYME IMMUNOASSAY OF EICOSANOIDS
29
TABLE I BINDING PARAMETERS OF ENZYME IMMUNOASSAYSOF EICOSANOIDS USING ACETYLCHOLINESTERASE AS LABEL Antiserum a Compound
IC~ob
Bo(%)
Final dilution
TxB2 6-Keto-PGFI~ PGE2 PGD2-MO PGF2~ Bicyclo-PGE2 11-dehydro-TxB2 dinor-TxB2
4 1 1 2 2 13 2 2.5
40 3 5 25 11 6 3 7
1/60,000 1/300,000 1/30,000 1/600,000 1/300,000 1/ 60,000 1/ 1,500,000 1/60,000
Final working dilution to obtain 40% of maximal binding. b Picogram of eicosanoids corresponding to 50% of initial tracer binding.
matic tracer added (a fraction of the tracer is added to designated wells after all washing steps); NSB, nonspecific binding, i.e., binding of tracer to wells without specific antibodies present; and S1-8, standard solutions.
Incubating the Standards and the Samples Because this method can be automated, we suggest a constant arrangement of the samples on the 96-well plate (Fig. 2) as shown in the tabulation below. With the exception of the B0, all points are run in duplicate.
Row/plate number
Preparation
1A to 1H 2A and 2B
Used for the blank of the Ellman's reagent (see below) Usually kept to have an evaluation of the total enzymatic activity; 5/~1 of tracer are added after washing the plate and before addition of the Ellman's reagent (NSB, nonspecific binding) 100/~1 of EIA buffer (Bo) 50/~1 of EIA buffer 50/zl of the least concentrated standard (S 8) 50/~1 of the next concentration ($7) 50/~i of the other 6 points of the standard curve (S 1-6) 50/zl of each sample (*) to be measured at the appropriate dilution
2C to 2D 2E to 2H 4G, 4H 4E, 4F 3A to 4D 5A to 12H
30
ASSAYS
1
2
3
4
5
6
[3l
7
8
9
10 11 12
A®O@®®®®®®®®C B®©@@®®®®®®®C e®@@®®®®®®®®¢
o®@@®®®®®®®®® E®@@@®®®®®®®® F®@@@®®®®®®®® G®@@®®®®®®®®®
"®@@®®®®®®®®® FIG. 2. Plate setup.
Add 50/zl of the enzyme tracer to each well from column 2 to 12 with the exception of row 2A,B. Then, add 50/zl of the diluted specific antieicosanoid antiserum from row 2E-H and in all subsequent rows. For the addition of tracer and antiserum, we recommend the use of a repeating dispenser (Multipet, Eppendorf, Hamburg, FRG) that allows good precision in the pipetting.
Measurement of the Solid-Phase Bound AChE (B) After completion of the competitive binding step, the plate is washed as described above in "preparation of the plates"; 5/zl of enzymatic tracer are added in wells 2A,2B (for total activity assessment) and 200/.d of the Ellman's reagent are added to all wells. The plate is agitated gently using a 96-well plate shaker. At this stage, the enzymatic reaction should be carried Out in the absence of uv light, including fluorescent tubes or direct sunlight. In order to achieve sufficient accuracy with the optical density reading, the absorption of the B0 should have reached at least 0.2 A at 412-414 nm (Fig. 3). This step usually takes approximately from 30 min to 2 hr depending on conditions. The blank of the reader (BK) is set to the absorbance of the wells in the first row (1A to 1H). Care should be taken to keep the optical surface (the bottom) of the microtiter plates clean. If the absorbance is too high, the plate can be washed and the enzymatic reaction restarted. Calculation of the Results. This is done following the classical protocol of competitive binding studies using a linear log-logit transformation.I° The mean of the nonspecific binding (NSB) is subtracted from all sample 10 D. W. Rodbard, W. Bridson, and P. Rayford, J. Lab. Clin. Med. 74, 770 (1969).
[3]
31
ENZYME IMMUNOASSAY OF EICOSANOIDS
40
N 30
.~
-o- Bo =0.215A ~
-4"- B0 = 1.338 Z
C g
(7o)
~
B° = 1.644A
20
10
0
"
|
0.1
!
. . . .
|
I
. . . . . . .
1
|
|
.
.
.
10
| | . 1
100
pg FIG. 3. Precision profile of the TxB2 assay at different stages of enzymatic reaction. The values of absorbance for Bo of colorimetric reaction are indicated in the box.
values, but should not exceed more than 0.1% of the total enzymatic activity. The mean of the enzymatic activity (A) of each replicate is calculated and the percentage of the bound fraction is calculated from the following equation: B/Bo (%) =
(A of sample) - (A NSB) × 100 (ABo) - (A NSB)
Fitting of the curves is done following Ref. 10. Assay Assessment Titer and Sensitivity o f Different Antisera Using Enzyme Tracer As we can see from Table I, the use of acetylcholinesterase-eicosanoid as a label for different eicosanoids permits the use of a high dilution of all antisera that have been tested. In addition, the sensitivity obtained with the various eicosanoids is in the low picogram range. In all cases when comparisons are made, sensitivity obtained using the AChE-eicosanoid tracer is equal to or exceeds that obtained using the high specific radioactivity ~zsI-labeled eicosanoids. H In addition, in most cases the sensitivity i~ j. Maclouf, M. Pradel, P. Pradelles, and F. Dray, Biochem. Biophys. Acta 431, 139 (1976).
32
ASSAYS
[3]
TABLE II E F F E C T O F A N T I S E R U M C O N C E N T R A T I O N ON S E N S I T I V I T Y OF
Antiserum dflution 1/60,000 1/120,000 1/240,000
TxB~A S S A Y a
Bo(%) 40.6 26.5 19.6
B/Bo,50%(pg) 7.8 5.4 3.6
a Immunological reaction was performed for 16 hr at room temperature. of these a s s a y s follow the f u n d a m e n t a l principles of competitive immunoassay which m a y be affected either b y diluting the specific antibody (Table II) or b y changes in the t e m p e r a t u r e o f the immunological reaction (Fig. 4).
Inter- and Intraassay Reproducibility Table I I I shows the reproducibility o f m e a s u r e m e n t of different eicosanoids at low, m e d i u m , and high concentration and a s s a y e d on different days. As can be seen, the coefficient of variation (CV) was less than 10% for m o s t s y s t e m s at m o s t concentrations.
100 90 80
70 60
~
÷
+5°
-~ +11 °
5o
-it- + 2 2 °
40
-A-
+37 °
30
20 tO 0
0.01
" ° "
0.I
1
10
100
pg FIG. 4. Effect of the temperature on the sensitivity of the TxB2assay. The antiserum was used at 1/150,000 and the immunological reaction performed for 18 hr.
[3]
ENZYME IMMUNOASSAY OF EICOSANOIDS
33
TABLE III INTERASSAY VARIATION OF ENZYME IMMUNOASSAYS AT L O W , MEDIUM, AND H I G H CONCENTRATION EICOSANOIDS
Eicosanoid TxB2 (n = 63)
6-Keto-PGFI,~ (n --- 8)
Bicyclo-PGE2 (n --- 8)
Concentration (pg)
Coefficient of variation (%)
12.5 - 0.5 5 +- 0.25 1.2+-0.1 4.8 +- 0.45 1.1 -+ 0.1 0.45 - 0.05 51 +- 2.5 13 -+ 0.75 2.6 - 0.15
8 5 4 13 9 9 5 6 5
Specificity of Assay: Validation with Gas ChromatographyMass Spectrometry The optimal way of establishing the specificity of an immunoassay should be performed by an independent assay method in the relevant biological fluid. Comparison with gas chromatography-mass spectrometry represents an excellent validation criterion. The results obtained after measuring the same rat lung extracts by these two methods is shown
300 0
1r
200
~
100
III I
r
0
O. 99
I00 200 6-keto-PGFta ng/Iun 9 GC/MS
300
FIG. 5. Correlation between EIA and GC-MS for the analysis of 6-keto-PGFt~ from rat lung samples (from Ref. 12 with permission).
34
ASSAYS
[4]
in Figure 5. As can be observed, there is a very good correlation between the measurements obtained by enzyme immunoassay and GC-MS. 12 Concluding Remarks The protocol presented here is a reliable nonisotopic alternative to radioimmunoassays. In situations where we have made a direct comparison with radioimmunoassays using I25I-labeled eicosanoids, a enzymatic tracers have provided sensitivity equal to or better than with radioactivity. With growing concerns of the use of radioactivity in the laboratory and its inherent costs, an enzyme label is an interesting option. Furthermore, the techniques described are technically straightforward and have a potential for automation, representing a major advantage over radioimmunoassay. Although the acetylcholinesterase tracers provide very high sensitivity, all guidelines of validation for immunoassays in a particular biological fluid should be strictly applied before one can rely on the quantitative information provided by this technique. 13 Acknowledgments These studies were made possible by financial support from Commissariat ~ l'Energie Atomique and grants from CNAMTS/INSERM and CNRS. 12 j. y . Wescott, S. Chang, M. Balazy, D. O. Stene, P. Pradeiles, J. Maclouf, N. F. Voelkel, and R. C. Murphy, Prostaglandins 32, 857 (1986). 13 C. Patrono, in "Radioimmunoassay in Basic and Clinical Pharmacology" (C. Patrono and B. A. Peskar, eds.), p. 213. Springer-Verlag, New York, 1987.
[4] Radioimmunoassay of 11-Dehydrothromboxane B2 B y G I O V A N N I C I A B A T T O N I , PAOLA PATRIGNANI, and CARLO PATRONO
Introduction 11-Dehydrothromboxane (TxB2) is a major enzymatic derivative of TxB2 identified in the circulating plasma and/or urine of several animal species, including the rat, rabbit, nonhuman primates, and humans. The rationale for measurement of 11-dehydro-TxB2 as a reflection of changes in TxA2 production in humans, as well as the experimental evidence supporting this contention, are reviewed elsewhere (Patrono et al. 1). In this chapi C. Patrono, G. Ciabattoni, and P. Patrignani, in "Platelets and Vascular Occlusion" (G. A. FitzGerald and C. Patrono, eds.), p. 193. Raven Press, New York, 1989.
METHODSIN ENZYMOLOGY,VOL. 187
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