82
ASSAYS
[10]
[10] E n z y m e I m m u n o a s s a y s for L e u k o t r i e n e s C4 a n d E 4 Using Acetylcholinesterase B y PHILIPPE PRADELLES, CATHERINE A N T O I N E , J E A N - P A U L L E L L O U C H E , and JACQUESM A C L O U F
Although quantitation of sulfidopeptide leukotrienes (LTs) can be achieved by the use of conventional bioassay, or gas chromatographymass spectrometry after catalytic desulfurization, 1 radioimmunoassay analyses have turned out to be the most convenient method to measure LTs. However, the generation of antisera against LTC4, LTD4, and LTE4 with sensitivities adapted to biological problems has remained difficult. This is due to several reasons including limitations in availability of synthetic material necessary to generate antibodies as well as the availability of 3H tracers with specific radioactivity limited to approximately 2.22 TBq/mmol. This latter feature limits radioimmunoassay sensitivity (IC50) to 0.3 pmol which should be able to be improved upon. 2-6 We have successfully developed acetylcholinesterase from Electrophorus electricus as a label for various eicosanoids, thereby providing an enzyme immunoassay with sensitivities equal to or superior to those achieved with ~25I radioactive tracers. 7'8 We have undertaken a similar approach for LTC4 and LTE4. However, because of specific problems inherent with these molecules, we have combined a dual strategy to prepare the protein-LT conjugates necessary for the generation of antibodies and corresponding enzyme tracers. We will describe the development of such assays below.
I M. Balazy and R. C. Murphy, Anal. Chem. 58, 1098 (1986). 2 L. Levine, R. A. Morgan, R. A. Lewis, K. F. Austen, D. A. Clark, A. Marfat, and E. J. Corey, Proc. Natl. Acad. Sci. U.S.A. 78, 7692 (1981). 3 E. C. Hayes, D. L. Lombardo, Y. Girard, A. L. Maycock, J. Rokach, A. L. Rosenthal, R. N. Young, R. W. Egan, and H. J. Zweering, J. Immunol. Methods 131, 429 (1983). 4 M. A. Wynalda, J. R. Brashler, M. K. Bach, D. R. Morton, and F. A. Fitzpatrick, Anal. Chem. 56, 1852 (1984). W. Aehringhaus, R. Wolbling, W. Konig, C. Patrono, B. M. Peskar, and B. A. Beskar, FEBS Lett. 146, 114 (1982). 6 j. Lindgren, S. Hammarstrom,and E. Goetzl, FEBS Lett. 152, 83 (1983). 7 p. Pradelles, J. Grassi, and J. Maclouf, Anal. Chem. 57, 1170 (1985). 8 p. PradeUes, J. Grass/, and J. Malouf, this volume, [31.
METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress, Inc. Allrightsof reproductionin any formreserved.
[10]
IMMUNOASSAY OF LEUKOTRIENES C 4 AND E4
83
Assay Procedure Reagents and Materials
Glutaraldehyde (Merck Darmstadt, FRG) N , N ' - D i c y c l o h e x y l c a r b o d i i m i d e (DCC), N-hydroxysuccinimide ester (NHS), S-acetylmercaptosuccinic anhydride (Sigma Chemicals, St. Louis, MO), succinimidyl-4-(N-maleimidomethyl)cyclohexane 1carboxylate (SMCC) (Pierce Chemicals, Rockford, IL) N-Succinimidyl-S-acetylthioacetate (SATA) (Calbiochem, San Diego, CA) Hydroxylamine-HC1 (Sigma) Dimethylformamide and methanol (Merck) kept anhydrous with molecular sieve Bovine serum albumin fraction V (Sigma) Buffers. Two buffers will be used for the couplings throughout the experiments and will be referred to as phosphate buffer (0.1 M potassium phosphate buffer, pH 7.4) and borate buffer (0.1 M borate buffer, pH 9). Enzyme. Acetylcholinesterase is prepared by a one-step affinity chromatography as described by Massouli6 and Bon. 9 The tetrameric form of the enzyme (G4 form) is obtained from the crude purified preparation by incubation with trypsin; its maleimidated form is prepared as described by McLaughlin et al. ~o LTC4 and LTE4 are synthesized as described by Corey et al. ~ and Rokach e t al. ~2 Gel filtration equipment. BioGel A15-m column (90 × 1.5 cm) (Bio-Rad, Richmond, CA); GF 0.5 column (20 × 1 cm) (IBF, France) NOTE. All the reagents, equipment, and other technical details necessary for the enzyme immunoassay are described in [3] of this volume. Coupling Procedures Production of Antisera
Peptido-LTs haptens require coupling to an antigenic molecule in order to elicit antibodies. Because LTs possess both amino and carboxylic 9j. Massouli6and S. Bon, Eur. J. Biochem. 68, 531 (1976). ~oL. Mc Laughlin, Y. Wei, P. T. Stockman, K. M. Leahy, P. Needleman, J. Grassi, and P. Pradelles, Biochem. Biophys. Res. Commun. 144, 469 (1987). H E. J. Corey, D. A. Clark, G. Goto, A. Marfat, C. Miokowski, B. Samuelsson, and S. Hammarstrfm, J. Am. Chem. Soc. 102, 1436(1980). ~zj. Rokach, Y. Girard, Y. Guindon, J. G. Atkinson, M. Larue, R. N. Young, P. Masson, and G. Holme, Tetrahedron Lett., p. 1485(1980).
84
ASSAYS
[10]
functions to which they can be attached to the carrier, the final selection of the method would depend on two important criteria: the uniqueness of the function carried on the LT (there is only I amino group/molecule of LT and 3 or 2 carboxylic groups for either LTC4 or LTE4) and the final yield of the coupling. In addition, we use bovine serum albumin as the carrier throughout these experiments. All tests are performed on LTE4 and the general approach extrapolated to LTC4. Coupling of LTE4 by Its Carboxylic Groups. LTE4 (25 nmol) is reacted overnight at room temperature in the dark with 250 nmol of NHS and 25 nmol of DCC in 60/~1 of dimethylformamide. The ester is then reacted with 2 nmol of bovine serum albumin in solution in 300/zl borate buffer. Coupling of LTE4 by Its Amino Group. LTE4 (25 nmol) and bovine serum albumin (2 nmol) in solution in 100/.d phosphate buffer, are reacted overnight at room temperature in the dark with 100/zl of a 0.12% glutaraldehyde solution. Coupling of LTE4 to Thiolated Albumin. Bovine serum albumin (0.83/.~mol) in 2 ml of borate buffer is reacted with 40 mmol of S-acetylmercaptosuccinic anhydride during 30 min at room temperature. The ester is then hydrolyzed using 3 ml of a I M hydroxylamine solution, pH 7.2, overnight at room temperature and the modified SH-albumin purified by gel filtration on a Sephadex G-25 (Pharmacia) column (1 × 30 cm). Colorimetric analysis at 414 nm reveals that approximately 7 thiols are present per molecule albumin. LTE4 (25 nmol), in 100/zl of phosphate buffer is mixed with 25 nmol of SMCC (in 5 p~ldimethylformamide). After 30 min at room temperature, thiolated albumin (3 nmol) is then added in 500/zl borate buffer for overnight reaction. The three conjugates are purified by gel filtration (GF 0.5 column 20 x 1 cm) to allow separation of free LTE4 from LT covalently linked to albumin. All fractions are measured for their immunoreactivity using an antiserum and enzyme tracer previously prepared (P. Pradelles and J. Maclouf, unpublished datal2a). Calculation of the percentage of bound LTE4 vs free showed that the yield of coupling is most efficient using glutaraldehyde (>86%) (Fig. 1), contrasting with less than 0.1% reaction by the carboxylic functions of the LT and undetectable reaction for the thiolated albumin. Because of the limited amount of our LTs supply, we did not investigate further these unsuccessful coupling approaches. Glutaraldehyde is thus selected for both LTC4 and LTE4 starting from 3.2 and 12a In preliminary experiments, a small amount of an immunogen of LTE4 (kindly provided by Drs. Rokach and Young, Merck Frosst, Montreal, Canada) allowed us to generate enough antiserum and corresponding immunoreactive tracer to perform the initial tests of immunoreactivity described in Fig. 1.
[10]
I M M U N O A S S AOF Y LEUKOTRIENES C4 AND E4
85
700 r
6oo t 500 [ 4-00 t 300 t
] t ~
200 t O L , . .
tO
,
free LTE4
-
20
30
40
50
fractions .-~
120 100
cq°¢ ~-~
60 40 2o
o. 10
30
.......
40
fractions Flo. 1. Purification of the albumin conjugates of LTE4 by gel filtration. Elution is performed using 0.1 M phosphate buffer, pH 7.4, containing 0.4 M NaC1, 0.01 M EDTA, bovine serum albumin 0.1% as described in the text. Top panel represents the immunoreactire LTE4 as a function of the fractions collected during the purification. Bottom panel represents the absorbance at 280 nm of the same fractions. (A. U., absorbance units.)
4.5/~mol of LT, respectively, using the ratio of the different reagents in the coupling process as previously found successful. Immunization. Immunization of rabbits followed the procedure described by Va'ftukaitis. 13The LT-conjugate (1 mg in 1 ml water) is emulsified with 1 ml of Freund's complete adjuvant (Difco, Detroit, MI) and injected subcutaneously to three rabbits. Six weeks later, the first booster is performed using the same dose of conjugate; rabbits are bled on a weekly basis. At each bleeding, the serum is analyzed for titer and sensitivity. When the titer drops, a new booster is performed and the same follow-up protocol used. Antisera are kept at 4 ° after addition of sodium azide (0.02% final).
Preparation of Tracers LTC4 and LTE4 can be coupled to the AChE taking advantage of the same structural moiety as that used to generate the immunogen. However, it is critical to assess whether the enzyme activity is impaired during the 13j. VaRukaitis, J. B. Robbins, and T. Ross, J. Clin. Endocrinol. 33, 988 (1971).
86
ASSAYS
[ 10]
coupling procedure (e.g., homo-bifunctional reagents such as glutaraldehyde polymerize the enzyme and may lead to a complete loss of activity). In addition, it is our experience that the coupling reagent should be different for the immunogen and for the tracer in order to minimize any undesirable recognition of the coupling moiety (i.e., spacer molecule) on the label by the antibodies. Therefore distinct methods of coupling are used for the preparation of immunogen and tracer. Finally, the stoichiometry of the reaction of the enzyme with the LT is ideally 1/1 in order to achieve the highest specific activity of the tracer and hence maximize sensitivity.
Coupling of LTE4 by Its Carboxylic Groups to Amino Groups of Enzyme. This reaction involves a first-stage formation of an activated ester (500 nmol of NHS and 50 nmol DCC in 20/zl are reacted with 50 nmol of LTE4 in 20/xl dimethylformamide). After 30 min at room temperature, 0.08 nmol of the G4 form is added in 300/zl of borate buffer for 1 hr at room temperature. Coupling of Thiolated LTE4 to Maleimidated Enzyme. LTE4 (8.5 nmol in 100/zl of borate buffer) is added to 17 nmol of SATA in 10/zl dimethylformamide and incubated at room temperature for 30 min. The resulting LT-thioester is hydrolyzed using 100/.d of 1 M hydroxylamine, pH 7, for 30 min and then added to the maleimidated G4 form (0.28 nmol in 800/xl 0.1 M phosphate buffer, pH 6, containing 5 mM EDTA). Coupling of LTE4 to Enzyme Using SMCC. LTE4 (100 nmol in 100 p.l phosphate buffer is added to 100 nmol of SMCC in 10/zl dimethylforo mamide during 30 min at room temperature in the dark. The G4 form (0.3 nmol in 500 t~l borate buffer is subsequently reacted overnight at room temperature. Each of these tracers is then purified by gel filtration on a BioGel A 15m column. For each of the conjugates, the fractions corresponding to the enzyme activity are pooled and their capacity to bind the antibodies is further tested as well as displacement of bound activity by unlabeled LTs. From these data, the coupling using SMCC is equivalent to the thiolated LTE4 method. However, the first method is much simpler to perform and is subsequently retained to prepare tracers. We could not obtain any results with the coupling by the carboxylic moieties of LTE4. However, as for the immunogen, insufficient material (i.e., LTE4) prevented further investigation of this approach.
Generation of Enzyme Immunoassay for Leukotrienes C4 and E4 Since the protocol of enzyme immunoassay for peptido-LTs as well as its practical aspects (materials, reagents, incubation) are identical to other
[10]
IMMUNOASSAY OF LEUKOTRIENES C4 AND
E4
87
0.75
0.50
-~ # 611 -.- # 81z + # 61a
A.U.
-~ B o o s t e r
0,25
0.00
.
.
2*3
.
.
4
"
5~r6
.
7
.
.
8
.
.
9"10
11
12
Time course of bleedings FIG. 2. Evolution of the titer of three different rabbits immunizedagainst LTE4-BSA as a function of the time course of immunization. The box indicates the rabbit number corresponding to each curve and the arrows the various boosters of immunizations. The numbers on the abscissa (1-12) represent the various bleedings performed after the first booster.
eicosanoids, the reader should consult chapter [3] of this volume for corresponding details.
Time-Course Evolution of Titer Figure 2 shows the absorbance units o f bound tracer ( L T E 4 acetylcholinesterase) for three different antisera used at 1/15,000 (final dilution) during the course o f immunization. For this experiment, incubation followed the protocol described for the titration o f antiserum ([3] in this volume). The absorbance in Fig. 2 represents the solid-phase bound e n z y m e activity and, as expected, the titer increased after each booster.
Sensitivity and Optimization Various procedures allow one to maximize sensitivity, 14 among these,
preincubation (i.e., incubation of antibody in the presence of the antigen and delayed addition o f labeled antigen) sometimes improves the sensitivity of various immunoassays. This procedure significantly improves the sensitivity o f the LTE4 system (i.e., IC5o = 10 pg for 1 hr preincubation as compared to 50 pg for the control curve) (Fig. 3). Similarly, the sensitivity z4 G. Ciabattoni, in "Radioimmunoassay in Basic and Clinical Pharmacology" (C. Patrono and B. A. Peskar, eds.), p. 181. Springer-Verlag, New York, 1987.
88
ASSAYS
[10]
90 80
-~ Control -A- tHr
70 6O
~ ~
O
50 40 30 20
to ! 0
1
,
I
a
I
. . . .
"
.
.
.
.
.
I0
.
.
.
"
100
.
.
.
.
.
.
.
.
"
I000
Pg FIO. 3. Effect of preincubation on the dose-response curve of LTE4. The anti-LTE4 is incubated 18 hr at 4° with the standard; the tracer is then added and incubated as indicated in the box. Separation of bound from free and enzymatic reaction are carried out as previously described ([3] elsewhere in this volume). The control curve is generated following the standard protocol in [3].
100
tO0
A
90
B ~ ~
90
LTB4
80
ASSAYS
[10]
[10] E n z y m e I m m u n o a s s a y s for L e u k o t r i e n e s C4 a n d E 4 Using Acetylcholinesterase B y PHILIPPE PRADELLES, CATHERINE A N T O I N E , J E A N - P A U L L E L L O U C H E , and JACQUESM A C L O U F
Although quantitation of sulfidopeptide leukotrienes (LTs) can be achieved by the use of conventional bioassay, or gas chromatographymass spectrometry after catalytic desulfurization, 1 radioimmunoassay analyses have turned out to be the most convenient method to measure LTs. However, the generation of antisera against LTC4, LTD4, and LTE4 with sensitivities adapted to biological problems has remained difficult. This is due to several reasons including limitations in availability of synthetic material necessary to generate antibodies as well as the availability of 3H tracers with specific radioactivity limited to approximately 2.22 TBq/mmol. This latter feature limits radioimmunoassay sensitivity (IC50) to 0.3 pmol which should be able to be improved upon. 2-6 We have successfully developed acetylcholinesterase from Electrophorus electricus as a label for various eicosanoids, thereby providing an enzyme immunoassay with sensitivities equal to or superior to those achieved with ~25I radioactive tracers. 7'8 We have undertaken a similar approach for LTC4 and LTE4. However, because of specific problems inherent with these molecules, we have combined a dual strategy to prepare the protein-LT conjugates necessary for the generation of antibodies and corresponding enzyme tracers. We will describe the development of such assays below.
I M. Balazy and R. C. Murphy, Anal. Chem. 58, 1098 (1986). 2 L. Levine, R. A. Morgan, R. A. Lewis, K. F. Austen, D. A. Clark, A. Marfat, and E. J. Corey, Proc. Natl. Acad. Sci. U.S.A. 78, 7692 (1981). 3 E. C. Hayes, D. L. Lombardo, Y. Girard, A. L. Maycock, J. Rokach, A. L. Rosenthal, R. N. Young, R. W. Egan, and H. J. Zweering, J. Immunol. Methods 131, 429 (1983). 4 M. A. Wynalda, J. R. Brashler, M. K. Bach, D. R. Morton, and F. A. Fitzpatrick, Anal. Chem. 56, 1852 (1984). W. Aehringhaus, R. Wolbling, W. Konig, C. Patrono, B. M. Peskar, and B. A. Beskar, FEBS Lett. 146, 114 (1982). 6 j. Lindgren, S. Hammarstrom,and E. Goetzl, FEBS Lett. 152, 83 (1983). 7 p. Pradelles, J. Grassi, and J. Maclouf, Anal. Chem. 57, 1170 (1985). 8 p. PradeUes, J. Grass/, and J. Malouf, this volume, [31.
METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress, Inc. Allrightsof reproductionin any formreserved.
[10]
IMMUNOASSAY OF LEUKOTRIENES C 4 AND E4
83
Assay Procedure Reagents and Materials
Glutaraldehyde (Merck Darmstadt, FRG) N , N ' - D i c y c l o h e x y l c a r b o d i i m i d e (DCC), N-hydroxysuccinimide ester (NHS), S-acetylmercaptosuccinic anhydride (Sigma Chemicals, St. Louis, MO), succinimidyl-4-(N-maleimidomethyl)cyclohexane 1carboxylate (SMCC) (Pierce Chemicals, Rockford, IL) N-Succinimidyl-S-acetylthioacetate (SATA) (Calbiochem, San Diego, CA) Hydroxylamine-HC1 (Sigma) Dimethylformamide and methanol (Merck) kept anhydrous with molecular sieve Bovine serum albumin fraction V (Sigma) Buffers. Two buffers will be used for the couplings throughout the experiments and will be referred to as phosphate buffer (0.1 M potassium phosphate buffer, pH 7.4) and borate buffer (0.1 M borate buffer, pH 9). Enzyme. Acetylcholinesterase is prepared by a one-step affinity chromatography as described by Massouli6 and Bon. 9 The tetrameric form of the enzyme (G4 form) is obtained from the crude purified preparation by incubation with trypsin; its maleimidated form is prepared as described by McLaughlin et al. ~o LTC4 and LTE4 are synthesized as described by Corey et al. ~ and Rokach e t al. ~2 Gel filtration equipment. BioGel A15-m column (90 × 1.5 cm) (Bio-Rad, Richmond, CA); GF 0.5 column (20 × 1 cm) (IBF, France) NOTE. All the reagents, equipment, and other technical details necessary for the enzyme immunoassay are described in [3] of this volume. Coupling Procedures Production of Antisera
Peptido-LTs haptens require coupling to an antigenic molecule in order to elicit antibodies. Because LTs possess both amino and carboxylic 9j. Massouli6and S. Bon, Eur. J. Biochem. 68, 531 (1976). ~oL. Mc Laughlin, Y. Wei, P. T. Stockman, K. M. Leahy, P. Needleman, J. Grassi, and P. Pradelles, Biochem. Biophys. Res. Commun. 144, 469 (1987). H E. J. Corey, D. A. Clark, G. Goto, A. Marfat, C. Miokowski, B. Samuelsson, and S. Hammarstrfm, J. Am. Chem. Soc. 102, 1436(1980). ~zj. Rokach, Y. Girard, Y. Guindon, J. G. Atkinson, M. Larue, R. N. Young, P. Masson, and G. Holme, Tetrahedron Lett., p. 1485(1980).
84
ASSAYS
[10]
functions to which they can be attached to the carrier, the final selection of the method would depend on two important criteria: the uniqueness of the function carried on the LT (there is only I amino group/molecule of LT and 3 or 2 carboxylic groups for either LTC4 or LTE4) and the final yield of the coupling. In addition, we use bovine serum albumin as the carrier throughout these experiments. All tests are performed on LTE4 and the general approach extrapolated to LTC4. Coupling of LTE4 by Its Carboxylic Groups. LTE4 (25 nmol) is reacted overnight at room temperature in the dark with 250 nmol of NHS and 25 nmol of DCC in 60/~1 of dimethylformamide. The ester is then reacted with 2 nmol of bovine serum albumin in solution in 300/zl borate buffer. Coupling of LTE4 by Its Amino Group. LTE4 (25 nmol) and bovine serum albumin (2 nmol) in solution in 100/.d phosphate buffer, are reacted overnight at room temperature in the dark with 100/zl of a 0.12% glutaraldehyde solution. Coupling of LTE4 to Thiolated Albumin. Bovine serum albumin (0.83/.~mol) in 2 ml of borate buffer is reacted with 40 mmol of S-acetylmercaptosuccinic anhydride during 30 min at room temperature. The ester is then hydrolyzed using 3 ml of a I M hydroxylamine solution, pH 7.2, overnight at room temperature and the modified SH-albumin purified by gel filtration on a Sephadex G-25 (Pharmacia) column (1 × 30 cm). Colorimetric analysis at 414 nm reveals that approximately 7 thiols are present per molecule albumin. LTE4 (25 nmol), in 100/zl of phosphate buffer is mixed with 25 nmol of SMCC (in 5 p~ldimethylformamide). After 30 min at room temperature, thiolated albumin (3 nmol) is then added in 500/zl borate buffer for overnight reaction. The three conjugates are purified by gel filtration (GF 0.5 column 20 x 1 cm) to allow separation of free LTE4 from LT covalently linked to albumin. All fractions are measured for their immunoreactivity using an antiserum and enzyme tracer previously prepared (P. Pradelles and J. Maclouf, unpublished datal2a). Calculation of the percentage of bound LTE4 vs free showed that the yield of coupling is most efficient using glutaraldehyde (>86%) (Fig. 1), contrasting with less than 0.1% reaction by the carboxylic functions of the LT and undetectable reaction for the thiolated albumin. Because of the limited amount of our LTs supply, we did not investigate further these unsuccessful coupling approaches. Glutaraldehyde is thus selected for both LTC4 and LTE4 starting from 3.2 and 12a In preliminary experiments, a small amount of an immunogen of LTE4 (kindly provided by Drs. Rokach and Young, Merck Frosst, Montreal, Canada) allowed us to generate enough antiserum and corresponding immunoreactive tracer to perform the initial tests of immunoreactivity described in Fig. 1.
[10]
I M M U N O A S S AOF Y LEUKOTRIENES C4 AND E4
85
700 r
6oo t 500 [ 4-00 t 300 t
] t ~
200 t O L , . .
tO
,
free LTE4
-
20
30
40
50
fractions .-~
120 100
cq°¢ ~-~
60 40 2o
o. 10
30
.......
40
fractions Flo. 1. Purification of the albumin conjugates of LTE4 by gel filtration. Elution is performed using 0.1 M phosphate buffer, pH 7.4, containing 0.4 M NaC1, 0.01 M EDTA, bovine serum albumin 0.1% as described in the text. Top panel represents the immunoreactire LTE4 as a function of the fractions collected during the purification. Bottom panel represents the absorbance at 280 nm of the same fractions. (A. U., absorbance units.)
4.5/~mol of LT, respectively, using the ratio of the different reagents in the coupling process as previously found successful. Immunization. Immunization of rabbits followed the procedure described by Va'ftukaitis. 13The LT-conjugate (1 mg in 1 ml water) is emulsified with 1 ml of Freund's complete adjuvant (Difco, Detroit, MI) and injected subcutaneously to three rabbits. Six weeks later, the first booster is performed using the same dose of conjugate; rabbits are bled on a weekly basis. At each bleeding, the serum is analyzed for titer and sensitivity. When the titer drops, a new booster is performed and the same follow-up protocol used. Antisera are kept at 4 ° after addition of sodium azide (0.02% final).
Preparation of Tracers LTC4 and LTE4 can be coupled to the AChE taking advantage of the same structural moiety as that used to generate the immunogen. However, it is critical to assess whether the enzyme activity is impaired during the 13j. VaRukaitis, J. B. Robbins, and T. Ross, J. Clin. Endocrinol. 33, 988 (1971).
86
ASSAYS
[ 10]
coupling procedure (e.g., homo-bifunctional reagents such as glutaraldehyde polymerize the enzyme and may lead to a complete loss of activity). In addition, it is our experience that the coupling reagent should be different for the immunogen and for the tracer in order to minimize any undesirable recognition of the coupling moiety (i.e., spacer molecule) on the label by the antibodies. Therefore distinct methods of coupling are used for the preparation of immunogen and tracer. Finally, the stoichiometry of the reaction of the enzyme with the LT is ideally 1/1 in order to achieve the highest specific activity of the tracer and hence maximize sensitivity.
Coupling of LTE4 by Its Carboxylic Groups to Amino Groups of Enzyme. This reaction involves a first-stage formation of an activated ester (500 nmol of NHS and 50 nmol DCC in 20/zl are reacted with 50 nmol of LTE4 in 20/xl dimethylformamide). After 30 min at room temperature, 0.08 nmol of the G4 form is added in 300/zl of borate buffer for 1 hr at room temperature. Coupling of Thiolated LTE4 to Maleimidated Enzyme. LTE4 (8.5 nmol in 100/zl of borate buffer) is added to 17 nmol of SATA in 10/zl dimethylformamide and incubated at room temperature for 30 min. The resulting LT-thioester is hydrolyzed using 100/.d of 1 M hydroxylamine, pH 7, for 30 min and then added to the maleimidated G4 form (0.28 nmol in 800/xl 0.1 M phosphate buffer, pH 6, containing 5 mM EDTA). Coupling of LTE4 to Enzyme Using SMCC. LTE4 (100 nmol in 100 p.l phosphate buffer is added to 100 nmol of SMCC in 10/zl dimethylforo mamide during 30 min at room temperature in the dark. The G4 form (0.3 nmol in 500 t~l borate buffer is subsequently reacted overnight at room temperature. Each of these tracers is then purified by gel filtration on a BioGel A 15m column. For each of the conjugates, the fractions corresponding to the enzyme activity are pooled and their capacity to bind the antibodies is further tested as well as displacement of bound activity by unlabeled LTs. From these data, the coupling using SMCC is equivalent to the thiolated LTE4 method. However, the first method is much simpler to perform and is subsequently retained to prepare tracers. We could not obtain any results with the coupling by the carboxylic moieties of LTE4. However, as for the immunogen, insufficient material (i.e., LTE4) prevented further investigation of this approach.
Generation of Enzyme Immunoassay for Leukotrienes C4 and E4 Since the protocol of enzyme immunoassay for peptido-LTs as well as its practical aspects (materials, reagents, incubation) are identical to other
[10]
IMMUNOASSAY OF LEUKOTRIENES C4 AND
E4
87
0.75
0.50
-~ # 611 -.- # 81z + # 61a
A.U.
-~ B o o s t e r
0,25
0.00
.
.
2*3
.
.
4
"
5~r6
.
7
.
.
8
.
.
9"10
11
12
Time course of bleedings FIG. 2. Evolution of the titer of three different rabbits immunizedagainst LTE4-BSA as a function of the time course of immunization. The box indicates the rabbit number corresponding to each curve and the arrows the various boosters of immunizations. The numbers on the abscissa (1-12) represent the various bleedings performed after the first booster.
eicosanoids, the reader should consult chapter [3] of this volume for corresponding details.
Time-Course Evolution of Titer Figure 2 shows the absorbance units o f bound tracer ( L T E 4 acetylcholinesterase) for three different antisera used at 1/15,000 (final dilution) during the course o f immunization. For this experiment, incubation followed the protocol described for the titration o f antiserum ([3] in this volume). The absorbance in Fig. 2 represents the solid-phase bound e n z y m e activity and, as expected, the titer increased after each booster.
Sensitivity and Optimization Various procedures allow one to maximize sensitivity, 14 among these,
preincubation (i.e., incubation of antibody in the presence of the antigen and delayed addition o f labeled antigen) sometimes improves the sensitivity of various immunoassays. This procedure significantly improves the sensitivity o f the LTE4 system (i.e., IC5o = 10 pg for 1 hr preincubation as compared to 50 pg for the control curve) (Fig. 3). Similarly, the sensitivity z4 G. Ciabattoni, in "Radioimmunoassay in Basic and Clinical Pharmacology" (C. Patrono and B. A. Peskar, eds.), p. 181. Springer-Verlag, New York, 1987.
88
ASSAYS
[10]
90 80
-~ Control -A- tHr
70 6O
~ ~
O
50 40 30 20
to ! 0
1
,
I
a
I
. . . .
"
.
.
.
.
.
I0
.
.
.
"
100
.
.
.
.
.
.
.
.
"
I000
Pg FIO. 3. Effect of preincubation on the dose-response curve of LTE4. The anti-LTE4 is incubated 18 hr at 4° with the standard; the tracer is then added and incubated as indicated in the box. Separation of bound from free and enzymatic reaction are carried out as previously described ([3] elsewhere in this volume). The control curve is generated following the standard protocol in [3].
100
tO0
A
90
B ~ ~
90
LTB4
80
