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Anal. Chem. 1984, 56, 1862-1865
Determination of Leukotriene C, by Radioimmunoassay with a Specific Antiserum Generated from a Synthetic Hapten Mimic M. A. Wynalda, J. R. Brashler, M. K. Bach, D. R. Morton, and F. A. Fitzpatrick* Pharmaceutical Research and Development, The Upjohn Company, 301 Henrietta Street, Kalamazoo, Michigan 49001
Rabblts were Inoculated with an lmmunogenlc conjugate composed of 7-cis-hexahydroieukotriene C, [(5S,6R)-Shydroxyl-&( S-glutathlonyi)-7-cls-eicosenolc acld] attached to the protein, keyhole limpet hemocyanln, vla the bifunctional coupllng reagent 6-N-maleknldohexandc acM chloride. The antiserum generated with this synthetlc hapten mimic was used to develop a radiolmrnunoassay for naturally occurrlng authentlc leukotriene Cq.At a final antiserum dilution of 1:lOO detedlon of 100 pg was possible. For the range 0.10-7.5 ng of leukotriene C,, the lntraassay variation was 1 1 to 1 1 2 % ( n = 3) and the interassay variation was f3 to 1 1 1 % ( n = 5). The antiserum bound leukotriene C4 with high spectficlty. Cross reactlon for 23 structurally relevant metabolltes was between 0.001 and 0.5%; cross reactlon for leukotrlenes D, and E, was 1.6 and 0.06%, respectively. Accuracy and speclflclty were established by comparatlve hlgkperformance llquld chromatographic analysis for selected samples.
The leukotrienes (LT) are a recently discovered class of biologically active lipids that originate from the oxidative metabolism of arachidonic acid and related polyunsaturated fatty acids (1-4). Among this class of substances, leukotriene C4 (LTC,) is notable as a biosynthetic precursor of other active leukotriene metabolites (5-7). Clarification of its role in allergic, bronchial, and inflammatory disorders requires accurate and reliable quantitative methods. Available methods for LTC4 determinations are deficient in some respects. For example, as a chemical constituent of the so-called slow-reacting substance of anaphylaxis (SRS-A) sensitive bioassay is possible (8). However, the bioassay, based on contraction of the guinea pig ileum, is nonspecific and it cannot distinguish among leukotriene C4 (LTC4) and its metabolites leukotriene D4 (LTD4) and leukotriene E4 (LTE4). Reversed-phase high-performance liquid chromatography (RP-HPLC) separation of individual leukotrienes offers improved assay specificity (9, IO), but conventional optical detection methods me insufficiently sensitive for some applications. Furthermore, the peptide-lipid attributes of LTC4 create unique problems for HPLC procedures (11). A few radioimmunoassays with subnanogram sensitivity for LTC4 have been reported; however, the specificity provisionally inherent in immunochemical methods has not been realized in practice (12-15). Specificity among these LTC, radioimmunoassays appears inadequate to permit its direct determination in samples containing mixtures of LTC4, LTD,, LTE,, and their 11-trans isomers. Analogous to the bioassay these radioimmunoassays cannot distinguish LTC4 from its metabolites without prior chromatographic purification. The origin of the poor specificity in LTC4 radioimmunoassays is uncertain. However, it occurred in every instance where antisera were obtained from immunogenic conjugates of naturally occurring leukotrienes, despite differences among the reagents and conditions used to attach the hapten to an immunogen (12-16). Precedents from the development of other eicosanoid radioimmunoassays suggested that chemical or metabolic instability of the conjugated hapten might cause 0003-2700/S4/0356-1862$01.50/0
poor antisera specificity (17,18). Substitution of a structurally related, synthetic hapten that chemically mimics the hapten of interest has previously solved such problems (19, 20). Consequently, we used this tactic to develop a radioimmunoassay for LTC,. The structural analogue, 7 4 s 9,10,11,12,14,15-hexahydroleukotriene C4was used as a hapten to produce an antiserum that recognized and bound authentic LTCb A sensitive, specific radioimmunoassay was developed with this antiserum.
EXPERIMENTAL SECTION Materials. Leukotriene A4 lithium salt [LTA4Lior (5S)-5,6oxido-7,9-trans-ll,14-cis-eicosatetraenoic acid lithium salt] (21), leukotriene B4 [LTB, or (5S712R)-5,12-dihydroxy-6,14-cis-8,10trans-eicosatetraenoic acid] (22), 6-trans-Ieukotriene B4 [6trans-LTB4or (5S,12R)-5,12-dihydroxy-6,8,10-trans-14-cis-eicosatetraenoic acid] (22), 6-trans-12-epi-leukotriene B4 [6-trans12-epi-LTB, or (5S,12S)-5,12-dihydroxy-6,8,lO-trans-14-cis-eicosatetraenoic acid] (22),(5S,12S)-DIHETE [ (5S,12S)-5,12-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid] (23),leukotriene C4 [LTC, or (5S,6R)-5-hydroxy-6-(S-glutathionyl)-7,9-trans11,14-cis-eicosatetraenoicacid] (24), leukotriene D4 [LTD4 or (5S,6R)-5-hydroxy-6-(glycyl-S-cysteinyl)-7,9-trans-ll,l4-cis-eicosatetraenoic acid] (25), leukotriene E4 [LTE, or (5S,6R)-5hydroxy-6-(S-cysteinyl)-7,9-trans-ll,14-cis-eicosatetraenoic acid] (26), and their corresponding 11-trans isomers and 7-cis9,10,11,12,14,15-hexahydroleukotrieneC4 (27) were provided by the Experimental Science 1 unit at The Upjohn Company and were synthesized as described in the literature. Synthetic methods for the preparation of the various leukotrienes have been recently 40 Ci/mmol (New England reviewed (2, 4 ) . [14,1F~-~Hl-LTc, Nuclear, Boston, MA), keyhole limpet hemocyanin and Freund's complete adjuvant (Calbiochem, LaJolla, CA), acetonitrile and tetrahydrofuran, HPLC grade (Burdick and Jackson, Muskegon, MI), dextran T70 (Pharmacia, Piscataway, NJ), and Norit A charcoal (Sigma, St. Louis, MO) were used as received. The coupling reagent 6-N-maleimidohexanoicacid chloride was freshly synthesized as described (16); (5RS)-5-hydroxy-6-trans-8,11,14cis-eicosatetraenoic acid (or B(RS)-HETE)was obtained by total synthesis (28), while (12S)-12-hydroxy-5,8,14-cis-l0-trans-eicosatetraenoic acid (or 12(S)-HETE) and (15S)-15-hydroxy5,8,11-cis-13-trans-eicosatetraenoic acid (or 15(S)-HETE)were prepared biosynthetically from arachidonic acid by using platelet and soybean lipoxygenases, respectively. Preparation of Immunogen. 7-cis-9,10,11,12,14,15-Hexahydroleukotriene C4 [7-cis-hexahydro-LTC, or (5S,6R)-5hydroxyl-6-(S-glutathionyl)-7-cis-eicosenoic acid] was attached to thiolated keyhole limpet hemocyanin with the bifunctional coupling reagent, 6-N-maleimidohexanoic acid chloride as described (16). Briefly, the hapten (5 mg), 0.5 MCi [14,15-3H]-LTC4, and triethylamine (80 pL) in anhydrous methanol (1.0 mL) were mixed with 6-N-maleimidohexanoic acid chloride (10 mg) in tetrahydrofuran (100 pL). Formation of a 6-N-maleimidohexanoic amide of hexahydro-7-cis-LTC4was >90% complete after stirring for 30 min at 25 OC. The reaction was monitored by high-performance liquid chromatography using a pBondapak C,, column eluted at 2.0 mL/min with 600/400 v/v 0.005 M tetrabutylammonium phosphate, pH 4/acetonitrile with UV detection at 220 nm. The starting material, 7-cis-hexahydro-LTC4eluted at 22 min under these conditions. The 6-N-maleimidohexanoicacid amide of the hapten was coupled to thiolated keyhole limpet hemocyanin (16). The immunogenic conjugate was isolated from low molecular weight reagents by ultrafiltration through an Amicon YMT membrane, with a molecular weight cutoff of 10000. 0 1984 Amerlcan Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
The epitope density (moles of hapten/mole protein) was estimated by monitoring the incorporation of [14,15-3H]-LTC4into the protein. Assuming equivalent reaction efficiencies for [3H]-LTC4 and the hapten, there were 10-15 mol of 7-cis-hexahydro-LTC4 attached per mol of keyhole limpet hemocyanin. The product (2 mg) was emulsified in sterile water (1.0 mL) and Freund's complete adjuvant (1.0 mL). Two albino New Zealand rabbits (4 kg) were inoculated intradermally with a portion of emulsion containing 1.0 mg of immunogen. After 45 days, the entire procedure was repeated and animals received a booster inoculation with 0.5 mg of immunogen. Blood was collected from the central ear artery biweekly after the latter inoculation. Application: Quantitation of LTC4 Biosynthesis and Metabolism. The radioimmunoassay was used to quantitate biosynthesis of LTC4by rat peritoneal macrophages. This system affords a rigorous evaluation of the assay sensitivity and selectivity because formation of subnanomole amounts of LTC4 is accompanied by its subsequent metabolism into related, potentially interferingleukotrienes. Briefly, rat peritoneal macrophages were isolated and purified as described (29). Leukotriene biosynthesis was initiated by addition of 0.2 M cysteine (50 pL) and calcium ionophore, A-23187 (1pg), to cells (7.5 X lo6 mL) suspended in Tyrode's buffer containing 1.8 mM Ca2+. After centrifugation to remove cells LTCI in the supernatant was determined by radioimmunoassay as described below. For certain samples LTC4 was quantitated by high-performance liquid chromatography for correlation with the radioimmunoassay results. Radioimmunoassay for LTCI. Antiserum, radiolabeled ligands, standards, and other reagents were all prepared in 0.10 M, pH 7.4 potassium phosphate buffer containing 0.9% w/v NaCl and 0.1% w/v NaNP Competitive binding equilibrium was established in a total assay volume of 0.18 mL at an antiserum dilution of 1:lOO. Samples (0.10 mL) or standards (0.10 mL) containing 0-10 ng of LTC4 and [ 14,15-3H]-LTC4(0.05 mL, 3000 dpm = 0.02 ng) were added to 12 X 75 mm tubes with LTCl antiserum (0.03 mL) diluted 1/18 v/v. Samples were mixed and centrifuged (200g)for 5 min. After incubation for 48 h at 4 "C unbound LTC4 was separated from antibody bound LTC4 by sequestration to dextran coated charcoal (30)'A suspension (500 mL) of Norit A charcoal (5 mg/mL) and dextran T70 (0.50 mg/mL) in 0.10 M, pH 7.4 potassium phospbte buffer was chilled to 0 "C. Assay tubes were also chilled for 30 min in an ice bath (0 "C). Charcoal suspension (1.0 mL) was added to each tube. After incubation for exactly 10.0 f 0.5 min, the tubes were centrifuged (100Og) for 10 min at 4 "C to sediment the charcoal. A portion (0.90 mL) of the supernatant from each tube was transferred to a scintillationvial. ACS scintillation fluid (10 mL) was added and the amount of antibody bound [3H]-LTC4in the supernatant was determined by @ counting for tritium. Data were calculated on a Hewlett-Packard 9830A computer using a logit transformation to delineate the relationship between the mass of LTC4 and the proportion of antibody-bound 13H]-LTC4. Standard curves were calculated as described (31). The amount (dpm) of antibody-bound (B) [3H]-LTC4was normalized relative to the amount bound (BO) in the absence of any competing LTC4 ligand. Comparative Determination of LTC, by Reversed-Phase High-Performance Liquid Chromatography. LTC4 in representative samples was determined by RP-HPLC for comparison with RIA results. For the RP-HPLC determinations a portion (0.05 mL) of supernatant from cell incubations was evaporated to dryness under nitrogen. The residue was reconstitpted in mobile phase (0.10 mL) and injected onto an IBV C18 column (5 pm, 250 X 6 X 4 mm) eluted (1.0 mL/min) with 0.005 M, pH 4 tetrabutylammonium phosphate/acetonitrile/tetrahydrofuran 600/400/1 v/v/v. The eluent was monitored at both 220 and 280 nm. Quantitation was based on the UV response at 280 nm, 0.01 absorbance unit. LTC4,LTD4, and LTE4 eluted with capacity factors (k') 13.0, 7.0, and 9.0, respectively. This procedure is an adaptation of that developed by Mathews et al. (9). The ion-pair mobile phase reduces peak distortion and adsorption effects reported for the original version (11).
RESULTS Within 3 months after inoculations with conjugates of 7-
OF
CALIBRATION CURVE: COMPETITIVE INHIBITION LTC4 BINDING AT 1:lOO ANTISERUM DILUTION
C~HJ
0 0
Y
2 Z
60
1863
Mean f SD (n=3) intra-assay Mean f SD (n=5) inter-assay a t weekly intervals
-
3
50%
0
rn
2
40-
2 3
20 -
c
1
0
0 .I
r
0.76 ng I
I
1
4
I
I , I ,
0.5
1
I
I
I I , ,
I
1.0
5.0
10.0
LTC4 (ng/tube)
Figure 1. Competitive inhibition of [3H]-LTC4bound to an antiserum produced by inoculation with an immunogenic conjugate of hexa-
hydro-74s -LTC4 bound to keyhole limpet hemocyanin. cis-hexahydro-LTC4 coupled to keyhole limpet hemocyanin, rabbit antiserum, at a 1:lOO dilution, bound 50 f 5% of [ 14,15-3H]-LTC4in the absence of competitive binding by unlabeled LTCI. Nonspecific binding was 6 f 1% Specific binding was inversely proportional to the antiserum dilution. At dilutions of 1:200,1:300, and 1:400 the antiserum bound 33 f 7%, 28 f 6%, and 22 f 4%, respectively, of the radiolabeled ligand. These values represent the mean f standard deviation of seven individual bleeding8 taken a t weekly intervals. Increasing amounts of LTC4 inhibited the binding of .114,15-3H]-LTC4in a competitive manner. Typical Calibration curves (Figure 1) indicated that the interassay reproducibility (n = 5 ) for experiments conducted at weekly intervals corresponded closely to the intraassay reproducibility (n = 3). The coeffiuents of variation in the'former case ranged from f 3 % to A l l % , and in the latter case from f l % to &12% for 0.10 to 7.5 ng of LTCI. The sensitivity of the assay, equivalent to the mass of LTC4 that reduced the binding of radiolabeled ligand from 100% to 85% bound/boundo, was 100 pg. Logit transformations of the calibration curves [bound/bound,, vs. logarithm LTC4 (ng)] were linear between 0.10 and 10.0 ng with correlation coefficients 20.99. The recovery of LTC4 added to platelet-rich human plasma was 95%. Twenty five compounds structurally and metabolically relevant to LTC4 analyses were evaluated as potential interferences. The ability of these heterologous ligands to displace antibody-bound [ 14,15-3H]-LTC4was inferior to that of the analyte, LTC4. The percentage of cross reaction (100 X ng of LTC4/ng of heterologous ligand) at 50% bound/ boundo was below 0.2% for 21 of these compounds. Six compounds, including the metabolite LTD4 and its 11trans-isomer, had cross reactions between 0.1% and 2 % . Cross-reaction was predictably higher, 6.1%, for the 11-trans isomer of LTC4. However, even this value indicates that the antiserum retains significant (16-fold) preference for the naturally occurring 11-cis isomer (Table I). Possible interference by the conjugate material is unlikely because hemocyanin, a protein from a marine organism, would seldom occur in samples commonly used to study leukotriene biosynthesis. Furthermore at concentrations ranging from 0.1 to 10 mg/mL hemocyapin did not displace [14,15-3H]-LTC4from the antiserum. Figure 2 depicts the cellular biosynthesis and metabolism of LTC, by rat peritoneal macrophages as a function of time,
.
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 11, SEPTEMBER 1984
Table I. Cross-Reaction of Heterologous Ligands with Antiserum That Binds Leukotriene C4 % cross reaction
compd
LTC4 11-trans-LTC, LTD4 11-trans-LTD, LTE4 11-trans-LTE, LTB4 6-trans-LTB4 6-trans-12-epi-LTB4 20-hydroxyl-LTB4 5(S),12(S)-DIHETEa 5(RS)-HETE 12(S)-HETE 15(S)-HETE 5(S),15(S)-DiHETE 8(R),15(S)-DiHETE 8(S),15(S)-DiHETE 5,6,15-TRI-HETE 11-glutathionyl-PGA, 14J5-DiHETE glutathione L-cysteine PGEz TxBz PGFZ. PGDz
sample
100.0 6.1 1.6
0.4 0.06 0.07
0.004 0.01 0.01
