Development of a highly sensitive ELISA for the determination of PBAN and its application to the analysis of hemolymph in Spodoptera littoralis.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 30:369-381 (1995) Development of a Highly Sensitive ELISA for the Determination of PBAN and Its Application to the Analysis of Hemolymph in Spodoptera littoralis M.-Pilar Marco, Gemma Fabriis, and Francisco Camps Department of Biological Organic chemistry, C.1.D.-C.S.I.C., Barcelona, Spain A highly sensitive enzyme linked immunosorbent assay (ELISA) for the determination of the pheromone biosynthesis activating neuropeptide (PBAN) has been developed. Six antisera have been obtained that recognize the carboxyl terminal side of this peptide. Two immunogens have been rationally designed and synthesized in order to direct antibody specificity, using as haptens PBAN or PBAN(20-33) with a Cys residue attached to their amino-terminal side. The Cys thiol group has been used to covalently bind the peptide to keyhole limpet hemocyanin (KLH) by using N-succinimidyl-4-(rnaleidimidomethyl)cyclohexane carboxylate (SMCC) as a convenient heterobifunctional cross-linker. Several usable competitive immunoassays have been obtained by synthesizing eight different coating antigens and screening the sera against all of them. The best assay was obtained with antibody 4 using Cys-Hez-PBAN(20-33) coupled to bovine serum albumin (BSA) through the Lys groups by using the homobifunctional cross-linker dimethylpimelidate dihydrochloride (DMP) as the coating antigen. The optimized assay allows to detect PBAN at concentrations as l o w as 1 fmol/well (Ijo = 2.5 fmol/well). An extraction procedure for the hemolymph has been developed that allows t o perform PBAN measurements in this tissue even after a tenfold dilution. In these conditions matrix effect is negligible. Preliminary results on the presence of PBANlike immunoreactivity (PBAN-IR) i n the hemolymph of Spodoptera littoralis females are reported. o 1995 WiIey-Liss, Inc. Key words: pheromone biosynthesis activating neuropeptide, enzyme linked immunosorbent assay, sex pheromone, Spodoptera littoralis Acknowledgments: We thank Isabel Milldn for rearing the insects used in this study and CICYT for financial support (grant AGF-95-0185). M.P.M. also thanks CSlC for a Contract as Research Associate in a Programme of the Spanish Ministry of Education and Science. Received February 14, 1995; accepted May 10, 1995. Address reprint requests to Dr. Gemma Fabrihs, Department of Biological Organic Chemistry, C.1.D.-C.S.I.C., Jorge Cirona, 18-26, 08034-Barcelona, Spain. 0 1995 Wiley-Liss, Inc. 370 Marco eta[. INTRODUCTION It is well known that in some species of Lepidoptera the production of the sex pheromone is controlled by PBAN*, a 33 aminoacid peptide that has been isolated and characterized from brains of Helicoverpa zea (Raina et al., 19891, Bornbyx mori (Kitamura et al., 1989), and Lyrnantria dispar (Masler et al., 1994). Research on PBAN biosynthesis, mode of action, and catabolism will open a new field of potential chemical agents for pest control. Unfortunately, the conclusions from the studies regarding PBAN target site and mode of action are at present still controversial (Raina, 1993). Some authors have suggested that PBAN, biosynthesized in the brain-subesophageal ganglion complex (BrSOG), is released into the hemolymph and acts directly on the pheromone gland. Alternatively, other researchers have reported that the presence of an intact ventral nerve cord (VNC) is necessary for PBAN to reach the terminal abdominal ganglia (TAG), which is its target organ. Octopamine is then presumably released from TAG neurons that innervate the pheromone gland, where it stimulates pheromone production. Questions such as identification of PBAN in the blood of adult female moths and localization and characterization of the receptors for PBAN would undoubtedly clarify important aspects of the physiological mode of action of PBAN. Immunoassays are sensitive, specific, rapid, and inexpensive techniques that have found a wide application in several areas of science. Intensive research devoted over the past several years to the development of immunoassays has often shown that appropriate immunogen design can control specificity and sensitivity of an immunoassay. However, in recent immunochemical studies, using a radioimmunoassay, Rafaeli et al. (1991) found PBAN-IR in the brain-subesophageal ganglion complex (Br-SOG), corpora cardiaca (CC), thoracic ganglia, and TAG of Heliothis arrnigera at selected times of the photoperiod, but not in blood extracts during the sampling periods. An ELISA has also been developed and used to quantify PBAN-IR in Heliothis peltigera (Gazit et al., 1992) and H. zea (Kingan et al., 1992). In the last article, although PBAN-IR was found in Br-SOG and CC, amounts in the *Abbreviations used: AKH = adipokinetic hormone; Anti@-AP = alkaline phosphatase-conjugated anti-rabbit IgG; AntilgG-HRP = horseradish peroxidase-conjugated anti-rabbit IgG; BrSOG = brain-subesophageal ganglion complex; BSA = bovine serum albumin; CC = corpora cardiaca; CONA = conalbumin; Cys-Hez-PBAN = Hez-PBAN with a Cys aminoacid attached to the N-terminal arninoacid; DEA = diethanolamine; DMF = dimethylformamide; D M P = dimethylpirnelidate dihydrochloride; ECDI = 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide; ELISA = enzyme linked immunosorbent assay; HRP = horseradish peroxidase; KLH = keyhole limpet hernocyanin; LPK = leukopyrokinin; OVA = ovalburnin; PBAN = pheromone biosynthesis activating neuropeptide; PBAN-IR = PBAN-like imrnunoreactivity; PNP = p-nitrophenol; SMCC = N-succinimidyl-4-(maleidimidomethyl)cyclohexane carboxylate; TAG = terminal abdominal ganglia; TMB = tetramethylbenzidine; VNC = ventral nerve cord. Protein conjugates are abbreviated: BSA(DMP)(20-33) = Cys-Hez-PBAN(20-33) conjugated to BSA with DMP; BSA(ECD1)(20-33) = Cys-Hez-PBAN(20-33) conjugated to BSA with ECDI; CONA(DMP)(20-33) = Cys-Hez-PBAN(2033) conjugated to CONA with DMP; CONA(ECD1)(20-33) = Cys-Hez-PBAN(20-33) conjugated to C O N A w i t h ECDI; CONA(SMCC) = Cys-Hez-PBAN conjugated to C O N A w i t h SMCC; CONA(SMCC)(20-33) = Cys-Hez-PBAN(20-33) conjugated to CONA with SMCC; KLH(SMCC) = Cys-Hez-PBAN conjugated to K L H with SMCC; KLH(SMCQ(20-33) = Cys-Hez-PBAN(20-33) conjugated to KLH with SMCC; OVA(DMP)(20-33) = Cys-Hez-PBAN(20-33) conjugated to OVA with DMP; OVA(ECD1)(20-33)= Cys-Hez-PBAN(20-33) conjugated to OVA with ECDI. ELISA Development for PBAN 371 TAG and hemolymph were at or below the level of sensitivity of the assay. The lack of PBAN-IR in the blood can be attributed to either an incorrect timing of sampling, to a lack of sensitivity of the techniques employed, or also because PBAN is not released into the blood in those cases. The aim of the present paper has been (1) to develop a highly sensitive ELISA by rational preparation of suitable protein conjugates and (2) to demonstrate the applicability of the method to analyze insect hemolymph. Evidence for the presence of PBAN-IR in the hemolymph of Spudoptera littoralis virgin females is reported. MATERIALS AND METHODS Materials Polystyrene microtiter plates (Maxisorb)were from NUNC (Roskilde, Denmark). Absorbances were measured using a Titertek Multiskan Plus ELISA plate reader (Labsystems, Helsinki, Finland). Curve adjustments were performed with a commercial package (Genesis, Labsystems, Helsinki, Finland) using a four parameter logistic equation. Chemicals Immunochemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Other chemical reagents were from Aldrich Chemical Co. (Milwaukee, WI). Hez-PBAN, Hez-PBAN with a Cys aminoacid attached to the N-terminal aminoacid (Cys-Hez-PBAN), and Cys-Hez-PBAN(20-30) were prepared by solid-phase synthesis at the Organic Chemistry Department (University of Barcelona). Bom-PBAN was purchased from Peninsula laboratories (Belmont, CAI, leucopyrokinin (LPK) and adipokinetic hormone I1 (AKH) were obtained from Sigma Chemical Co. Oxidized Hez-PBAN was prepared as reported (Kitamura et al., 1989). Buffers PBS is 0.2 M phosphate buffer, 0.8% NaC1, pH 7.5. Borate buffer is 0.2 M boric acid-sodium borate, pH 8.7. Coating buffer is 0.5 M carbonate-bicarbonate buffer, pH 9.6. PBST is 0.05% Tween 20 in PBS. DEA buffer is 10% diethanolamine (DEA) buffer, pH 9.8. Citrate buffer is a 0.1 M solution of sodium citrate, pH 5.5. Insects S. littoralis larvae were reared on a wheat germ diet at 25 f 1"C, with a 1ight:dark cycle of 16h:8h. Sexes were separated as pupae, which were then transferred to a reversed photoperiod chamber with the same photoregime. Adults were segregated daily before the onset of the scotophase and fed with a 5% sucrose solution. Preparation of the Protein Conjugates SMCC-conjugates. To prepare the immunogens, Cys-Hez-PBAN (6.6 mg, 1.6 pmol) or Cys-Hez-PBAN(20-33) (3.4 mg, 1.6 pmol) were coupled to KLH (10 mg, 3.3 nmol, 4.7 pmol Lys) using SMCC (53 pL, 1.6 pmol, 10 mg/mL in 372 Marco et al. dimethylformamide [DMF])as described (Van Regenmortel et al., 1988).The conjugates were purified on a Sephadex G-50 column, lyophilized and stored dry at -80°C. The same procedure was applied to prepare the SMCC-conjugates for coating from conalbumin (CONA) (1 mg, 13 nmol), SMCC (10 pL, 0.29 pmol, 10 mg/mL in DMF), and Cys-Hez-PBAN (1.17 mg, 0.29 pmol) or Cys-Hez-PBAN(20-33) (0.60 mg, 0.29 pmol). The solutions were dialyzed extensively against 0.01 M PBS (4 x 5 L) and milliQ water (5 L) at 4"C, lyophilized and stored at -80°C. ECDI-conjugates. Cys-Hez-PBAN(20-33) (1.2 mg, 0.56 pmol, 1.16 pmol Lys, 1 mg/mL in borate buffer), previously treated with citraconic anhydride (7.8 mg, 69.6 pmol, 100 mg/mL in borate buffer) to protect the peptide Lys amino groups, was reacted with l-(3-dimethylaminopropyl)-3-ethyl cabodiimide (ECDI) (33.3pmol, 6.3 mg, freshly prepared solution 20 mg/mL in borate buffer) and the resulting adduct was then coupled to 2 mg of protein, BSA (29 nmol), CONA (26 nmol), or ovalbumin (OVA) (25 nmol), following standard procedures (Van Regenmortel et al., 1988). The conjugates were finally dialyzed for 3.5 h at room temperature against 1.5 L of 5% acetic acid and extensively against 0.01 M PBS (4 x 5 L) and milliQ water (5 L) at 4"C, lyophilized and stored at -80°C. DMP-conjugates. A freshly prepared solution of DMP (497 pg, 1.92 pmol, 1 mg/mL of borate buffer) was added to a solution of Cys-Hez-PBAN(20-33) (1 mg, 0.48 pmol) and the protein, BSA (1.4 mg, 21 nmol), CONA (1.7 mg, 22 nmol), or OVA (1 mg, 22 nmol), in 0.8 mL of borate buffer. The mixture was stirred for 4 h at room temperature and overnight at 4°C. The solution was extensively dialyzed against PBS 0.01 M (4 x 5 L) and milliQ water (2 x 5 L) at 4"C, lyophilized and stored at -80°C. Immunization Protocol and Collection of the Antisera Six female white New Zealand rabbits (2-3 kg) were used to raise antibodies using Cys-Hez-PBAN(20-33)conjugated to KLH with SMCC [KLH(SMCC)(2033)l (rabbits 1, 2, and 3) and Cys-Hez-PBAN conjugated to KLH with SMCC [KLH(SMCC)I (rabbits 4, 5 and 6) as immunogens. The immunogens were dissolved in 0.2 M PBS, mixed 1:l with Freund's complete adjuvant, and 300 pg were injected intradermally at 10 sites on the back of the animals. Booster injections were started 1 month after the initial immunization, and repeated every 4-5 weeks, with 150 pg of the same conjugate solutions mixed 1:l with Freund's incomplete adjuvant. Animals were bled 10 days after each boost (3 to 5 mL) from the marginal ear vein. Ten days after the last boost the rabbits were exsanguinated under deep anesthesia, the blood was allowed to clot and the clear serum was collected, mixed with NaN3 (final concentration 0.02%),and stored in aliquots at -80°C. Antibody Titer Test Plates were coated with Cys-Hez-PBAN(20-33)conjugated to CONA with SMCC [CONA(SMCC)(20-33)](2.5 pg/mL in coating buffer, 100 pL/well), sealed with adhesive plate sealers, and incubated overnight at 4°C. The following day the plates were washed 5 times with PBST and serum from rabbits 1 to 6, at 8 different dilutions (1/1,000 to 1/64,000 and 0 in PBST), was ELISA Development for PBAN 373 added to the wells (100 pL/well) and incubated for 1 h at room temperature. Plates were washed again 5 times with PBST and alkaline phosphatase-conjugated anti-rabbit IgG (antiIgG-AP) was added (115,000 in PBST, 100 pL/ well) and incubated for 1 h more at room temperature. The plates were washed again, and the substrate (p-nitrophenol phosphate (PNP), 1 mg/mL in DEA buffer, 100 pL/well) was added and incubated for 30 min at room temperature. Absorbance values were read at 405 nm. Screening of the Antisera and Coating Antigens A two-dimensional titration protocol was used for the screening and determination of the optimum concentrations of both coating antigens and antisera to be used later in the competitive experiments. Following the procedure described for titre analysis, CONA(SMCC)(20-33), Cys-HezPBAN conjugated to CONA with SMCC [CONA(SMCC)I, Cys-HezPBAN(20-33) conjugated to BSA [BSA(DMP)(20-33)1, CONA [CONA (DMP)(20-33)], or OVA [OVA(DMP)(20-33)] with DMP and Cys-HezPBAN(20-33) conjugated to BSA [BSA(ECDI)(20-33)1, CONA [CONA (ECDI)(20-33)],or OVA [OVA(ECDI)(20-33)1with ECDI were used to coat plates at 6 different concentrations (from 6 to 0.07 pg/mL and 0 in coating buffer, 100 pL/well). The plates were washed the next day and the antiserum from rabbits 1 to 6 was checked at different dilutions (1/1,000 to 1/64,000 and 0 in PBST) against the coating antigens. Plates were then developed as previously described. Optimal conditions were chosen to produce absorbances around 0.6 U in 30 min. Screening of the competitive experiments was performed as reported (Marco et al., 1993). Optimized ELISA Protocol: Competitive Experiment Microtiter plates were coated with BSA(DMPl(20-33)(0.3 pg/mL in coating buffer, 50 pL/well) overnight at 4°C. The following day, the plates were washed 5 times with PBST and blocked with 3% nonfat milk for 1 h at room temperature. The plates were washed again and the samples and standards (eight different concentrations from 10,000 to 3.2 pM and 0), which had been preincubated with the antibody 4 (1/25,000 diluted in PBST) overnight at 4"C, were added to the wells (50 pL/well) and incubated for 1 h more at room temperature. After another washing step horseradish peroxidase-conjugated anti-rabbit IgG (antiIgG-HRP) (116,000 in PBST, 50 pL/well) was added and incubated for an additional hour at room temperature. Plates were washed and a solution of the substrate (tetramethylbenzidine [TMBI 0.01%, H2020.004% in citrate buffer) was added (50 pL/well). The enzyme reaction was stopped after 30 min at room temperature with 25 pL of 4 M H2S04and the absorbances were read at 450 nm. Cross-Reactivity Studies Standard curves of Cys-Hez-PBAN, Cys-Hez-PBAN(20-33), oxidized HezPBAN, Bom-PBAN, LPK, and AKH (10 pM to 0.01 pM) were prepared in PBST and used for ELISA determination to study the specificity of the assay. Cross-reactivity values were calculated as follows: (IsoHez-PBAN /I5opeptide) x 100. 374 Marco et al. Hemolymph Collection and Sample Preparation Insects were decapitated and placed into a 500 pL Eppendorf tube with the bottom tip severed, which was inserted into a 1.5 mL Eppendorf tube. The Eppendorf tube was centrifuged at 2,000 rpm for 5 min and obtained 10 to 20 pL of clear hemolymph per female. Blood (20 pL) was collected with a 10 pL microcapillary glass pipette, diluted with ethano1:water (5:4,180 pL), the mixture was vortexed for about 5 s and then centrifuged at 12,000 rpm for 5 min. The clear supernatant (150-160 pL) was collected and dried down in a SpeedVac rotatory evaporator. Dry samples were stored at -80°C for ELISA analyses. Prior to ELISA determination, samples were redissolved in the same volume of the solution of antibody (1/25,000 diluted in PBST). Hemolymph was taken from 2-day-old adult virgin females either 2 h before or 2 h after the onset of the scotophase. One group of animals were decapitated 2 h before lights off and hemolymph was collected 2 h into darkness. Hemolymph extracts were prepared from one to two individuals. PBAN Recovery Studies Photophase hemolymph samples (20 pL) were diluted with ethano1:water (5:4, 180 pL) containing Hez-PBAN to a final concentration of 100 pM, processed as described above, and used for ELISA determination. Hemolymph extracts without Hez-PBAN and Hez-PBAN solutions in the absence of hemolymph were also run simultaneously. Matrix Effect Studies Second-photophasehemolymph extracts, prepared as described above, were serially diluted with PBST, spiked with Hez-PBAN to a final concentration of 100 pM, and measured by ELISA to determine the optimum blood dilution factor. Then, Hez-PBAN standard curves were prepared in PBST and 1/10 PBST diluted hemolymph to determine the parallelism of the curves. RESULTS Immunoassay Development As outlined in the discussion, two different immunogens were prepared to obtain antibodies directed to the C-terminal part of PBAN. The first immunogen, KLH(SMCC),was synthesized by coupling Hez-PBAN, with an extra Cys residue attached to the N-terminal aminoacid, to the carrier protein using SMCC as cross-linker. SMCC reacts with the Lys amino acids of the carrier protein through the activated acid moiety and, in a second step, it reacts specifically with the Cys residue of the peptide through the maleidimido group. A second immunogen was prepared following the same methodology using Cys-Hez-PBAN(20-33) as hapten. Because of the enhanced immunogenicity of KLH conjugated PBAN, maximum antibody titers were already obtained after the second boost, and they remained almost constant after subsequent boostings. The development of a two-step competitive ELISA based on the coating antigen format was accomplished by screening a library of coating antigens ELISA Development for PBAN 375 and searching for a suitable competitor. For the preparation of these coating antigens, we used the less expensive hapten, Cys-Hez-PBAN(20-33),BSA, CONA, or OVA as carrier proteins and SMCC, ECDI, or DMP as cross-linkers. The titration experiments performed with all these coating antigens (Table 1) revealed that acceptably high antibody titers were obtained with both immunogens, allowing a considerable dilution of the antisera. Only the protein conjugates prepared using ECDI gave slightly lower antibody titers. Optimal antibody dilution and coating antigen concentration with all the possible antibody/protein conjugate combinations were determined by a twodimensional titration experiment (Table 1).A tiered system was used starting with a broad screening and finally resulting in one antibody/coating antigen combination for full evaluation. Most of the assays showed moderate sensitivity and some of them presented high slope values, indicating significant antibody affinity. Among the coating antigens prepared with ECDI, only OVA(ECDI)(20-33)gave competitive assays with two of the antisera, although maximal absorbances were very low (data not shown). Although CONA(SMCC)(20-33)gave competitive assays with three of the antisera tested, the maximal absorbance/background signal ratios were never satisfactory (data not shown). In contrast, DMP coating antigens gave the most competitive assays (Table 2). Among the different combinations, CONA(DMP)(20-33) and BSA(DMP)(20-33)and antiserum 4 showed the best slopes, 15,, values, and maximal absorbance/background signal ratios. Consequently, we focused our attention on the optimization of the ELISA with these immunoreactants. Blocking of the plates after the coating step along with preincubation of the antibody with Hez-PBAN before the competition step, improved the maximal absorbance/background signal ratio and sensitivity in both assays. However, the best values were obtained with BSA(DMP)(20-33),which was selected for routine use. Finally, the optimized assay, whose features are specified in the four parameter equation in Figure 1, allows the measurement of PBAN in a concentration range between 20 to 500 pM with an 150= 53 k 12 pM (2.6 k 0.6 fmol/well, n = 5). Cross-reactivity studies were conducted to determine the specificity of the assay by monitoring recognition by the antibody of a series of peptides structurally similar to PBAN. As shown in Table 3, similar binding affinities were observed for both Bom-PBAN and Hez-PBAN, which makes the assay suitable for the analysis of PBAN from several sources. Obviously, haptens used for immunization were detected by the antibody with a higher sensitivity. Oxidized Hez-PBAN was also recognized by the same assay with a crossreactivity value of 64% and the octapeptide LPK exhibited a 27% cross-reactivity. It is important to note that the slope is slightly lower in the assay for LPK, which indicates a decreased affinity of the antibodies to this peptide. Evidently, AKH, a peptide with a completely different aminoacid sequence, was not recognized by the antibody. A series of experiments were performed to evaluate the effectiveness and reliability of the developed ELISA to determine PBAN in the insect hemolymph, following our optimized extraction procedure. First, photophase hemolymph extracts were serially diluted with PBST and spiked with a known amount of Hez-PBAN. These experiments showed that the lowest dilution factor that M L L M M M M L M M L M H H H H H H H H H H M H 1 2 3 4 5 6 L L L L M L n.t. H M M H M M n.t. L L M M M n.t. H H H H *H, M, and L indicate the serum dilution range that gave absorbances of 0.6-0.7 after 30 min. H, more than 1/32,000; M, between 1/32,000 and 1 / 8,000; L, less than 1/8,000. n.t., not tested. The concentration of the coating antigens selected was that which ensured an optimum coverage of the solid phase, ranging from 0.1 to 1 pg/mL. KLH(SMCC) KLH(SMCC) (20-33) Immunogen Coating antigen CONA CONA BSA CONA OVA BSA CONA OVA Antisera (SMCC) (SMCC)(20-33) (ECDI)(20-33) (ECDI)(20-33) (ECDI)(20-33) (DMP)(20-33) (DMP)(20-33) (DMP)(20-33) TABLE 1. Titer of Antisera Measured in the Presence of Different Coating Antigens* ELISA Development for PBAN 377 TABLE 2. Features of the Most Competitive Assays Obtained During the Screening of the Different AntibodylCoating Antigen Combinations* Coating antigen BSA(DMP)(20-33) CONA(DMP)(20-33) OVA(DMP)(20-33) Antiserum, dilution factor A/D Is,, (pM) 1,1/20,000 3,l /80,000 4,1/25,000 5,1/25,000 6, 1 /80,000 4,1/25,000 1,1/10,000 4,1/5,000 5,1/10,000 6,1/20,000 2.3 3.3 4.8 5.5 2.1 3.2 2.1 2.5 2.1 2.5 296 6429 223 389 1138 217 2790 551 2635 1737 Slope A max 0.6 0.4 1.o 0.5 0.9 0.8 0.4 1.4 0.4 0.8 0.801 0.197 0.266 0.331 0.263 0.751 0.551 0.398 0.731 0.538 r 0.965 0.936 0.968 0.987 0.925 0.947 0.954 0.962 0.963 0.851 *Maximal absorbance (A), slope (B), Is0 (C), and minimal absorbance (D) are the values of the four parameter equation calculated for each assay: y = (A-D)/[I+(X/C)~]+D.Three well replicates were used for each concentration of PBAN in the standard curves. gave no matrix effect was 1/10. In a second set of experiments, serial dilutions of Hez-PBAN were prepared in both a hemolymph extract diluted 1/ 10 and PBST. The sigmoid curves obtained for this peptide in both cases showed identical slope and sensitivity (Fig. 1). Finally, photophase hemolymph extracts were prepared in the presence of known amounts of Hez-PBAN to determine the recovery of this peptide, which was 82 16% (n = 7). It is worth noting that no protease inhibitors had to be added to the extraction solvent, as long as the samples were rapidly processed. Determination of PBAN-IR in the Hemolymph In order to demonstrate the applicability of this ELISA, we determined PBAN-IR in S. littoralis hemolymph (Table 4). Hemolymph samples taken 1 h 0.2 Fig. 1. Standard curve for Hez-PBAN performed in PBST buffer (filled circles) or hemolymph diluted 1/10 in PBST buffer (open circles) showing the absence of matrix effect. Analysis was performed by the two-step competitive ELISA using 8 serial dilutions, in duplicate, of synthetic Hez-PBAN, ranging from 0.1 KM to 0.01 pM. Four parameter logistic equation of an optimized routine assay is as follows: y = 0.495/[1 + (~/48.7)'.*~] + 0.044. For general equation, see Table 2. 378 Marco eta!. TABLE 3. Cross-Reactivity of PBAN Antiserum With Selected Peptides, Peptide Hez-PBAN Cys-Hez-PBAN Cys-Hez-PBAN(20-33) Oxidized Hez-PBAN Bom-PBAN LPK AKH 150(pM) Slope % Cross-reactivity 68 59 46 106 72 252 n.d. 1.1 1.2 0.9 1.1 1.2 0.7 100 115 148 64 95 27 n.c. *Cross-reactivitydata were obtained by preparing standard curves in PBST. Two or three well replicates were used for each concentration (10 pM to 0.01 pM) of the peptides. Is0 and slope values were obtained from the four parameter equation of every standard curve. Cross reactivity values were calculated according to the equation: (I50H ~ Z - P B A N / peptide) I~~ x 100. n.d., not detected at concentrations below 10 pM; n.c., no cross-reactivity. before lights off were always below the detection limit of the assay (200 pM when measuring 1/10 diluted hemolymph). Conversely, all the samples taken 2 h into the dark period could be measured with concentration values around 600 pM. In contrast, concentrations of PBAN-IR in all hemolymph samples taken 2 h into the scotophase from females that had been decapitated 2 h before darkness were below the detection limit of the assay. DISCUSSION Since small amounts of PBAN can elicit a pheromonotropic response, physiological studies about PBAN mode of action rely on the availability of an analytical tool able to detect trace amounts of this peptide. Immunoassays are rapid, selective, and very sensitive analytical techniques that have proven to be useful in several areas of pharmacology, toxicology, etc. In contrast with other analytical procedures, small sample sizes are sufficient to perform quantification and clean-up procedures can often be avoided. However, the existence of PBAN-IR in the blood could not be confirmed with either of the three immunoassays previously reported (Gazit et al., 1992; Kingan et al., 1992; Rafaeli et al., 1991), probably because they lacked the required sensitivity. The first aim of this paper was the development of a highly sensitive ELISA TABLE 4. PBAN-IR of Hemolymph Extracts of S. littoralis Virgin Females* [PBAN-IR]" Scotophase Photophase <200 (7) Intact Decauitated * 665 89 (20) <200 (8) *Extraction of hemolymph and preparation of samples were carried out as described in Materials and Methods. Analyses were performed by the two-step competitive ELISA using 8 serial dilutions, in duplicate, of synthetic Hez-PBAN, ranging from 10 nM to 3.2 pM. A dilution of hemolymph extract of 1/10 was used, in duplicate. "Data are expressed in pM and are the mean k SEM of the number of replicates given within parentheses. ELISA Development for PBAN 379 by rational preparation of suitable protein conjugates to be used as both immunogens and coating antigens. One of the most important stages in immunoassay development is the design of appropriate immunogens to obtain a suitable antiserum. In order to enhance immunogenicity, small molecules, which usually do not stimulate immunological response, are attached to a carrier protein. The position of covalent coupling of the hapten to the carrier protein has a strong influence on the selectivity and sensitivity of the immunoassay. The spacer arm used as cross-linker is also important because, due to the shielding effect of the carrier macromolecule, it directs antibody specificity so that the immunodominant part of the molecule is that situated farthest from the attachment point. In the development of this ELISA, the immunogens were designed taking into account that the pheromonotropic response may not be prompted by the complete 33 amino acid PBAN, as smaller peptides with the C-terminal sequence of PBAN are also active (Fonagy et al., 1992; Gazit et al., 1990; Raina and Kempe,1990). Therefore, we directed antibody specificity towards the C-terminal part of PBAN, so that any pheromonotropic peptide present in the blood could be detected. With the above considerations in mind, this was achieved by covalently attaching the carrier protein, KLH, to the N-terminal side of the peptide so that its C-terminus would be highly exposed for antibody recognition. Coupling of KLH to the N-terminal portion of Hez-PBAN was accomplished through an extra Cys residue, bound to the N-terminal amino acid of PBAN, which specifically reacted with the sulfhydryl directed moiety of the cross-linker, SMCC. Although in most of the immunoassays the entire target structure is included in the immunizing hapten, a characteristic fragment can often be sufficient to generate antibodies with high affinity to bind the whole compound. Such an approach is very useful if the target substance is unstable, toxic, difficult, or expensive to obtain. Therefore, we prepared a second immunogen using a 15 amino acid peptide with the Cterminal sequence of Hez-PBAN, functionalized with an extra Cys residue bound to the N-terminal amino acid. The development of competitive immunoassays require the preparation of suitable coating antigens or enzyme tracers, depending on the format chosen. Since adsorption of peptides to the plates can lead to a lack of reproducibility of the assay due to their small size (Van Regenmortel et al., 1988), in our coating antigen ELISA we used a protein conjugate as competitor, in contrast to the ELISAs previously described (Gazit et al., 1992; Kingan et al., 1992). From the screening experiments we could corroborate that heterologous ELISAs give the most competitive experiments (Harrison et al., 1991). In heterologous ELISA systems recognition of the coating antigen is weaker than that of the target compound because a different protein, hapten, coupling position, or procedure from that employed in the preparation of the immunogen is used in its preparation. As DMP reacts with Lys y-amino groups under mild conditions with a high degree of specificity, in the DMP-conjugates covalent coupling probably occurred mainly through the. 27Lysamino acid of Hez-PBAN(20-33), near to the C-terminal amino acid, thereby providing, besides protein and bridge, also site heterology affording the most effective competitive assays. In contrast, ECDI-conjugates did not always provide 380 Marco et al. site heterology, whereas SMCC coating antigens did not fulfill either site or bridge heterology, resulting in more deficient assays. The optimized ELISA herein described can detect trace amounts of PBAN, with a detection limit of 1 fmol/well. As we had planned, the assay is aimed at the recognition of the C-terminal part of the peptide, as concluded from the fact that LPK still cross-reacted with the antiserum. Although only 27% of cross-reactivity was observed for this fragment, recognition (Iso = 252 pM, 12.6 fmol/well) is still higher than that for PBAN in the ELISA previously reported (Gazit et al., 1992).The lack of specificity of this assay is convenient in that PBAN and also shorter fragments derived from it, which could be responsible for the pheromonotropic activity, are detected. However, it should be mentioned that the assay might also detect fragments that are not active, since Raina and Kempe (1990) reported that PBAN(25-33)and PBAN(29-33) are much less active than PBAN at a dose of 10 pmol in H . zea. On the other hand, the fact that the myotropic peptides of the FXPRL-NH2family are also recognized raises the need for confirmatory methods to ensure the identity of the peptide responsible for that immunoreactivity. Using this ELISA, the presence of PBAN-IR in hemolymph of S. littoralis virgin females was detected during the scotophase, at the time of pheromone production. In contrast, no PBAN-IR was found in blood samples taken during the photophase, when only very low amounts of pheromone are present in the gland. Likewise, no PBAN-IR was detected in blood samples taken during the scotophase from females that had been decapitated during the light period. This last result agrees with the observation that decapitation during the photophase abolished normal sex pheromone production, probably because PBAN had not yet been released into the circulatory system (Martinez and Camps, 1988). These results suggest that a pheromonotropic peptide, either PBAN or a shorter fragment with the same C-terminal sequence, is present in the blood of S. littoralis virgin females at the time of pheromone production. Since five peptides with almost identical terminal sequences are coded by the PBAN gene (Ma et al., 19941, it is possible that the five peptides are released into the blood and detected by this ELISA. However, given the lack of specificity of this assay, we cannot disregard the possibility that an immunoreactive peptide without pheromonotropic activity is coincidentally present in the hemolymph at the time of pheromone production. Future studies will be conducted to clarify these points. In summary, the rational design of immunogens and coating antigens has led us to the development of a very sensitive ELISA for PBAN, with a detection limit of 1 fmol/well. The assay has been characterized and we have demonstrated its utility to determine PBAN-IR in the hemolymph. An extraction procedure has been developed that requires little or almost no sample cleanup to analyze the samples, thus diminishing the probabilities of the peptide to be enzymatically or chemically degraded prior to the analysis. This extraction procedure affords high PBAN recoveries and reliable measurements with samples diluted 1/10. The application of this ELISA to S. littoralis virgin females indicates the presence of PBAN-IR in the hemolymph of pheromoneproducing insects. Further determinations aimed at clarifying the physiological mode of action of PBAN in this species will be reported elsewhere. ELISA Development for PBAN 381 LITERATURE CITED Fonagy A, Schoofs L, Matsumoto S, De Loof A, Mitsui T (1992): Functional cross-reactivities of some locustamyotropins and Bornbyx pheromone biosynthesis activating neuropeptide. J Insect Physiol38:651-657. Gazit Y, Dunkelblum E, Benichis M, Altstein M (1990): Effect of synthetic PBAN and derived peptides on sex pheromone biosynthesis in Heliothis peltigeru (Lepidoptera: Noctuidae). Insect Biochem 20:853-858. Gazit Y, Dunkelblum E, Ben-Aziz 0,Altstein M (1992): Immunochemical and biological analysis of pheromone biosynthesis activating neuropeptide in Heliothis peltigeru. Arch Insect Biochem Physiol19:247-260. 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