Sensitive enzyme immunoassay for Manduca allatotropin and the existence of an allatotropin-immunoreactive peptide in Periplaneta americana.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 23:99-109 ( 1 993) Sensitive Enzyme lmmunoassay for Manduca Allatotropin and the Existence of an Allatotropin-lmmunoreactive Peptide in Periplaneta americana Jan A. Veenstra and Henry H. Hagedorn Deyartmezt of Enlnrtro2ogy and Center for [tisect Sctence, Th2 L f n i r ~ s z t of y Anzann, T~KSQ+I ,Intisera to Manduca scxta allatotropin were raised in rabbits and were used to develop a competitive enzyme immunoassay for this neuropeptide. The detection limit of the assay is less than 2 fmol/well. A useful quantification can be obtained from 2 to 30 fmol/well. No cross-reactivity was observed with several insect ppptirles, but the enzyme-linked immunosorbent assay does recognize [ A h 6 , Leu7, Ser']-allatotropin, a myotropin recently isolated from Locusta migratoria The assay was used to study the distribution of allatotropin within the nervous system of Manduca sexta. The peptide is present in the retrocerebral complex, the brain, and the ventral nerve cord of this species, in quantities of respectively 0.01, 1.2, d i d 1 . 7 pmol per insect. An allatotropiti-imniutioredctive peptide W ~ found in the nervous system of Periplanera americana. It is present in the ventral nerve cord ( 3 . 3 pmol/insect), brain (1.9 pmol/insect), and retrocerebral complex iO.09 pmoliinsect). These data suggest that peptides of this family are generally present in insects. 0 1993 W r y - L i s s , Inc S Key wnrds: juvenile hormone, allatotropin, myotropin, ELISA, Manduca sexta, f ocusta migratoria INTRODUCTION Juvenile hormone regulates a number of important physiological processes in insects. The regulation of its hernolymph titers and the rate of juvenile hormone synthesis by the corpora allata have been studied extensively. The activity of the corpora allata is regulated by the central nervous system, via both nervous and humoral signals [I]. Scvcral putative corpus allatum-reguAcknowledgments: We thank RenC Feyereisen for his generow gifts of peptide,, use of his ELISA-reader, and critical reading of the manuscript. This work was supported by a grant from the Agricultural Experiment Station at The University of Arizona (ARZT436317-H-3 1-02 I ). Received June 12, 1992; accepted October 14, 1592 Address reprint requests to ].A. Veenstra, Department of Entomology and Center for Insect Science. The Uiiiversily d Ariiona, 410 Forbes, Building #36, Tucson, AZ 85721. o 1993 Wiley-Liss, Inc. 100 Veenstra and Hagedorn latory factors have been shown to be small peptides, and a few of them have now been fully identified. They include several allatostatins from the cockroach Diplopteru puncfufu [ 2 4 ] and an allatostatin and an ailatotropin from the moth Manducu sexta [5,6].From the locust, Locusta migrutoria, the [Ala', Leu7,Ser8]-homologue of Manduca allatotropin was isolated from male accessory glands on the basis of its myotropic effects on the oviduct in the same species 171. A fast and relatively simple, sensitive assay to quantitate a neuropeptide is essential to study its distribution and physiology. Quantitative immunoassays are often the only practical solution, and for this reason antisera were raised against Muvlduca allatotropin. This report describes a sensitive ELISA*for this peptide. MATERIALS AND METHODS Insects Manducu sextu were obtained from a colony maintained in the Division of Neurobiology of the Arizona Research Laboratories at The University of Arizona. Peviplunetu amevicanu were from a laboratory culture raised on dog chow and water. Peptides Mnnducu allatotropin was synthesized using solid phase technology on a n Applied Biosystems model 430A automated peptide synthesizer (Foster City, CA) by the Division of Biotechnology of The University of Arizona. The crude peptide was analyzed by HPLC on a Vydac C-18 column (Hesperia, CA) using a water and acetonitrile gradient with TFA as the pairing ion. The reduced and three oxidized allatotropins were tentatively identified by their elution positions, and their identification was confirmed by total and partial oxidation with hydrogen peroxide of these peaks and analysis of the oxidized peptides. The allatotropin which had both methionines in the reduced form was then purified on the same column. Munduca allatotropin (with a known peptide content used for quantification in the assay), Locustu migrutoria accessory gland myotropin I, and their C-terminal analogues were a gift from Dr. Rene Feyereisen. Corazonin, vasopressin, and proctolin were purchased from Sigma Chemical Company (St. Louis, MO). Conjugates Two milligrams of purified allatotropin (see above) were coupled to 5 mg of thyroglobulin using glutaraldehyde as described . This preparation was used as antigen. The conjugate for coating the ELISA plates was made by coupling 0.2 mg allatotropin to 5 mg BSA using DFDNB 191. *Abbreviations used: BSA = bovine serum albumin; ELISA = enzyme-linked immunosorbentassay; DFDNB = .I,5-difluoro-2,4-dinitrobenzene; HPLC = high-performanceliquid chromatography; IgG = immunoglobulin G; NGS = normal goat serum; PBS = phosphate buffered saline (1 50 mM NaCI, 10 mM phosphate, pH = 7.4); TFA = trifluoroacetic acid. ELISA for Manduca Allatotropin 101 Antisera Antigen was mixed with RIBI adjuvant system (complete Freunds adjuvant in which the tubercle bacilli have been replaced by trehalose dimycolate; from RIBI ImmunoChem Research, Inc., Hamilton, MT) and 200 pg injected at six sites on the backs of two New Zealand White rabbits. At intervals of 6 weeks to 4 months booster injections were given, containing 125 pg of the conjugate in incomplete Freund’s. Rabbits were bled 7 days after each booster injection and serum was stored frozen. ELISAs The ELISA was performed as described [10,11]. Two nanograms of the allatotropin-DFDNB-BSA conjugate in 50 pl PBS were plated per well in an “Easy Wash” microtiter plate (Corning Glass Works, Corning, NY). After shaking for 3 h at 3 7 T , the wells were rinsed with 0.05% Tween 20 in PBS and 200 pl of 1%NGS were added to each well. The plates were shaken at room temperature for 1h to blockthe remaining binding sites. The plates were then rinsed with 0.05% Tween 20 in PBS and known amounts of allatotropin NGS in PBS were then (in triplicate) or test samples (in duplicate) in 50 ~ 1 1 % added to each well, followed by 50 pl of anti-allatotropin antiserum diluted in 1%NGS in PBS, usually 1:500,000, to yield a final concentration in the well of 1:1,000,000. The plates were then sealed and gently shaken at 4°C overnight. The next morning the plates were moved to room temperature and were shaken for 1 h before rinsing the wells three times for 5 min with 0.05% Tween in PBS. A peroxidase-conjugated affinity purified goat-anti-rabbit IgG (Kirkegaard & Perry, Gaithersburg, MD) was diluted to 1:1,000 in 1%NGS in PBS, and 70 p1 were added to each well. The incubation at room temperature was then continued for 1 h on a shaker. The plates were washed four times for 5 min with 0.05% Tween 20 in PBS and developed for 30 min with 75 pl 3,3‘5,5’-tetramethylbenzidineand H202 (Kirkegaard & Perry) at room temperature. The enzyme reaction was stopped and the color was intensified by adding 75 cl.1 1 M H3P04 per well. The absorbance was read at 450 nm in a microtiter plate reader. This is the ELISA that has been used for all data presented in this paper. In a few experiments the peroxidase-conjugated goat anti-rabbit IgG was replaced by an alkaline phosphatase-conjugated goat anti-rabbit IgG (Pierce, Rockford, IL), which was diluted 1:1,000 in 1%NGS in PBS. The plates were similarly incubated, but washed only twice with 0.05% Tween in PBS after removal of the enzyme-conjugated second antibody. The plates were then processed as described by Johannsson et al. . They were rinsed twice for 5 min with alkaline phosphatase buffer, i.e., 0.1 mM ZnCL2,l mM MgC12,15 mM NaN3, and 50 mM diethanolamine adjusted to pH 9.5 with diluted HCl and filtered through a 0.22 pm filter. They were subsequently incubated with 100 pl of 0.1 mM NAD-free NADP (Boehringer Mannheim, Indianapolis, IN) in the same buffer for 45 min at room temperature. The generated NAD was then enzymatically amplified with 200 p1 of amplifier buffer containing alcohol dehydrogenase (Sigma) and diaphorase (Boehringer Mannheim) titered to the appropriate concentrations to yield a final absorbance of 2 at 490 nm after 30 102 Veenstra and Hagedorn min when 200 pl of amplifier were added to 100 pl of 300 nM NAD in alkaline phosphatase buffer. Amplifier buffer consisted of 4% ethanol, 5 mg/mI BSA, and 0.55 mM p-iodonitrotetrazolium violet (Sigma) in 20 mM phosphate buffer, pH 7.2. Tissue Extraction and HPLC After dissection in saline, tissues were immediately frozen on dry ice. Tissues were extracted in 1 ml of Bennett’s mixture  in a glass-glass homogenizer. The extracts were centrifuged for 5 min at 12,OOOg and the supernatants loaded on a Cis Sep-Pak (Waters Associates, Milford, MA) containing 300 mg material, which had previously been activated and equilibrated with 0.1% TFA in water. The Sep-Pak was washed with 5 ml of water containing 0.1% TFA and 5 ml10% acetonitrile in water containing 0.1% TFA (these fractions did not contain allatotropin-immunoreactivity as measured by ELISA). The peptides were eluted with 4 ml of 65% acetonitrile containing 0.1% TFA. RIA-grade BSA (100 pg) was added to samples to be analyzed in the ELISA; the samples were then lyophilized. Fifty microliters of a 25% solution of thiodiglycol were added to samples to be analyzed by HPLC. These samples were reduced in volume by vacuum centrifugation to about 1.5 ml, diluted with 0.1% TFA to about 3.5 ml total volume, and injected onto a Beckman C18 column and precolumn that had been equilibrated with 13% acetonitrile in 0.1% TFA. After injection the column was eluted isocraticaily for 20 min with 13% acetonitrile in 0.1% TFA followed by a linear gradient over 40 min to 32.5% acetonitrile in 0.1% TFA. The flow rate was 1 ml/min and the eluant was monitored at 214 and 280 nm. Fractions (1 min) were collected and aliquots lyophilized with 100 pg RIA-grade BSA for analysis in the ELISA. RESULTS Antiserum from rabbit 1 obtained after the fifth bleeding was selected for routine use as it had the lowest detection limit and the steepest slope for the standard curve. The ELISA did not cross-react with 100 pmol corazonin, vasopressin, leucokinin IV, or proctolin (data not shown). There is, however, significant cross-reactivity with the myotropin recently isolated from the male accessory gland of the locust Locustu miptoriu (Fig. 1). The N-terminally truncated &13-analogue of Munducu allatotropin was almost equally immunoreactive in the ELISA as the intact peptide. The same was found for the 6-13-analogue of the locust myotropin with respect to the intact locust myotropin (Fig. 1). The ELISA was used to study the distribution of allatotropin within the nervous and endocrine systems of Maizducu. Brains, retrocerebral complexes, and ventral nerve cords were dissected out, extracted, and separately processed by HPLC. The results showed only a single allatotropin-immunoreactive peak in each of these tissues, which co-eluted with synthetic allatotropin in which both methionines are in the reduced form (Fig. 2). The amounts in the ventral nerve cord (1.7 pmol/insect) and the brain (1.2 pmol/insect) were ELISA for Manduca Allatotropin 0 .I 1 10 103 100 1,00010,000 100,000 ferntomoles Peptide per well Fig. 1 . Allatotropin ELISA performed as described in Materials and Methods. Manduca allatotropin (a),Manduca allatotropin 6-13 (O), Locustd myotropin (m), and Locusta myotropin 6 1 3 (0). Samples were assayed in duplicate to allow all samples to be run on a single plate. Standard deviations were smaller than the data points. similar, while the amounts in the retrocerebral complex (0.014 pmolhnsect) were much smaller. This experiment was repeated with the American cockroach, Periplaneta arnericana, to determine whether or not allatotropin-immunoreactive peptides were also present in a hemimetabolous insect species (Fig. 3). The relative abundancies of allatotropin-immunoreactive material in the three tissues resembled those in Manduca. There was a single immunoreactive peak, which had the same retention time in each of the three tissues. This immunoreactive peak eluted 2 min earlier than M a ~ d u c aallatotropin. Serial dilutions of the immunoreactive peptide from Periplaneta yielded the same slope as the Locusfa peptide, which is not as steep as the slope obtained with Manduca allatotropin (Fig. 1). The locust peptide was therefore used to calculate the amounts of peptide present in the cockroach extracts. Although only 8 fmol allatotropin per well are sufficient for a 50% response in the ELISA, two attempts were made to further improve its sensitivity. In the first, the antibody bound in the well was visualized using the enzyme amplification system of Johannsson et al. . Although the system detects much smaller amounts of antibody (the anti-allatotropin serum had to be diluted 1:20,000,000 at these conditions), the sensitivity of the final alla- 104 Veenstra and Hagedorn I I 0 $.I 30 I 60 min 0 6 D 60 min r I 0 20 I 60 min Figure 2 . ELISA for Manduca Allatotropin 105 totropin assay increased only twofold. The concomitant increase in the variability together with the tediousness in preparing the enzyme amplifier are, however, two major disadvantages of this system. A similar twofold increase in sensitivity could be obtained more easily by reducing the volume containing the peptide and the anti-allatotropin serum in each well to 20 ~ 1The . antiserum concentration had then to be increased to 1:250,000. DISCUSSION A sensitive ELISA for Manduca allatotropin is described here, which, with one exception, does not appear to cross-react with other identified insect peptides. The exception is a myotropin isolated from the male accessory glands from Locusfa rnigratoria [ 7 ] .The structure of the locust myotropin is sufficiently similar to Manduca allatotropin to suggest that the two peptides are homologues. It is likely that the immunoreactive peptide in Periplaneta is also a homologue. Since few insect peptides have so far been fully identified, it is possible that other unknown peptides might cross-react, but no other cross-reactive peptides were found on analysis of extracts from the central nervous system of either Manduca or Peripluneta. The present data allow some conclusions regarding the requirements for immunoreactivity in the ELISA. The almost total cross-reactivity of allatotropin 6-13 suggests that the N-terminal five amino acids are not important for recognition. This conclusion is reinforced by the very similar cross-reactivities between the locust myotropin and its 613-analogue. The relatively strong cross-reactivity of the locust myotropin, which differs from Munducu allatotropin only in amino acids 6, 7, and 8, suggests that the contribution of these three amino acids to the antigen-antibody interaction is limited, but is more important than the five N-terminal amino acids. This indicates that the five C-terminal amino acids form the major part of the molecule that is recognized by the antiserum. This ELISA is very sensitive and compares favorably to other recently described quantitative immunoassays for insect neuropeptides [e.g., 14-18]. It is not as sensitive as a similar ELISA for corazonin, which has a detection limit of less than 300 attomoles per well [lo]. It has been suggested that ELISAs are more sensitive than radioimmunoassays [191, because the detection limits that can be achieved by enzymatic methods are inherently lower than those of radioisotopes. However, for ELISAs, in which limited amounts of bound Fig. 2 . Chromatograms of ventral nerve cords (A), brains (B), and retrocerebral complexes (C) of 10 adult Manduca sexta. Tissues were prepurified on a CIS Sep-Pak and separated by HPLC as described in Materials and Methods. Acetonitrilegradient as indicated in D. Aliquots of 1 ml fractions were assayed in the allatotropin-ELISA and the amount of allatotropin-immunoreactivitycalculated per fraction as described. Arrows in A, B, and C indicate the elution time of synthetic Manduca allatotropin in which both methionines are in the reduced form. Arrows in D numbered 1 through 4 indicate the elution time of synthetic Manduca allatotropin in which both ( I ) , one (2 and 3), or none (4) of the methionines are in the oxidized form. w d altatotropin mnoi) - 0 I 0 I 6 I 0 hl (pmoi) allatot ropin 3. 3 -.3 3 0- Q, 0 Q, 0- w 0 w 0 W -.L 0- 0 0 i 0 0 0 1 I 6 atlatotropin cpmoi I 0 h) 1 +*I \ I 0 0 ELISA for Manduca Allatotropin 107 and free antigen compete for a limited amount of specificantibodies, or where enzyme-labeled antigen competes with unlabeled antigen for a limited amount of specific antibody bound to a solid phase, sensitivity, as in radioimmunoassay , is necessarily limited by the affinity constant of the antibody for the antigen. The best detection limits reported for competitive ELISAs are about 300400 attomoles, when colorimetric methods for the detection of horseradish peroxidase are used . This can be increased slightly either by using fluorescent substrates , or, as was done here, by using enzymatic amplification. If antisera could be raised with sufficiently high affinity constants, competitive assays should be able to detect less than 1 attomole, as demonstrated for the biotin-avidin model . However, affinity constants as high as the one M) cannot be realistically characterizing the biotin-avidin interaction expected for peptide-antibody interactions; 10l1 M is generally considered very good. Nevertheless, if the affinity constant is the limiting factor and not the possibility to detect label, then it should be possible to increase the sensitivity by decreasing the volume of the incubation of the peptide sample with the specific antiserum. By decreasing the volume to 20 ~ 1 a, twofold increase in sensitivity was obtained for the allatotropin ELISA, and a similar increase was observed for the corazonin ELISA (unpublished observations). Further decreases in incubation volume are likely to increase relative errors, due to the difficulty in pipetting volumes much smaller than 20 pl accurately and repetitively as well as to a further increase in signal to noise ratio. Further increases of sensitivity will therefore need other types of enzyme immunoassays, e.g., sandwich enzyme immunoassays or sandwich transfer immunoassays . The recent isolation of a myotropin from the male accessory glands from Locusfa, and the elucidation of its structure as [Ala', Leu7, Ser8]-allatotropin  and the detection described here of an allatotropin-immunoreactive peptide in Peviplaneta, suggests that related peptides are generally present in insects. The structures of several insect neuropeptides have been sufficiently preserved during evolution to be biologically active in other insect species. Furthermore, the members of the rapidly evolving adipokinetic hormone family are very often physiologically active in other species . In contrast, the allatotropin from Manduca does not stimulate juvenile hormone biosynthesis in cockroaches, locusts, or the beetle Teizebrio nzolitor IS]. The function of the allatotropin-immunoreactive peptide in Periplmeta remains therefore to be elucidated. The widespread distribution in the nervous system of both Manduca and Periplaneta suggests that these peptides may have more than one function. Fig. 3 . Chromatograms of ventral nerve cords (A), brains (B), and retrocerebral complexes (C)of 10 adult Periplarieta arnericma. Tissues were prepurified on a C," Sep-Pak and separated by HPLC as described in Materials and Methods. Acetonitrile gradient as indicated in D. Aliquots of 1 rnl fractions were assayed in the allatotropin-ELISA and the amount of allatotropin-irnmunoreactivity calculated per fraction as described in Results. Short arrows in A, 6, C, and D indicate the elution time of synthetic Locusta myotropin, while the long arrow in D indicates the elution time of synthetic Manducd allatotropin in which both methionines are in the reduced form. 108 Veenstra and Hagedorn LITERATURE CITED 1. Khan MA: Brain-controlled synthesis of juvenile hormone in adult insects. Entomol Exp Appl46, 3 (1988). 2. Woodhead AP, Stay B, Seidel SL, Khan MA, Tobe SS: Primary structure of four allatostatins: Neuropeptide inhibitors of juvenile hormone synthesis. Proc Natl Acad Sci USA 86, 5997 (1989). 3. Pratt GE, Farnsworth DE, Siegel NR, Fok KF, Feyereisen RE: Identification of an allatostatin from adult Diphptera punctata. Biochem Biophys Res Comrnun 163, 1243 (1989). 4. Pratt GE, Farnsworth DE, Fok KF, Siegel NR, McCormack AL, Shabanowits J, Hunt DF, Feyereisen RE: Identity of a second type of allatostatin from cockroach brains: An octadecapeptide amide with a tyrosine-rich address sequence. 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