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Sensitive enzyme immunoassay for Manduca allatotropin and the existence of an allatotropin-immunoreactive peptide in Periplaneta americana.

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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
Key wnrds: juvenile hormone, allatotropin, myotropin, ELISA, Manduca sexta, f ocusta
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.
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
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.
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).
Two milligrams of purified allatotropin (see above) were coupled to 5 mg
of thyroglobulin using glutaraldehyde as described [8]. 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
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.
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. [12]. 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
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 [13] 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.
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
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. [12]. 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-
Veenstra and Hagedorn
60 min
60 min
60 min
Figure 2 .
ELISA for Manduca Allatotropin
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.
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.
allatot ropin
cpmoi I
+*I \
ELISA for Manduca Allatotropin
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 [20], 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 [21]. This can be increased slightly either by using fluorescent
substrates [22], 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 [23]. 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 [24].
The recent isolation of a myotropin from the male accessory glands from
Locusfa, and the elucidation of its structure as [Ala', Leu7, Ser8]-allatotropin
[7] 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 [25]. 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
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.
Veenstra and Hagedorn
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