Determination of ecdysteroid titers by a double-antibody precipitation in the tropical warehouse moth, Ephestia cautella (Lepidoptera, phycitidae)

Lazarovici, Philip; Shapira, D; Shaaya, Eli

doi:10.1016/0300-9629(83)90542-x

Cited by 10 publications

(3 citation statements)

References 22 publications

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“…Also, an ecdysteroid peak of about 10 nglg seems to appear shortly prior to the prepupal stage. However, the appearance of this peak is strongly dependent on the synchronization status of the insect's batch, and it was not detected in our previous study [8]. So, we have to assume that this peak is released during a short period of time.…”

Section: Ecdysteroid Titers In Control and Methoprene-treated Larvaementioning

confidence: 64%

“…A double-antibody RIA as described previously by Lazarovici et a1 [8] was used to measure the ecdysteroid level, The difference between this method and the single-antibody RIA [9] lies in the means of separating the antigenantibody complex. In the double-antibody RIA, the desalting step by ammo-nium sulphate was omitted and the antigen-antibody complex was precipitated by goat antirabbit IgG.…”

Section: Extraction and Measurement Of Ecdysteroidsmentioning

confidence: 99%

“…All experiments were performed in duplicate, using different concentrations. Standard curves and evaluation of RIA data were performed as described previously by Lazarovici et a1 [8] . For measuring the radioactivity each sample was placed in a scintillation vial together with 200 pl toluene (Packard, Holland) and 4 ml scintillation fluid (4 g PPO, 0.4 g POPOP in 1 liter toluene).…”

Section: Extraction and Measurement Of Ecdysteroidsmentioning

confidence: 99%

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Ecdysteroid level and the change in sensitivity to a juvenile hormone analogue in Ephestia cautella larvae (Lepidoptera, Phycitidae)

Lazarovici

Shapira

Pisarev

et al. 1984

Arch Insect Biochem Physiol

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A dou ble-antibody ecdysone-specific radioimmunoassay was used to clarify whether the effects on metamorphosis of the juvenile hormone analogue methoprene are correlated with changes in ecdysteroids level. It appears that a small ecdysteroids peak, 5 days before pupation, i s responsible for the transition from inhibition to defective metamorphosis. Study of the changes in ecdysteroid titer in last-instar larvae treated with the JHA 2 days prior to the appearance of the above small ecdysteroids' peak showed an immediate reduction in ecdysteroid level, followed by cyclic, successively reduced titer for about 20 days. After this period the larvae ceased to feed and entered to a diapauselike stage which ended in the death of the larvae. A similar effect on ecdysteroid titer and developmental arrest was exhibited by JHA-treated first-instar larvae. The mechanism of the interactions between JHA and ecdysteroid level deserves further investigation.

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Section: Ecdysteroid Titers In Control and Methoprene-treated Larvaementioning

confidence: 64%

Section: Extraction and Measurement Of Ecdysteroidsmentioning

confidence: 99%

Section: Extraction and Measurement Of Ecdysteroidsmentioning

confidence: 99%

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Ecdysteroid level and the change in sensitivity to a juvenile hormone analogue in Ephestia cautella larvae (Lepidoptera, Phycitidae)

Lazarovici

Shapira

Pisarev

et al. 1984

Arch Insect Biochem Physiol

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Radioimmunoassay: Ecdysteroids

Warren¹,

Gilbert²

1998

Springer Series in Experimental Entomology

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Simplification of the ecdysteroid radioimmunoassay by the use of protein A fromStaphylococcus aureus

1984

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Summary. Three methods of separating antibody-bound ligand from free ligand were compared for an ecdysteroid radioimmunoassay. Ecdysteroid antibody concentration and ligand specific radioactivity were adjusted to measure 2 ranges of ecdysone concentrations (0.0t-2.0 ng and 0.25 32.0 ng). In comparison with the traditional separating agent, ammonium sulfate, neither protein A nor polyethylene glycol altered sensitivity or specificity of the radioimmunoassays. The protein A immunoglobulin precipitation method is quick and simple, making it a preferred alternative protocol for terminating ecdysteroid radioimmunoassays.One of the principal methodological advances in the field of insect endocrinology was the development of the ecdysteroid radioimmunoassay (RIA) by Borst and O'Connor 2. This RIA has resulted in a plethora of papers in which insect molting hormone has been quantified during specific developmental stages, e.g. in Manduca sexta 3, Calpodes ethlius 4. In addition, it has proven useful in identifying ecdysteroids collected from high performance liquid chromatography (HPLC) s and has resulted in the development of a specific and reproducible in vitro assay for the insect prothoracicotropic hormone ~. Separation of antibody-bound from free ligand for the ccdysteroid RIA has been achieved in various ways including equilibrium dialysis 7, double-antibody precipitation ~, gel filtration 9, dextran-coated charcoal m, and ammonium sulfate 2. Due to the high cost and/or labor requirements of the first 3 of these methods, and the adverse effects of charcoal absorption on antibody ligand equilibria l~, ammonium sulfate precipitation is most commonly empToyed as a separating agcnt for ecdysteroid RIA. This method, however, is time-consuming for large scale processing of samples, such as has recently become necessary in our laboratory following HPLC of biological material. In an attempt to further simplify the ecdystcroid RIA, we have tested 2 additional methods of scparating bound from free ligand: polyethylene glycol (PEG) 6000, recently reported to be of use for separating bound from free eedysteroids u, and immobilized protein A, currently in common use for vertebrate peptide radioimmunoassay .3 ts. Materials and methods. Ecdysone and 20-hydroxyecdysone were purchased from Rohto and Sigma while [3H]-ecdysone (~ 60 Ci/mmol) was obtained from New England Nuclear Corp. The latter was repurified by HPLC when necessary. 3-Epi-ccdysonc was a generous giI't from Drs J. Svoboda and M. Thompson

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Determination of ecdysteroid titers by a double-antibody precipitation in the tropical warehouse moth, Ephestia cautella (Lepidoptera, phycitidae)

Cited by 10 publications

References 22 publications

Ecdysteroid level and the change in sensitivity to a juvenile hormone analogue in Ephestia cautella larvae (Lepidoptera, Phycitidae)

Ecdysteroid level and the change in sensitivity to a juvenile hormone analogue in Ephestia cautella larvae (Lepidoptera, Phycitidae)

Radioimmunoassay: Ecdysteroids

Simplification of the ecdysteroid radioimmunoassay by the use of protein A fromStaphylococcus aureus

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