A simple and sensitive radioimmunoassay of insect Juvenile hormone using an iodinated tracer

Baehr, J.C.; Pradelles, P.; Lebreux, C.; Cassier, Pierre; Dray, F.

doi:10.1016/0014-5793(76)80667-9

Cited by 40 publications

(17 citation statements)

References 8 publications

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“…Moreover, the conjugate (10R) JH 111-TG did not induce an enantiomerically directed antiserum. Curiously, no antisera cross-reacted with (10R) JH I11 acid; in previous studies with similarly conjugated immunogens, JH and JH acids displayed identical displacement activity (Baehr et al, 1976;Kimura et al, 1989). *One microliter of [10-3H]-JH I (12,000 dpm) was placed in conical vials (n = 3) and 1 ml of the appropriate solvent added.…”

Section: Discussionmentioning

confidence: 93%

Recent advances in radioimmunoassay technology for the juvenile hormones

Goodman

Orth

Toong

et al. 1995

Arch Insect Biochem Physiol

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Recent refinements in juvenile hormone radioimmunoassay technology now make this method significantly more sensitive and easier to use. Rabbit polyclonal antisera against (10R) JH III and racemic JH II have been developed to determine hemolymph hormone titers in the low picogram range. The antisera display minimal cross-reactivity with JH metabolites, JH analogs, and hemolymph lipids. One antiserum recognizes racemic JH I, II, and (10R) III almost equivalently, exhibiting 50% displacement between 100 and 130 pg per tube. Another antiserum is JH II-specific and exhibits 50% displacement at 35 pg per tube. Assay sensitivity has been enhanced by using (10R,11S) [methyl-3H]-JH II of very high specific activity (> 80 Ci/mmol) generated with Hyalophora cecropia accessory gland S-adenosylmethionine transferase and S-[methyl-3H]-adenosyl-L-methionine. Preparation of biological samples has been simplified with overall recoveries of JH from hemolymph ranging between 60 and 75%.

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Section: Discussionmentioning

confidence: 93%

Recent advances in radioimmunoassay technology for the juvenile hormones

Goodman

Orth

Toong

et al. 1995

Arch Insect Biochem Physiol

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“…When the RIA for JH, was being developed, each step in the preparation of the immunogen and the iodinated tracer could be controlled by using a tritiated JH 1 tracer (JH,,NEN,13 Ci/mM) (Baehr et al, 1976 (Giese, Spindler and Emmerich, 1977). This adsorption was reduced in our experiments by carrying out the assay in an aqueous medium using a 0.02M phosphate buffer containing 0.1 p.100 of BSA (fraction V).…”

Section: Methods and Resultsmentioning

confidence: 99%

“…Attemps to improve RIA sensitivity and specificity. -A study of the cross reactions of the antiserum directed against JH 1 showed that the JH 1 -histamine complex had a cross-reaction of 210 p. 100 as compared with the JH, (Baehr et al, 1976). The greater affinity for the iodinated tracer than for the JH, resulted in an RIA of lower sensitivity than that which could be achieved using a tracer of lower affinity but with the same specific radioactivity.…”

Section: Methods and Resultsmentioning

confidence: 99%

“…1) (Lauer et al, 1974), which used a tritiated tracer, was not sufficiently sensitive to measure the low levels of hormone present. We have developed a RIA for JH 1 (Baehr et al, 1976), and more recently for JH, and JH,, using high specific activity l2s l_labelled tracers. The present work describes the preparation of the immunogens and the iodinated tracers, the methods used for the assays, and their sensitivity and specificity.…”

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A radioimmunological assay for naturally occurring insect juvenile hormones using iodinated tracers : Its use in the analysis of biological samples

Baehr¹,

Pradelles²,

Dray³

1979

Ann. Biol. anim. Bioch. Biophys.

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Radioimmunological assays (RIA) are useful tools in investigative research, and are commonly used in the field of vertebrate physiology. In insect physiology,radioimmunoassays for ecdysteroids are used routinely in many laboratories. However, the first RIA described for JH's ( fig. 1) (Lauer et al., 1974), which used a tritiated tracer, was not sufficiently sensitive to measure the low levels of hormone present. We have developed a RIA for JH 1 (Baehr et al., 1976), and more recently for JH, and JH,, using high specific activity l2s l_labelled tracers. The present work describes the preparation of the immunogens and the iodinated tracers, the methods used for the assays,

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“…However, the cross reactivity of the JH antisera for their respective JH acids is well documented [18,19] From the above considerations we hypothesized that prepupal CA secrete JH acid which is then taken up and methylated to JH in target tissues and subsequently released into the hemolymph [16]. Two aspects of this hypothesis were tested, and the results are reported in the present paper.…”

Section: Introductionmentioning

confidence: 93%

Juvenile hormone acid methyltransferase activity in imaginal discs of Manduca sexta prepupae

Sparagana

Bhaskaran

Barrera

1985

Arch Insect Biochem Physiol

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The occurrence of a peak of juvenile hormone (JH) during the prepupal period has been noted in several lepidopterans. In Manduca sexta and Hyalophora cecropia this peak is known to prevent the precocious onset of adult differentiation in imaginal tissues. However, it has previously been observed in our laboratory that corpora allata (CA) of this age are incapable of making JH owing to a lack of the terminal synthetic enzyme, juvenile hormone acid methyltransferase (JHAMT). Since the CA are required for normal pupation, it is likely that JH acid is the product released by the prepupal CA. Therefore, we analyzed whether JH acid treatment would prevent precocious adultoid differentiation in allatectomized Msexta larvae.J H acid injections were found to be as effective as JH in normalizing pupation, and acted in a time-and dose-dependent manner. This finding led to a question of whether injected or endogenous JH acid could be methylated to JH. Homogenates of several tissues from prepupae were assayed for the presence of JHAMT. Of the tissues assayed, only imaginal discs possessed significant levels of the enzyme. These results support our previously proposed mechanism for production of the prepupal JH peak in M. sexta.Key words: juvenile hormone acid methyltransferase, juvenile hormone acid, juvenile hormone, imaginal disc, Manduca sexta Acknowledgments: We thank Drs. Gijnter Weirich and Judith Willis for reviewing the manuscript. We gratefully acknowledge the advice of Dr. Karl H. Dahm during the course of this work. This work was supported by Organized Research at Texas A&M University. This paper is a portion of the Master of Science thesis of Steven P. Sparagana.*Abbreviations: accessory sex glands = ASG; corpora allata = CA; hi h pressure liquid chromatography = HPLC; juvenile hormone = JH; juvenile hormone acicf methyltransferase = JHAMT; radioimmunoassay = RIA; S-adenosylmethionine = AdoMet; S-benzyl-O-ethyl phosphorarnidothiolate = BEPAT; thin-layer chromatography = TLC.

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A simple and sensitive radioimmunoassay of insect Juvenile hormone using an iodinated tracer

Cited by 40 publications

References 8 publications

Recent advances in radioimmunoassay technology for the juvenile hormones

Recent advances in radioimmunoassay technology for the juvenile hormones

A radioimmunological assay for naturally occurring insect juvenile hormones using iodinated tracers : Its use in the analysis of biological samples

Juvenile hormone acid methyltransferase activity in imaginal discs of Manduca sexta prepupae

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