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Microtitre plate enzyme immunoassay of oestradiol-17β
133 ETERMINATS~NOF DEXAMETHASONE (DXM) LEVELSIN
PLASMA BY HPLCANDRIA; Lejeune-Lenain,CX;Bosson, DX;Beyloos, M*;Copinschi, G+; Mascart, PhX; Franckson, J.R.MX. DeFree University of partments of Medical ChemistryXand Internal Medicine*; St PierreHospital; Brussels; BELGIUM. to measure with precision low DXMconcentrations in routine laThe method was developed boratory practice, in spite of the poor specificity of commercially available antisera. It uand especially its correct ses a fully-automated HPLCprocedure which allows DXMisolation separation from cortisol, major interference in RIA. Plasma (250 ~1 or less) was extracted by a mixture of (hexane : ethylacetate) (3 : 7)(V : V) and chromatographied on a lichrospher-diol Eluates were collected on a precolumn using a (hexane : 2-propanol)(88 : 12) mobile phase. Radioprogrammed fraction collector. RIA was performed on aliquots of the selected fraction. active corticosterone was added to each sample to monitor the procedural losses. Lowest detectable amount averaged 0.1 ng/ml. Linearity (logit-log transform) was achieved and intraassay coefficients of variation up to 10 ngfml fSb=7%).BetWeen 0.5 and 5 ng/ml,interfell to 5X or less. Omission of the chromatographic step prior to RIA raised the lowest detectable amount from 0.1 to 1.0 ng/ml. Within the Z-10 ng/ml concentrations range, a tight correlation
was observed with non chromatographic techniques. Owing to these characteristics, the present method appears particularly useful to monitor plasma DM levels during DXMsuppression tests or treatments and to compare them with those of adrenal hormones. Indeed in these tests,plasma samplesintendedto estimateadrenal inhibition (detection of Gushing’s syndrome) or 17 hrs (detection of rate are usually collected 10 hrs primary depression) after oral administration of 1 mg DXM. Under these conditions, plasma DXM levels ranged between 3 and 0.5 ng/ml, i.e. in the accurate part of the present assay.
_
134
IMPROVFMENT OF ANTIBODYBINDINGOF IODINAW EsrTw)IOL TRACERS for Reactor Research; lingen; ~TZ~
Tiefenauer,L; Frei, W; Andres,R; Swiss Federal Institute The chemical
bridge
5303 Wiiren-
linking
hapten molecule with the carrier protein is often recognizedby the is the cause that iodinated tracers bearing a ~logous bridge are nxore avidily hound than the snalyte itself. This results in a reduced sensitivity of the radio’ -say (RIA). Previously we have denrnnstxated that this pr&lem can be solved by shortening the bridge in the jantigen (1). Carrie et al. have altered the bridge in a progesterone tracer and thereby increas-
antibody.This so calledbridge effect
kd
the sensitivity of the RIA dramatically12). /Wehave tested two differentestradiol tracers (estradiol-6-(O-carbcarymethyl)axime-[125~)-iodo[histamine and estradiol-6-amido-[ 1251]-iodo-p-hydroxyphenylpropionic acid) with 40 antibodies producedagainst an estradiol-6(C-carboxyrsathyl )oxime-bovine serum albumine conjugate. Titer, sensitivityand slope of the standardcurve were caqxired. We conclude,that an insensitiveRI.Afor estradiolc&n be improvedby alteringthe bridge in the tracer even when the stereccheznistry at the site of attachersent changes.
(1) Tiefenauer,L; Andres,R; J. Imnunol.Ivktthods 74, 293-298 (1984) 12) Corrie,J et al.; Clin. Chem. -27, 594-599 (198E
135 ‘I?iCROT~RE~~%i%E
IMMUNOASSAY OF OESTRADIOL176,; Station de Physiologie Laurel, M.C.; Labrouaae, Ii. ; Terqui, M; Avrameas, S. tion, I.N.R.A., 37380 Nouzilfy; and lLaboratoire d’Immunocytochimie, Inetitut 28 rue du Dr. Roux, 75015 Paris; FRANCE
de la ReproducPasteur,
A rapid and sensitive solid phase microtitre plate enzyme immunoassay for oestradiol-176 (E2) has been established using fluorescence detection a6 the end-point. The tracer is an E2-6-carboxymethyl oxime-8 galactoaidaae conjugate. Sheep antiserum was raised against an E -6 CMO-BSAconjugate and the IgG fraction purified by DEAEcolumn chromatography. This IgG frac e* ion was passively adeorbed onto the surface of wells of Luxlon microtitre plates. After washing, a two-step8 procedure was used. First, E2 samples in wells were evaporated to dryness, then the tracer E -8 galactoaidase was added and incubated for 1 l/2 hours at +37’C. Unbound E, and E -8 galact 8aidaee were eliminated by washinf and the 4_methylumbellifcryl@-galactoside (4%RJG) wk used aa the substrate with a reaction time of 2 H at +42*C. The fluorescence intensity of the 4 MU formed waa determinated using an automatic microtitre plate reader. This assay was very sensitive since 0,l pglwell wae detectably differentfrom 0 p&/well (2081585absolute fluorescence units (f.u.) ~6 2407f150 f.u. respectively; m+sd). The imeasurable range was from 0,l pg to 10 ng/well. Intra and inter-assay coeffitient of variation were 2.5-8.13 (n-4) and 6+7X (n-4) respectively at the 0,l pg/well level. Thus this technique imay be able to measure very low levels of oestkadiol-178 in, for example, the blood of idomestic animals.
I
__-_--__48s
PLASMA BY HPLCANDRIA; Lejeune-Lenain,CX;Bosson, DX;Beyloos, M*;Copinschi, G+; Mascart, PhX; Franckson, J.R.MX. DeFree University of partments of Medical ChemistryXand Internal Medicine*; St PierreHospital; Brussels; BELGIUM. to measure with precision low DXMconcentrations in routine laThe method was developed boratory practice, in spite of the poor specificity of commercially available antisera. It uand especially its correct ses a fully-automated HPLCprocedure which allows DXMisolation separation from cortisol, major interference in RIA. Plasma (250 ~1 or less) was extracted by a mixture of (hexane : ethylacetate) (3 : 7)(V : V) and chromatographied on a lichrospher-diol Eluates were collected on a precolumn using a (hexane : 2-propanol)(88 : 12) mobile phase. Radioprogrammed fraction collector. RIA was performed on aliquots of the selected fraction. active corticosterone was added to each sample to monitor the procedural losses. Lowest detectable amount averaged 0.1 ng/ml. Linearity (logit-log transform) was achieved and intraassay coefficients of variation up to 10 ngfml fSb=7%).BetWeen 0.5 and 5 ng/ml,interfell to 5X or less. Omission of the chromatographic step prior to RIA raised the lowest detectable amount from 0.1 to 1.0 ng/ml. Within the Z-10 ng/ml concentrations range, a tight correlation
was observed with non chromatographic techniques. Owing to these characteristics, the present method appears particularly useful to monitor plasma DM levels during DXMsuppression tests or treatments and to compare them with those of adrenal hormones. Indeed in these tests,plasma samplesintendedto estimateadrenal inhibition (detection of Gushing’s syndrome) or 17 hrs (detection of rate are usually collected 10 hrs primary depression) after oral administration of 1 mg DXM. Under these conditions, plasma DXM levels ranged between 3 and 0.5 ng/ml, i.e. in the accurate part of the present assay.
_
134
IMPROVFMENT OF ANTIBODYBINDINGOF IODINAW EsrTw)IOL TRACERS for Reactor Research; lingen; ~TZ~
Tiefenauer,L; Frei, W; Andres,R; Swiss Federal Institute The chemical
bridge
5303 Wiiren-
linking
hapten molecule with the carrier protein is often recognizedby the is the cause that iodinated tracers bearing a ~logous bridge are nxore avidily hound than the snalyte itself. This results in a reduced sensitivity of the radio’ -say (RIA). Previously we have denrnnstxated that this pr&lem can be solved by shortening the bridge in the jantigen (1). Carrie et al. have altered the bridge in a progesterone tracer and thereby increas-
antibody.This so calledbridge effect
kd
the sensitivity of the RIA dramatically12). /Wehave tested two differentestradiol tracers (estradiol-6-(O-carbcarymethyl)axime-[125~)-iodo[histamine and estradiol-6-amido-[ 1251]-iodo-p-hydroxyphenylpropionic acid) with 40 antibodies producedagainst an estradiol-6(C-carboxyrsathyl )oxime-bovine serum albumine conjugate. Titer, sensitivityand slope of the standardcurve were caqxired. We conclude,that an insensitiveRI.Afor estradiolc&n be improvedby alteringthe bridge in the tracer even when the stereccheznistry at the site of attachersent changes.
(1) Tiefenauer,L; Andres,R; J. Imnunol.Ivktthods 74, 293-298 (1984) 12) Corrie,J et al.; Clin. Chem. -27, 594-599 (198E
135 ‘I?iCROT~RE~~%i%E
IMMUNOASSAY OF OESTRADIOL176,; Station de Physiologie Laurel, M.C.; Labrouaae, Ii. ; Terqui, M; Avrameas, S. tion, I.N.R.A., 37380 Nouzilfy; and lLaboratoire d’Immunocytochimie, Inetitut 28 rue du Dr. Roux, 75015 Paris; FRANCE
de la ReproducPasteur,
A rapid and sensitive solid phase microtitre plate enzyme immunoassay for oestradiol-176 (E2) has been established using fluorescence detection a6 the end-point. The tracer is an E2-6-carboxymethyl oxime-8 galactoaidaae conjugate. Sheep antiserum was raised against an E -6 CMO-BSAconjugate and the IgG fraction purified by DEAEcolumn chromatography. This IgG frac e* ion was passively adeorbed onto the surface of wells of Luxlon microtitre plates. After washing, a two-step8 procedure was used. First, E2 samples in wells were evaporated to dryness, then the tracer E -8 galactoaidase was added and incubated for 1 l/2 hours at +37’C. Unbound E, and E -8 galact 8aidaee were eliminated by washinf and the 4_methylumbellifcryl@-galactoside (4%RJG) wk used aa the substrate with a reaction time of 2 H at +42*C. The fluorescence intensity of the 4 MU formed waa determinated using an automatic microtitre plate reader. This assay was very sensitive since 0,l pglwell wae detectably differentfrom 0 p&/well (2081585absolute fluorescence units (f.u.) ~6 2407f150 f.u. respectively; m+sd). The imeasurable range was from 0,l pg to 10 ng/well. Intra and inter-assay coeffitient of variation were 2.5-8.13 (n-4) and 6+7X (n-4) respectively at the 0,l pg/well level. Thus this technique imay be able to measure very low levels of oestkadiol-178 in, for example, the blood of idomestic animals.
I
__-_--__48s