James R. Soares

Direct redioimmune assay of 16-glucosiduronate metabolites of estriol in human plasma and urine

It has been established that the 16-glucosiduronate metabolites of estriol comprise the major estriol component in pregnancy plasma [ 1 ] , urine [ 1,2] and amniotic fluid [3]. However, little is known about their physiological or pathological correlations. One reason is the fact that assays for individual metabolites of estriol either do not exist or are unsuitable for practical processing of large numbers of clinical samples. The possibility that immune assays might provide a useful means of measuring individual estriol metabolites prompted us to develop and investigate various antigens. A non-specific 16a-glucosiduronate antiserum has been reported [4]. This report describes radioimmune experiments in which picogram quantities of the 16-glucosiduronate metabolites of estriol were measured in the presence of vast excesses of estriol, other estrogens and/or their metabolites. Antibody specificity together with the natural preponderance of the 16-glucosiduronate metabolites made possible their direct measurement in plasma and urine.

Separate radioimmune measurements of body fluid Δ9-THC and 11-nor-9-carboxy-Δ9-THC

Abstract Simultaneous native molecule and discrete metabolite immune assays were performed after exposure of subjects to standardized Δ9-THC cigarettes. Plasma (and urine) 11-nor-9-carboxy-Δ9-THC remains elevated long after Δ9-THC becomes scant or undetectable enabling simple radioimmune determination of recent versus distant exposure to multiple cigarettes.

MEASUREMENT OF DELTA 9-TETRAHYDROCANNABINOL (THC) IN WHOLE BLOOD SAMPLES FROM IMPAIRED MOTORISTS

The major psychoactive cannabinoid in marihuana, delta 9-tetrahydrocannabinol (THC) was measured in 1792 randomly selected blood specimens from erratic motorists arrested for impairment who submitted to blood alcohol sampling. Of these specimens, 14.4% were positive for THC (greater than or equal to 5.5 ng/mL). In those erratic driver specimens negative for alcohol THC positives rose to 23%. Drivers who used marihuana covered a broad age range. Aliquots of hemolyzed blood (10 microL) were analyzed by a sensitive radioimmunoassay (RIA) not requiring extraction. RIA accuracy and specificity were validated by gas liquid chromatography/mass spectroscopy (GLC/MS) split pair analysis (correlation coefficient = 0.93). This initial experience should facilitate and amplify a program designed to set forth the epidemiology of marihuana use in motorists and possible behavioral correlates.

Radioimmunoassay of estriol-16-glucuronide using tritiated and radioiodinated radioligands: direct radioimmunoassay of urinary estriol-16-glucuronide during the menstrual cycle.

Abstract Estriol-16-glucuronide-[ 125 I]-iodohistamine (E 3 -16G-[ 125 I]) was prepared and utilized in a radioimmunoassay (RIA) in conjunction with anti-estriol-16-glucuronide-bovine serum albumin (E 3 -16G-BSA) serum (RIA No. 1). This RIA was then compared with two other RIA methods employing [ 3 H]-labeled estriol-16-glucuronide (E 3 -16G) together with either anti-E 3 -16G-BSA serum (RIA No. 2) or antiestriol-16-glucuronide-2-azobenzoic acid-bovine serum albumin (E 3 -16G-2-ABA-BSA) serum (RIA No. 3). All three RIAs were accurate and precise, however, they differed in assay sensitivity and specificity. Most sensitive was RIA No. 1 and least sensitive was RIA No. 3. Both RIAs No. 1 and 2 were less specific than RIA No. 3 since unconjugated estriol and estrone-3-sulfate exhibited large and comparable cross-reactions in RIAs No. 1 and 2, averaging 50% and 41%, respectively. Furthermore, measurement of E 3 -16G in 10 different urine aliquots collected from non-pregnant women employing RIAs No. 1–3 showed that RIAs No. 1 and 2 yielded comparable results, however, the results obtained by RIA No. 3 were, on the average, 26% lower than the mean of the values measured with RIAs No. 1 and 2. Consequently, RIA No. 3 was used to measure daily 24-hour urinary E 3 -16G excretion in 7 women throughout an entire menstrual cycle. These data are in agreement with colorimetric and fluorimetric estimates of 24-h urinary estriol values throughout the menstrual cycle. The preovulatory rise of urinary E 3 -16G excretion, as quantitated by this RIA, is comparable to that of serum estradiol measured in the same 7 women, but peaks 1 to 2 days later than the serum estradiol surge.

Separation of high affinity hapten specific and crossreacting IgG populations

Abstract Antisera to an azohapten (2-(4′-carboxyphenylazo)-estriol) contains several IgG populations indistinguishable by their association constants for estriol—the homologue. However, significantly different relative affinities for a slightly altered hapten (estriol-3-sulfate) were shown. Immunosorption of crude antisera with an estriol-3-glucuronide-aminoethyl cellulose sorbant selectively removed antibody populations with highest affinity for haptens containing a blocked C-3 phenolic function. The residual antibody pool contained IgG with highest specificity for unaltered hapten.

Antibody competition for plasma protein-bound estriol.

Plasma unconjugated estriol (E3) is now a more generally accepted indicator of feto-placental function than urinary E3-metabolite excretion [1,2]. Of the numerous methods which have been used to measure estrogens only radioreceptor assay (RRA) and especially radioimmune assay (RIA) easily quantitate estriol. Most current methods utilize ether extracts of nonpolar plasma estrogens. Assays performed with nonspecific antisera or steroid binding plasma protein (SBP) require chromatography of plasma extracts to .separate estriol (E3) from estradiol (E2) and estrone (Et) [2,3]. We have described specific azoestriol antiserum for direct plasma E3 assay which reliably measures E3 even in the presence of El, E2 and E3 metabolites [4,5]. It had previously been th9ught that direct plasma E3 assay might not accurately quantitate serum E3 because most plasma estriol is protein bound [6,10]. The present report describes quantitative experiments in which high affinity specific estriol antibody overwhelms binding of E3 by non-specific serum proteins, notably steroid binding globulin (SBG).