Monoclonal antibodies to pregnanediol-3-glucuronide: application to a direct enzyme-linked immunosorbent assay of urine John G. Lewis, Janet K. Clifford, and Peter A. Elder Steroid Unit, Department New Zealand

of Clinical





Monoclonal antibodies to pregnanediol-3-glucuronide were produced and characterized. One of three clones investigated provided antibody suitable for a direct urinary enzyme-linked immunosorbent assay (ELISA). The ELISA uses a pregnanediol-thyroglobulin conjugate adsorbed onto the wells of a standard 96well microtiter plate. Pregnanediol-3-glucuronide in standards or diluted urine competes with the immobilized steroid for antibody-binding sites. After washing, mouse monoclonal antibody bound to the plate is probed with antimouse immunoglobulin peroxidase. After further washing, o-phenylenediamine substrate is added and, @ally, the absorbance is read at 492 nm. The ELISA shows excellent performance and agreement with the previous gas chromatographic method. The ELISA is ideal for aiding the assessment of ovarian function in the routine laboratory. (Steroids 55:314-318, 1990)


steroids; ELISA; pregnanediol-3-glucuronide;

Introduction Measurement of the excretion of pregnanediol-3-glucuronide, the major metabolite of progesterone in urine, has proved useful and valid for detecting ovulation’s* and aiding in the definition of the fertile/infertile stages for natural family planning,3 as well as assisting in the documentation of the transition to menopause.4 Although radioimmunoassays5,6 and other immunoassays’s8 have been reported for pregnanediol-3-glucuronide, this metabolite was previously measured in our hospital using a laborious gas chromatographic method.9 The introduction of enzyme-linked immunosorbent assay (ELISA) and monoclonal antibody technology to our laboratory, for steroid hormone assays, has many advantageslO*l* and should also lead to efficiency gains in a pregnanediol-3gIucuronide ELISA-not only over the existing gas chromatographic method, but also over other immunoassaysdue to the ease of automation, the avoidance of radia-

Address reprint requests to Dr. John G. Lewis, Steroid Unit, Department of Clinical Biochemistry, Christchurch Hospital, Christchurch, New Zealand. Received September 13, 1989; accepted January 22, 1990.



1990, vol. 55, July

monoclonal antibodies

tion hazards, and less complex synthetic reactions. We report the production and characterization of monoclonal antibodies to pregnanediol-3-glucuronide and the development of a direct semiautomated urinary ELISA using an immobilized steroid-thyroglobulin conjugate.

Experimental Materials Steroids were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and all other chemicals were of analytic grade. 20a-Hydroxy-SP-pregnan-3-one was used as starting material for the synthesis of pregnanediol3-(0-carboxymethyl)oxime,l* which was then coupled to bovine thyroglobulin by the mixed anhydride method.i3 This method was also used to couple pregnanediol-3-glucuronide to bovine serum albumin (BSA). Goat antimouse Ig-peroxidase and the mouse monoclonal antibody isotyping kit were from Amersham Corp. (Amersham, UK).

Immunization and cell fusion Four male RBF/DN mice were each immunized intraperitioneally with 10 to 100 pg of pregnanediol-3-glu0 1990 Buttetworth-Heinemann

Lewis et al.

hlonoclonal antibodies to pregnanediol-3-glucuronide:

curonide-BSA in SAFl adjuvant14 at four weekly intervals. Seven days after the fourth injection, spleens were aseptically excised and the splenic lymphocytes fused with FOX-NY mouse myeloma cells at a ratio of 5 : 1, respectively, using 50% polyethylene glycol and Taggart Hybridoma Technology*5 from Hyclone Laboratories (Logan, UT, USA). After fusion, the cells were resuspended to 1OVml in RPM1 containing 10% fetal calf serum (v/v), 2 mM rghnamine, and AAT (adenine/aminopterin/thymidine; 75 : 0.08 : 16 x 10V6M, respectively). They were plated at 1oZcells/well in 96-well culture plates and grown under standard conditions with regular media changes. Positive hybrids were cloned twice by limiting dilution using RBF/DN spleen cells as a feeder layer (100 x l@/well). After cloning, the cells were grown in 50-ml culture flasks and supernatants centrifuged before use. Immunoglobulin class and subclass were determined using the isotyping kit. Screening of supernatants ELISA plates (Falcon 3912 Microtest III, Becton Dickinson, USA) were coated overnight at 4°C with 100 ~1 of pregnanediol-thyroglobulin conjugate/well (500 ng/ml) in aqueous 6 M guanidine hydrochloride. The plates were then washed four, times with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (v/v) and blocked with assay buffer (150 @well) for 1 hour at 20°C. Assay buffer was PBS containing 0.1% Tween 20 (v/v) and 0.1% gelatin (w/v). The plates were emptied, 50 ~1 of assay buffer was added to each well followed by 50 ~1 of supernatant, and the plates were incubated for 2 hours at 20°C. The plates were then washed four times, and goat antimouse Igperoxidase was added (100 pi/well at 1: 2,000 in assay buffer) for 1 hour at 20°C. The plates were finally washed and substrate was added. Substrate solution was freshly prepared from 40 mg o-phenylenediamine in 100 ml of 50 mM Na2HPQ and 25 mM citric acid buffer, pH 5.0. Immediately before use, Hz02 (60 ~1) was added. Color development was terminated by the addition of 100 ~1 of 1.25 M H#04/well and the absorbance read at 492 nm. Dispensing steps (with the exception of samples and standards), washing, and photometry were carried out using the Behring ELISA Processor II (Hoechst, FRG). Cross-reactivity

assay buffer. Occasionally, samples required further dilution for interpolation on the standard curve. Seven different doses of standard were also prepared in assay buffer (0 to 250 pg/50 4). After the addition of samples (50 ~1) or standards (50 ~1) to the wells of a pregnanediol-thyroglobulin-coated and blocked plate, supematant from clone CT diluted 1: 200 in assay buffer was added to each well (50 ~1) for a 2-hour incubation at 20°C. The plate was then washed, antimouse Ig-peroxidase was added (1: 2,000 in assay buffer), and the plate was further processed as described. Recovery was carried out by the addition of increasing doses of pregnanediol-3glucuronide to normal male urine, which was subsequently assayed. Pregnanediol-3-glucuronide was also assayed using the gas chromatographic method described by MetcaKg

Rl?SUltS Screening and subsequent cloning resulted in three clones secreting antibody to pregnanediol3glu curonide worthy of further investigation. Cross-reactivity profiles of these antibodies, calculated at 50% displacement, are shown in Table 1. Supematant derived from clone CS was subsequently used for the pregnanediol-3glucuronide ELISA. A typical standard curve is shown in Figure 1. Urine samples displayed parallelism, and results are shown at various dilutions in Table 2. The sensitivity at two standard deviations from zero was less than 5 pg. Comparison of urine pregnanediol-3glucuronide levels by ELISA and the gas chromatography method is shown in Figure 2. Excretion data for three normal cycling women are shown in Figure 3. Excretion over 24 hours was calculated from its concentration relative to creatinine.16 The precision was assessed by repeated analysis of four urine pools. Within-assay variation (mean it


Cross-reactivity studies were carried out by testing each steroid at seven different doses from 5 pg to 5,000 ng/well. Steroid in assay buffer (50 ~1) was added to the wells of a coated and blocked plate followed by 50 ~1 of supematant, from the appropriate clone, diluted in assay buffer. Subsequent processing was as described. Pregnanediol-3-glucuronide


Overnight urine samples were diluted 1: 200 in distilled water and then diluted a further 1: 10 or 1: 20 in

o,. j





150 200


250 (pg/5OyL)

Figure 1 Standard curve for urinary pregnanedioWglucuronide ELISA. Values are the mean of duplicate determinations. Standard deviations are not shown as they are within the confines of the symbol 0.

Steroids, 1990, vol. 55, July


Papers Table 1


(%) of supernatants

as determined

by ELISA Supernatant




Pregnanediol-3-glucuronide 56-Pregnane-3a,20cY-diol 5/3-Pregnane-3a,206-diol 20a-Hydroxy4-pregnen-3-one 5/3-Pregnane-3a,17a,2Oa-triol 5a-Pregnane-3,20-dione 3a,l16,17a-Trihydroxy-56-pregnan-20-one 3/3-Hydroxy-5-pregnen-20-one 3P-Hydroxy-56-pregnan-20-one Progesterone Cortisol ll-Dehydrocorticosterone Corticosterone 21-Deoxycortisone 17~OH-Progesterone 11 -Deoxycortisol Deoxycorticosterone Testosterone Estradiol Estrone Estriol Estrone-3-glucuronide

100 19.3 co.1 13.7 2.1

Monoclonal antibodies to pregnanediol-3-glucuronide: application to a direct enzyme-linked immunosorbent assay of urine.

Monoclonal antibodies to pregnanediol-3-glucuronide were produced and characterized. One of three clones investigated provided antibody suitable for a...
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