Zoo Biology 5: 1-6 (1986)

RESEARCH ARTICLES

The Development and Application of an Enzyme lmmunoassay for Urinary Estrone Conjugates N.M. Czekala, S. Gallusser, J.E. Meier, and B.L. Lasley

Research Department (N. M. C., S. G., 6. L. L.) and Veterinary Services Department (J. E. M.), San Diego Zoo, San Diego

An enzyme immunoassay (EIA) for estrone conjugates is described and applied to urine samples from a female Indian rhinoceros, a female gorilla, and a female lion-tailed macaque. Concomitant measures of estrone conjugates in the same sample are compared to the values obtained with radioimmunoassay. High corre- lation coefficients for values obtained from each assay indicate that EIA measure- ments provide information that is comparable to values obtained by radioimmunoassay . EIA methods for urinary steroid conjugates can provide a practical tool to evaluate female reproductive status of zoo species without the need for a traditional endocrine laboratory.

Key words: urinary hormones, enzyme assays, estrone conjugates

INTRODUCTION

One of the limitations of endocrine monitoring is that actual analyses must be performed in specialized laboratories, often some distance from the study site. The need for such centralized facilities stems from the use of radioisotopes and expensive equipment necessary in the employment of radiolabeled materials. The development of enzyme-linked immunoassays provides a potential alternative that removes this limitation since enzymes instead of radiolabels are used as endpoint indicators.

This report describes the development and application of an enzyme immunoas- say (EIA), which was designed with zoo-related endocrine studies of female repro- duction in mind. This assay measures estrone conjugate levels in unprocessed urine samples throughout the reproductive cycle of most mammalian species. Urinary estrogen profiles representing normal ovarian function in the Indian rhinoceros (Rhi- noceros unicornis), lowland gorilla (Gorilla gorilla gorilla), and lion-tailed macaque

Received for publication June 13, 1985; accepted August 10, 1985.

Address reprint requests to N.M. Czekala, Research Department, San Diego Zoo, P.O. Box 551, San Diego, CA 92112.

0 1986 Alan R. Liss, Inc.

2 Czekala et al

(Mucucu silenus) are reported here to demonstrate the application of the enzyme assay system to a wide range of species. EIA results are compared to similar measurements obtained by radioimmunoassay .

MATERIALS AND METHODS Urine Collection

Daily random urine samples (3-5 ml) were obtained from a female Indian rhinoceros (Rhinoceros unicornis), a lowland gorilla (Gorilla gorilla gorilla), and a lion-tailed macaque (Mucucu silenus). Urines were aspirated from the enclosure floor with a syringe and immediately frozen until assayed.

Radioimmunoassa y Estrone conjugate concentrations were evaluated by radioimmunoassay as re-

ported elsewhere [Shideler et al, 19831. Briefly, estrone glucuronide antiserum ( I 5,000; D. Collins, Emory University, Atlanta, GA) was combined with tritiated estrone sulfate (7,000 CPM, specific activity, 55 Ci/mM; New England Nuclear, Boston, MA) and unlabeled estrone sulfate (5 ng - 0.03 ng) or urine (gorilla, 0.01 ml; lion-tailed macaque, 0.02 ml; rhinoceros, 0.00005 ml) in an assay buffer of 0.1 M Tris, 0.9% NaC1, 0.1 % NaN3, and 0.1 % gelatin, pH 8.4.

After incubation at 15°C for 1 h, antibody-bound hormone was separated from unbound steroid by the addition of 0.3 ml charcoal dextran (0.065 charcoal, 0.00065% dextran in 0.1 M phosphate buffer), incubated at 15°C for 30 min, and centrifuged for 10 min. Supernatants were counted for 5 min with scintillation cocktail (Ready- Solv, HP Beckman, Fullerton, CA).

Enzyme lmmunoassay All buffer and wash solutions were those used by Munro and Stabenfeldt [ 19841.

Estrone-3-glucuronide (Sigma, St. Louis, MO) was conjugated to bovine serum albumin (Sigma) [Kellie et al, 19741, which was then used as the antigen in rabbits to produce estrone conjugate antisera [Vaitukaitis et al, 19711. The gammaglobulin fraction of the rabbit serum was purified with repeated precipitation using sodium sulfate (18, 14, and 12%) and dialyzed to remove salts. The antiserum was subse- quently adsorbed with bovine serum albumin (BSA) to eliminate antibodies directed against BSA [Munro and Stabenfeldt, 19841. Cross-reactivity of the antisera was 100% to both estrone sulfate and estrone glucuronide.

Estrone-3-glucuronide was conjugated to horseradish peroxidase (HRP, type VI, Rz 3.0; Sigma) and purified by the method of Kellie et a1 [1974]. Column eluates of the purified enzyme conjugate were diluted (1 : l0,OOO) and used directly in the assay system. Ninety-six-well flat-bottomed microtiter plates (Immunlon I; Dynatech, Alexandria, VA) were coated with 0.2 ml purified estrone-3-glucuronide antiserum (1:1,600 in coating buffer, pH 9.6). Each plate was sealed to eliminate evaporation and incubated overnight at 15°C. Following the incubation, the plate was inverted and washed five times with wash solution to remove the antisera not bound to the plate. The plate was then inverted on paper toweling and allowed to dry.

Estrone-3-glucuronide-HRP (0.1 ml; 1 : 10,000 in phosphate buffer) was added to each well using a multichannel pipette. A standard curve (estrone sulfate 5 pg/O. 1 ml to 625 pg/O.1 ml) was analyzed in parallel and in triplicate. Blank wells (no antisera) and zero wells (no standard or urine) were measured in triplicate. Urine

EIA for Urinary Estrogens 3

samples were diluted with enzyme assay buffer (rhinoceros, 1 : 10,000; gorilla, 1 : 100; lion-tailed macaque, 1 : 100) according to the concentration of estrone conjugates in the urine. These dilutions were determined by dose response analysis in the RIA at an earlier time. The diluted urine (0.1 ml) was added to wells in triplicate and sealed. Following a 2-h room temperature incubation, the plate was inverted and washed five times with wash solution to remove unbound steroid. Two hundred microliters of the substrate solution [0.250ml40mM 2,2’-azinobis (3-ethylbenz-thiazolinesulfonic acid) (ABTS), 80 p10.5 M Hz02, 24.6 mIO.05 M citrate, pH 4.01 was added to each well and sealed. After a 2-h incubation, absorbance was measured at 410 nm in the Dynatech MR600. Blank absorbance was subtracted from each reading. The zero wells were considered 100% binding of enzyme conjugate to the antiserum, and all standards and unknowns were divided by the mean zero reading (percentage bound). A dose response curve of the standards was prepared by plotting the percentage bound against the concentration of estrone sulfate added. The unknown percentage bound values were compared to the dose response curve and values calculated from it.

All steroid levels were indexed by creatinine levels in the same sample [Taussky, 19541.

RESULTS

Estrone conjugate values determined by radioimmunoassay and enzyme immu- noassay were compared in daily urines from a female gorilla, Indian rhinoceros, and lion-tailed macaque. The correlation coefficient of the 12 gorilla samples, which included the late follicular phase, ovulation, and early luteal phase, was 0.71. Linear regression between the RIA and EIA of the gorilla samples had a slope of 0.89 k 0.27 and an intercept of 45.57 =F 5.14 ng f SD. In the lion-tailed macaque, 14 urine samples included the late follicular phase, ovulation, and the luteal phase. They had a correlation coefficient of 0.71, a linear regression slope of 0.96 f 0.26, and an intercept of 12.95 f 9.26 ng 1- SD. The 16 rhinoceros urine samples included the preovulatory phase and estrous period and produced a correlation coefficient of 0.90 between the two assays. Linear regression of the rhinoceros values gave a slope of 0.37 k 0.053 and an intercept of 1,151 f 150 ng 1- SD.

Figure 1 depicts the concomitant measure of urinary El conjugate with RIA and EIA. There was a slight overestimation by the EIA as compared to RIA for the gorilla cycle (157% f 11.2), whereas for the rhinoceros the EIA gave lower results than the RIA (49% i 13.8). For the lion-tailed macaque urine samples, the results between EIA and RIA were nearly indistinguishable (p = 0.71).

DISCUSSION

Endocrine monitoring of exotic animals has proven to be a useful tool in zoos for animal management, clinical evaluation, and comparative scientific information [Lasley, 19851. For the zoo veterinarian and animal manager, the ability to monitor the reproductive status of exotic animals simply, noninvasively, and economically has great clinical significance. Periods of estrus, pregnancies, reproductive cycle abnor- malities, and impending parturition can be predicted. Animal care and improved neonatal survival rates should result when animal caretakers can predict and prepare for reproductive events. The recent application of steroid conjugate radioimmunoas- says to unprocessed urine has enabled endocrine laboratories to speed processing and

4 Czekala et al

t 0

\ cn E

3-

El CONJUGATES w EIA *-* RIA

Gorilla

r - .7l

Lion -Tailed ,400 in Macaque .300

,200

,100

r = .7l

Rhinoceros

= .90

OAYS

Fig. 1. Daily urine samples from gorilla (top), a lion-tailed macaque (middle), and an Indian rhinoceros (bottom) were evaluated for estrone conjugates using either a radioimmunoassay (0) or an enzyme immunoassay (0). Correlation coefficients (r) for the two measures are shown in each panel.

reduce costs [Shideler et al, 1983, 1985; Evans et al, 19841. The limitations of all radioimmunoassay, however, are the requirements for sophisticated laboratories, radioactivity, and organic solvents.

An alternative to the radioimmunoassay is the enzyme immunoassay (EIA). EL4 techniques for monitoring serum progesterone [Munro and Stabenfeldt, 19841 and milk progesterone [Chang and Estergreen, 19831 have been reported. The tech- nique and data presented here demonstrate that an enzyme immunoassay technique can be applied to unprocessed urine to measure steroid conjugates. This approach retains all of the advantages of the direct radioirnmunoassay measure of steroid conjugates in unprocessed urine. Furthermore, this technique provides a test that can be implemented outside of the traditional endocrine laboratory and should ultimately

EIA for Urinary Estrogens 5

lead to the development of field test kits. Field test kits will make these techniques widely available to those who do not have access to radioimmunoassay. Potentially, the test kits would provide sophisticated reproductive monitoring to anyone who could collect urine samples and follow standard laboratory procedures.

The approach to the development of the EIA reported here is less complicated than most other enzyme-linked assay systems. The only requirement to convert the radioimmunoassay to the EIA for estrone conjugates was the conjugation of HRP to the glucuronide moiety of estrone glucuronide. The antisera previously used to detect both estrone glucuronide and estrone sulfate were directed against estrone glucuron- ide-bovine serum albumin. The relative small size of the HRP as well as its position on the glucuronide moiety allows the original antiserum to bind the enzyme conjugate. This one-step conversion of radioimmunoassay to EIA will allow most laboratories to convert to or institute EIA for the first time without large investments of time or money. As this procedure becomes available in laboratories, its use has the potential to influence the survival of many exotic animal species in captivity.

The EIA and radioimmunoassay produce the same qualitative profile for each representative of three diverse species (Fig. 1). Absolute values for each assay are similar for the gorilla and lion-tailed macaque. In the rhinoceros, the values are lower for the EIA than for the radioimmunoassay; however, the RIA analysis was an independent study performed earlier. There are several possible explanations for this discrepancy although no attempt was made to reconcile the two measurements in this report. The most likely reason for the larger discrepancy in rhinoceros samples compared to primate samples is that the high levels of estrone conjugates in this species requires a larger dilution (l:lO,OOO). Since this was performed at separate times it is possible that these independent dilutions account for differences in the results obtained.

ACKNOWLEDGMENTS

The authors acknowledge the assistance of the keepers and staff at the Toledo Zoo, San Diego Zoo, and Miami Zoo for collection of samples; Lonnie Kasman for RIA evaluation of rhinoceros samples; and Sharon Dinwiddie for preparation of the manuscript. The research reported here was supported by a grant from the Bay Foundation through the American Association of Zoo Veterinarians.

REFERENCES

Chang, C.F.; Estergreen, V.L. Development of a Lasley, B.L. Endocrinology and reproduction in direct enzyme immunoassay of milk progesterone exotic species, pp. 209-228 VETERINARY SCI- and its application to pregnancy diagnosis in ENCE AND COMPARATIVE MEDICINE. Vol. COWS. STEROIDS 41:173-195, 1983. 30. C. Cornelius, ed. Florida, Academic Press,

Evans, K.L.; Hughes, J.P.; Couto, M.; Kasman, 1985. L.H.; Lasley, B.L. Pregnancy diagnosis in the Munro, C.; Stabenfeldt, G. Development of mi- domestic horse through direct urinary estrone crotiter plate enzyme immunoassay for the deter- conjugate analysis. THERIOGENOLOGY mination of progesterone. JOURNAL OF 22:615- ENDOCRINOLOGY 101:4149, 1984. 620, 1984. Shideler, S.E. ; Czekala, N.M. ; Kasman, L.H. ;

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Lasley, B.L.: Monitoring luteal function in the Vaitukaitis, J . ; Robbins, J.B.; Nieschlag, E.; Ross, lion-tailed macaque (Mucacu silenus) through G.T. A method for producing specific antisera urinary progesterone measurement. ZOO BIOL- with small doses of immunogen. JOURNAL OF OGY 4:65-73, 1985. CLINICAL ENDOCRINOLOGY AND ME-

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Taussky, H . H . A micrometric determination of TABOLISM 33:988-99 1, 1971.