Use of enzyme-immunoassay for oestradiol-17β and progesterone quantification in canine serum

Animal Reproduction Science 69 (2002) 53–64

Use of enzyme-immunoassay for oestradiol-17␤ and progesterone quantification in canine serum H.N. Ververidis a , C.M. Boscos a,∗ , A. Stefanakis b , E. Krambovitis b a b

Clinic of Obstetrics & Artificial Insemination, Faculty of Veterinary Medicine, Aristotle University of Thessaloniki, 11 Stavrou Voutyra Str., 546 27 Thessaloniki, Greece Department of Applied Biochemistry & Immunology, Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology, P.O. Box 1527, Heraklion, 711 10 Crete, Greece Received 2 January 2001; received in revised form 12 July 2001; accepted 22 August 2001

Abstract The objective of this investigation was to develop and evaluate competitive inhibition-enzymeimmunoassays for canine serum oestradiol-17␤ (E2 ) and progesterone (P4 ) quantification. Sera from 56 healthy bitches at various stages of oestrus cycle and pregnancy were tested. For E2 measurement, each sample (0.4 ml) was extracted with diethyl ether and after solvent evaporation the resultant hormone was reconstituted to one-fifth of the original sample volume in aqueous buffer. Each reconstitute (30 ␮l) was assayed for E2 to estimate respective serum concentration. For P4 , each sample (10 ␮l) was directly assayed without extraction. The classic cyclic hormonal pattern during the oestrus cycle of the bitch was observed. The brief, sharp dominance of E2 during the follicular phase was followed by the long-lasting dominance of P4 during the luteal phase (late oestrus, dioestrus or pregnancy). During the anoestrus phase both hormones were found at basal levels, with the exception of E2 during late anoestrus which appeared to be rising. Both assays had acceptable specificity (cross-reactions ≤ 10%), precision (coefficient of variation (C.V.) < 7%) and accuracy (E2 recovery: 97%; P4 recovery: 104.7%). The sensitivity of E2 and P4 assay was 4 pg ml−1 and 0.28 ng ml−1 , respectively. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Oestradiol-17␤; Progesterone; Enzyme-immunoassay; Oestrus cycle; Pregnancy; Dog

1. Introduction Oestradiol-17␤ (E2 ) and progesterone (P4 ) are of major importance during most of the canine reproductive events or disorders. Oestradiol-17␤ signals the stages of proestrus and ∗ Corresponding author. Present address: 1 Sthenonos Str., 546 33 Thessaloniki, Greece. Tel.: +30-31-994471; fax: +30-31-994470. E-mail address: [email protected] (C.M. Boscos).

0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 1 ) 0 0 1 7 1 - 3

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oestrus (Hadley, 1975; Wildt et al., 1979; Silva et al., 1995) or induces signs of hyperoestrogenism as a sequel to some ovarian (Fiorito, 1992) or testicular disorders (Feldman and Nelson, 1996). Progesterone dominates during pregnancy and long-lasting dioestrus (Concannon et al., 1975, 1989; Weilenmann et al., 1993). During oestrus, increasing P4 indicates the degree of follicular maturation and the approximate time of ovulation (Wright, 1991). Furthermore, P4 is partly implicated in the aetiology of canine pyometra (Threlfall, 1995), pseudopregnancy (Okkens et al., 1997) and complicated pregnancies (Estill, 1998). A variety of radio-immunoassays (RIA) (Edqvist et al., 1975; Wildt et al., 1979; Olson et al., 1982; Weilenmann et al., 1993; Badinand et al., 1993; Hayer et al., 1993; Hoffmann et al., 1996) including double antibody RIA (Silva et al., 1995) have been previously described to estimate E2 and/or P4 in canine serum or plasma. Other described RIA were less specific, estimating total unconjugated (Hadley, 1975) or totally extracted (Concannon et al., 1975, 1989) oestrogen and P4 . Although, enzyme-immunoassays (EIA) are the most popular alternatives to RIA, their use for canine E2 or P4 estimation is still limited. So far, only two E2 EIA have been used in dog, a microparticle EIA adapted for an automated analyser (De Cock et al., 1997) and a luminescence-enhanced immunoassay (Forsberg et al., 1993). The few described quantitative (Eckersall and Harvey, 1987; England et al., 1989; Günzel-Apel et al., 1990b; Arbeiter, 1993; Dietrich and Möller, 1993; Forsberg et al., 1993) or qualitative P4 EIA (Günzel-Apel et al., 1990a; Arbeiter, 1993; Forsberg et al., 1993; Manothaiudom et al., 1995) have been mainly employed during canine follicular phase to estimate ovulation time and succeed fertile mating or artificial insemination. The development of a competitive inhibition EIA for the measurement of residual E2 in oestrogen receptors (Krambovitis et al., 1995) and for P4 (Hatzidakis et al., 1993), allowed us to adapt the assays for estimating these steroid hormones in canine serum. In the present study, we are reporting the results from the evaluation of the assays and the hormone profiles obtained from bitches at various stages of oestrus cycle and pregnancy. 2. Materials and methods 2.1. Animals Fifty six healthy bitches, irrespective of breed, were used in this study. All the animals were in reproductive age (1–10 years old) and were presented at the Faculty of Veterinary Medicine, Thessaloniki, Greece, for elective ovariohysterectomy. Some of them (n = 15) were not submitted to ovariohysterectomy due to stage contra-indications (proestrus, oestrus and late pregnancy). 2.2. Sampling Blood samples were collected once from each animal, by jugular venepuncture, using anticoagulant-free tubes. Blood was allowed to clot at 8 ◦ C for 15 min and centrifuged at 3000 rpm for 10 min. Serum was collected and stored at −20 ◦ C until assayed for E2 and P4 .

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2.3. Classification and grouping of the animals The stage of the oestrus cycle of each bitch was determined by: (a) reproductive history; (b) clinical examination of external genitalia; (c) vaginal swab cytology (Olson et al., 1984); (d) macroscopic inspection of the ovaries and uterus, when ovariohysterectomy was performed; (e) the subsequent interval between sample collection and parturition (in late pregnancies). Thus, bitches and their corresponding samples were grouped in the following stages: proestrus (n = 4); oestrus (n = 6); first half of dioestrus (n = 12); second half of dioestrus (n = 8); early anoestrus (n = 8); late anoestrus (n = 7); middle third of pregnancy (n = 6); final third of pregnancy (n = 5). Dioestrus and pregnancy were supposed to last 60 days. The first 60 days post-partum or post-dioestrus were regarded as early anoestrus stage, while the rest of anoestrus as late anoestrus stage. 2.4. Extraction of E2 from the serum Because of the low E2 levels in the canine sera, the hormone was extracted by adding 1.2 ml diethyl ether to 0.4 ml serum in a 2 ml polypropylene vial. This mixture was vigorously shaken for 3 min, centrifuged (9000 rpm for 3 min) and allowed to freeze (−80 ◦ C for 10 min). The liquid ether fraction containing the hormone was poured into a new 2 ml polypropylene vial and the ether was evaporated to dryness. The same extraction procedure was performed once more using the same vials. A volume of 80 ␮l PBS containing 0.1% (w/v) BSA was added to the second vial and this was vigorously shaken for 4 min to re-dissolve the hydrophobic fraction including the hormone. The resultant five-fold concentrated E2 could be reliably detected as it was well within the sensitivity limits of the assay. 2.5. Enzyme-immunoassay The measurement of E2 and P4 was performed by using a competitive inhibition-EIA essentially as previously described (Hatzidakis et al., 1993). Briefly, flat-bottomed microtitration plates (Maxisorb, Nunc) were coated overnight at 4 ◦ C, with 100 ␮l per well with rabbit anti-E2 (antibodies raised against 3-hemisuccinate-oestradiol) diluted 1/1500 (v/v) in carbonate–bicarbonate buffer (50 mM; pH 9.6) or anti-P4 (antibodies raised against 7␣-carboxyethyl thioether-progesterone) (Hatzidakis et al., 1993) diluted 1/1000 (v/v) in the same buffer. Excess antibody was washed off with distilled water containing 0.05% Tween 20 (Merck) and the coated wells were blocked with 200 ␮l per well of 1% (w/v) BSA in PBS for 1 h at room temperature. Blocking buffer was discarded and the plates were dried, sealed and stored at 4 ◦ C until use. Duplicate wells were used for each sample, in the assays. Test samples (30 ␮l serum extract for E2 and 10 ␮l direct serum for P4 ) or the corresponding standards of E2 (0, 20, 50, 150 and 500 pg ml−1 in PBS containing 0.1% BSA) and P4 (0, 1, 5, 30 ng ml−1 in inactivated canine serum) were placed into each antibody-coated well. For E2 , 180 ␮l of 25 pM 3-hemisuccinate-oestradiol-peroxidase conjugate at working strength (v/v: 1/50 000), whereas, for P4 , 200 ␮l of 12.5 pM 6␤-hemisuccinate-progesterone-peroxidase, (v/v: 1/100 000), were added to the appropriate wells. The mixture was incubated in a humid

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chamber at 37 ◦ C for 2 and 1 h, respectively. After three washings with distilled water containing 0.05% Tween 20, the antibody–antigen reaction was revealed by adding 50 ␮l of substrate (0.03% (v/v) hydrogen peroxidase in 50 mM sodium acetate buffer; pH 5.2, Boehringer Mannheim) and 50 ␮l chromogen (0.25 M 3,3 ,5,5 -tetramethylbenzidine, Sigma) into each well. After a 15 min incubation in darkness, at room temperature, the reaction was terminated by the addition of 50 ␮l sulphuric acid (2 M) per well. The absorbance from the reaction was measured at 450 nm (Multiscan, Flow, Finland). The absorbance obtained from the standards was used to plot the calibration curve (Hatzidakis et al., 1993) (Fig. 1). For E2 , the estimations from the standard curve were divided by five in order to determine the true hormone concentration in the serum. Since the assays were adapted to canine sera either by ether extraction and concentration for E2 or directly for P4 , their appropriateness was evaluated in terms of sensitivity, specificity, precision and hormone recovery. Additionally, seven random canine samples were assayed for E2 , by both the present E2 assay and a commercially available solid-phase RIA for human serum or plasma E2 (Coat-A-Count® , Estradiol, DPC). Linear regression analysis was used to compare the results. 2.6. Statistical analysis Resultant E2 and P4 concentrations are depicted as box and whisker plots in Fig. 2. Data were subjected to Kruskal–Wallis test. The mean ranks of the groups were compared by Dunn’s test corrected for ties (Zar, 1996). Probability levels less than 0.05 were considered significant.

3. Results 3.1. E2 assay characteristics Sensitivity was found at 4 pg ml−1 (Table 1). Considerable antiserum cross-reactions were detected with oestrone (10%) and oestrone sulfate (8%) (Table 2) (nonetheless, oestrone sulphate could not influence the estimations, since the used ether soluble portion was devoid of this hormone). The mean intra- and inter-assay coefficient of variation (C.V.) were 2.26 and 5.49%, respectively (Table 3). The mean recovery was 97% (Table 4). A correlation coefficient r = 0.913 (P < 0.005) was detected between results (E2 concentration

Table 1 E2 and P4 EIA sensitivity Assay

Zero standard replicates

Sensitivitya

E2 P4

n = 12 n = 14

4 pg ml−1 0.28 ng ml−1

a

Concentration of hormone detected at three standard deviations of the zero standard.

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Table 2 E2 and P4 EIA specificity Steroida

% Antiserum cross-reactivityb

Oestriol Oestrone Oestrone sulphate E2 Oestradiol-17␣ P4 11␣-Hydroxyprogesterone 5␣-Pregnane-3,20-dione 5␤-Pregnane-3,20-dione Pregnanolone Desoxycorticosterone 17␣-Hydroxyprogesterone Corticosterone Cortisol Desoxycorticosterone acetate Androstenedione Androstenolone Dehydroisoandrosterone Testosterone

E2 assay

P4 assayc

0.8 10 8 100 <0.1 nt nt nt nt nt nt nt nt nt nt nt <0.1 <0.1 <0.1

<0.1 <0.1 ntd <0.1 nt 100 1 2.8 10 4 1.2 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 nt <0.1

a

Steroids commercially available (Steraloids, Wilton). Cross-reactivity was defined as the amount of the tested steroid required to reduce label binding by 50%. c Tested previously (Hatzidakis et al., 1993). d nt: not tested. b

Table 3 E2 and P4 EIA precision Assay (units)

Hormone concentration in samples

Intra-assay

Inter-assay

Repl.a

Repl.

C.V.% Mean

No. of plates

Overall mean

C.V.% Mean

Overall mean

E2 (pg ml−1 )

2.0, 2.4, 3.4 4.0, 4.6, 5.4 7.4, 24

10

2.45 1.96 2.41

2.26

10

5

4.12 6.13 6.49

5.49

P4 (ng ml−1 )

0.1, 0.54 2.4, 3.2, 4.8 6.9, 8.5, 25

8

5.00 4.17 5.91

4.98

6

3

3.00 3.73 2.89

3.26

a

Repl.: Replicate estimations of each sample.

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Table 4 Recovery of authentic E2 and P4 added into random canine sera Assay (units)

Hormone added

E2 (pg ml−1 )

0.25, 5.2, 17, 22

20b 50b

19.0 ± 8.9 49.5 ± 17.4

94.9 99.1

97

P4 (ng ml−1 )

0.71, 1.17, 1.63, 2.5

1 10

1.04 ± 0.28 10.58 ± 1.38

103.6 105.8

104.7

a b

Hormone difference detected (Mean ± S.D.)

Recovery %a

Initial concentration in samples

Mean

Overall mean

(Hormone difference detected/hormone added) × 100. Authentic E2 , added prior to extraction.

range: 2.80–64 pg ml−1 ) obtained by the present method and those given by Coat-A-Count® method (= 0.941 × EIA + 5.187 pg ml−1 ). 3.2. P4 assay characteristics The P4 assay sensitivity was found at 0.28 ng ml−1 (Table 1). Considerable antiserum cross-reactions were detected with 5␤-pregnane-3,20-dione (10%) and of pregnanolone (4%) (Table 2). The mean intra- and inter-assay C.V. were 4.98 and 3.26%, respectively (Table 3) and the mean recovery was 104.7% (Table 4). 3.3. Detected canine serum E2 and P4 concentrations Proestrus E2 median was significantly greater than the E2 medians of dioestrus first half (P < 0.05), mid-pregnancy (P < 0.05) and early anoestrus (P < 0.01). Oestrus E2 median was significantly greater than the E2 medians of dioestrus first-half (P < 0.05) and early anoestrus (P < 0.01) (Fig. 2). Progesterone median was found significantly greater during dioestrus first-half (P < 0.001), dioestrus second-half (P < 0.01) and middle or late pregnancy (P < 0.05) than during proestrus and anoestrus periods (Fig. 2).

4. Discussion 4.1. E2 assay Normal concentration of E2 in the serum of bitches has been found ranging from 5 to 70 pg ml−1 by Olson et al. (1982), from 9 to 90 pg ml−1 by Weilenmann et al. (1993) and from 7 to 100 pg ml−1 by Edqvist et al. (1975). This E2 concentration range (although not absolutely established) is lower than the corresponding range in women (levels of 20–500 pg ml−1 ) (Martini and Welch, 1998), therefore, requiring very sensitive assays to accurately detect the hormone. Most of the previously employed canine E2 RIA or EIA

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Fig. 2. Box and whisker plots of serum E2 and P4 concentration detected during various stages of canine oestrus cycle and pregnancy. Lines inside boxes: medians; box boundaries: 25th and 75th percentiles; whiskers: 5th and 95th percentiles; o: outliers; ∗ : extremes; n: number of cases; (a–c): different superscript letters: significant difference (P < 0.05).

needed well-trained personnel and/or expensive specialist equipment (De Cock et al., 1997; Forsberg et al., 1993). Furthermore, the E2 EIA method used by De Cock et al. (1997), resulted in E2 concentrations exceedingly higher (37–189 pg ml−1 range) than the ones previously described in dog (Edqvist et al., 1975; Olson et al., 1982; Weilenmann et al., 1993). The present E2 competitive inhibition-EIA, can be easily performed with standard laboratory equipment. The extraction of canine sera is considered necessary by the

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authors in order to avoid unpredictable canine serum-dependent E2 -like background reactions in the E2 assay and also to concentrate the hormone five-fold and bring it within the sensitivity limits of the assay (4 pg ml−1 ). The concentrated E2 levels remain proportional to their initial E2 concentrations and are precisely detected by the assay. The serum volume (0.4 ml) required for a duplicate estimation is not unacceptably large. Assay characteristics were considered satisfactory (Tables 1–4). Results obtained by this method correlated well (r = 0.913) with those given by an E2 RIA method. Preliminary trials (data not shown) indicate that the same method can be easily applied for the estimation of E2 in feline, ovine, bovine or other animal sera which display relatively low normal concentration range. 4.2. Progesterone assay Previously described RIA demonstrated normal canine serum or plasma P4 concentrations ranging between 0.8 or less, to 40 ng ml−1 by Wildt et al. (1979), to 50 ng ml−1 by Edqvist et al. (1975), to 70 ng ml−1 by Weilenmann et al. (1993) or to 90 ng ml−1 by Concannon et al. (1989). Higher detected limits depended mainly on the assay used, while the same P4 oestrus cycle pattern was observed by most methods. Most of the previously described quantitative or qualitative canine P4 EIA have been designed for and employed during proestrus and oestrus (P4 useful maximum level of 10 ng ml−1 ) (Eckersall and Harvey, 1987; England et al., 1989; Dietrich and Möller, 1993; Günzel-Apel et al., 1990a; Manothaiudom et al., 1995). As far as the authors know, only three canine P4 EIA have incorporated standards presenting P4 concentration greater than 10 ng ml−1 , but their use was restricted to: (a) serial samples from a single bitch during follicular phase followed by a sole sample during early dioestrus (Forsberg et al., 1993); (b) some anovulatory ovarian cycles (Arbeiter, 1993); (c) serial samples from 13 bitches during proestrus and oestrus (Günzel-Apel et al., 1990b), thus, missing progesterone peaks. Furthermore, the EIA described by Forsberg et al. (1993) and Arbeiter (1993), as well as RIA methods, required well-trained personnel and/or expensive specialist equipment. The present P4 EIA incorporates a P4 standard of 30 ng ml−1 permitting the detection of high P4 concentrations observed during canine luteal phase. The assay requires only the standard laboratory equipment, a minimal volume of canine serum (10 ␮l per well) and a short incubation period. Assay characteristics were all considered satisfactory (Tables 1–4), when compared to the ones of other quantitative P4 EIA (Eckersall and Harvey, 1987; Forsberg et al., 1993; Arbeiter, 1993; Dietrich and Möller, 1993). Quantitative results were obtained by use of a photometer. The same P4 assay may also be used semi-quantitatively for an optical estimation of P4 concentration, by relating colour intensity of samples to the one closest to an appropriate standard. Thus, a rapid, rough distinction is possible, at least between the significantly different canine luteal and anoestrus phase or proestrus. 4.3. Application of the assays in measuring serum E2 and P4 during stages of canine oestrus cycle and pregnancy Detected E2 and P4 concentrations (Fig. 2) were stage-reasonable and comparable to previous RIA canine E2 or P4 estimations.

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Oestradiol-17␤ reached its highest concentration during proestrus, while it remained high during oestrus. These follicular phase E2 peaks are a result of follicular growth and maturation and have been previously observed by Wildt et al. (1979), Olson et al. (1982) and Hayer et al. (1993). During the other stages, E2 remained at low-almost basal levels, as described earlier by Concannon et al. (1975), Edqvist et al. (1975) and Weilenmann et al. (1993), with the exception of a slight but not significant rise during second half of dioestrus, late anoestrus and late pregnancy. The late anoestrus E2 rise might be attributed to the reactivation of follicular development for the oncoming proestrus and has also been observed by Edqvist et al. (1975), Olson et al. (1982), Jeffcoate (1993) and Hoffmann et al. (1996). During proestrus, P4 concentration was found at basal levels, in accordance to Olson et al. (1982). During oestrus a slight (although not significant) increase was noticed, as it has been extensively studied previously by Wildt et al. (1979), Badinand et al. (1993) and De Cock et al. (1997), representing the early pre- or post-ovulation follicular luteinisation. During dioestrus or pregnancy, P4 concentration was constantly high as a product of active corpora lutea. During early and late anoestrus, P4 concentration progressively decreased due to luteal regression, as reported by Olson et al. (1982), Jeffcoate (1993) and Hoffmann et al. (1996).

5. Conclusions The present EIAs can be easily, quickly and safely used with minimal demands in laboratory equipment and experience. As an alternative to RIAs, these can be applied for the determination of serum E2 and P4 concentrations during the various stages of canine oestrus cycle and pregnancy.

Acknowledgements The authors express their thanks to Z. Vlata, M. Siumbara and G. Hatzidakis for their technical assistance and advise during the immunoassay development. The work was supported in part by the Regional Secretariat General of Crete (Grant 40/94) and by the State Scholarship Foundation of Greece (scholarship granted to H.N. Ververidis). References Arbeiter, K., 1993. Anovulatory ovarian cycles in dogs. In: Concannon, P.W., England, G.C.W., Verstegen, J.P., Russell, H.A. (Eds.), Fertility and infertility in dogs, cats and other carnivores. J. Reprod. Fertil. (Suppl. 47) 453–456. Badinand, F., Fontbonne, A., Maurel, M.C., Siliart, B., 1993. Fertilization time in the bitch in relation to plasma concentration of oestradiol, progesterone and luteinizing hormone and vaginal smears. In: Concannon, P.W., England, G.C.W., Verstegen, J.P., Russell, H.A. (Eds.), Fertility and infertility in dogs, cats and other carnivores. J. Reprod. Fertil. (Suppl. 47) 63–67. Concannon, P.W., Hansel, W., Visek, W.J., 1975. The ovarian cycle of the bitch: plasma estrogen, LH and progesterone. Biol. Reprod. 13, 112–121.

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