33
DIRECT RADIOIMMUNOASSAY OF PROGESTERONE IN MARE PLASMA H.P. Mathieu, C. Mathieu-Nast and C. Vrignaud Laboratoire de Physique Pharmaceutique, UER de Pharmacie Place de la Victoire, 33000 BORDEAUX - FRANCE RECEIVED 113177
ABSTRACT
A rapid and low cost radioimmunologicprocedure for progesterone assay in mare plasma is proposed. Radioimmunoassay is performed directly on 10 pl of unextracted plasma. Free progesterone is adsorbed on dextran-charcoal, then the aqueous phase is decanted and extracted by 1 ml of scintillation fluid. Counting is performed directly on this two-phase system. Results are comparable to those obtained with radioimmunoassaysusing extracted plasma. INTRODUCTION Mare plasma progesterone assay is helpful in studying luteal activity and for early diagnosis of pregnancy. This assay has been previously performed by competitive protein-binding or radioimmunological procedures after extraction of plasma with hexane or petroleum ether (I to 7). To increase assay speed and to reduce its cost, we studied a radioimmunoassay on unextracted plasma along with a micro two-phase liquid scintillation counting method. MATERIALS AND METHODS Reagents - Phosphate buffer : 0.1 M sodium phosphate, pH 7.4, 0.9 % Na Cl, 0.1 % Na azide, 0.1 % gelatin. - Dextran-charcoal solution : Norit A (Prolabo) 2.5 g, Dextran T 70 (Pharmacia) 0.25 g in 1,000 ml of phosphate buffer. - Petroleum ether (40-60') Merck. - Scintillation fluid : PPO (Merck) 5 g, POPOP (Merck) 0.025 g in 1,000 ml of toluene. Mare plasma pool with very low progesterone concentration : due to their very low level of progesterone, plasmas from mares without ovarian activity were selected. Small aliquots (lml) were frozen at - 20°C until used. Progesterone level, determined by radioimmunoassay of extracted plasma, was lower than 0.1 ng/ml. Antisera : lyophilized rabbit anti-progesterone (llp-succinyloxyprogesterone BSA conjugate) antisera were prepared by Institut Pasteur and New England Nuclear Corporation. It was dissolved in buffer to achieve an initial binding of SO-70 % of 3H-progesterone.
Steroids : 1,2,6,7- 3H-progesterone, supplied by NEN, had a specific activity of 96 Ci/mmole and a radiochemical purity greater than 97 %, checked every 6 months by paper chromatography using hexanelformamide solvent system. Stock solution (10 pCi/ml) was prepared in benzene and stored at 7°C. For radioimmunoassay, aliquots were evaporated to dryness and redissolved in buffer to produce a solution containing 0.004 pCi/O.lO ml. Nonlabelled progesterone was supplied by Ikapharm. Radioimmunoassay - Standard curve : 0.10 ml of ethanolic progesterone solution (25 ng/ml) were evaporated to dryness under nitrogen in a heating block at 6O'C. Pool plasma (0.50 ml) was added and the tubes were gently shaken for good redissolution. This solution was diluted to 215,115, l/10,1/25 and l/50 with the same plasma. Standard curves were obtained from assays performed on 10 pl of each dilution (500,200,100,50,20 and 10 pg, respectively). - Assay procedure : the assays were carried out in duplicate. H-Progesterone solution (0.10 ml) was added to all tubes. Tubes T and blank received 0.20 ml buffer and 10 ul plasma pool. B, received 0.10 ml buffer, 0.10 ml antiserum and 10 pl plasma pool. Standards and samples received 0.10 ml buffer, 0.10 ml antiserum and 10 pl plasma standard or sample. The tubes were vortexed and incubated at 4°C for 2 to 12 hours. Then they were left IO min in an ice bath. We added I ml of ice-cold dextran-coated charcoal suspension, except to tube T which received I ml of buffer. The tubes were quickly vortexed, left for a further 10 min in the ice bath, and then centrifugated for 5 min. The supernatant was decanted into 65 x 10 mm (ID) glass tubes containing 1 ml toluene scintillator. These tubes were stoppered with a polypropylene cap entering the tube up to 9 mm and placed on a horizontal shaker (120 excursions per minute) : the tube's axis must be horizontal and perpendicular to the movement in order to avoid emulsion formation. After shaking for 30 min at room temperature and standing for one hour in a rack, the tubes were counted for 10,000 counts in a scintillation spectrometer (Coruflow with Scaler SC 711 M - ICN). For this, each tube was positioned vertically in a 20 ml glass counting vial. 3
- Assays with plasma extraction : 0.50 ml of plasma were extracted with 5 ml petroleum ether ; aliquots of 0.10 ml to 0.40 ml of the organic phase were evaporated to dryness in reaction tubes. Radioimmunoassays were performed as for B, tubes, adding the same reagents. RESULTS 3
H-Progesterone
fixation by mare plasma
The progesterone binding ability of mare plasma was checked by a series of "blanks" using 10 different plasmas (IO pl). The difference in binding of the blanks, in the presence of plasma, from those without plasma was 1.2 z 0.3 %. Moreover, between the duplicate procedures of
S
WEEOXDI
+ each assay, the derived standard deviation was - 0.2 X, thus showing 3 slight variation between the mare plasmas. Binding of H-progesterone by 0.10 ml of mare plasma (12-14 X) was not reduced by addition of 5 ng progesterone : protein binding of progesterone is nonspecific. Standard curve A mean standard curve is represented on figure I. Intra-assay standard deviation has been plotted for each point.
I 100
200
300
400
500
pg Progesterone Figure 1.
Mean standard curve (n=12).
Two standard curves, with and without plasma, have been compared : all the values of the second curve were within the 95 X confidence limits of the first one. Precision The precision of the method has been determined on 2 plasma pools whose progesterone levels were 3.3 ng/ml and 9.6 ng/ml : intra-assay coefficients of variation were t 4.8 % (n =16) and z 3.6 X (n -10) respectively ; inter-assay coefficients of variation were t 6 % (n -11) and + 5.7 % (n =7). Accuracy Two and 10 ng/ml of progesterone have been added to the 3.3 ng/ml
plasma pool to determine the accuracy. Recoveries were, respectively, 101.5 + 9 % (n =lO) and 100.6 4 9.3 % (n =lO). Sensitivity The mean binding of B, tube was 63.2 + 1.6 %. Sensitivity, defined as the smallest concentration
able to give a response significantly
different from B. (-2SD), has been determined from a standard curve obtained with 2,5,10 and 20 pg. Its value was 4 pg in the reacting tube, i.e., 0.4 ng/ml in the mare's plasma. Specificity Results obtained for mares without ovarian activity or in follicular phase were close to or lower than the minimal detectable concentration
: there was no important cross-reactivity.
cortisol determinations gave values between
assayed by a radiocompetition
Several plasma
procedure
10 and 30 ngfml. The cross-reactivity
(8)
percentage at
50 % displacement binding was 0.01 for Cortisol with this antiserum ;it did not cross-react
Y
.
significantly.
I IO -9 --
8 -7 --
5 --
n = 30. Y = 0.2 + 1.02 x r = 0.98. p(O.001.
ng/ml progesterone.Extracted Figure 2.
plasma.
Comparison of progesterone concentrations measured with (X) and without (Y) extraction of mare plasma.
S
IDEOXDI
37
Progesterone radioimmunoassay of mare plasma using the same antiserum and a single hexane extraction has been described (7).
Therefore,
the results of the present procedure were compared with those using the extraction method. (fig. n'2). The correlation is significant. Liquid scintillation counting Counting efficiency was approximatly 50 %. Counting precision was determined on tubes T with 0.10 ml antiserum and without decantation : the coefficient of variation was +_2 % (n 310). 3H-progesterone extraction by scintillation liquid was 96.3 + 0.7 % ; it was not reduced 3 by antiserum. Pipeting error of H-progesterone solution, determined by weighing, , was + 1 % ; statistical error on radioactivity was + 1 %. DISCUSSION Horse serum has been used for specific radioassay of cortisol (S,9,10) : progesterone competition for cortisol binding sites on horse transcortin is very weak. The results we obtained indicate that 3 H-progesterone binding of mare plasma is weak, constant and nonspecific ; therefore, direct radioimmunoassay of mare progesterone in unextracted plasma is possible. Non-specific binding is reduced to a minimum by taking 10 1.11 of plasma, and corrected by adding to standard tubes the same volume from a plasma pool containing a low level of progesterone. Radioimmunoassay is not disturbed by mare plasma. With our procedure, progesterone levels may be determined with suitable precision from 1 to 50 ng/ml, allowing study of mare luteal function and gestation. In the follicular phase, progesterone levels are close to or lower than radioassay sensitivity ; this sensitivity may be reduced to 0.2 ng/ml by antiserum dilution up to 30-40 % binding for B. tube. Plasma sample volume can be increased while performing a blank for each plasma. Liquid scintillation counting has been modified. Counting is generally performed in a homogeneous solution of aqueous phase in the scintillation fluid : this is necessary when the solubility of radioactive product in the organic phase is poor, or when a mixture of radioactive products with different physical properties must be counted. For a unique radioactive product that is soluble in organic phase,
however, direct extraction of the aqueous phase by scintillation fluid seems the better way. Two-phase liquid scintillation counting has already been used in radioimmunoassay,
for extraction of free hormone either directly(lO,ll,
12) or after precipitation
of globulins by ammonium sulfate (13,14,15).
Scintillation fluid volume used is generally IO ml, a volume very much greater than the aqueous phase volume. Liquid scintillation counting with a 1 ml homogeneous
liquid phase has been also described
(16).
The proposed procedure is a two-phase micromethod with a scintillation fluid volume reduced to 1 ml, offering advantages of the two precedent technics : no quenching and scintillation fluid saving. 3 H-Progesterone bound to antibodies is extracted by scintillation fluid after free hormone adsorption on dextran-charcoal
: assay precision is
little influenced by the extraction procedure, the efficiency of which is not reduced by antiserum and plasma. Our tube agitation procedure, involving perpendicular movement in reference to the axis of the tube, avoids the formation of a stable emulsion produced by parallel movement. This former does not reduce the counting efficiency but will slightly decrease precision. This radioimmunoassay
of progesterone
in mare plasma has been used
during two breeding seasons for early diagnosis of gestation and for study of luteal function with concordant results (17). Note : Since sending this manuscript,
a similar liquid scintil-
lation counting method has been proposed for estradiol by Kandell, et -al. (Steroids -28 : 755, 1976).
ACKNOWLEDGEMENTS We wish to thank D.W. Bruneteau, M.D., and F. Laulan for their technical assistance.
REFERENCES I.
2. 3.
Smith I.D., Basset J.M., and Williams T. VIIth Int. Congr.Anim. Reprod. Artif. Insemination. Munchen. 2 : 1190 (1972). Stabenfeldt G.H., Hughes J.P., and Evans J.W. Endocrinology 90 : 1379 (1972). 33 : 535 (1973). Sharp D.C., and Black D.L. J. Reprod. Fert. -
4. 5. 6. 7.
8. 9. 10.
11. 12. 13. 14. 15. 16. 17.
Robertson HA., and Sarda I.R. J. Endocr. 49 : 407 (1971). Thimonier J. Rec. Med. Vet. Alfort. 149 : 503 (1973). Prod. Sci. l : 197 Palmer E., Thimonier J., and Lemon MTivest. (1974). Palmer E., and Jousset B. J. Reprod. Fert. 2 : 214 (1974). Murphy B.E.P. J. Clin. Endocr. Met. 41 : 1050 (1975). Fischer M., Curtis G.C., Gaujam V.K.,Joshlin L., and Perry S. Clin. Chem. 19 : 511 (1973). P.M. Keane, x Stuart, J. Mender, S. Barbadozo, and W.H.C. Walker Clin. Chem. 21 : 1474 (1975). Castanier M., and SchollerR. C.R. Acad. Sci. Paris. 271 : 1787 (1970). Jowett T.P., Slater J.D.H., Piyasena R.D., and Ekins R.P. Clin. Sci. Mol. Med. 45 : 607 (1973). Mayes D.M., and%gent C.A. Steroids. 15 : 389 (1970). Kream J., Hellman L., and Rosenfeld R.K Steroids. 27 : 727 (1976). Kurtz A.B., and Bartter F.C. Steroids. 28 : 133 (19%). Tyler J.P.P., Hennam J.P., Newton J.R.,and Collins W.P. Steroids. 22 : 871 (1973). xrchhoff J.L. These Dr. Vet. Toulouse (1977) in press.
DIRECT RADIOIMMUNOASSAY OF PROGESTERONE IN MARE PLASMA H.P. Mathieu, C. Mathieu-Nast and C. Vrignaud Laboratoire de Physique Pharmaceutique, UER de Pharmacie Place de la Victoire, 33000 BORDEAUX - FRANCE RECEIVED 113177
ABSTRACT
A rapid and low cost radioimmunologicprocedure for progesterone assay in mare plasma is proposed. Radioimmunoassay is performed directly on 10 pl of unextracted plasma. Free progesterone is adsorbed on dextran-charcoal, then the aqueous phase is decanted and extracted by 1 ml of scintillation fluid. Counting is performed directly on this two-phase system. Results are comparable to those obtained with radioimmunoassaysusing extracted plasma. INTRODUCTION Mare plasma progesterone assay is helpful in studying luteal activity and for early diagnosis of pregnancy. This assay has been previously performed by competitive protein-binding or radioimmunological procedures after extraction of plasma with hexane or petroleum ether (I to 7). To increase assay speed and to reduce its cost, we studied a radioimmunoassay on unextracted plasma along with a micro two-phase liquid scintillation counting method. MATERIALS AND METHODS Reagents - Phosphate buffer : 0.1 M sodium phosphate, pH 7.4, 0.9 % Na Cl, 0.1 % Na azide, 0.1 % gelatin. - Dextran-charcoal solution : Norit A (Prolabo) 2.5 g, Dextran T 70 (Pharmacia) 0.25 g in 1,000 ml of phosphate buffer. - Petroleum ether (40-60') Merck. - Scintillation fluid : PPO (Merck) 5 g, POPOP (Merck) 0.025 g in 1,000 ml of toluene. Mare plasma pool with very low progesterone concentration : due to their very low level of progesterone, plasmas from mares without ovarian activity were selected. Small aliquots (lml) were frozen at - 20°C until used. Progesterone level, determined by radioimmunoassay of extracted plasma, was lower than 0.1 ng/ml. Antisera : lyophilized rabbit anti-progesterone (llp-succinyloxyprogesterone BSA conjugate) antisera were prepared by Institut Pasteur and New England Nuclear Corporation. It was dissolved in buffer to achieve an initial binding of SO-70 % of 3H-progesterone.
Steroids : 1,2,6,7- 3H-progesterone, supplied by NEN, had a specific activity of 96 Ci/mmole and a radiochemical purity greater than 97 %, checked every 6 months by paper chromatography using hexanelformamide solvent system. Stock solution (10 pCi/ml) was prepared in benzene and stored at 7°C. For radioimmunoassay, aliquots were evaporated to dryness and redissolved in buffer to produce a solution containing 0.004 pCi/O.lO ml. Nonlabelled progesterone was supplied by Ikapharm. Radioimmunoassay - Standard curve : 0.10 ml of ethanolic progesterone solution (25 ng/ml) were evaporated to dryness under nitrogen in a heating block at 6O'C. Pool plasma (0.50 ml) was added and the tubes were gently shaken for good redissolution. This solution was diluted to 215,115, l/10,1/25 and l/50 with the same plasma. Standard curves were obtained from assays performed on 10 pl of each dilution (500,200,100,50,20 and 10 pg, respectively). - Assay procedure : the assays were carried out in duplicate. H-Progesterone solution (0.10 ml) was added to all tubes. Tubes T and blank received 0.20 ml buffer and 10 ul plasma pool. B, received 0.10 ml buffer, 0.10 ml antiserum and 10 pl plasma pool. Standards and samples received 0.10 ml buffer, 0.10 ml antiserum and 10 pl plasma standard or sample. The tubes were vortexed and incubated at 4°C for 2 to 12 hours. Then they were left IO min in an ice bath. We added I ml of ice-cold dextran-coated charcoal suspension, except to tube T which received I ml of buffer. The tubes were quickly vortexed, left for a further 10 min in the ice bath, and then centrifugated for 5 min. The supernatant was decanted into 65 x 10 mm (ID) glass tubes containing 1 ml toluene scintillator. These tubes were stoppered with a polypropylene cap entering the tube up to 9 mm and placed on a horizontal shaker (120 excursions per minute) : the tube's axis must be horizontal and perpendicular to the movement in order to avoid emulsion formation. After shaking for 30 min at room temperature and standing for one hour in a rack, the tubes were counted for 10,000 counts in a scintillation spectrometer (Coruflow with Scaler SC 711 M - ICN). For this, each tube was positioned vertically in a 20 ml glass counting vial. 3
- Assays with plasma extraction : 0.50 ml of plasma were extracted with 5 ml petroleum ether ; aliquots of 0.10 ml to 0.40 ml of the organic phase were evaporated to dryness in reaction tubes. Radioimmunoassays were performed as for B, tubes, adding the same reagents. RESULTS 3
H-Progesterone
fixation by mare plasma
The progesterone binding ability of mare plasma was checked by a series of "blanks" using 10 different plasmas (IO pl). The difference in binding of the blanks, in the presence of plasma, from those without plasma was 1.2 z 0.3 %. Moreover, between the duplicate procedures of
S
WEEOXDI
+ each assay, the derived standard deviation was - 0.2 X, thus showing 3 slight variation between the mare plasmas. Binding of H-progesterone by 0.10 ml of mare plasma (12-14 X) was not reduced by addition of 5 ng progesterone : protein binding of progesterone is nonspecific. Standard curve A mean standard curve is represented on figure I. Intra-assay standard deviation has been plotted for each point.
I 100
200
300
400
500
pg Progesterone Figure 1.
Mean standard curve (n=12).
Two standard curves, with and without plasma, have been compared : all the values of the second curve were within the 95 X confidence limits of the first one. Precision The precision of the method has been determined on 2 plasma pools whose progesterone levels were 3.3 ng/ml and 9.6 ng/ml : intra-assay coefficients of variation were t 4.8 % (n =16) and z 3.6 X (n -10) respectively ; inter-assay coefficients of variation were t 6 % (n -11) and + 5.7 % (n =7). Accuracy Two and 10 ng/ml of progesterone have been added to the 3.3 ng/ml
plasma pool to determine the accuracy. Recoveries were, respectively, 101.5 + 9 % (n =lO) and 100.6 4 9.3 % (n =lO). Sensitivity The mean binding of B, tube was 63.2 + 1.6 %. Sensitivity, defined as the smallest concentration
able to give a response significantly
different from B. (-2SD), has been determined from a standard curve obtained with 2,5,10 and 20 pg. Its value was 4 pg in the reacting tube, i.e., 0.4 ng/ml in the mare's plasma. Specificity Results obtained for mares without ovarian activity or in follicular phase were close to or lower than the minimal detectable concentration
: there was no important cross-reactivity.
cortisol determinations gave values between
assayed by a radiocompetition
Several plasma
procedure
10 and 30 ngfml. The cross-reactivity
(8)
percentage at
50 % displacement binding was 0.01 for Cortisol with this antiserum ;it did not cross-react
Y
.
significantly.
I IO -9 --
8 -7 --
5 --
n = 30. Y = 0.2 + 1.02 x r = 0.98. p(O.001.
ng/ml progesterone.Extracted Figure 2.
plasma.
Comparison of progesterone concentrations measured with (X) and without (Y) extraction of mare plasma.
S
IDEOXDI
37
Progesterone radioimmunoassay of mare plasma using the same antiserum and a single hexane extraction has been described (7).
Therefore,
the results of the present procedure were compared with those using the extraction method. (fig. n'2). The correlation is significant. Liquid scintillation counting Counting efficiency was approximatly 50 %. Counting precision was determined on tubes T with 0.10 ml antiserum and without decantation : the coefficient of variation was +_2 % (n 310). 3H-progesterone extraction by scintillation liquid was 96.3 + 0.7 % ; it was not reduced 3 by antiserum. Pipeting error of H-progesterone solution, determined by weighing, , was + 1 % ; statistical error on radioactivity was + 1 %. DISCUSSION Horse serum has been used for specific radioassay of cortisol (S,9,10) : progesterone competition for cortisol binding sites on horse transcortin is very weak. The results we obtained indicate that 3 H-progesterone binding of mare plasma is weak, constant and nonspecific ; therefore, direct radioimmunoassay of mare progesterone in unextracted plasma is possible. Non-specific binding is reduced to a minimum by taking 10 1.11 of plasma, and corrected by adding to standard tubes the same volume from a plasma pool containing a low level of progesterone. Radioimmunoassay is not disturbed by mare plasma. With our procedure, progesterone levels may be determined with suitable precision from 1 to 50 ng/ml, allowing study of mare luteal function and gestation. In the follicular phase, progesterone levels are close to or lower than radioassay sensitivity ; this sensitivity may be reduced to 0.2 ng/ml by antiserum dilution up to 30-40 % binding for B. tube. Plasma sample volume can be increased while performing a blank for each plasma. Liquid scintillation counting has been modified. Counting is generally performed in a homogeneous solution of aqueous phase in the scintillation fluid : this is necessary when the solubility of radioactive product in the organic phase is poor, or when a mixture of radioactive products with different physical properties must be counted. For a unique radioactive product that is soluble in organic phase,
however, direct extraction of the aqueous phase by scintillation fluid seems the better way. Two-phase liquid scintillation counting has already been used in radioimmunoassay,
for extraction of free hormone either directly(lO,ll,
12) or after precipitation
of globulins by ammonium sulfate (13,14,15).
Scintillation fluid volume used is generally IO ml, a volume very much greater than the aqueous phase volume. Liquid scintillation counting with a 1 ml homogeneous
liquid phase has been also described
(16).
The proposed procedure is a two-phase micromethod with a scintillation fluid volume reduced to 1 ml, offering advantages of the two precedent technics : no quenching and scintillation fluid saving. 3 H-Progesterone bound to antibodies is extracted by scintillation fluid after free hormone adsorption on dextran-charcoal
: assay precision is
little influenced by the extraction procedure, the efficiency of which is not reduced by antiserum and plasma. Our tube agitation procedure, involving perpendicular movement in reference to the axis of the tube, avoids the formation of a stable emulsion produced by parallel movement. This former does not reduce the counting efficiency but will slightly decrease precision. This radioimmunoassay
of progesterone
in mare plasma has been used
during two breeding seasons for early diagnosis of gestation and for study of luteal function with concordant results (17). Note : Since sending this manuscript,
a similar liquid scintil-
lation counting method has been proposed for estradiol by Kandell, et -al. (Steroids -28 : 755, 1976).
ACKNOWLEDGEMENTS We wish to thank D.W. Bruneteau, M.D., and F. Laulan for their technical assistance.
REFERENCES I.
2. 3.
Smith I.D., Basset J.M., and Williams T. VIIth Int. Congr.Anim. Reprod. Artif. Insemination. Munchen. 2 : 1190 (1972). Stabenfeldt G.H., Hughes J.P., and Evans J.W. Endocrinology 90 : 1379 (1972). 33 : 535 (1973). Sharp D.C., and Black D.L. J. Reprod. Fert. -
4. 5. 6. 7.
8. 9. 10.
11. 12. 13. 14. 15. 16. 17.
Robertson HA., and Sarda I.R. J. Endocr. 49 : 407 (1971). Thimonier J. Rec. Med. Vet. Alfort. 149 : 503 (1973). Prod. Sci. l : 197 Palmer E., Thimonier J., and Lemon MTivest. (1974). Palmer E., and Jousset B. J. Reprod. Fert. 2 : 214 (1974). Murphy B.E.P. J. Clin. Endocr. Met. 41 : 1050 (1975). Fischer M., Curtis G.C., Gaujam V.K.,Joshlin L., and Perry S. Clin. Chem. 19 : 511 (1973). P.M. Keane, x Stuart, J. Mender, S. Barbadozo, and W.H.C. Walker Clin. Chem. 21 : 1474 (1975). Castanier M., and SchollerR. C.R. Acad. Sci. Paris. 271 : 1787 (1970). Jowett T.P., Slater J.D.H., Piyasena R.D., and Ekins R.P. Clin. Sci. Mol. Med. 45 : 607 (1973). Mayes D.M., and%gent C.A. Steroids. 15 : 389 (1970). Kream J., Hellman L., and Rosenfeld R.K Steroids. 27 : 727 (1976). Kurtz A.B., and Bartter F.C. Steroids. 28 : 133 (19%). Tyler J.P.P., Hennam J.P., Newton J.R.,and Collins W.P. Steroids. 22 : 871 (1973). xrchhoff J.L. These Dr. Vet. Toulouse (1977) in press.
