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A radioimmunoassay of 15 (S) 15 methyl prostaglandin F2α
A RADIOIMMUNOASSAY METHYL
John A. Salmon,
OF 15 (S) 15
PROSTAGLANDIN
Fza
B.C. Ng and S. M. M. Karim
Department of Obstetrics and Gynaecology, Un ivers ity of Singapore, Kandang Kerbau Hospital, Singapore 8.
ABSTRACT A sensitive and relatively specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F has been developed to enable the measurements of the concentrations of the dr%g in biological fluids after its administration for therapeutic abortion. The precision, accuracy and specificity of the assay are described.
ACKNOWLEDGEMENTS The prostaglandins used in this study were generously supplied by the Upjohn Company of Canada. Part of the work was carried out under U.S.A. I.D. Contract No. AID/cm/pha - C - 73/ 36. Financial assistance from the Wellcome Trust is gratefully acknowledged.
PROSTAGLANDINS FEBRUARY
1975
VOL. 9 NO. 2
339
PROSTAGLANDINS
INTRODUCTION by 15 (S) 15 methyl prostaglandin F2o (15 me F ) was synthesized Bundy et al (1) and was shown to be less susceptible P” o enzymatic degradation (PGF ). Consequently this analog is (inactivation) than prostaglandin F more potent than PGF and has a 5”onger J”uratton of action in stimulating the pregnant human uteru? In preliminary studies the drug has been adm inistered by the intravenous, intra-muscular, vaginal, extra-amniotic and intraamniotic routes and the efficacy as an abortifacient has been demonstrated (2-6). Extended clinical trials in several countries are planned. In order to develop optimal dose-schedules it would be useful to determine blood and amniotic fluid concentrations of the analog after administration by various routes. The present investigation was undertaken to develop a sensitive and specific radioimmunoassay for 15 me F 20’
MATERIALS
study
AND METHODS
All the prostaglandins used as reference were supplied by the Upjohn Co. of Canada.
and standards
in this
Due to the unavailability of radioactively labelled 15-methyl PGF20 (15 Ci/m Mol; Amersham) was used in the assay.
tritiated
PGF2c,
Antisera 15-methyl PGF20 free acid was conjugated to bovine serum albumin using the water soluble carbodiimide method previously employed to synthes ise a similar conjugate of PGF 2pl (7,8). After the purification of the conju; gate by repeated dialysis it was injected (2 mg in Freunds adjuvant) subcutaneously into New Zealand white rabbits. Weekly injections were made for 6 weeks and thereafter once a month. Blood was withdrawn periodically from the marginal ear vein and tested for antibody activity.
goats Assay
The second antibody, as previously described
anti-rabbit (7).
gamma
globulin,
was raised
in
Procedure
The final assay procedure was similar to that described for PGF2,, (7). The anti-15 methyl PGF serum (UR # 13) was used at a final dilutton of 1:2000 in tyrode buffer (p I? 4.8) and this was capable of binding 50% of the added tracer. Incubation of the anti-serum (0.5 ml) with the radioactive PGFZa! (0. lml of approximately 12,500 dpm / 0. lml) and sample (or standard) at room
340
FEBRUARY
1975
VOL. 9 NO. 2
PROSTAGLANDINS
temperature was continued for 1 hour after which time the second antibody (0. lml) was added. Incubation at 0 - 4’C was continued for a further 18 hours. The insoluble complex was separated by centrifugation at 3000 rpm for 30 min in the cold. An aliquot (0.5 ml) of the supernatant was counted in a toluene-triton X-100 scintillation fluid using a Nuclear Chicago Mark II Liquid Scintillation counter. The instrument was equipped with a photon monitor which rejected The effic iency of counting trit ium in samples with high chemiluminescence. was reprothis system was 49.9 + 0.2%(n = 22). As the counting efficiency ducible the results were calculated from c. p.m. data. Specific ity The cross reaction of a series of prostaglandins and related comA comparison of 15-methyl pounds was assessed in the above assay system. PGFZo, free acid and 15-methyl PGF2a! methyl ester was included. Accuracy Varying concentrations of 15-methyl PGF20 free acid were prepared in amniotic fluid collected from a patient in the second trimester of pregnancy. Aliquots (0. lml) of these solutions were assayed in duplicate by the RIA. Reproducibility
10 times;
Three concentrations of 15-methyl PGFpa! free acid were each assayed the standard deviation and standard error of the mean were calculated.
RESULTS PGF ,Ryl;T;;a’ezeyhibited All rabbits injected with the 15-methyl antibody activity. The animal with the highest titre throughout the study. Using the conditions described in the assay procedure 320 pg 15 methyl PGF20 (0. lml of 3200 pg /ml) could be detected. The anti-sera was very specific for prostaglandins there was significant cross reaction with PGF with other prostaglandins are listed in Table
FEBRUARY
1975
VOL. 9 NO. 2
of the F series
but
341
PROSTAGLANDINS
Fig, l
Cross reactions of various prostagland[ns with ant[-15-methyl PGF2a serum (UR ~t 13) (See Table I for the code)
1002
2
4
~
SO.
g i3
13 hll
I I ng;rml .
t~ ngttml
I
I
100 ng/ml
,
1Asg/ml
Concentretion of Prostag|andin$
342
F E B R U A R Y 1975
VOL. 9 NO. 2
PROSTAGLANDINS
Table
Cross
reaction
anti-IS-methyl
Code
of various
Prostaglandin
Prostaglandin
20 ethyl PGF2@ 15-epi-PGF2@ 15 (S) 15 methyl
PGF**
15 (R) 15 methyl PGF2a
7 8 9 10
with
(UR #
4. 7
PGFIU
15iketo
prostaglandins FZa! serum
Mass required to displace 50% of bound
PGF2a,
6
I
FGFZa
PGEl PGE2 PGA2
11
PGB2 PGBl
12
13,14 dihydro
PGF2a,
13
13,14 dihydro
15-keto PGFZa
1975
VOL. 9 NO. 2
Relative Cross Reaction (%)
36
20
8
39
4.3
169
1
1. 7 85
100 2
206
0.6
142
1.2
206
0.6
206
0.6
206
0.6
206
0.6
78 206
* There was no significant difference of displacement methyl ester of 15 (S) 15 methyl PGF2a.
FEBRUARY
13)
2 0.6
by the acid or
343
PROSTAGIANDINS
of the method, as assessed by the comparison of the in amniotic fluid with the amount added, was satis-
The accuracy
measured concentration factory (Table II).
Table
II
Radioimmunoassay of known amounts of PGF2o added to amniotic fluid.
l&Methyl PGF (nglm1j20
Assayed Concentration of 15-Methyl FGF20 (ngl ml)
Added
6.4
5.8
12.8
11. 0
20. 5
23.2
25.6
27. 1
34. 1
33.5
51. 2
46.0
The reproducibility
of the assay Table
is reported
in Table III.
III
Reproducibility of the radioimmunoassay at different concentrations of added 15-Methyl PGF2a!
15-Methyl PGF Added (ng 1 ml)20
Detezizft (n)
ions
Assayed Concentration of 15-Methyl PGF20
S. D.
S.E.M.
0.363 3. 107 11.102
0. 115
(ng/ ml) 12.8 25.6 51.2
344
10 10 10
13. 7 29. 4 55. 1
FEBRUARY
1975
0.983 3.511
VOL. 9 NO. 2
PROSTAGLANDINS
DISCUSSION has been A sensitive and specific assay for &methyl PGF developed to enable reliable measurements of the compoun 2z in bio logical We have already fluids after its administration for therapeutic abortion. established that an accurate assay of the drug added to amniotic fluid is possible. The specificity of the assay will permit the measurement of 15-methyl in amniotic fluid without chromatography. The influence of natural PGF2a prostaglandin and its metabolites should be insignificant when, for example, amniotic fluid is assayed after the intra-amniotic administration of 500 pg or 1 mg IS-methyl PGF2o. PGF in blood is not possible using The determination of 15-methyl of the the assay conditions described in this paper Joue to the poor sensitivity The sens it ivity of the assay can be improved by using a more dilute assay. of high specific antibody solution and less tritiated prostaglandin. 3H-PGF activity 0175 Ci/m Mel) is now available and its use in th?assay of 15methyl PGF should permit more sensitive analyses. Also, sensitivity and specificity o2i”the assay would probably improve if radioactive 15-methyl PGF20 itself was employed in the assay. Extraction and chromatographic separation of the analog from plasma will undoubtedly be required prior to radioimmunoassay. There was no significant difference in the reaction of 15-methyl PGF20 methyl ester or 15-methyl PGF free acid in the radioimmunoassay system. This confirms the theory that t.P”ts the sites in the parent molecule furthest from the point of conjugation which are critical in its reaction with the antibody. 15 (R) 15 methyl PGF only cross reacts 2% in the assay thus illustrating the influence of con f2” igurational modifications in the antigen molecule in its reaction with the antibody. We are at present employing the assay to monitor methyl PGF2o free acid and methyl ester from the amniotic administration for therapeutic termination of pregnancy.
the decay of 15fluid after its
REFERENCES 1.
Bundy, G. I. , Lincoln, F. , Nelson, N. , Pike, Novel Prostaglandin syntheses. Ann. N. Y. Acad. Sci. 180 : 76 1971
FEBRUARY
1975
VOL. 9 NO. 2
J. , Schneider,
W.
345
PROSTAGLANDINS
2.
3.
Karim, S.M.M. and Sharma, S.D. Termination of second trimester pregnancy with 15-methyl prostaglandins F2 and F . J. Obstet. Gynaec. Br. eommonw. 79 : 737, 1972.
analogues
Karim, S. M. M., Sharma, S.D. and Filshie, G. M. Termination of pregnancy with 15-methyl analogues of prostagiandins and F . The P%taglandins - Clinical Applications in Human Reproduction. Edited by E. M. Southern. Futura, Mount Kisco, P. 307.
of
E2
4.
Karim, S. M. M. , Sharma, S. D. , Filshie, G. M., Salmon, J. A. and Adaikan, Ganesan, P. Termination of pregnancy with prostaglandin analogs. Advances in Biosciences, 9 - International Conference on Prostaglandins. Edited by S. BergstrBm and S. Bernhard. Pergamon Press - Vieweg, Oxford P. 811.
5.
Toppozada, M., Bkguin, F., Bygdeman, M. and W iqvist, N. Response of the mid-pregnant human uterus to systemic administration of 15 (S) 15 methyl prostaglandin F . Prostaglandins 2 : 239, 1972. 2.
6.
Wiqvist, N., Bkguin, F., Bygdeman, M. and Toppozada, M. 15 (S) 15 methyl Prostaglandin F : Myometrial response. Advances in the Biosciences 9 J%ternational Conference on Prostaglandins. Edited by S. Bergstrijm and S. Bernhard. Pergamon Press - Vieweg, Oxford P. 831.
7.
Salmon, J.A. and Amy, J. J. Levels of prostaglandin FZO in amniotic labour. Prostaglandins 4 : 523, 1973.
8.
fluid during pregnancy
Caldwell, B. V., Burstein, S., Breck, W.A. Radioimmunoassay of the F Prostaglandins. J. Clin. Endocrinol. Metab. 33 : 171, 1971.
and
FEBRUARY
Speroff,
1975
and
L.
VOL. 9 NO. 2
John A. Salmon,
OF 15 (S) 15
PROSTAGLANDIN
Fza
B.C. Ng and S. M. M. Karim
Department of Obstetrics and Gynaecology, Un ivers ity of Singapore, Kandang Kerbau Hospital, Singapore 8.
ABSTRACT A sensitive and relatively specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F has been developed to enable the measurements of the concentrations of the dr%g in biological fluids after its administration for therapeutic abortion. The precision, accuracy and specificity of the assay are described.
ACKNOWLEDGEMENTS The prostaglandins used in this study were generously supplied by the Upjohn Company of Canada. Part of the work was carried out under U.S.A. I.D. Contract No. AID/cm/pha - C - 73/ 36. Financial assistance from the Wellcome Trust is gratefully acknowledged.
PROSTAGLANDINS FEBRUARY
1975
VOL. 9 NO. 2
339
PROSTAGLANDINS
INTRODUCTION by 15 (S) 15 methyl prostaglandin F2o (15 me F ) was synthesized Bundy et al (1) and was shown to be less susceptible P” o enzymatic degradation (PGF ). Consequently this analog is (inactivation) than prostaglandin F more potent than PGF and has a 5”onger J”uratton of action in stimulating the pregnant human uteru? In preliminary studies the drug has been adm inistered by the intravenous, intra-muscular, vaginal, extra-amniotic and intraamniotic routes and the efficacy as an abortifacient has been demonstrated (2-6). Extended clinical trials in several countries are planned. In order to develop optimal dose-schedules it would be useful to determine blood and amniotic fluid concentrations of the analog after administration by various routes. The present investigation was undertaken to develop a sensitive and specific radioimmunoassay for 15 me F 20’
MATERIALS
study
AND METHODS
All the prostaglandins used as reference were supplied by the Upjohn Co. of Canada.
and standards
in this
Due to the unavailability of radioactively labelled 15-methyl PGF20 (15 Ci/m Mol; Amersham) was used in the assay.
tritiated
PGF2c,
Antisera 15-methyl PGF20 free acid was conjugated to bovine serum albumin using the water soluble carbodiimide method previously employed to synthes ise a similar conjugate of PGF 2pl (7,8). After the purification of the conju; gate by repeated dialysis it was injected (2 mg in Freunds adjuvant) subcutaneously into New Zealand white rabbits. Weekly injections were made for 6 weeks and thereafter once a month. Blood was withdrawn periodically from the marginal ear vein and tested for antibody activity.
goats Assay
The second antibody, as previously described
anti-rabbit (7).
gamma
globulin,
was raised
in
Procedure
The final assay procedure was similar to that described for PGF2,, (7). The anti-15 methyl PGF serum (UR # 13) was used at a final dilutton of 1:2000 in tyrode buffer (p I? 4.8) and this was capable of binding 50% of the added tracer. Incubation of the anti-serum (0.5 ml) with the radioactive PGFZa! (0. lml of approximately 12,500 dpm / 0. lml) and sample (or standard) at room
340
FEBRUARY
1975
VOL. 9 NO. 2
PROSTAGLANDINS
temperature was continued for 1 hour after which time the second antibody (0. lml) was added. Incubation at 0 - 4’C was continued for a further 18 hours. The insoluble complex was separated by centrifugation at 3000 rpm for 30 min in the cold. An aliquot (0.5 ml) of the supernatant was counted in a toluene-triton X-100 scintillation fluid using a Nuclear Chicago Mark II Liquid Scintillation counter. The instrument was equipped with a photon monitor which rejected The effic iency of counting trit ium in samples with high chemiluminescence. was reprothis system was 49.9 + 0.2%(n = 22). As the counting efficiency ducible the results were calculated from c. p.m. data. Specific ity The cross reaction of a series of prostaglandins and related comA comparison of 15-methyl pounds was assessed in the above assay system. PGFZo, free acid and 15-methyl PGF2a! methyl ester was included. Accuracy Varying concentrations of 15-methyl PGF20 free acid were prepared in amniotic fluid collected from a patient in the second trimester of pregnancy. Aliquots (0. lml) of these solutions were assayed in duplicate by the RIA. Reproducibility
10 times;
Three concentrations of 15-methyl PGFpa! free acid were each assayed the standard deviation and standard error of the mean were calculated.
RESULTS PGF ,Ryl;T;;a’ezeyhibited All rabbits injected with the 15-methyl antibody activity. The animal with the highest titre throughout the study. Using the conditions described in the assay procedure 320 pg 15 methyl PGF20 (0. lml of 3200 pg /ml) could be detected. The anti-sera was very specific for prostaglandins there was significant cross reaction with PGF with other prostaglandins are listed in Table
FEBRUARY
1975
VOL. 9 NO. 2
of the F series
but
341
PROSTAGLANDINS
Fig, l
Cross reactions of various prostagland[ns with ant[-15-methyl PGF2a serum (UR ~t 13) (See Table I for the code)
1002
2
4
~
SO.
g i3
13 hll
I I ng;rml .
t~ ngttml
I
I
100 ng/ml
,
1Asg/ml
Concentretion of Prostag|andin$
342
F E B R U A R Y 1975
VOL. 9 NO. 2
PROSTAGLANDINS
Table
Cross
reaction
anti-IS-methyl
Code
of various
Prostaglandin
Prostaglandin
20 ethyl PGF2@ 15-epi-PGF2@ 15 (S) 15 methyl
PGF**
15 (R) 15 methyl PGF2a
7 8 9 10
with
(UR #
4. 7
PGFIU
15iketo
prostaglandins FZa! serum
Mass required to displace 50% of bound
PGF2a,
6
I
FGFZa
PGEl PGE2 PGA2
11
PGB2 PGBl
12
13,14 dihydro
PGF2a,
13
13,14 dihydro
15-keto PGFZa
1975
VOL. 9 NO. 2
Relative Cross Reaction (%)
36
20
8
39
4.3
169
1
1. 7 85
100 2
206
0.6
142
1.2
206
0.6
206
0.6
206
0.6
206
0.6
78 206
* There was no significant difference of displacement methyl ester of 15 (S) 15 methyl PGF2a.
FEBRUARY
13)
2 0.6
by the acid or
343
PROSTAGIANDINS
of the method, as assessed by the comparison of the in amniotic fluid with the amount added, was satis-
The accuracy
measured concentration factory (Table II).
Table
II
Radioimmunoassay of known amounts of PGF2o added to amniotic fluid.
l&Methyl PGF (nglm1j20
Assayed Concentration of 15-Methyl FGF20 (ngl ml)
Added
6.4
5.8
12.8
11. 0
20. 5
23.2
25.6
27. 1
34. 1
33.5
51. 2
46.0
The reproducibility
of the assay Table
is reported
in Table III.
III
Reproducibility of the radioimmunoassay at different concentrations of added 15-Methyl PGF2a!
15-Methyl PGF Added (ng 1 ml)20
Detezizft (n)
ions
Assayed Concentration of 15-Methyl PGF20
S. D.
S.E.M.
0.363 3. 107 11.102
0. 115
(ng/ ml) 12.8 25.6 51.2
344
10 10 10
13. 7 29. 4 55. 1
FEBRUARY
1975
0.983 3.511
VOL. 9 NO. 2
PROSTAGLANDINS
DISCUSSION has been A sensitive and specific assay for &methyl PGF developed to enable reliable measurements of the compoun 2z in bio logical We have already fluids after its administration for therapeutic abortion. established that an accurate assay of the drug added to amniotic fluid is possible. The specificity of the assay will permit the measurement of 15-methyl in amniotic fluid without chromatography. The influence of natural PGF2a prostaglandin and its metabolites should be insignificant when, for example, amniotic fluid is assayed after the intra-amniotic administration of 500 pg or 1 mg IS-methyl PGF2o. PGF in blood is not possible using The determination of 15-methyl of the the assay conditions described in this paper Joue to the poor sensitivity The sens it ivity of the assay can be improved by using a more dilute assay. of high specific antibody solution and less tritiated prostaglandin. 3H-PGF activity 0175 Ci/m Mel) is now available and its use in th?assay of 15methyl PGF should permit more sensitive analyses. Also, sensitivity and specificity o2i”the assay would probably improve if radioactive 15-methyl PGF20 itself was employed in the assay. Extraction and chromatographic separation of the analog from plasma will undoubtedly be required prior to radioimmunoassay. There was no significant difference in the reaction of 15-methyl PGF20 methyl ester or 15-methyl PGF free acid in the radioimmunoassay system. This confirms the theory that t.P”ts the sites in the parent molecule furthest from the point of conjugation which are critical in its reaction with the antibody. 15 (R) 15 methyl PGF only cross reacts 2% in the assay thus illustrating the influence of con f2” igurational modifications in the antigen molecule in its reaction with the antibody. We are at present employing the assay to monitor methyl PGF2o free acid and methyl ester from the amniotic administration for therapeutic termination of pregnancy.
the decay of 15fluid after its
REFERENCES 1.
Bundy, G. I. , Lincoln, F. , Nelson, N. , Pike, Novel Prostaglandin syntheses. Ann. N. Y. Acad. Sci. 180 : 76 1971
FEBRUARY
1975
VOL. 9 NO. 2
J. , Schneider,
W.
345
PROSTAGLANDINS
2.
3.
Karim, S.M.M. and Sharma, S.D. Termination of second trimester pregnancy with 15-methyl prostaglandins F2 and F . J. Obstet. Gynaec. Br. eommonw. 79 : 737, 1972.
analogues
Karim, S. M. M., Sharma, S.D. and Filshie, G. M. Termination of pregnancy with 15-methyl analogues of prostagiandins and F . The P%taglandins - Clinical Applications in Human Reproduction. Edited by E. M. Southern. Futura, Mount Kisco, P. 307.
of
E2
4.
Karim, S. M. M. , Sharma, S. D. , Filshie, G. M., Salmon, J. A. and Adaikan, Ganesan, P. Termination of pregnancy with prostaglandin analogs. Advances in Biosciences, 9 - International Conference on Prostaglandins. Edited by S. BergstrBm and S. Bernhard. Pergamon Press - Vieweg, Oxford P. 811.
5.
Toppozada, M., Bkguin, F., Bygdeman, M. and W iqvist, N. Response of the mid-pregnant human uterus to systemic administration of 15 (S) 15 methyl prostaglandin F . Prostaglandins 2 : 239, 1972. 2.
6.
Wiqvist, N., Bkguin, F., Bygdeman, M. and Toppozada, M. 15 (S) 15 methyl Prostaglandin F : Myometrial response. Advances in the Biosciences 9 J%ternational Conference on Prostaglandins. Edited by S. Bergstrijm and S. Bernhard. Pergamon Press - Vieweg, Oxford P. 831.
7.
Salmon, J.A. and Amy, J. J. Levels of prostaglandin FZO in amniotic labour. Prostaglandins 4 : 523, 1973.
8.
fluid during pregnancy
Caldwell, B. V., Burstein, S., Breck, W.A. Radioimmunoassay of the F Prostaglandins. J. Clin. Endocrinol. Metab. 33 : 171, 1971.
and
FEBRUARY
Speroff,
1975
and
L.
VOL. 9 NO. 2