- Email: [email protected]
A simple radioimmunoassay for 3′,5′, cyclic adenosine monophosphate
A Simple 3’,5’,
Cyclic
Radioimmunoassay Adenosine
for
Monophosphate
ROBERT W. FARMER, CHARLES A. HARRINGTON, AND DOUGLASS H. BROWN
Received
June 2 I. I Y74: accepted
October
15, I974
A radioimmunoassay for 3’.5’ cAMP has been developed in which [‘H]3’.5’ cAMP is the radioligand. Antibody-bound and free fractions are separated with dextran-coated charcoal. The sensitivity of the assay is tJ.03 pmoles and antiserum specificity is 7 orders with respect to other adenine nucleotideb. Samples are prepared by ethanol precipitation. Tissue levels of 3’2’ CAMP are comparable to thobe reported by others.
Several methods have been reported for the measurement of 3’2’ cyclic adenosine monophosphate (3’2’ CAMP) including radioimmonoassay (RlA) (I). The most frequently used RIA is that of Steiner et crl. (3) who used an iodinated tyrosine methyl ester as the radioligand. The affinity constant of the antibody for 3’.S’ CAMP in their system was about lox M-'. Cailla et LI/. (3) reported a system in which both the radioligand and substrate were derivatized. In their system, the affinity of the antibody for the derivative was I .25 X 10”’ M- ‘. It appeared to us that the lower affinity of Steiner’s system was due to the lesser ability of 3’,5’ CAMP per se to compete with the higher affinity derivative, assuming the antibody was the same in both cases. We elected to investigate the possibility of developing a highly sensitive system in which [:jH]3’,5’ CAMP is used as radioligand such as is done routinely with steroids. While neither the use of tritiated 3’.5’ CAMP or charcoal separation are totally unique (4.5). the conditions described herein offer improved sensitivity and a simple purification procedure. MATERIALS
AND
METHODS
Antisera were generated to the 2-O’ succinyl-BSA derivative prepared as described by Steiner rr trl. (3). [:(H]3’.5’ cyclic adenosine monophosphate (3 1.3 Cilmmole) was purchased from New England Nuclear and used without purification. Antiserum was diluted in 0.05 M sodium borate buffer (pH 8.9) and had a titer of I :400. [:*H]3’.5’ CAMP and unlabeled 3’S CAMP were diluted in buffer to add 0.3 pmoles of [:‘H]3’.5’ CAMP and an appropriate amount of standard in 50 ~1.
456
FARMER,
HARRINGTON
AND
BROWN
Samples of tissue for two dose comparisons were prepared by exposing pentobarbital, morphine-blocked rats to microwave radiation for 30 sec. The tissues were then homogenized in IO-20 vol of absolute ethanol and centrifuged. Appropriate aliquots of supernatants were then transferred to assay tubes and dried in a vacuum desiccator for assay. Deproteinization techniques were compared using aliquots of common homogenates of spleen, adrenals, and muscle recovered from etheranesthetized rats. Perchlorate deproteinization was effected by adding 200 ~1 of each homogenate to 700 ~1 water and adding 100 ~1 of 30% perchloric acid followed by vigorous Vortexing and centrifuging. The supernatant is recovered, chilled to 4°C in iced water, and 50 ~1 of 10 N potassium hydroxide is added. On standing in the cold, potassium perchlorate precipitates and the clear supernatant is recovered after centrifugation. The supernatant was neutral to litmus paper. Trichloroacetic acid deproteinization was performed as described by Steiner et al. (2). Whole adrenal cell incubates (250 ~1) were transferred to 1 ml of cold absolute ethanol, Vortexed vigorously and centrifuged. One-half-milliliter aliquots transferred to assay tubes and dried in a vacuum desiccator for assay. To assay, 50 ~1 each of [“H]3’.5’ CAMP and standard or sample in buffer, and 0.5 ml of diluted antiserum were added to 12 X 75-mm culture tubes. Samples were incubated for 30 min at 21°C and 30 min at 4°C. Free and bound nucleotide were separated by adding 50 ~1 of dextran-charcoal (0.5% and 5% w/v) that was suspended in water and stirred constantly, vortexing, and allowing to stand for 8 min. Tubes were centrifuged at 900 g to settle out the charcoal. One-half milliliter of supernatant was transferred to counting vials along with scintillation cocktail and counted to 2% counting error. RESULTS Figure 1 shows a typical standard curve. Sensitivity as judged by 2 S.D. about the zero was 0.03 pmoles. Figure 2 shows a Scatchard plot which was linear indicating the homogeneity of the antibody. This antibody showed no more than 0.03 pmoles deflection when 300 nmoles of 5’-ATP. 3’-AMP, 5’-AMP, 2’-AMP. or 2’,3’ CAMP were added, i.e.. it has at least 7 orders of specificity. Figure 3 shows the tissue levels of 3’,5’ CAMP in the pentobarbitalanesthetized, morphine-blocked rats. This preparation was chosen to attain minimal stimulation of the adrenal from endogenous ACTH (6). ACTH (100 mU and glucagon 100 pg) were injected in two rats to obtain stimulated levels in adrenal and liver, respectively. This figure clearly shows stimulation of the adrenal and liver. More importantly,
RADIOIMMUNOASSAY
FOR
3l.5’.
CAMP
315’ CAMP RABBIT 2 l/8/74
08 07 OE 05 i k E
04
5 g
03 0; 0
I MASS I-I<,.
2. Scatchard
2 BOUND
(MOLES
plot
of data
3
4
~10~‘~)
shown
in I-if.
I
457
458
FARMER.
HARRINGTON
AND
LIVER
BROWN
ADRENAL
0 Olr-
5
IO
TISSUE
15
I
EQUIVALENT
2
ASSAYED
3 Cmg)
FIG. 3. Concentration of 3’.5’ CAMP in adrenal gland and liver anesthetized, morphine-blocked rats prior iA) and 5 min after (0) ACTH ( 100 mU) and glucagon ( 100 pg).
from pentobarbitalthe iv injection
of
this figure shows that two doses of homogenate gave similar values. This indicated that nonspecific interference in the assay was minimal. Table 1 shows a comparison of deproteinizing agents. In our hands, perchlorate have a lower extraction efficiency than did trichloracetic acid (TCA) or ethanol. Ethanol has equivalent or higher values than did TCA. The recovery of labeled 3’,5’ CAMP added to tissue was 99-100%. The blank was less than 0.03 pmoles in all assays performed to date. Figure 4 shows the response of isolated adrenocortical cells (7) to TABLE COMPARISONOFPERCHLORICACID. EX~RACTIONOF
3’ . 5’ CAMP Perchlorate
Spleen Adrenal Muscle Mean Percent” ?-SD ‘I Expressed
as percentage
1
TRICHLOROACETICACID,
93.0 78.2 13.3 92.3 6.68 of TCA
extract
(pg/mg
ANDETHANOLFORTHE
wet TCA
weight)
~---
105.6 X8.1 13.3 too -
ETOH 105.6 117.4 13.3 lll.I 19.23
RADIOIMMUNOASSAY
FOR
3’,5’,
CAMP
459
lh
MINUTES
POST
STIMULATION
FIG. 4. 3’,5’ CAMP in isolated adrenalcorticoid cell incubates (cell plus media) in response to 1000 ~Uiml ACTH in the absence I A) and presence K:) of 100 pg/ml methadone. Fifty thousand cells were incubated in I ml of Krebs-Ringer bicarbonate buffer containing I mg/ml glucose and 4% bovine serum albumin for I hr.
ACTH in the presence and absence of methadone. Only 50,000 cells were used per incubation vessel. With a standard curve sensitivity of 0.03 pmoles, the assay gave an over-all sensitivity of 0.3 pmoles when 10% of each cell incubate was measured. In this incubation, the basal levels of 3’2’ CAMP in the absence of ACTH were 0.14 pmoleslml and 0.08 pmoles/ml in the absence and presence of methadone, respectively. By virtue of it’s sensitivity, this assay system permits simultaneous measurement of several steroid parameters by radioimmunoassay in addition to 3’.5’ CAMP, with a relatively small number of cells. DISCUSSION This assay system is simple, highly sensitive, and reproducible in our hands. Ethanol deproteinization as a method of preparing tissues is simple and given 99-100% recovery. The ethanol per se has a very low blank and in our hands. is far superior to other reported extraction systems in this respect. The behavior of the antibody was as predicted and gave standard curves which compared with the steroid assays with which we are most familiar. The possibility that our antibody is of higher affinity than anyone else’s seems remote except to note that prolonged
460
FARMER,
HARRINGTON
AND
BROWN
immunization induced in one donor rabbit a low affinity protein which had a short half-life, and disappeared in 2-3 wk. Insofar as the tissue levels reported herein are at least as low as those reported by other workers, the specificity of the assay is quite adequate. Attempts to verify the lack of nonspecific interference by further purification have thus far met with failure. The columns used (Dowex and Alumina) have had high and variable blanks which precluded their use. However, the levels obtained in adrenals of pentobarbital-anesthetized morphine-blocked rats are quite low as would be expected in the absence of ACTH and increased to quite high levels after the injection of ACTH. Similarly. the low basal levels in liver increased markedly in response to glucagon. Insofar as this system uses tritiated radioligand. which is highly stable, and an ethanol precipitation, it is simpler than those reported previously and in our hands has a lower blank. The single disadvantage is the larger amount of antibody required. We think this system to be superior to others reported to date by virtue of its simplicity, sensitivity (except ref. 3) and dependability. REFERENCES I. Greengard, P.. and Robison. G. A. (1972) Advances in Cyclic Nucleotide Research, Vol. II. Raven Press, New York. 7. Steiner, A. L., Wehmann, R. E., Parker. C. W., and Kipnis. D. M. (1972) irt Advances in Cyclic Nucleotide Research. (Greengard, P.. and Robison, G. A., eds.). Vol. II. p. 5 I. Raven Press, New York. 3. Cailla, H. L., Racine-Weisbuch. M. S.. and Delaage, M. A. (1973) Antrl. Bioclrem. 56, 394. 4. Weinryk. I.. Michel. I. M.. and Hess. S. M. ( 1971) Allnl. Biocllr~n. 45, 659. 5. Brown, B. L., Albano. J. D. M.. Ekins. R. P.. and Sgherzi. A. M. (1971) Biochem. ./. 121, 561. 6. Arimura, A., Saito, T.. and Schally. A. V. (I 967) End~xrino/,~fi~ 81. 235. 7. Swallow, R. L.. and Sayers. G. ( 1969) P,uK. Sot. X%/I. Biol. Med. 131, I.
Cyclic
Radioimmunoassay Adenosine
for
Monophosphate
ROBERT W. FARMER, CHARLES A. HARRINGTON, AND DOUGLASS H. BROWN
Received
June 2 I. I Y74: accepted
October
15, I974
A radioimmunoassay for 3’.5’ cAMP has been developed in which [‘H]3’.5’ cAMP is the radioligand. Antibody-bound and free fractions are separated with dextran-coated charcoal. The sensitivity of the assay is tJ.03 pmoles and antiserum specificity is 7 orders with respect to other adenine nucleotideb. Samples are prepared by ethanol precipitation. Tissue levels of 3’2’ CAMP are comparable to thobe reported by others.
Several methods have been reported for the measurement of 3’2’ cyclic adenosine monophosphate (3’2’ CAMP) including radioimmonoassay (RlA) (I). The most frequently used RIA is that of Steiner et crl. (3) who used an iodinated tyrosine methyl ester as the radioligand. The affinity constant of the antibody for 3’.S’ CAMP in their system was about lox M-'. Cailla et LI/. (3) reported a system in which both the radioligand and substrate were derivatized. In their system, the affinity of the antibody for the derivative was I .25 X 10”’ M- ‘. It appeared to us that the lower affinity of Steiner’s system was due to the lesser ability of 3’,5’ CAMP per se to compete with the higher affinity derivative, assuming the antibody was the same in both cases. We elected to investigate the possibility of developing a highly sensitive system in which [:jH]3’,5’ CAMP is used as radioligand such as is done routinely with steroids. While neither the use of tritiated 3’.5’ CAMP or charcoal separation are totally unique (4.5). the conditions described herein offer improved sensitivity and a simple purification procedure. MATERIALS
AND
METHODS
Antisera were generated to the 2-O’ succinyl-BSA derivative prepared as described by Steiner rr trl. (3). [:(H]3’.5’ cyclic adenosine monophosphate (3 1.3 Cilmmole) was purchased from New England Nuclear and used without purification. Antiserum was diluted in 0.05 M sodium borate buffer (pH 8.9) and had a titer of I :400. [:*H]3’.5’ CAMP and unlabeled 3’S CAMP were diluted in buffer to add 0.3 pmoles of [:‘H]3’.5’ CAMP and an appropriate amount of standard in 50 ~1.
456
FARMER,
HARRINGTON
AND
BROWN
Samples of tissue for two dose comparisons were prepared by exposing pentobarbital, morphine-blocked rats to microwave radiation for 30 sec. The tissues were then homogenized in IO-20 vol of absolute ethanol and centrifuged. Appropriate aliquots of supernatants were then transferred to assay tubes and dried in a vacuum desiccator for assay. Deproteinization techniques were compared using aliquots of common homogenates of spleen, adrenals, and muscle recovered from etheranesthetized rats. Perchlorate deproteinization was effected by adding 200 ~1 of each homogenate to 700 ~1 water and adding 100 ~1 of 30% perchloric acid followed by vigorous Vortexing and centrifuging. The supernatant is recovered, chilled to 4°C in iced water, and 50 ~1 of 10 N potassium hydroxide is added. On standing in the cold, potassium perchlorate precipitates and the clear supernatant is recovered after centrifugation. The supernatant was neutral to litmus paper. Trichloroacetic acid deproteinization was performed as described by Steiner et al. (2). Whole adrenal cell incubates (250 ~1) were transferred to 1 ml of cold absolute ethanol, Vortexed vigorously and centrifuged. One-half-milliliter aliquots transferred to assay tubes and dried in a vacuum desiccator for assay. To assay, 50 ~1 each of [“H]3’.5’ CAMP and standard or sample in buffer, and 0.5 ml of diluted antiserum were added to 12 X 75-mm culture tubes. Samples were incubated for 30 min at 21°C and 30 min at 4°C. Free and bound nucleotide were separated by adding 50 ~1 of dextran-charcoal (0.5% and 5% w/v) that was suspended in water and stirred constantly, vortexing, and allowing to stand for 8 min. Tubes were centrifuged at 900 g to settle out the charcoal. One-half milliliter of supernatant was transferred to counting vials along with scintillation cocktail and counted to 2% counting error. RESULTS Figure 1 shows a typical standard curve. Sensitivity as judged by 2 S.D. about the zero was 0.03 pmoles. Figure 2 shows a Scatchard plot which was linear indicating the homogeneity of the antibody. This antibody showed no more than 0.03 pmoles deflection when 300 nmoles of 5’-ATP. 3’-AMP, 5’-AMP, 2’-AMP. or 2’,3’ CAMP were added, i.e.. it has at least 7 orders of specificity. Figure 3 shows the tissue levels of 3’,5’ CAMP in the pentobarbitalanesthetized, morphine-blocked rats. This preparation was chosen to attain minimal stimulation of the adrenal from endogenous ACTH (6). ACTH (100 mU and glucagon 100 pg) were injected in two rats to obtain stimulated levels in adrenal and liver, respectively. This figure clearly shows stimulation of the adrenal and liver. More importantly,
RADIOIMMUNOASSAY
FOR
3l.5’.
CAMP
315’ CAMP RABBIT 2 l/8/74
08 07 OE 05 i k E
04
5 g
03 0; 0
I MASS I-I<,.
2. Scatchard
2 BOUND
(MOLES
plot
of data
3
4
~10~‘~)
shown
in I-if.
I
457
458
FARMER.
HARRINGTON
AND
LIVER
BROWN
ADRENAL
0 Olr-
5
IO
TISSUE
15
I
EQUIVALENT
2
ASSAYED
3 Cmg)
FIG. 3. Concentration of 3’.5’ CAMP in adrenal gland and liver anesthetized, morphine-blocked rats prior iA) and 5 min after (0) ACTH ( 100 mU) and glucagon ( 100 pg).
from pentobarbitalthe iv injection
of
this figure shows that two doses of homogenate gave similar values. This indicated that nonspecific interference in the assay was minimal. Table 1 shows a comparison of deproteinizing agents. In our hands, perchlorate have a lower extraction efficiency than did trichloracetic acid (TCA) or ethanol. Ethanol has equivalent or higher values than did TCA. The recovery of labeled 3’,5’ CAMP added to tissue was 99-100%. The blank was less than 0.03 pmoles in all assays performed to date. Figure 4 shows the response of isolated adrenocortical cells (7) to TABLE COMPARISONOFPERCHLORICACID. EX~RACTIONOF
3’ . 5’ CAMP Perchlorate
Spleen Adrenal Muscle Mean Percent” ?-SD ‘I Expressed
as percentage
1
TRICHLOROACETICACID,
93.0 78.2 13.3 92.3 6.68 of TCA
extract
(pg/mg
ANDETHANOLFORTHE
wet TCA
weight)
~---
105.6 X8.1 13.3 too -
ETOH 105.6 117.4 13.3 lll.I 19.23
RADIOIMMUNOASSAY
FOR
3’,5’,
CAMP
459
lh
MINUTES
POST
STIMULATION
FIG. 4. 3’,5’ CAMP in isolated adrenalcorticoid cell incubates (cell plus media) in response to 1000 ~Uiml ACTH in the absence I A) and presence K:) of 100 pg/ml methadone. Fifty thousand cells were incubated in I ml of Krebs-Ringer bicarbonate buffer containing I mg/ml glucose and 4% bovine serum albumin for I hr.
ACTH in the presence and absence of methadone. Only 50,000 cells were used per incubation vessel. With a standard curve sensitivity of 0.03 pmoles, the assay gave an over-all sensitivity of 0.3 pmoles when 10% of each cell incubate was measured. In this incubation, the basal levels of 3’2’ CAMP in the absence of ACTH were 0.14 pmoleslml and 0.08 pmoles/ml in the absence and presence of methadone, respectively. By virtue of it’s sensitivity, this assay system permits simultaneous measurement of several steroid parameters by radioimmunoassay in addition to 3’.5’ CAMP, with a relatively small number of cells. DISCUSSION This assay system is simple, highly sensitive, and reproducible in our hands. Ethanol deproteinization as a method of preparing tissues is simple and given 99-100% recovery. The ethanol per se has a very low blank and in our hands. is far superior to other reported extraction systems in this respect. The behavior of the antibody was as predicted and gave standard curves which compared with the steroid assays with which we are most familiar. The possibility that our antibody is of higher affinity than anyone else’s seems remote except to note that prolonged
460
FARMER,
HARRINGTON
AND
BROWN
immunization induced in one donor rabbit a low affinity protein which had a short half-life, and disappeared in 2-3 wk. Insofar as the tissue levels reported herein are at least as low as those reported by other workers, the specificity of the assay is quite adequate. Attempts to verify the lack of nonspecific interference by further purification have thus far met with failure. The columns used (Dowex and Alumina) have had high and variable blanks which precluded their use. However, the levels obtained in adrenals of pentobarbital-anesthetized morphine-blocked rats are quite low as would be expected in the absence of ACTH and increased to quite high levels after the injection of ACTH. Similarly. the low basal levels in liver increased markedly in response to glucagon. Insofar as this system uses tritiated radioligand. which is highly stable, and an ethanol precipitation, it is simpler than those reported previously and in our hands has a lower blank. The single disadvantage is the larger amount of antibody required. We think this system to be superior to others reported to date by virtue of its simplicity, sensitivity (except ref. 3) and dependability. REFERENCES I. Greengard, P.. and Robison. G. A. (1972) Advances in Cyclic Nucleotide Research, Vol. II. Raven Press, New York. 7. Steiner, A. L., Wehmann, R. E., Parker. C. W., and Kipnis. D. M. (1972) irt Advances in Cyclic Nucleotide Research. (Greengard, P.. and Robison, G. A., eds.). Vol. II. p. 5 I. Raven Press, New York. 3. Cailla, H. L., Racine-Weisbuch. M. S.. and Delaage, M. A. (1973) Antrl. Bioclrem. 56, 394. 4. Weinryk. I.. Michel. I. M.. and Hess. S. M. ( 1971) Allnl. Biocllr~n. 45, 659. 5. Brown, B. L., Albano. J. D. M.. Ekins. R. P.. and Sgherzi. A. M. (1971) Biochem. ./. 121, 561. 6. Arimura, A., Saito, T.. and Schally. A. V. (I 967) End~xrino/,~fi~ 81. 235. 7. Swallow, R. L.. and Sayers. G. ( 1969) P,uK. Sot. X%/I. Biol. Med. 131, I.