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125I-radioimmunoassay for antipyrine
Pergamon Press
Life Sciences Vol . 22, pp . 855-858 Printed in the U.S .A .
125 1-RADIOIMMUNOASSAY FOR ANTIPYRINE R . L . Chang, A . W . Wood, and W . R . Dixon Department of Biochemistry and Drug Metabolism Hoffmann-LaRoche, Inc . Nutley, New Jersey 07110 Received in final form January 23, 1978)
Abstract A radioimmunoassay (RIA) for the determination of antipyrjne directly in plasma and saliva has been developed using 4-1 5I-iodoantipyrine as the radioligand . The method showed excellent agreement (r=0 .98) with a recently reported RIA for antipyrine using 3Hrantipyrine as the radioligand . The inter5 I-RIA did not exceed assay coefficient of variation for the 7 .6% and the mean recovery of antipyrine added to plasma or saliva was 101±1 .73 (S .E .) over a range of 1 .5 to 30 pg/ml . By virtue of its simplicity and low cost, the 1251-RIA for antipyrine offers an attractive method for the routine determination of antipyrine levels and subsequent calculation of its half-life in man . Hepatic drug metabolizing enzyme activity in man is commonly assessed by measurement of the plasma antipyrine half-life . Since the physico-chemical assays for the determination of antipyrine in blood (1,2,3) are either time consuming or lack sensitivity, we recently developed a radioimmunoassay (RIA) for the determination of antipyrine levels in both plasma and saliva (4) using 3H-antipyrine (3H-AP) as the radioligand . Although the 3H-RIA is simple and sensitive, a procedure using an 1251-radioli and would be more desirable in terms of cost and high sample through-put (5) . The use of a gamma emitting radioligand avoids the use of expensive liquid scintillation fluids and counting vials necessary for the 3H-RIA procedure . The present study reports he successful utilization of commercially available 4-1251-iodoantipyrine ( 15 1-AP) in the RIA for antipyrine . The high precision of the original assay is maintained under conditions which result in a significant saving of antiserum and time . Materials and Methods 4-1251-iodoantipyrine (specific activity 2 .6 Ci/mmole) was obtained from the New England Nuclear Corp ., Boston, Mass . The components of the RIA utilizing 1251-AP were essentially the same as the original RIA utilizing 3H-AP (4) except that each assay tube contained
0300-9653/78-0313-0897$02,00/0 Copyright © Pergamon Press
856
125 1-Radioimmunoassay for Antipyrine
Vol . 22, No . 10, 1978
10,000 CPM of 125,-AP and 0 .1 ml of antipyrine antiserum diluted 1 :1000 . The final volume of the assay mixture was 0 .5 ml . Antibody bound and free radioligand were separated using saturated ammonium sulfate (pH 7 .4) and the initial precipitate was washed once with 1 ml of 50% saturated ammonium sulfate . The radioactivity in the final precipitate was determined by counting the assay tube directly in a gamma counter . All samples were assayed in duplicate and the data calculated as outlined by Robard (6) using a digital computer for iterative weighted linear regression analysis of B/Bo versus log dose, where B and B o are the amounts of antibody bound radioligand in the presence and absence of unlabeled antipyrine respectively . Results and Discussion A linear calibration curve was obtained when logit B/B was plotted against the log concentration of antipyrine over a range of 1 to 108 ng of antipyrine per 0 .1 ml plasma . The percentage inhibition of binding (10%) due to 1 ng of antipyrine was statistically significant ; p < 0 .01 . The accuracy and interassay precision of the RIA are shown in Table I . The mean recoveries of 1 .5 to 30 ug/ml of antipyrine ranged from 98 to 113% and the interassay coefficients of variation were between 1 .3 and 7 .6% . The intraassay precision was evaluated with human plasma samples . The mean and standard deviation of 6 replicate determinations were calculated and the intra-assay coefficients of variation were between 1 .5 to 7 .5% over the range of 10 .7 to 29 .5 ug/ml of antipyrine (data not shown) . In order to directly compare the two RIA methods, 27 different plasma samples from four subjects who had each received a single 18 mg/kg dose of antipyrine were obtained 4 to 48 hours after drug ingestion and assayed . These samples were assayed for antipyrine by the 3H-RIA and by the newly developed 1251-RIA (Fig . 1) . The results indicate a very high correlation between the two assays when analyzed by the straight line method of Wald (7) . The correlation coefficient was 0 .98 . The fitted intercept was 0 .5 ug/ml and the slope was 0 .97 . The 125 1-RIA retains all the advantages of high sensitivity, accuracy and In addition, the convenience of our previously reported RIA which used 3H-AP . new assay affords several significant improvements . First, there is a consider able saving in time and cost of the assay since the need for liquid scintillation counting is eliminated . The original RIA procedure required the supernate to be decanted into counting vials and extracted into scintillation counting solution . The new method eliminates these steps and radioactivity can be counted directly . Secondly, there is a dramatic reduction in the amount of antiserum needed, under conditions of comparable sensitivity . The new procedure requires only 1/20th the amount of antiserum . Finally, the new assay shows significantly less cross reactivity with the major metabolite of antipyrine, 4-hydroxyantipyrine . Nine times as much 4-hydroxyantipyrine as antipyrine is needed to cause 50% inhibition of binding of the 1251 radioligand .
125
Vol . 22, No . 10, 1978
I-Radioi-unoassay for Antipyrine
857
30 25 20
10 5 0
5
10
15 20 25 les I_RIA (Kg/m1)
30
FIG . 1
Comparison of 125 I-RIA and 3H-RIA of Antipyrine in Plasma . Twenty-seven plasma samples obtained from four subjects 4 to 48 hours after administration of antipyrine were assayed by both methods . The line drawn from slope and intercept values was calculated by the method of Wald (7) . Table I Accuracy and Inter-assay Precision of the RIA for Antipyrine in Plasma and Saliva
Amount Antipyrine added(ug/ml)
Plasma
Saliva
Antipyrine recovery (vg/ml) Exp 1
Exp 2
Exp 3
Exp 4
Mean Recovery (vg/ml)
Mean % of Recovery
Coefficient of Variation (I)
1 .5
1 .5
1 .5
1 .6
1 .5
1 .5
100
3.3
7.5
7.6
7 .4
7.4
7 .5
7 .5
100
1 .3
15 .0
16 .1
14 .6
15 .0
13 .9
14 .9
99
6 .2
30 .0
30 .7
30 .3
30 .5
27 .9
29 .9
99
4.4
1 .5
1 .7
1 .6
1 .8
1 .5
1 .7
113
7.6
7.5
7.0
7.5
7.7
7 .2
7 .4
98
4.2
15 .0
14 .4
15 .8
16 .0
14 .3
15 .1
100
6.0
30 .0
31 .8
31 .5
29 .3
27 .0
29 .9
99
7.5
Control plasma containing the indicated amounts of antipyrine was assayed immediately after addition of the drug (Exp . 1) and after storing the samples at -20° for 2, 7 and 14 days in Exp . 2, 3 and 4, respectively .
858
125
I-Radioimmunoassay for Antipyrine
Vol . 22, No . 10, 1978
References 1. 2. 3. 4. 5. 6. 7.
B . B . BRODIE, J . AXELROD, R . SOBELMAN, AND B . B . LEVY ., J . Biol . Chem . 179 25-29 (1949) . L . F . PRESCOTT, K . K . ADJEPON-YAMOAH, and E . ROBERTS, J . Pharm . Pharmacol 25 205-207 (1973) . R. M . WELCH, R . L . DE ANGELIS, M . WINGFIELD, and T . W . FARMER, Clin . Pharmacol . Ther . 18 249-258 (1975) . R . L . CHANG,A . W . WOOD, W . R . DIXON, A . H . CONNEY, K . E . ANDERSON, J . EISEMAN, and A . P . ALVARES, Clin . Pharmacol . Ther . 20 219-226 (1976) . W . M . HUNTER, Brit Med . Bull . 30 18 (1974) . D . ROBARD, Cli n. C em . 20 1255-1270 (1974) . A . Wald, Ann . Math . Stat . 11 284-300 (1940) .
Life Sciences Vol . 22, pp . 855-858 Printed in the U.S .A .
125 1-RADIOIMMUNOASSAY FOR ANTIPYRINE R . L . Chang, A . W . Wood, and W . R . Dixon Department of Biochemistry and Drug Metabolism Hoffmann-LaRoche, Inc . Nutley, New Jersey 07110 Received in final form January 23, 1978)
Abstract A radioimmunoassay (RIA) for the determination of antipyrjne directly in plasma and saliva has been developed using 4-1 5I-iodoantipyrine as the radioligand . The method showed excellent agreement (r=0 .98) with a recently reported RIA for antipyrine using 3Hrantipyrine as the radioligand . The inter5 I-RIA did not exceed assay coefficient of variation for the 7 .6% and the mean recovery of antipyrine added to plasma or saliva was 101±1 .73 (S .E .) over a range of 1 .5 to 30 pg/ml . By virtue of its simplicity and low cost, the 1251-RIA for antipyrine offers an attractive method for the routine determination of antipyrine levels and subsequent calculation of its half-life in man . Hepatic drug metabolizing enzyme activity in man is commonly assessed by measurement of the plasma antipyrine half-life . Since the physico-chemical assays for the determination of antipyrine in blood (1,2,3) are either time consuming or lack sensitivity, we recently developed a radioimmunoassay (RIA) for the determination of antipyrine levels in both plasma and saliva (4) using 3H-antipyrine (3H-AP) as the radioligand . Although the 3H-RIA is simple and sensitive, a procedure using an 1251-radioli and would be more desirable in terms of cost and high sample through-put (5) . The use of a gamma emitting radioligand avoids the use of expensive liquid scintillation fluids and counting vials necessary for the 3H-RIA procedure . The present study reports he successful utilization of commercially available 4-1251-iodoantipyrine ( 15 1-AP) in the RIA for antipyrine . The high precision of the original assay is maintained under conditions which result in a significant saving of antiserum and time . Materials and Methods 4-1251-iodoantipyrine (specific activity 2 .6 Ci/mmole) was obtained from the New England Nuclear Corp ., Boston, Mass . The components of the RIA utilizing 1251-AP were essentially the same as the original RIA utilizing 3H-AP (4) except that each assay tube contained
0300-9653/78-0313-0897$02,00/0 Copyright © Pergamon Press
856
125 1-Radioimmunoassay for Antipyrine
Vol . 22, No . 10, 1978
10,000 CPM of 125,-AP and 0 .1 ml of antipyrine antiserum diluted 1 :1000 . The final volume of the assay mixture was 0 .5 ml . Antibody bound and free radioligand were separated using saturated ammonium sulfate (pH 7 .4) and the initial precipitate was washed once with 1 ml of 50% saturated ammonium sulfate . The radioactivity in the final precipitate was determined by counting the assay tube directly in a gamma counter . All samples were assayed in duplicate and the data calculated as outlined by Robard (6) using a digital computer for iterative weighted linear regression analysis of B/Bo versus log dose, where B and B o are the amounts of antibody bound radioligand in the presence and absence of unlabeled antipyrine respectively . Results and Discussion A linear calibration curve was obtained when logit B/B was plotted against the log concentration of antipyrine over a range of 1 to 108 ng of antipyrine per 0 .1 ml plasma . The percentage inhibition of binding (10%) due to 1 ng of antipyrine was statistically significant ; p < 0 .01 . The accuracy and interassay precision of the RIA are shown in Table I . The mean recoveries of 1 .5 to 30 ug/ml of antipyrine ranged from 98 to 113% and the interassay coefficients of variation were between 1 .3 and 7 .6% . The intraassay precision was evaluated with human plasma samples . The mean and standard deviation of 6 replicate determinations were calculated and the intra-assay coefficients of variation were between 1 .5 to 7 .5% over the range of 10 .7 to 29 .5 ug/ml of antipyrine (data not shown) . In order to directly compare the two RIA methods, 27 different plasma samples from four subjects who had each received a single 18 mg/kg dose of antipyrine were obtained 4 to 48 hours after drug ingestion and assayed . These samples were assayed for antipyrine by the 3H-RIA and by the newly developed 1251-RIA (Fig . 1) . The results indicate a very high correlation between the two assays when analyzed by the straight line method of Wald (7) . The correlation coefficient was 0 .98 . The fitted intercept was 0 .5 ug/ml and the slope was 0 .97 . The 125 1-RIA retains all the advantages of high sensitivity, accuracy and In addition, the convenience of our previously reported RIA which used 3H-AP . new assay affords several significant improvements . First, there is a consider able saving in time and cost of the assay since the need for liquid scintillation counting is eliminated . The original RIA procedure required the supernate to be decanted into counting vials and extracted into scintillation counting solution . The new method eliminates these steps and radioactivity can be counted directly . Secondly, there is a dramatic reduction in the amount of antiserum needed, under conditions of comparable sensitivity . The new procedure requires only 1/20th the amount of antiserum . Finally, the new assay shows significantly less cross reactivity with the major metabolite of antipyrine, 4-hydroxyantipyrine . Nine times as much 4-hydroxyantipyrine as antipyrine is needed to cause 50% inhibition of binding of the 1251 radioligand .
125
Vol . 22, No . 10, 1978
I-Radioi-unoassay for Antipyrine
857
30 25 20
10 5 0
5
10
15 20 25 les I_RIA (Kg/m1)
30
FIG . 1
Comparison of 125 I-RIA and 3H-RIA of Antipyrine in Plasma . Twenty-seven plasma samples obtained from four subjects 4 to 48 hours after administration of antipyrine were assayed by both methods . The line drawn from slope and intercept values was calculated by the method of Wald (7) . Table I Accuracy and Inter-assay Precision of the RIA for Antipyrine in Plasma and Saliva
Amount Antipyrine added(ug/ml)
Plasma
Saliva
Antipyrine recovery (vg/ml) Exp 1
Exp 2
Exp 3
Exp 4
Mean Recovery (vg/ml)
Mean % of Recovery
Coefficient of Variation (I)
1 .5
1 .5
1 .5
1 .6
1 .5
1 .5
100
3.3
7.5
7.6
7 .4
7.4
7 .5
7 .5
100
1 .3
15 .0
16 .1
14 .6
15 .0
13 .9
14 .9
99
6 .2
30 .0
30 .7
30 .3
30 .5
27 .9
29 .9
99
4.4
1 .5
1 .7
1 .6
1 .8
1 .5
1 .7
113
7.6
7.5
7.0
7.5
7.7
7 .2
7 .4
98
4.2
15 .0
14 .4
15 .8
16 .0
14 .3
15 .1
100
6.0
30 .0
31 .8
31 .5
29 .3
27 .0
29 .9
99
7.5
Control plasma containing the indicated amounts of antipyrine was assayed immediately after addition of the drug (Exp . 1) and after storing the samples at -20° for 2, 7 and 14 days in Exp . 2, 3 and 4, respectively .
858
125
I-Radioimmunoassay for Antipyrine
Vol . 22, No . 10, 1978
References 1. 2. 3. 4. 5. 6. 7.
B . B . BRODIE, J . AXELROD, R . SOBELMAN, AND B . B . LEVY ., J . Biol . Chem . 179 25-29 (1949) . L . F . PRESCOTT, K . K . ADJEPON-YAMOAH, and E . ROBERTS, J . Pharm . Pharmacol 25 205-207 (1973) . R. M . WELCH, R . L . DE ANGELIS, M . WINGFIELD, and T . W . FARMER, Clin . Pharmacol . Ther . 18 249-258 (1975) . R . L . CHANG,A . W . WOOD, W . R . DIXON, A . H . CONNEY, K . E . ANDERSON, J . EISEMAN, and A . P . ALVARES, Clin . Pharmacol . Ther . 20 219-226 (1976) . W . M . HUNTER, Brit Med . Bull . 30 18 (1974) . D . ROBARD, Cli n. C em . 20 1255-1270 (1974) . A . Wald, Ann . Math . Stat . 11 284-300 (1940) .