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Comparative evaluation of antisera for 5α-dihydrotestosterone radioimmunoassay
COMPARATIVE EVALUATION OF ANTISERA FOR 5ceDIHYDROTESTOSTERONE RADIOIMMUNOASSAY C. RICHARD PARKER JR., J. 0. ELLEGOODand V. B. MAHESH Department of Endocrinology, Medical College of Georgia, Augusta, Georgia 30902. U.S.A. (Recented 3 May 1974) SUMMARY A comparison was made between two antisera, one (A-3) which was prepared in our laboratory and another (S-741) which was purchased to be used for the assay of both testosterone and k-dihydrotestosterone (DHT). The comparison was made to evaluate the usefulness of these two antisera to be used for DHT assay in the context of a multiple steroid radioimmunoassay system. We wish to report that considerable interference by such compounds as androstenedione and dehydroepiandrosterone (DHA) was encountered when using S-741 whiie there was little interference when A-3 was utilized for DHT assay. Differences in plasma levels of DHT obtained using antisera A-3 and S-741 are explained by cross react;vity findings in this study.
ml; they were @4 ml when A-3 was employed for radioimmunoassays. Blood samples to be analyzed were extracted twice with 2 vol. of ether after addition of tracer amounts of [‘HI-DHT. Extracts were then evaporated under vacuum and redissolved in 05ml isooctane. Chromatography was performed using 5 cm tall celite columns (stationary phase ethylene glycol-propylene glycol 1: 1 v/v). After application of the extract to the columns. elution was carried out under nitrogen gas (23 psi). Mobile solvents were 7.0 ml of isooctane which eluted progesterone and androstenedione followed by 40 ml of 5% ethyl acetate in isooctane which eluted both DHT and DHA. Multiple samples were collected. each sample being 0.5 ml in vol. Column samples 17 24. which contained DHT and DHA were collected together and evaporated. This fraction was then dissolved in buffer for subsequent radioimmun~ssay. Assays were performed as follows: @4 or 05 ml of standard or unknown were incubated with 0.1 ml antiserum solution (A-3 or S-741) for 1 h at 4’C. Then 0.1 ml of E3H)-DHT was added and tubes were incubated for an additional h at 4°C. Dextran coated charcoal suspension (0.2 ml) was then added, tubes mixed and incubated for 10 min at 4°C after which they were centrifuged at 2000 rev./min for 10 min at 4’C. The supernatants were then decanted and counted after addition of 10 ml of counting solution (@So/, POPGP in toluene). Unknown concentrations were determined through the use of a standard line and were corrected for recovery of internal tracer and blood volume assayed. When performed, DHA assays were the same
INTRODUCITON With the advent of procedures for multiple steroid measurement on a single blood sample [IN] using
radioimmunoassays it is becoming increasingly important that cross reactivity evaluations of antisera used be exhaustively performed. This is essential primarily because in such methods, one often isolates in a single chromatographic fraction more than one compound for subsequent assay, The quantity of one steroid in such a fraction may be IOO-fold higher than the other steroid and therefore even minor cross reactivity may be of major consequence. One such example is the mixture of DHA and DHT. In this report, we have compared two antisera which are presently being utilized for DHT radioimmunoas~y. The comparisons involve cross reactivity studies based upon the ability of steroids other than DHT to interfere in the radioimmunoassay of this steroid under several conditions.
MATERIALS AND METHODS Ether, isooctane and ethyl acetate were distilled prior to use; toluene, ethylene glycol and propylene glycolwere used without further purification. 1,2-c3H]-
DHT (44 Ci/mmol) and 7-C3H]-DHA (21 Ci/mmol) were purified by paper chromatography as described earlier 141. Antiserum A-3 was dissolved in 0.1 moljl phosphate buffer while antiserum S-741 (purchased from Dr. G. E. Abraham), in addition to steroid standards and radioactive steroids were dissolved in a 1% gelatin phosphate buffer. When S-741 was used assay volumes for both standards and unknowns were 0.5 9
I(1
<‘. R. PAKU K JI<.. J. 0. ELI.I.GOOI) and V. B. MAIII SII
signi~catit cross reactivity was found for both testosterone and DHT. WC’tested its suitability for LISCin the assay of tcstosteronc as well as DHT. When standard lines were run using a variety ofsteroid standards and C3H]-DHT as tracer. this antiserum uas found to have minimal affinity for steroids other than DHT and testosterone as shown in Table I, Since these two steroids arc easily separated from one another on cclite columns [4. 61. this antiserum has been utilized for the ;~ssay of both compounds. The cross r’eaction of this antiserum cvith the epimcrs cti‘ 3,171i-;1ndrostaliediol is of no conscquencc since these compounds arc not cluted from the column in tho DHT or testosterone fraction.
(A-3)
RESC’LTS
its described
for DHT with the exception that [3H]DHA and a DHA specific antisera were used.
The ~-~-carl~oxymethyl oxime of testosterone was prepared by the method of Erlanger CJ~a/.[51 and covalently bound to bovine serum albumin (BSA). Immunilation of rabbits was performed as described for our cstradiol antiserum-RPPil 147. Originally. this antiserum was designed to be used for testosterone radioimmunoassay. However. when Cross b-o)Assoy (e-.)Assay
reactivity
Standard tines, using [-‘HI-DHT as radioli~~nd were established for DHT. DHA and androstenedionc with both antisera. A-3 and S-741. The r-esults of this comparison are shown in Fig. I. 11 is apparent that DHA and androstcnedione arc potentially interfering substances when DHT is assayed with antiserum S-741 whereas there is minimal cross reaction with these stcroids when antiserum A-3 is used. Since androstenedione is well separated from the DHA,!DHT fraction in the chromatographic system which we used [4]. only the interference of DHA w/as studied in detail for these antisera. To invcstigatc further the potential for DHA interferencc in our assay procedure. two plasma pools (peri-
evoiuations
on cintlsero
t+A-3ond
S-741
us,ng #A-3 using S-741
c
‘g
loor-
ml I i d
Q
Elo-
c;
v
5
60-
2 $
40-
2 + s zoLY
DHA
I 1000 Pg standard Fig. I. Cross reactivity evaluations on antisera A-3 and S-741. Incubation procedures (time. temperature, standard volumes and antiserum volumes) were as described previously. Each ~ln~is~rurn was reacted with various standard amounts of DHT. DHA or nndrostcnedione; then reacted with [“HI-DHT. For case graphicalrepresentation. standard lines are expressed as lop dose vs relative percentage hound c.p.m. [“HI-DHT. Following ~pplic~ltion of il togit tr~r~sform~tion~ these standard lines are ltnear with coetficients of variation (r) grwter than 0.990.
of
Antisera for .5a-dihydrotestosterone radioimmunoassay pheral and adrenal vein) were extracted and chromatographed as described earlier with the following exception: several 05 ml column fractions [17--243 which would ordinarily be pooled were collected separately. The fractions were then evaporated and reconstituted in assay buffer. Aliquots of each column fraction were then analyzed to determine the recovery of C3HJ-DHT, DHA and DHT. DHT was assayed using both antisera, A-3 and S-741, while DHA was assayed using an antiserum specific for DHA [4]. The results of this study are shown in Fig. 2 and 3. When the peripheral plasma pool was analyzed, the elution of DHT, as measured with antiserum A-3 paralleled the recovery of the radioactive tracer, C3H]DHT. However, the elution of DHT as measured by anti~runl S-741 paralleled the elution of both [“HIDHT and DHA (Fig. 2). Fraction 21, which contained only 1% of the [3H]-DHT appeared to contain more DHT than fraction 20 which contained 127; of the recovered C3H]-DHT when assayed using S-741. The apparent recovery of DHT in fraction 21 is due to the interference with this antiserum by 8 ng of DHA which was found to be present in this fraction. Column fraction 22, which contained no c3H]-DHT but contained 8 -&Recovery of +DtiT
4--" ::, ;’:
0
(x--a)DHA content g gy rodioimunounoy30 z
/ 'x I \ \ \ : \ I \ I I
:: L t -*O a z
16 17 18 19 20 21 22 23 24 25 Fractcon number
Fig. 2. Fractional recovery of DHT. C3H]-DHT and DHA from a 10 ml peripheral plasma pool. The plasma sample was extracted and chromatographed using the modified column A system; fractions 16-25 (0.5 ml each) were eluted and collected separately. Each fraction was evaporated, reconstituted in buffer and evaluated for DHA content by radioimmunoassay. C3H]-DHT content and DHT content using both antisera. S-741 and A-3, for radioimmunoas~y.
Fraction
11
number
Fig. 3. Fractional recovery of DHT, [‘HI-DHT and DHA from a5 ml adrenal vein plasma pool. Extraction, chromatography and assay procedures were as described for Fig. 2.
30 ng of DHA, appeared to contain over 0.3 ng of DHT when assayed using antiserum S-741. There was no measurable DHT in this fraction (22) when antiserum A-3 was employed. It seems that as little as 8 ng of DHA can interfere significantly with the measurement of DHT when antiserum S-741 is used while there is no detectable interference with antiserum A-3 by as much as 30 ng of DHA. When the adrenal vein plasma pool was extracted and chromatographed there was highly significant interference by DHA in the assay of DHT when S-741 was used (Fig. 3). Additionally. A-3 showed some cross reactivity for DHA in this plasma pool, Column fraction 20, when analyzed was found to contain no c3H]DHT, which was eluted in fractions 17 and I8 primarily, but did contain 34 ng of DHA. The DHT content of this fraction as measured using S-741 was nearly 0.4 ng while it was only @025 ng when assayed using antiserum A-3. Fraction 21, which also contained no r3H]DHT but contained more than 50 ng of DHA appeared to contain 1.3 ng of DHT when antiserum S741 was used while only 0.06 ng of DHT was recorded with A-3. It is apparent that even a highly specific antiserum such as A-3, in the presence of very large quantities of substances which interfere to a slight degree,
I2
C. R. PARKER JK.. J. 0. ELLEG~OII and V. B. MAIII SH Table 7. Comparison of plasma Peripheral plasma sample I
3 ; 4
.s Mean Adrenal
+ _ SE.
DHT (ng;ml) s-741 0.x’)
I
using A-? and S-741 DHA (ng lllll
DHT (@ml) Corrected S-741
DHT (ng ml) A-2
00x5
IO.4
0.042
0~0’)‘) 0. I70 0~046
om1 0.227
0465 0.2:-l
10,: ?,-I
0.I 66
0.212 0. I ox 0, I x0 0~209 0~20.3 & 0.03
DHT concentrations
0 I I? f 0,035
-1.7 5.5
0.12 I _t 0.035
vein plasma sample
I 2
may result in obtaining erroneous data. There was, however, much less interference by DHA when DHT was assayed using A-3 than determinations made using s-741. As a final evaluation of these antisera, several plasma samples with varying DHA concentrations were extracted and chromatographed using celite columns as described earlier. Column fractions 17724 were pooled as usual whereby DHT and DHA were isolated together for assay. The concentrations of DHT were higher when assayed with S-741 than levels obtained on the same plasma samples assayed with A3. The DHT concentrations found using S-741 could be corrected for DHA interference. A comparison of values obtained using both antisera and the corrected values using S-741 are shown in Table 2. Included in this table are the DHA concentrations of each plasma sample. Several separate evaluations were made to determine the cross reactivity of DHA (I 15 ng) with S-741. A mean value for cross reactivity of 1.2% was obtained from these studies and applied to correct the DHT concentrations of plasma samples described above. The formula used was as follows: Corrected DHT (S-741) = DHT (S-741) (0.012 x DHA concentration). When applied to peripheral plasma samples (l-5. Table 2) with DHA concentrations less than I5 ng/ml, this correction resulted in similar DHT concentrations as measured by A-3 and S-741. Additionally. when applied to adrenal vein plasma samples (1 and 2) this correction also proved useful. That this correction worked with plasma samples with extremely high DHA concentrations may have been coincidental. We present these data to support our contention that high values of DHT found using S-741 are due to interference by DHA in the radioimmunoassay of DHT. We do not recommend the use of this correction for routine assays.
The results of this study demonstrate the ncod I& steroid specific antisera when multiple determinations or even single steroid measurements arc to be made on a plasma sample. This is especially important when more than one steroid is isolated in ;I single chromatographic fraction, regardless of the chromatographic method. for subsequent radioimmunoassa~. WC II;ILL’ shown that when antiserum S-711 is LISC~ for DHT assay. physiological concentrations of DHA can intcrfcre in this assay when the two compounds arc not separated by chromatographic prnccdurcs. When antrserum A-3 was tested, there was no intcrfcrcncc bq DHA except in pharmacological concentrations III the assay of DHT. The results thus indicate that antiacrum A-3 is a suitable antiserum to USC for the radioimmunoassay of DHT from most plasma samples C\CII when isolated in the presence of DHA. We have noted differences with respect to DHT concentrations in blood of men and women [-I1 ;IS IXILC other investigators [X I I]. Their findings agree with ours as to actual concentrations of DHT in such suhjects but differ from the findings of Coyotupa cjf tr/.[7] who have reported concentrations of DHT which [II-C‘ twice as high for both men and women. Antiserum S741 is very similar to the antiserum which Co>otupa c’t tr/_[7] used in so lar as cross reactivit) is conccrncd. Thus the differences het\+een our findings and theirs may well be due to intcrferencc 1~~DHA and androstcnedione which are both isolated with DHT in the chromatographic method which the! employed L7].
Ack,Iowl~,dgenlr,rts-This research wits bupportrd lby NIH Grant Number 5 ROI HD 04626-I 3 of the National Institutes of Health, Public Health Service. We uish to thank D-. T. G. Muldoon for his assistance in synthesillng the tcstostcrone-?-BSA conjugate.
Antisera for Sa-dihydrotestosterone REFERENCES Dupon C., Hosseinian A. and Kim M. H.: Steroids 22 (1973)47&l. Concolino G. and Marocchi A.: J. steroid Biochem. 3 (1972) 125-733. West C. D., Mahajan D. K., Chavre V. J., Nabois C. J. and Tyler F. H.: J. C’lin. Endocr. Metab. 36 (1973) 1230-1236. Parker C. R., Jr., Ellegood J. 0. and Mahesh V. B.: J. steroid Rio&m. 6 (1975). Erlanger B. F. Borek F., Beiser S. M. and Lieberman S. J.: J. hiol, Chm. 234 (1959) 109~1~4.
radioimmunoassay
13
6. Buster J. E. and Abraham G. E.: Anaiyt. Mt. 5 (1972) 105-l 13. 7, Coyotupa J., Parlow A. F. and Abraham G. E.: Annlyt. I.&t. 5 (19721329-340.
g, Ito T. and Horton R.: J. clin. Endocr. Mrtah. 31 (1970) 362-368. 9. Paetzmann H., Elaesser F., Hailer J. and Haffele R.: Actu Endocr. Copmh. Suppl. 173 (1973) 170. IO. Beitins I. Z.. Bayard F., Kowarski A. and Migeon C. J.: J. chin. Etzdocr. ~~t~~. 35 (1972) 101-107. I I. Barberio J. M. and Thornecroft I. H.: Strrolds 23 (1974) 157. 766.
ml; they were @4 ml when A-3 was employed for radioimmunoassays. Blood samples to be analyzed were extracted twice with 2 vol. of ether after addition of tracer amounts of [‘HI-DHT. Extracts were then evaporated under vacuum and redissolved in 05ml isooctane. Chromatography was performed using 5 cm tall celite columns (stationary phase ethylene glycol-propylene glycol 1: 1 v/v). After application of the extract to the columns. elution was carried out under nitrogen gas (23 psi). Mobile solvents were 7.0 ml of isooctane which eluted progesterone and androstenedione followed by 40 ml of 5% ethyl acetate in isooctane which eluted both DHT and DHA. Multiple samples were collected. each sample being 0.5 ml in vol. Column samples 17 24. which contained DHT and DHA were collected together and evaporated. This fraction was then dissolved in buffer for subsequent radioimmun~ssay. Assays were performed as follows: @4 or 05 ml of standard or unknown were incubated with 0.1 ml antiserum solution (A-3 or S-741) for 1 h at 4’C. Then 0.1 ml of E3H)-DHT was added and tubes were incubated for an additional h at 4°C. Dextran coated charcoal suspension (0.2 ml) was then added, tubes mixed and incubated for 10 min at 4°C after which they were centrifuged at 2000 rev./min for 10 min at 4’C. The supernatants were then decanted and counted after addition of 10 ml of counting solution (@So/, POPGP in toluene). Unknown concentrations were determined through the use of a standard line and were corrected for recovery of internal tracer and blood volume assayed. When performed, DHA assays were the same
INTRODUCITON With the advent of procedures for multiple steroid measurement on a single blood sample [IN] using
radioimmunoassays it is becoming increasingly important that cross reactivity evaluations of antisera used be exhaustively performed. This is essential primarily because in such methods, one often isolates in a single chromatographic fraction more than one compound for subsequent assay, The quantity of one steroid in such a fraction may be IOO-fold higher than the other steroid and therefore even minor cross reactivity may be of major consequence. One such example is the mixture of DHA and DHT. In this report, we have compared two antisera which are presently being utilized for DHT radioimmunoas~y. The comparisons involve cross reactivity studies based upon the ability of steroids other than DHT to interfere in the radioimmunoassay of this steroid under several conditions.
MATERIALS AND METHODS Ether, isooctane and ethyl acetate were distilled prior to use; toluene, ethylene glycol and propylene glycolwere used without further purification. 1,2-c3H]-
DHT (44 Ci/mmol) and 7-C3H]-DHA (21 Ci/mmol) were purified by paper chromatography as described earlier 141. Antiserum A-3 was dissolved in 0.1 moljl phosphate buffer while antiserum S-741 (purchased from Dr. G. E. Abraham), in addition to steroid standards and radioactive steroids were dissolved in a 1% gelatin phosphate buffer. When S-741 was used assay volumes for both standards and unknowns were 0.5 9
I(1
<‘. R. PAKU K JI<.. J. 0. ELI.I.GOOI) and V. B. MAIII SII
signi~catit cross reactivity was found for both testosterone and DHT. WC’tested its suitability for LISCin the assay of tcstosteronc as well as DHT. When standard lines were run using a variety ofsteroid standards and C3H]-DHT as tracer. this antiserum uas found to have minimal affinity for steroids other than DHT and testosterone as shown in Table I, Since these two steroids arc easily separated from one another on cclite columns [4. 61. this antiserum has been utilized for the ;~ssay of both compounds. The cross r’eaction of this antiserum cvith the epimcrs cti‘ 3,171i-;1ndrostaliediol is of no conscquencc since these compounds arc not cluted from the column in tho DHT or testosterone fraction.
(A-3)
RESC’LTS
its described
for DHT with the exception that [3H]DHA and a DHA specific antisera were used.
The ~-~-carl~oxymethyl oxime of testosterone was prepared by the method of Erlanger CJ~a/.[51 and covalently bound to bovine serum albumin (BSA). Immunilation of rabbits was performed as described for our cstradiol antiserum-RPPil 147. Originally. this antiserum was designed to be used for testosterone radioimmunoassay. However. when Cross b-o)Assoy (e-.)Assay
reactivity
Standard tines, using [-‘HI-DHT as radioli~~nd were established for DHT. DHA and androstenedionc with both antisera. A-3 and S-741. The r-esults of this comparison are shown in Fig. I. 11 is apparent that DHA and androstcnedione arc potentially interfering substances when DHT is assayed with antiserum S-741 whereas there is minimal cross reaction with these stcroids when antiserum A-3 is used. Since androstenedione is well separated from the DHA,!DHT fraction in the chromatographic system which we used [4]. only the interference of DHA w/as studied in detail for these antisera. To invcstigatc further the potential for DHA interferencc in our assay procedure. two plasma pools (peri-
evoiuations
on cintlsero
t+A-3ond
S-741
us,ng #A-3 using S-741
c
‘g
loor-
ml I i d
Q
Elo-
c;
v
5
60-
2 $
40-
2 + s zoLY
DHA
I 1000 Pg standard Fig. I. Cross reactivity evaluations on antisera A-3 and S-741. Incubation procedures (time. temperature, standard volumes and antiserum volumes) were as described previously. Each ~ln~is~rurn was reacted with various standard amounts of DHT. DHA or nndrostcnedione; then reacted with [“HI-DHT. For case graphicalrepresentation. standard lines are expressed as lop dose vs relative percentage hound c.p.m. [“HI-DHT. Following ~pplic~ltion of il togit tr~r~sform~tion~ these standard lines are ltnear with coetficients of variation (r) grwter than 0.990.
of
Antisera for .5a-dihydrotestosterone radioimmunoassay pheral and adrenal vein) were extracted and chromatographed as described earlier with the following exception: several 05 ml column fractions [17--243 which would ordinarily be pooled were collected separately. The fractions were then evaporated and reconstituted in assay buffer. Aliquots of each column fraction were then analyzed to determine the recovery of C3HJ-DHT, DHA and DHT. DHT was assayed using both antisera, A-3 and S-741, while DHA was assayed using an antiserum specific for DHA [4]. The results of this study are shown in Fig. 2 and 3. When the peripheral plasma pool was analyzed, the elution of DHT, as measured with antiserum A-3 paralleled the recovery of the radioactive tracer, C3H]DHT. However, the elution of DHT as measured by anti~runl S-741 paralleled the elution of both [“HIDHT and DHA (Fig. 2). Fraction 21, which contained only 1% of the [3H]-DHT appeared to contain more DHT than fraction 20 which contained 127; of the recovered C3H]-DHT when assayed using S-741. The apparent recovery of DHT in fraction 21 is due to the interference with this antiserum by 8 ng of DHA which was found to be present in this fraction. Column fraction 22, which contained no c3H]-DHT but contained 8 -&Recovery of +DtiT
4--" ::, ;’:
0
(x--a)DHA content g gy rodioimunounoy30 z
/ 'x I \ \ \ : \ I \ I I
:: L t -*O a z
16 17 18 19 20 21 22 23 24 25 Fractcon number
Fig. 2. Fractional recovery of DHT. C3H]-DHT and DHA from a 10 ml peripheral plasma pool. The plasma sample was extracted and chromatographed using the modified column A system; fractions 16-25 (0.5 ml each) were eluted and collected separately. Each fraction was evaporated, reconstituted in buffer and evaluated for DHA content by radioimmunoassay. C3H]-DHT content and DHT content using both antisera. S-741 and A-3, for radioimmunoas~y.
Fraction
11
number
Fig. 3. Fractional recovery of DHT, [‘HI-DHT and DHA from a5 ml adrenal vein plasma pool. Extraction, chromatography and assay procedures were as described for Fig. 2.
30 ng of DHA, appeared to contain over 0.3 ng of DHT when assayed using antiserum S-741. There was no measurable DHT in this fraction (22) when antiserum A-3 was employed. It seems that as little as 8 ng of DHA can interfere significantly with the measurement of DHT when antiserum S-741 is used while there is no detectable interference with antiserum A-3 by as much as 30 ng of DHA. When the adrenal vein plasma pool was extracted and chromatographed there was highly significant interference by DHA in the assay of DHT when S-741 was used (Fig. 3). Additionally. A-3 showed some cross reactivity for DHA in this plasma pool, Column fraction 20, when analyzed was found to contain no c3H]DHT, which was eluted in fractions 17 and I8 primarily, but did contain 34 ng of DHA. The DHT content of this fraction as measured using S-741 was nearly 0.4 ng while it was only @025 ng when assayed using antiserum A-3. Fraction 21, which also contained no r3H]DHT but contained more than 50 ng of DHA appeared to contain 1.3 ng of DHT when antiserum S741 was used while only 0.06 ng of DHT was recorded with A-3. It is apparent that even a highly specific antiserum such as A-3, in the presence of very large quantities of substances which interfere to a slight degree,
I2
C. R. PARKER JK.. J. 0. ELLEG~OII and V. B. MAIII SH Table 7. Comparison of plasma Peripheral plasma sample I
3 ; 4
.s Mean Adrenal
+ _ SE.
DHT (ng;ml) s-741 0.x’)
I
using A-? and S-741 DHA (ng lllll
DHT (@ml) Corrected S-741
DHT (ng ml) A-2
00x5
IO.4
0.042
0~0’)‘) 0. I70 0~046
om1 0.227
0465 0.2:-l
10,: ?,-I
0.I 66
0.212 0. I ox 0, I x0 0~209 0~20.3 & 0.03
DHT concentrations
0 I I? f 0,035
-1.7 5.5
0.12 I _t 0.035
vein plasma sample
I 2
may result in obtaining erroneous data. There was, however, much less interference by DHA when DHT was assayed using A-3 than determinations made using s-741. As a final evaluation of these antisera, several plasma samples with varying DHA concentrations were extracted and chromatographed using celite columns as described earlier. Column fractions 17724 were pooled as usual whereby DHT and DHA were isolated together for assay. The concentrations of DHT were higher when assayed with S-741 than levels obtained on the same plasma samples assayed with A3. The DHT concentrations found using S-741 could be corrected for DHA interference. A comparison of values obtained using both antisera and the corrected values using S-741 are shown in Table 2. Included in this table are the DHA concentrations of each plasma sample. Several separate evaluations were made to determine the cross reactivity of DHA (I 15 ng) with S-741. A mean value for cross reactivity of 1.2% was obtained from these studies and applied to correct the DHT concentrations of plasma samples described above. The formula used was as follows: Corrected DHT (S-741) = DHT (S-741) (0.012 x DHA concentration). When applied to peripheral plasma samples (l-5. Table 2) with DHA concentrations less than I5 ng/ml, this correction resulted in similar DHT concentrations as measured by A-3 and S-741. Additionally. when applied to adrenal vein plasma samples (1 and 2) this correction also proved useful. That this correction worked with plasma samples with extremely high DHA concentrations may have been coincidental. We present these data to support our contention that high values of DHT found using S-741 are due to interference by DHA in the radioimmunoassay of DHT. We do not recommend the use of this correction for routine assays.
The results of this study demonstrate the ncod I& steroid specific antisera when multiple determinations or even single steroid measurements arc to be made on a plasma sample. This is especially important when more than one steroid is isolated in ;I single chromatographic fraction, regardless of the chromatographic method. for subsequent radioimmunoassa~. WC II;ILL’ shown that when antiserum S-711 is LISC~ for DHT assay. physiological concentrations of DHA can intcrfcre in this assay when the two compounds arc not separated by chromatographic prnccdurcs. When antrserum A-3 was tested, there was no intcrfcrcncc bq DHA except in pharmacological concentrations III the assay of DHT. The results thus indicate that antiacrum A-3 is a suitable antiserum to USC for the radioimmunoassay of DHT from most plasma samples C\CII when isolated in the presence of DHA. We have noted differences with respect to DHT concentrations in blood of men and women [-I1 ;IS IXILC other investigators [X I I]. Their findings agree with ours as to actual concentrations of DHT in such suhjects but differ from the findings of Coyotupa cjf tr/.[7] who have reported concentrations of DHT which [II-C‘ twice as high for both men and women. Antiserum S741 is very similar to the antiserum which Co>otupa c’t tr/_[7] used in so lar as cross reactivit) is conccrncd. Thus the differences het\+een our findings and theirs may well be due to intcrferencc 1~~DHA and androstcnedione which are both isolated with DHT in the chromatographic method which the! employed L7].
Ack,Iowl~,dgenlr,rts-This research wits bupportrd lby NIH Grant Number 5 ROI HD 04626-I 3 of the National Institutes of Health, Public Health Service. We uish to thank D-. T. G. Muldoon for his assistance in synthesillng the tcstostcrone-?-BSA conjugate.
Antisera for Sa-dihydrotestosterone REFERENCES Dupon C., Hosseinian A. and Kim M. H.: Steroids 22 (1973)47&l. Concolino G. and Marocchi A.: J. steroid Biochem. 3 (1972) 125-733. West C. D., Mahajan D. K., Chavre V. J., Nabois C. J. and Tyler F. H.: J. C’lin. Endocr. Metab. 36 (1973) 1230-1236. Parker C. R., Jr., Ellegood J. 0. and Mahesh V. B.: J. steroid Rio&m. 6 (1975). Erlanger B. F. Borek F., Beiser S. M. and Lieberman S. J.: J. hiol, Chm. 234 (1959) 109~1~4.
radioimmunoassay
13
6. Buster J. E. and Abraham G. E.: Anaiyt. Mt. 5 (1972) 105-l 13. 7, Coyotupa J., Parlow A. F. and Abraham G. E.: Annlyt. I.&t. 5 (19721329-340.
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