- Email: [email protected]
Monoclonal antibodies for use with 125iodine-labeled radioligands in progesterone radioimmunoassay
J. steroid Biochem. Vol. 21, No. 4, pp. 405411, Printed in Great Britain. All rights reserved
1984 Copyright 0
0022-4731/84 $3.00 + 0.00 1984 Pergamon Press Ltd
MONOCLONAL ANTIBODIES FOR USE WITH ‘2510DINE-LABELED RADIOLIGANDS IN PROGESTERONE RADIOIMMUNOASSAY MICHELE BROCHU, RAYMONDE VEILLEUX, ANNE LORRAIN
Department
and
ALAIN BELANGER*
of Molecular Endocrinology, Le Centre Hospitalier de 1’UniversitC Lava], Qukbec GIV 4G2, Canada (Received 19 December 1983)
Summary-The interactions of heterologous and homologous “‘1-iodinated radioligand with polyclonal and monoclonal antibodies directed against 1I-hydroxyprogesterone hemisuccinate conjugated to bovine serum albumin were compared. Our data show that, with a polyclonal antibody, the use of the same bridge in the tracer and the antigen results in a low sensitivity while a heterologous tracer can decrease markedly the titer of antibody but increases the sensitivity of the radioimmunoassay. Due to the high specificity of monoclonal antibodies, the interaction with heterologous and homologous iodinated tracers is extremely variable from one antibody to another but the present results demonstrate that radioimmunoassays of very high sensitivity can be obtained with homologous and heterogenous tracers. However, an appropriate tracer has to be selected to meet the requirement of such an assay.
INTRODUCTION
The disadvantage of using homologous radioiodinated labels for steroid radioimmunoassay arises from the affinity that the antibody may show not only for the steroid but for the bridge through which it was attached to the carrier protein [l-5]. Consequently, assays using iodinated labels in which a bridge homologous to that in the immunogen is present are frequently much less sensitive than their tritiated counterparts. The best method that have been used to overcome the bridge-binding problem is the reduction of antibody recognition of the tracer bridge by making changes in the nature or the position of the bridge [5,6]. Recent reports have demonstrated the feasibility of raising monoclonal antibodies against steroid [7-91. It has been shown that the affinity and the specificity of monoclonal antibody against the steroid is extremely variable from one antibody to another suggesting that for some antibodies the bridge-binding phenomenom may not be present. In the present report, we have examined the interaction of [‘251]iodo-labeled steroid derivatives as radioligands with monoclonal progesterone antibodies developed against 1la: -hydroxyprogesterone hemisuccinate conjugated to BSA. MATERIALS AND METHODS
Reagents
Non-radioactive steroids and 1l-hydroxyprogesterone hemisuccinate conjugated to BSA were obtained from Research Plus Inc. Bayonne, New *To whom correspondence should be addressed. 405
Jersey, U.S.A., and [1,2,6,7-3H (N)lprogesterone (90-l 15 Ci/mmol) (Tracer A) from New England Nuclear, Boston, MA. Bovine serum albumin (fraction V) was obtained from Sigma, St Louis, U.S.A. Mouse IgG was purchased from Miles Biochemicals. Sheep anti-mouse IgG and anti-rabbit IgG sera were produced following monthly injection of 1 mg IgG subcutaneously in Freund’s adjuvant. L-Tyrosine derivatives of 1I-hydroxyprogesterone hemisuccinate (tracer B) and progesterone-12(0carboxymethyl)-oxime (tracer D) were used for iodination. Preparation of “‘I-steroids has been already described [lo]. 1I-Hydroxyprogesterone glucuronide [‘251]iodotyramine (tracer C) was purchased from Radiochemical Center, Amersham. Immunization
For the production of monoclonal antibodies, 6 female BALB/c mice of 15-20 g were immunized with 40 pg of 1I-hydroxyprogesterone hemisuccinateBSA subcutaneously in Freund’s complete adjuvant (DIFCO Laboratories). At intervals of 2 weeks for the first and the second month, booster injections were given exactly as above except incomplete Freund’s adjuvant was used. At the third month, a similar amount of antigen in saline was given as intravenous boost. Three days after this boost, the spleen was removed for the cell fusion. For the production of polyclonal antibodies, rabbits were immunized with 150 pg 1l-hydroxyprogesterone hemisuccinate-BSA subcutaneously in Freund’s complete adjuvant. The injection was repeated in incomplete Freund’s adjuvant at intervals of 2 weeks during the first 2 months and at monthly interval for the third and the fourth month. Seven
MICHELE BROCHU et al.
406
days after the last boost, the rabbits were sacrificed and the antiserum was collected.
Table 2. Affinity constants of monoclonal and polyclonal antibodies to progesterone
Cell fusion
Antibody
The method used was based on that of Galfrt and Milstein[ 111. The mouse myeloma cells used for fusion was NSO/l generously provided by Dr C. Milstein. Cell fusion was induced by polyethylene glycol 4000 (BDH Chemicals Canada Ltd). Cells after fusion were suspended in selective HAT medium prepared as described in GalfrC and Milstein[l 1] but instead of DMM, we used Iscove medium (Flow Laboratories). This suspension of cells was distributed into eight 96-well tissue culture plates (0.2 ml/well). Supernatants were tested for antibody production after 10-14 days, and positive cultures were transfered to 2 ml wells in HAT medium. Cells lines were routinely cloned by limiting dilution at least twice, and then grown to large volume in tissue culture flasks or as tumors in mice [1 11. Detection of antibody activity
Culture fluids (0.1) ml) were incubated with 0.1 ml of BSA buffer (Na,HPO,, 0.1 M, pH 7.2, BSA, 0. lx, NaN,, O.l%, NaCl, 0.15 M), 0.1 ml of ‘25iodinated radioligand and 0.2 ml of anti-mouse IgG. After 2 h at room temperature, 0.2 ml of PEG 20% was added and tubes were centrifuged at 3000 rpm for 15 min. The supernatant was decanted and the pellets counted for radioactivity. For the tritiated tracer, 0.1 ml BSA buffer, 0.1 ml culture fluid and 0.1 ml of tritiated progesterone were incubated for 105 min at room temperature and for 15min at 4°C. Dextran-coated charcoal (0.2 ml) was added and the incubation mixture was centrifuged at 3000 rpm for 15 min. The supernatants were collected and counted.
RESULTS
All rabbits produced antisera of high titers when using the homologous ‘25iodinated tracer in the radioimmunoassay. Table 1 shows the titers obtained with the tritiated and ‘251-iodinated radioligands from one of these antisera (147A). As previously shown, a change of the structure of the bridge leads to a lower titer than is observed with the homologous tracer,
Affinity constant* (K,) l/m01 0.088 0.308 1.04 0.238 1.66 0.26
P-l P-2 P-3 P-4 P-5 147-A *Calculated according ref. [12].
x x x x x x
10” IO’” IO” IO’” 10’” 10”
to the
method in.
and, moreover, the use of a tritiated tracer further markedly decreases the dilution of the antisera. Hybrid growth was observed in 50-60x of wells following fusion. While l&15% of antibodyproducing clones were obtained from these fusions, several clones stopped antibody production before cloning or showed very low binding to radioligand A and B and were therefore not cloned. Five stable monoclonal antibodies to progesterone were isolated from three independent hybridizations. When injected into pristane-treated irradiated mice, the hybridomas secreted high amounts of progesterone antibodies into the ascitic fluid of these mice. The titers of each antibody when using the tritiated and 12’iodinated tracer are shown in Table 1. It can be seen that contrary to the well-known interaction of polyclonal antibodies with the homologous and heterologous tracer, monoclonal antibodies can bind a heterologous tracer at a higher titer than that observed with the homologous radioligand. However, while the interaction with the tritiated and the ‘25iodineligand A,B and C is present for the five antibodies, three of these antibodies do not bind the tracer D. Figures l-4 shows that standard curves constructed using the antibody dilution as indicated in Table 1. Concentrations of cold progesterone from O-10000 picograms per tube were added. It is shown that, using the ‘25iodinated tracers, high sensitivity radioimmunoassay can be obtained with both monoclonal and polyclonal antibodies. However, contrary with the well-known interaction with polyclonal antibody, the present data indicate that with monoclonal antibodies, the use of heterologous homologous tracer may generate a better titer of the antibody and also a better sensitivity. Moreover, it can be seen in Table
Table 1. Titers of oolvclonal and monoclonal antibodies with tritiated and ‘*‘-iodinated tracers at 30-35% binding 1
,
Tracer ‘2SI_ iodinated Pro-12CMO Pro-l l-HS Pro-l 1G Tritiated Tracer
Polyclonal antibody
Monoclonal antibodies PI
P2
P3
Pi-
~- P5 ~~
1/4&o 1/250000
1/7500000 1/300000 ljlOOO0
1/10000 l/l 10000
l/2000 l/40000 l/250000
l/l0000 l/90000
1/10000
l/210000
l/SW00
l/24000
l/66000
147-A
l/80000 I /300000 l/80000 l/12000
Pro-12-CMO: ‘*‘iodo progesterone-12-(O-carboxymethyl)-oxime tyrosine methyl ester Pro-l I-HS: ‘“‘iodo-1 I-hydroxyprogesterone hemisuccinate-tyrosine methyl ester. Pro-l IG: ‘*‘iodo-1 1-hydroxyprogesterone glucuronide tyramine.
Monoclonal antibodies to progesterone POLYCLONAL 100
ANTIBODY,
147-A
A
75
50
!?=325
25
0 ~
1OC
0
75
4 I!!& %=”
50
25
0
1
PROGESTERONE
ANTISODY~
100
10'
10’ 10’
10’
(Pgltuba)
Fig. 1. MONOCLONAL
10'
Pl
B
75
F=”
50
25
0
100
75
50
25
0
C
1
NC BINDING
P =19
1
10'
1v
lry
1w
10’
PROGESTERONE
Fig. 2.
WITH THE TRACER
I
1
(Pgmbe,
10’ 1w 10’ 10’ 10’
40X MON~CLONAL
ANTIBODY
~2
100
75
50
25
0
100
75
50
25
0
PROGESTERONE
(i'Q&k)
Fig. 3. M0N0CL0NAL
100
ANTIBOOY.
‘zi”=”
Y=215
50
25
100
75
50
25
0
p3
B
A
75
0
I I 1
~
a
0
C
NO BINDING
Y=25
WITH THE TRACER
9
1
10’
w
101 IV
1w
PROGESTERONE
Fig. 4.
(pg4ibe)
Monoclonal
antibodies MONOCLCNAL
to progesterone ANTIBODY.
409
P4
A S
?=” 9
4.
= 212
1
100
C
75
i, Bo-503
50
_L 2
25
0
1
PROGESTERONE
10’ IO’ IO’ 10’ IO’
(Pgltube)
Fig. 5. -LCNAL loo
75
50
25
0
100
75
ANTIBODY:
P5
A
Lsl!L L.L ILF=207
?=52
C
D
No BINDING
50
25
0
B =26
WITH THE TRACER
0
1
IO'
1w
101 IO’
10)
PROOESTERCNE
1 10'10
103 IO’ 10’
(Pgltube)
Fig. 6.
Figs. 1-6. Standard curves using rabbit antiserum 147-A (Fig. 1) and monoclonal antibodies PI-5 (Figs 2-6) with tritiated progesterone (A), ‘?odo- I I-hydroxyprogesterone hemisuccinate tyrosine methyl ester ‘*5iodo-I I-hydroxyprogesterone glucuronide tyramine (C) and (B), ‘*%odoprogesterone-12(o-carboxymethyl)-oxime tyrosine methyl ester (D).
MICHELEBROCHU et
410
al
Tab!e 3. Specificity of monoclonal and polyclonal antibodies raised against 1la-hydroxyprogesterone serum albumin
PI
Steroid
P2
1 1-Hydroxyprogesterone
100 83
100 321
hemisuccinate I I-Hydroxyprogesterone SLY.-Pregnane-3,20-dione 17-Hydroxyprogesterone Sa-Pregnan-3 a-ol-20-one Sa-Pregnan-3 /3-ol-20-one Androsterone Androstenedione Androstanedione 20B-Dihvdrooroeesterone 20k-Dih;droprogesterone Sa-Pregnane-3a,20a-dial Pregnenolone 17-Hydroxypregnenolone Testosterone Dihydrotestosterone Androstane-3a,l7fi-dial Androstane-3B,178-diol Androst-5-ene-38,178-diol Cholesterol Estrone Estradiol Estriol Cortisol
5.4 I1 2.4 8.6 2.3 3.7 2.5 1.7 0.6 < 0.03 0.4 0.3 < 0.03 < 0.03 < 0.02 < 0.02 < 0.02 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03
65 29 19 < 0.4 < 0.4 < 0.4 0.4 0.4 0.7 0.1 0.4 0.65 0.1 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.2
Progesterone
2 that antibody P-l which shows the lowest affinity constant for progesterone lead to the highest sensitivity when using the appropriate iodinated tracer. Both polyclonal and monoclonal antibodies were assessed for specificity using a range of steroids from 10 to 100,000 pg/tube. The cross-reactivities of the two types of antibodies are shown in Table 3. While the radioligands giving the best sensitivity of radioimmunoassay are used to determine the specificity, we have shown that almost similar specificity is obtained when other tracers are used (data not shown). DISCUSSION
In order to achieve a sensitive steroid radiousing polyclonal antibodies and immunoassay ‘?odinated tracer, it is well established that heterologous tracer must be used. We have shown in the present study that a sensitive assay can be obtained with a polyclonal antibody developed against 1la-hydroxyprogesterone hemisuccinate-BSA either by a change in the structure of the bridge or by using a bridge attached at a position different from that in the immunogen. However, the titer of the antibody is markedly reduced when heterologous tracer is used. We have demonstrated that assays of high sensitivity may be obtained by using homologous and heterologous tracers with monoclonal antibody, but the system is unpredictable. In some cases, a sensitive standard curve can be obtained with a given monoclonal antibody and a homogenous bridge or with a bridge that only the structure has been changed while a tracer with a bridge at a position different from that in the immunogen does not bind to this antibody.
%Cross-reaction Monoclonal P3 P4 100 142 < 0.03 0.48 3.4 2.1 I.1 3.4 1.5 0.5 73 < 0.03 41 0.5 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 0.07
hemisuccinate conjugated to bovine
Polyclonal 147-A
PS
100 219
100 204
100 129
104 55 I.1 0.9 3.9 1.5 0.6 1.2 1.0 0.1 0.4 0.6 < 0.06 < 0.06 < 0.2 < 0.06 < 0.06 c 0.06 < 0.06 < 0.06 < 0.06 < 0.06 < 0.06
0.13 27 14 21 13 0.1 0.1 9.6 1.2 < 0.03 46 0.4 < 0.03 < 0.03 < 0.03 < 0.03 7.4 0.09 < 0.03 < 0.03 < 0.03 < 0.03 < 0.07
22 35 3.6 0.07 0.61
However, we have also demonstrated that a monoclonal antibody can bind a heterologous tracer at a different position and give a highly sensitive assay. The use of heterologous tracer clearly increases the sensitivity of the assay but, contrary to observations with polyclonal antibody, the titer is also increased, suggesting that low amount of monoclonal antibody is important in order to achieve the highest sensitive assay. The present data indicate that monoclonal antibodies are suitable for radioimmunoassay of steroids with lz5-iodinated ligands and have specificities comparable to those obtained with conventional techniques. However, it should be emphasized that the appropriate tracer has to be selected in order to meet the requirements of each specific monoclonal antibody and in order to reach the highest sensitivity. Moreover, our data also suggest that the screening test of monoclonal antibody has to be performed with more than one ‘25-iodinated ligand having either an homologous or heterologous bridge.
REFERENCES 1. Corrie J. E. T., Hunter W. M. and Macpherson A strategy for radioimmunoassay of plasma gesterone with use of a homologous-site radioligand. Clin. Chem. 27 (1981) 594-599.
J. S.: pro‘%labeled
2. Nordblum G. D., Counsel1 R. E. and England B. G.: Ligand specificity and bridging phenomenon in hapten radioimmunoassays. Lignnd Q. 2 (1979) 34-36. 3. Allen R. M. and Redshaw M. R.: The use of homologous and heterologous ‘251-radioligands in the radioimmunoassay of progesterone. Steroids 32 (I 978) 467486. 4. Scarisbrick
J. J. and Cameron
E. M. D.: Radio-
Monoclonal antibodies to progesterone immunoassay of progesterone: comparison of [1,2,6,7-‘HI-progesterone and progesterone-[‘251]-iodohistamine radioligands. J. steroid Biochem. 6 (1975)
9.
51-56. 5. Peele B. and Holmes A. L.: Potential
[‘251]iodinated tracers for the radioimmunoassay of dehydroepiandrosterone. J. steroid Biochem. 14 (1981) 1197-1200. 6. Norblom G. D., Webb R., Consell R. E. and England B. G.: A chemical approach to solving bridging phenomena in steroid radioimmunoassays. Steroid 38 (1981) 161-173. 7. Kohen F., Lichter S., Eshar Z. and Lindner H. R.: Preparation of monoclonal antibodies able to discriminate between testosterone and Sa-dihydrotestosterone. Steroids 39 (1982) 453459. 8. Fantl V. E., Wang D. Y. and Knyba
R.E.:
The
10.
11.
12.
411
production of high affinity monoclonal antibodies to progesterone. J. steroid Biochem. 17 (1982) 125-130. White A., Anderson D. C. and Daly J. R. : Production of a highly specific monoclonal antibody to progesterone. J. clin. Endocr. Metab. 54 (1982) 205-207. Belanger A., Caron S. and Picard V.: Simultaneous radioimmunoassay of progestin, androgen and estrogen in adult rat testis. J. steroid Biochem. 13 (1979) 185-l 90. Galfrt G. and Milstein C.: Preparation of monoclonal antibodies: strategies and procedures. In Methods of Enzymology (Edited by M. De Luca). Academic Press, New York, Vol. 73 (1975) p. 3. Abraham G. E.: Characterization of anti-steroid antisera. in Steroid Immunoassay (Edited by E. H. D. Cameron, S. G. Hillies and K. Griffiths). Alpha Omega, Cardiff Wales (1974) pp. 67-79.
1984 Copyright 0
0022-4731/84 $3.00 + 0.00 1984 Pergamon Press Ltd
MONOCLONAL ANTIBODIES FOR USE WITH ‘2510DINE-LABELED RADIOLIGANDS IN PROGESTERONE RADIOIMMUNOASSAY MICHELE BROCHU, RAYMONDE VEILLEUX, ANNE LORRAIN
Department
and
ALAIN BELANGER*
of Molecular Endocrinology, Le Centre Hospitalier de 1’UniversitC Lava], Qukbec GIV 4G2, Canada (Received 19 December 1983)
Summary-The interactions of heterologous and homologous “‘1-iodinated radioligand with polyclonal and monoclonal antibodies directed against 1I-hydroxyprogesterone hemisuccinate conjugated to bovine serum albumin were compared. Our data show that, with a polyclonal antibody, the use of the same bridge in the tracer and the antigen results in a low sensitivity while a heterologous tracer can decrease markedly the titer of antibody but increases the sensitivity of the radioimmunoassay. Due to the high specificity of monoclonal antibodies, the interaction with heterologous and homologous iodinated tracers is extremely variable from one antibody to another but the present results demonstrate that radioimmunoassays of very high sensitivity can be obtained with homologous and heterogenous tracers. However, an appropriate tracer has to be selected to meet the requirement of such an assay.
INTRODUCTION
The disadvantage of using homologous radioiodinated labels for steroid radioimmunoassay arises from the affinity that the antibody may show not only for the steroid but for the bridge through which it was attached to the carrier protein [l-5]. Consequently, assays using iodinated labels in which a bridge homologous to that in the immunogen is present are frequently much less sensitive than their tritiated counterparts. The best method that have been used to overcome the bridge-binding problem is the reduction of antibody recognition of the tracer bridge by making changes in the nature or the position of the bridge [5,6]. Recent reports have demonstrated the feasibility of raising monoclonal antibodies against steroid [7-91. It has been shown that the affinity and the specificity of monoclonal antibody against the steroid is extremely variable from one antibody to another suggesting that for some antibodies the bridge-binding phenomenom may not be present. In the present report, we have examined the interaction of [‘251]iodo-labeled steroid derivatives as radioligands with monoclonal progesterone antibodies developed against 1la: -hydroxyprogesterone hemisuccinate conjugated to BSA. MATERIALS AND METHODS
Reagents
Non-radioactive steroids and 1l-hydroxyprogesterone hemisuccinate conjugated to BSA were obtained from Research Plus Inc. Bayonne, New *To whom correspondence should be addressed. 405
Jersey, U.S.A., and [1,2,6,7-3H (N)lprogesterone (90-l 15 Ci/mmol) (Tracer A) from New England Nuclear, Boston, MA. Bovine serum albumin (fraction V) was obtained from Sigma, St Louis, U.S.A. Mouse IgG was purchased from Miles Biochemicals. Sheep anti-mouse IgG and anti-rabbit IgG sera were produced following monthly injection of 1 mg IgG subcutaneously in Freund’s adjuvant. L-Tyrosine derivatives of 1I-hydroxyprogesterone hemisuccinate (tracer B) and progesterone-12(0carboxymethyl)-oxime (tracer D) were used for iodination. Preparation of “‘I-steroids has been already described [lo]. 1I-Hydroxyprogesterone glucuronide [‘251]iodotyramine (tracer C) was purchased from Radiochemical Center, Amersham. Immunization
For the production of monoclonal antibodies, 6 female BALB/c mice of 15-20 g were immunized with 40 pg of 1I-hydroxyprogesterone hemisuccinateBSA subcutaneously in Freund’s complete adjuvant (DIFCO Laboratories). At intervals of 2 weeks for the first and the second month, booster injections were given exactly as above except incomplete Freund’s adjuvant was used. At the third month, a similar amount of antigen in saline was given as intravenous boost. Three days after this boost, the spleen was removed for the cell fusion. For the production of polyclonal antibodies, rabbits were immunized with 150 pg 1l-hydroxyprogesterone hemisuccinate-BSA subcutaneously in Freund’s complete adjuvant. The injection was repeated in incomplete Freund’s adjuvant at intervals of 2 weeks during the first 2 months and at monthly interval for the third and the fourth month. Seven
MICHELE BROCHU et al.
406
days after the last boost, the rabbits were sacrificed and the antiserum was collected.
Table 2. Affinity constants of monoclonal and polyclonal antibodies to progesterone
Cell fusion
Antibody
The method used was based on that of Galfrt and Milstein[ 111. The mouse myeloma cells used for fusion was NSO/l generously provided by Dr C. Milstein. Cell fusion was induced by polyethylene glycol 4000 (BDH Chemicals Canada Ltd). Cells after fusion were suspended in selective HAT medium prepared as described in GalfrC and Milstein[l 1] but instead of DMM, we used Iscove medium (Flow Laboratories). This suspension of cells was distributed into eight 96-well tissue culture plates (0.2 ml/well). Supernatants were tested for antibody production after 10-14 days, and positive cultures were transfered to 2 ml wells in HAT medium. Cells lines were routinely cloned by limiting dilution at least twice, and then grown to large volume in tissue culture flasks or as tumors in mice [1 11. Detection of antibody activity
Culture fluids (0.1) ml) were incubated with 0.1 ml of BSA buffer (Na,HPO,, 0.1 M, pH 7.2, BSA, 0. lx, NaN,, O.l%, NaCl, 0.15 M), 0.1 ml of ‘25iodinated radioligand and 0.2 ml of anti-mouse IgG. After 2 h at room temperature, 0.2 ml of PEG 20% was added and tubes were centrifuged at 3000 rpm for 15 min. The supernatant was decanted and the pellets counted for radioactivity. For the tritiated tracer, 0.1 ml BSA buffer, 0.1 ml culture fluid and 0.1 ml of tritiated progesterone were incubated for 105 min at room temperature and for 15min at 4°C. Dextran-coated charcoal (0.2 ml) was added and the incubation mixture was centrifuged at 3000 rpm for 15 min. The supernatants were collected and counted.
RESULTS
All rabbits produced antisera of high titers when using the homologous ‘25iodinated tracer in the radioimmunoassay. Table 1 shows the titers obtained with the tritiated and ‘251-iodinated radioligands from one of these antisera (147A). As previously shown, a change of the structure of the bridge leads to a lower titer than is observed with the homologous tracer,
Affinity constant* (K,) l/m01 0.088 0.308 1.04 0.238 1.66 0.26
P-l P-2 P-3 P-4 P-5 147-A *Calculated according ref. [12].
x x x x x x
10” IO’” IO” IO’” 10’” 10”
to the
method in.
and, moreover, the use of a tritiated tracer further markedly decreases the dilution of the antisera. Hybrid growth was observed in 50-60x of wells following fusion. While l&15% of antibodyproducing clones were obtained from these fusions, several clones stopped antibody production before cloning or showed very low binding to radioligand A and B and were therefore not cloned. Five stable monoclonal antibodies to progesterone were isolated from three independent hybridizations. When injected into pristane-treated irradiated mice, the hybridomas secreted high amounts of progesterone antibodies into the ascitic fluid of these mice. The titers of each antibody when using the tritiated and 12’iodinated tracer are shown in Table 1. It can be seen that contrary to the well-known interaction of polyclonal antibodies with the homologous and heterologous tracer, monoclonal antibodies can bind a heterologous tracer at a higher titer than that observed with the homologous radioligand. However, while the interaction with the tritiated and the ‘25iodineligand A,B and C is present for the five antibodies, three of these antibodies do not bind the tracer D. Figures l-4 shows that standard curves constructed using the antibody dilution as indicated in Table 1. Concentrations of cold progesterone from O-10000 picograms per tube were added. It is shown that, using the ‘25iodinated tracers, high sensitivity radioimmunoassay can be obtained with both monoclonal and polyclonal antibodies. However, contrary with the well-known interaction with polyclonal antibody, the present data indicate that with monoclonal antibodies, the use of heterologous homologous tracer may generate a better titer of the antibody and also a better sensitivity. Moreover, it can be seen in Table
Table 1. Titers of oolvclonal and monoclonal antibodies with tritiated and ‘*‘-iodinated tracers at 30-35% binding 1
,
Tracer ‘2SI_ iodinated Pro-12CMO Pro-l l-HS Pro-l 1G Tritiated Tracer
Polyclonal antibody
Monoclonal antibodies PI
P2
P3
Pi-
~- P5 ~~
1/4&o 1/250000
1/7500000 1/300000 ljlOOO0
1/10000 l/l 10000
l/2000 l/40000 l/250000
l/l0000 l/90000
1/10000
l/210000
l/SW00
l/24000
l/66000
147-A
l/80000 I /300000 l/80000 l/12000
Pro-12-CMO: ‘*‘iodo progesterone-12-(O-carboxymethyl)-oxime tyrosine methyl ester Pro-l I-HS: ‘“‘iodo-1 I-hydroxyprogesterone hemisuccinate-tyrosine methyl ester. Pro-l IG: ‘*‘iodo-1 1-hydroxyprogesterone glucuronide tyramine.
Monoclonal antibodies to progesterone POLYCLONAL 100
ANTIBODY,
147-A
A
75
50
!?=325
25
0 ~
1OC
0
75
4 I!!& %=”
50
25
0
1
PROGESTERONE
ANTISODY~
100
10'
10’ 10’
10’
(Pgltuba)
Fig. 1. MONOCLONAL
10'
Pl
B
75
F=”
50
25
0
100
75
50
25
0
C
1
NC BINDING
P =19
1
10'
1v
lry
1w
10’
PROGESTERONE
Fig. 2.
WITH THE TRACER
I
1
(Pgmbe,
10’ 1w 10’ 10’ 10’
40X MON~CLONAL
ANTIBODY
~2
100
75
50
25
0
100
75
50
25
0
PROGESTERONE
(i'Q&k)
Fig. 3. M0N0CL0NAL
100
ANTIBOOY.
‘zi”=”
Y=215
50
25
100
75
50
25
0
p3
B
A
75
0
I I 1
~
a
0
C
NO BINDING
Y=25
WITH THE TRACER
9
1
10’
w
101 IV
1w
PROGESTERONE
Fig. 4.
(pg4ibe)
Monoclonal
antibodies MONOCLCNAL
to progesterone ANTIBODY.
409
P4
A S
?=” 9
4.
= 212
1
100
C
75
i, Bo-503
50
_L 2
25
0
1
PROGESTERONE
10’ IO’ IO’ 10’ IO’
(Pgltube)
Fig. 5. -LCNAL loo
75
50
25
0
100
75
ANTIBODY:
P5
A
Lsl!L L.L ILF=207
?=52
C
D
No BINDING
50
25
0
B =26
WITH THE TRACER
0
1
IO'
1w
101 IO’
10)
PROOESTERCNE
1 10'10
103 IO’ 10’
(Pgltube)
Fig. 6.
Figs. 1-6. Standard curves using rabbit antiserum 147-A (Fig. 1) and monoclonal antibodies PI-5 (Figs 2-6) with tritiated progesterone (A), ‘?odo- I I-hydroxyprogesterone hemisuccinate tyrosine methyl ester ‘*5iodo-I I-hydroxyprogesterone glucuronide tyramine (C) and (B), ‘*%odoprogesterone-12(o-carboxymethyl)-oxime tyrosine methyl ester (D).
MICHELEBROCHU et
410
al
Tab!e 3. Specificity of monoclonal and polyclonal antibodies raised against 1la-hydroxyprogesterone serum albumin
PI
Steroid
P2
1 1-Hydroxyprogesterone
100 83
100 321
hemisuccinate I I-Hydroxyprogesterone SLY.-Pregnane-3,20-dione 17-Hydroxyprogesterone Sa-Pregnan-3 a-ol-20-one Sa-Pregnan-3 /3-ol-20-one Androsterone Androstenedione Androstanedione 20B-Dihvdrooroeesterone 20k-Dih;droprogesterone Sa-Pregnane-3a,20a-dial Pregnenolone 17-Hydroxypregnenolone Testosterone Dihydrotestosterone Androstane-3a,l7fi-dial Androstane-3B,178-diol Androst-5-ene-38,178-diol Cholesterol Estrone Estradiol Estriol Cortisol
5.4 I1 2.4 8.6 2.3 3.7 2.5 1.7 0.6 < 0.03 0.4 0.3 < 0.03 < 0.03 < 0.02 < 0.02 < 0.02 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03
65 29 19 < 0.4 < 0.4 < 0.4 0.4 0.4 0.7 0.1 0.4 0.65 0.1 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.2
Progesterone
2 that antibody P-l which shows the lowest affinity constant for progesterone lead to the highest sensitivity when using the appropriate iodinated tracer. Both polyclonal and monoclonal antibodies were assessed for specificity using a range of steroids from 10 to 100,000 pg/tube. The cross-reactivities of the two types of antibodies are shown in Table 3. While the radioligands giving the best sensitivity of radioimmunoassay are used to determine the specificity, we have shown that almost similar specificity is obtained when other tracers are used (data not shown). DISCUSSION
In order to achieve a sensitive steroid radiousing polyclonal antibodies and immunoassay ‘?odinated tracer, it is well established that heterologous tracer must be used. We have shown in the present study that a sensitive assay can be obtained with a polyclonal antibody developed against 1la-hydroxyprogesterone hemisuccinate-BSA either by a change in the structure of the bridge or by using a bridge attached at a position different from that in the immunogen. However, the titer of the antibody is markedly reduced when heterologous tracer is used. We have demonstrated that assays of high sensitivity may be obtained by using homologous and heterologous tracers with monoclonal antibody, but the system is unpredictable. In some cases, a sensitive standard curve can be obtained with a given monoclonal antibody and a homogenous bridge or with a bridge that only the structure has been changed while a tracer with a bridge at a position different from that in the immunogen does not bind to this antibody.
%Cross-reaction Monoclonal P3 P4 100 142 < 0.03 0.48 3.4 2.1 I.1 3.4 1.5 0.5 73 < 0.03 41 0.5 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 < 0.03 0.07
hemisuccinate conjugated to bovine
Polyclonal 147-A
PS
100 219
100 204
100 129
104 55 I.1 0.9 3.9 1.5 0.6 1.2 1.0 0.1 0.4 0.6 < 0.06 < 0.06 < 0.2 < 0.06 < 0.06 c 0.06 < 0.06 < 0.06 < 0.06 < 0.06 < 0.06
0.13 27 14 21 13 0.1 0.1 9.6 1.2 < 0.03 46 0.4 < 0.03 < 0.03 < 0.03 < 0.03 7.4 0.09 < 0.03 < 0.03 < 0.03 < 0.03 < 0.07
22 35 3.6 0.07 0.61
However, we have also demonstrated that a monoclonal antibody can bind a heterologous tracer at a different position and give a highly sensitive assay. The use of heterologous tracer clearly increases the sensitivity of the assay but, contrary to observations with polyclonal antibody, the titer is also increased, suggesting that low amount of monoclonal antibody is important in order to achieve the highest sensitive assay. The present data indicate that monoclonal antibodies are suitable for radioimmunoassay of steroids with lz5-iodinated ligands and have specificities comparable to those obtained with conventional techniques. However, it should be emphasized that the appropriate tracer has to be selected in order to meet the requirements of each specific monoclonal antibody and in order to reach the highest sensitivity. Moreover, our data also suggest that the screening test of monoclonal antibody has to be performed with more than one ‘25-iodinated ligand having either an homologous or heterologous bridge.
REFERENCES 1. Corrie J. E. T., Hunter W. M. and Macpherson A strategy for radioimmunoassay of plasma gesterone with use of a homologous-site radioligand. Clin. Chem. 27 (1981) 594-599.
J. S.: pro‘%labeled
2. Nordblum G. D., Counsel1 R. E. and England B. G.: Ligand specificity and bridging phenomenon in hapten radioimmunoassays. Lignnd Q. 2 (1979) 34-36. 3. Allen R. M. and Redshaw M. R.: The use of homologous and heterologous ‘251-radioligands in the radioimmunoassay of progesterone. Steroids 32 (I 978) 467486. 4. Scarisbrick
J. J. and Cameron
E. M. D.: Radio-
Monoclonal antibodies to progesterone immunoassay of progesterone: comparison of [1,2,6,7-‘HI-progesterone and progesterone-[‘251]-iodohistamine radioligands. J. steroid Biochem. 6 (1975)
9.
51-56. 5. Peele B. and Holmes A. L.: Potential
[‘251]iodinated tracers for the radioimmunoassay of dehydroepiandrosterone. J. steroid Biochem. 14 (1981) 1197-1200. 6. Norblom G. D., Webb R., Consell R. E. and England B. G.: A chemical approach to solving bridging phenomena in steroid radioimmunoassays. Steroid 38 (1981) 161-173. 7. Kohen F., Lichter S., Eshar Z. and Lindner H. R.: Preparation of monoclonal antibodies able to discriminate between testosterone and Sa-dihydrotestosterone. Steroids 39 (1982) 453459. 8. Fantl V. E., Wang D. Y. and Knyba
R.E.:
The
10.
11.
12.
411
production of high affinity monoclonal antibodies to progesterone. J. steroid Biochem. 17 (1982) 125-130. White A., Anderson D. C. and Daly J. R. : Production of a highly specific monoclonal antibody to progesterone. J. clin. Endocr. Metab. 54 (1982) 205-207. Belanger A., Caron S. and Picard V.: Simultaneous radioimmunoassay of progestin, androgen and estrogen in adult rat testis. J. steroid Biochem. 13 (1979) 185-l 90. Galfrt G. and Milstein C.: Preparation of monoclonal antibodies: strategies and procedures. In Methods of Enzymology (Edited by M. De Luca). Academic Press, New York, Vol. 73 (1975) p. 3. Abraham G. E.: Characterization of anti-steroid antisera. in Steroid Immunoassay (Edited by E. H. D. Cameron, S. G. Hillies and K. Griffiths). Alpha Omega, Cardiff Wales (1974) pp. 67-79.