Vol. 168, No. 3, 1990 May 16, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1020-1026
A MONOCLONALANTI-PEPTIDE ANTIBODY FUXDGNIZES THE ADRBNOCORTICOTROPICRECEPTOR B.L.
Clarke and K.L. Bost
University of Alabama at Birmingham Department of Physiology and Biophysics Birmingham, Alabama Received
March
20,
1990
We have produced a monoclonal antibody that specifically recognizes the adrenocorticotropic receptor on rat adrenal cells. The immunogen was designed from an RNA sequence complementary to the mRNA coding for This complementary peptide, termed HTCA, has been shown to ACTH,-,,. specifically bind ACTH and was proposed to mimic the ACTH binding site of the hormone receptor. The monoclonal anti-HTCA antibody recognized a restricted domain of the HTCA peptide, bound to Y-l adrenal cells with a KD of 1.8 nM, and blocked the binding of 'lsI-ACTH to rat adrenal cells. These findings show that anti-HTCA competes with ACTH for binding to the ACTH receptor. 0 1990 Academic Press, Inc.
Owing to the difficult task of isolating the ACTH receptor by conventional procedures we have used a novel technique to mimic the receptor binding site. Blalock and coworkers hypothesized that a complementary copy from the mRNA coding for a peptide hormone may code for a peptide having similar binding properties as the native receptor (1). Although the physical principal for the specific binding interactions are as yet unknown, there are several examples for demonstrating this type of molecular mimicry such as insulin (2), fibronectin (3), opiates (4). luteinizing hormone releasing hormone (5,6). myelin basic protein (7), vasopressin (9), angiotensin II (lo), and ribonuclease S peptide (11). In a previous study, Bost and Blalock (12) produced a rabbit polyclonal antibody to the ACTH receptor by synthesizing the complementary peptide to the ACTH,-,, analogue. This polyclonal anti-HTCA antibody was found to specifically bind to Y-l cells and could be used to immunopurify a protein complex of 225,000 with an ACTH binding subunit of 83,000 Mr. We decided to produce a monoclonal antibody given the possibility that a even if monospecific, polyclonal antibody, may have a subpopulation of antibodies that could crossreact with several protein species. Since ACTH and its complementary peptide HTCA both consist of 24 residues, a priori there will be a heterogeneous population of epitopes recognized by the polyclonal antibody: by contrast a monoclonal antibody would recognize a single epitope. The availability of such a monoclonal antibody would provide a valuable probe for studying the biochemical and structural properties of the ACTH receptor. In this report, we describe the binding characteristics 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
1020
Vol.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of one such monoclonal antibody that was generated against the peptide HTCA representing the complementary peptide to the peptide hormone ACTH,-,,. This monoclonal antibody was found to bind adrenal cells at an ACTH specific site. MATERIALS AND METHODS Peptide probes. The peptides PHE'-NLE'-ACTH,_,, (ACTH,-,,). HTCA1_,,. and LYS-GLU-ARG-ILE-LELJ-LEU (LERILL) were synthesized as previously described (13). Iodinated ACTH,-,, or Protein G was prepared using iodo-beads (Pierce Chemical) and Na "'1 (NEN DuPont) as previously described (13). Monoclonal antibody production. The immunogen was produced by conjugating HTCA to keyhole limpet hemocyanin (Calbiochem) using gluteraldehyde (13) with a coupling efficiency of 40% based on uncoupled peptide recovery. Several BALB/c mice were inoculated with 100 Irg of conjugated peptide prepared in an emulsification of Freund's complete adjuvant (Sigma). Mice developing antibodies capable of binding HTCA in an ELISA, rounding Y-l cells (14) and blocking "'I-ACTH binding were fused to SP2/0-Ag14 (ATCC CRL 1581) myeloma cells using polyethylene glycol (Boehringer Mannheim) as previously described (15). After 11 days, 768 wells were screened by ELISA for reactivity against HTCA, and 82 were determined to be positive as judged by at least a three-fold increase in absorbance over media controls. Cells were cloned in soft agar, and supemates from clones were retested for reactivity against HTCA and further tested for their ability to block 1"51-ACTH binding to Y-l adrenal cells. Clones most effective in blocking binding were injected into pristane primed BALB/c mice, and ascitic fluid collected. As a routine, hybridomas were periodically subcloned and selected for HTCA binding to ensure an optimal in vivo production of monoclonal antibody. One hybridoma subclone, designated 3/l-8, was chosen for further characterization of agonist activity based on optimal induction of Y-l adrenal cell rounding and is the subject of this report. Cell culture. The adrenal glands from decapitated male Sprague-Dawley rats (150-2OOg) were rapidly excised and stored in sterile RPMI-1640 plus 1% BSA on ice. The adipose tissue was removed prior to squeezing the medulla and fasciculata-reticularis layers away from the capsule. The inner adrenal tissue was suspended in 1 ml of RPMI-1640/l% BSA per two glands, then minced by opposing slices with two razor blades, and then collagenase type IV (Sigma) and DNase I (Sigma) were added to a final concentration of 1 mg/ml and 0.1 mg/ml, respectively. The digestion mix was incubated for 15 min at 37OC with mild agitation, then spun to a pellet and resuspended in fresh RPMI-1640/l% BSA, and finally poured through a sterile (50 mesh) sieve. Viable cells were isolated by centrifugation on Hypaque-Ficoll and cultured for 3-5 days in RPMI-1640/l% FCS prior to use in binding assays. Binding assays using mouse Y-l adrenal cells were performed in 24-well Cells were grown in RPMI-1640/l% fetal calf serum to plates (Costar, Mark II). a density of approximately 500,000 per well prior to measuring antibody binding. All chemicals were of reagent grade unless otherwise specified. General. Protein content was determined by the Bradford method (16). Purified immunoglobulin was quantitated by absorbance at 280 nM using 1.3 as the absorbtivity in mg/ml per absorbance units. Centrifugations were performed on either a Sorvall RT6000B refrigerated table-top unit or Sorvall RC5B refrigerated superspeed centrifuge and a Sorvall SS34 rotor. RESULTS
!Che anti-Hl'CA antibody binds specifically to the ETCA peptide. Specificity for the 3/l-8 monoclonal antibody was demonstrated using an ELISA Microtiter plates coated with BSA conjugated peptides and washed technique. Bound to remove nonbound protein were tested for binding to anti-HTCA. 1021
Vol.
168,
No.
3, 1990
2 31.0 5 . 25 L
BIOCHEMICAL
AND
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RESEARCH
COMMUNICATIONS
A
3.5
1.0
-
0.8
-
,0.5
0 20 40 BSA-HTCA
60 80 (ug/mi)
P g s
0.6~
s f z 0
0.4
-
ANTIBODY
(ug/ml)
Figure 1. Binding of anti-EICA to El!CA. Microtiter plates were coated with BSA conjugated peptides and assayed by an BLI8A procedure. Panel A shows the effects from various concentrations of soluble BSA-BTCA on the binding of anti-El'CA to solid phase BSA-BTCA. Panel B shows the bind1 3/l-8 to wells coated with BSA-BTCA (0). BSA-LBBILL (A>, BSA-A normal non-e mouse IgG was also checked for binding to BSA-ElKA Thekdndingofanti-EICA to &fferentEl!CApeptidefragmente BTCA consisting of residues l-24 (0) lo-24 (0). 15-24 6-18 a) were used in a solid p&&assay to determine us fragments of BTCA on binding to anti-ETCA.
complexes were then detected using an anti-mouse IgG antisera conjugated to alkaline phosphatase. Only the wells coated with BSA-HTCA showed significant binding to anti-HTCA with proportional binding starting at a protein concentration of 16 &ml of anti-HTCA and attaining saturation by 50 &ml (Figure 1). Soluble BSA-HTCA competed with the binding of anti-HTCA to the solid phase BSA-HTCA (Figure 1). Binding by the constant regions of anti-HTCA was deemed insignificant since a nonimmune mouse IgG showed no binding above background levels at protein concentrations of 50 &ml or less, and an additional increase to 160 &ml raised the binding to only 30% of anti-HTCA level. A binding analysis using fragments of the HTCA peptide demonstrated that the anti-HTCA monoclonal antibody recognizes a specific domain (Figure 2). Various fragments of HTCA were conjugated to BSA and used to assay for anti-HTCA binding by the ELISA method. The fragments containing only residues 1-11 and 6-18 were poor ligands for the anti-HTCA antibody, in comparison the fragments containing residues 15-24 and lo-24 bound well to anti-HTCA antibody. 1022
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168, No. 3, 1990
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Table 1 ANTI-HTCABINDING TO RAT ADRENALCELLS
Treatment 3/l-8
3/l-8 3/l-8 3/l-8
BSA-HTCA ACTH
NIgG
Concentration
(&ml)
Radioactivity
0.2 1.0 5.0 25.0 25.0 25.0 25.0
(CPM)
416 484
1,056
3,740 1,027
949
949
Isolated rat adrenal cells were treated with various concentrations of protein G purified antibody for 60 min at room temperature. The cells were then washed two times with RF'MI-1640 and then approximately 100.000 cpm of "'I-labeled protein G wss added and incubated for an additional 60 min at room temperature. The cells were finally washedthree times with RPMI-1640 and then assayed for cell-associated radioactivity.
The mmoclonal mtibody auti-3TCA recognizes ACTH receptors. Direct binding of anti-HTCA to the cell surface was measured using "'I-labeled protein G (Table 1). This probe forms high affinity complexes with immunoglobulins including mouse IgG, (17). In preliminary experiments, we determined that protein G binding to the cell surface of rat adrenal cells was stoichiometrically related to the binding of anti-HTCA to the cell surface, and in the absence of antibody there was virtually no cell surface bound protein G (data not shown). However, a low level of cell-associated radioactivity was detected in the presence of normal mouse immunoglobulin. Both excess free ACTH and free BSA-HTCA could compete away similar amounts of anti-HTCA binding at similar protein concentrations. Presumably under these conditions the ACTH is binding to the receptor, while free HTCA is binding to anti-HTCA thus prohibiting antibody binding to the cell. The binding of "'I-protein G was proportional to the amount of anti-HTCA. A nonimmune IgG at 25 fig/ml showed only 17% of the cell-associated radioactivity compared to an equivalent amount of anti-HTCA. The anti-HTCA titers in ascities fluid could be increased by 180-fold A further purification after eluting over a protein G sepharose column. using an HTCA sepharose column recovered 34% of the total protein with a This protein G corresponding enrichment of blocking activity by 3-fold. to isolated rat purified material competitively blocked 'a51-ACTH binding adrenal ce.lls in a dose dependent manner with a 50% blocking titer at 1.6 purified antibody, the dissociation N/ml (Figure 3). Using an HTCA-affinity constant was calculated to be 1.8 nM and the antibody recognized 64,000 dissociation binding sites per cell (Fig. 4).In comparison, the high affinity constant of la'I-ACTH was 0.69 nM and recognized 85,000 of the high affinity sites on Y-l adrenal cells (data not shown). DISCUSSION Observations made during earlier polyclonal antisera to HTCA suggested 1023
studies on the development of a that the development of an ACTH
vol. 168,
No.
3. 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
25
t m
I
0
3
I
0.16 0.016 ANTIBODY
I
&$I-;
I
16.0
10-
04 0 O
100 BOUND
I, 200
1
300
(fmol/106
*
'00 P
cells)
Fiaure 3. Anti-H!KA blocks the binding of r*'I-ACTE to the ACT8 receptor. Isolated adrenal cells were incubated with "'I-ACT8 at 800 nM. in the presence of various concentrations of anti-8TCA for 30 min at room temperature. The cells were then washed prior to assaying for cell-associated radioactivity. Each symbol represents the mean of three values plus the S.E.M. Figure 4. adrenal cells.
A Scatchard analysis
of "'I-anti-EEA
binding
to
moue.
Y-l
mimicking antibody would be better accomplished using monoclonal antibodies. the anti-receptor titer from one animal to the next varied First, considerably due in part to genetic variability since the rabbits were Second, anti-HTCA antibodies could interact with rabbit ACTH outbred. induce an autoimmune response (unpublished receptors and possibly observation). Considering the findings of ACTH receptors on lymphocytes (18) and that high concentrations of ACTH will down-regulate antibody production (19) would suggest that the anti-ACTH receptor antibodies may limit antibody the anti-HTCA antibodies that are production. In support of this notion, specific for the receptor were found to transiently appear and then disappear even though the titers for anti-HTCA remained high suggesting a selective down regulation was occurring. This last observation should be viewed in light of the HTCA used as an immunogen was a 24 residue peptide and would, therefore, generate antibodies of differing specificity and affinities. The production of monoclonal antibodies therefore provides an unlimited source with known specificity. The anti-HTCA monoclonal antibody was found to be selective towards HTCA The binding while showing no binding towards ACTH nor an Irrelevant peptide. of anti-HTCA was freely competitive since soluble BSA-HTCA blocked binding to the solid phase BSA-HTCA in a dose-dependent pattern. Also binding was through the epitope and not by some structural domain of the immunoglobulin 1024
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168, No. 3, 1990
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
since a nonimmune IgG showed very little interaction with BSA-HTCA. The important property for anti-HTCA is that the antibody does recognize the ACTS receptor as demonstrated by: 1) the direct competition of anti-HTCA with '"I-ACTH binding to rat adrenal cells; 2) a concentration dependent binding of "'I-anti-HTCA to Y-l cells: and 3) indirect determination by binding "'I-protein G to anti-HTCA labeled adrenal cells. Also the binding of anti-HTCA could be disrupted by the presence of BSA-HTCA to block antibody binding or ACTH to block ACTH receptor binding, and therefore negating any arguments that surface entrapment may be occurring. In addition, this monoclonal antibody was able to recognize an anti-ACTH antibody in an fashion idiotypic/anti-idiotypic (manuscript in preparation). ThiS observation is consistent with investigations where anti-idiotypic antibodies made against a particular anti-ligand antibody have been used to recognize that ligand's receptor (20). The anti-HTCA antibody bound to Y-l cells with relatively high affinity; lasI-ACTH binding was only about a 2.6 times greater affinity than the In addition to having comparable anti-HTCA antibody for ACTH receptors. binding KD's, both ACTH and anti-HTCA recognize similar numbers of surface the competitive inhibition study receptors and therefore supports demonstrating a common binding site. Developing antibodies directed against a receptor binding site is, in general, a difficult task. Here we have used a novel technique to generate monoclonal antibodies against the adrenal ACTH receptor. Although this antibody will certainly be useful for further characterizing of ACTH receptors, a more important consideration is the application of the molecular The ability to make anti-receptor antibodies without recognition hypothesis. first purifying the receptor offers a significant advantage to conventional methods. Furthermore, this body of work may have implications about the in general, and the possible encoding nature of protein-protein interactions, of this information in the mRNA. We would like to thank Diane Weigent for help in Acknowledgments. preparation of this manuscript, Todd McBurnett for expert technical assistance, and David Pascual for insightful suggestions. This research was supported by NIDDKD grant DK 39299. REFERENCES
::
3. 4. 5. 6.
Bost, K. L. and Blalock. J. E. (1989) Meth.. 168, 16-28. Knutson, V. P. (1988) J. Biol.. 263, 14146-14151. Brentani, R. R.. Ribeiro. S. F.. Potochjak. P.. Pasqualini. R., Lopes, J. D. and Nakaie, C. R. (1988) Proc.Natl. Sci. USB 85, 364-367. Carr, D. J. J., Bost, K. L., and Blalock. J. E. (1986) J. e. 12, 329-337. Mulchahey. J. J.. Neill. J. D.. Dion, L. D., Best. K. L.. and Blalock, J. E. (1986) Prop. Natl.d. Sci. &A 83. 9714-9718. P. E.. Coy, D. H. and Arimura. A. (1986) Gores. T. J.. Gottschall.
7. 8.
Knigge, K. M., Piekut, 269-271.
Walker D. P.. Goodin. R. and Benveniste, 157-i66 D. T. and Berlove', D. (1988) bleurosci. I&t. 86. 1025
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AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Vol.
168, No. 3, 1990
9. 10.
Johnson, H. M. and Torres. B. A. (1988) ;I.. 141, 2420-2423. Elton, T. S., Dion. L. D., Bost. K. L., Oparil, S.. and Blalock, J. E. (1988) Boc. Natl. 85, 2518-2522. Shai, Y., Flashner. M. and Chaiken, I. M. (1987) U. 26, 669-675. Bost, K. L. and Blalock, J. E. (1986) Mol. Cell.. 44, l-9. Bost. K. L.. Smith, E. M., and Blalock. J. E. (1985) Prop. Natl. Arad, sci. 82. 1372-1375. Mattson, P.. and Kowal, J. (1980) Tissue 12, 685-701. Carr. D.J.J., Blalock, J.E. and Bost, K.L. (1989) m. 20, 181-186. Bradford. M. (1976) Anal.. 72, 248-254. Bjorck. L. and Knonvall. G. (1984) J.. 133, 969-974. Clarke, B. L. and Bost, K. L. (1989) J,. 2~3, 464-469. Johnson, H. M., Smith. E. M., Torres, B. A., and Blalock, J. E. (1982) Eror. lYuLAAcad.Scl._USA 79, 4171-4174. Gaulton, G.N. and Greene, M.I. (1986) Am, Rev, m. 4. 253-280.
::: 13. 14.
15. 16. 17. 18.
19. 20.
1026
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1020-1026
A MONOCLONALANTI-PEPTIDE ANTIBODY FUXDGNIZES THE ADRBNOCORTICOTROPICRECEPTOR B.L.
Clarke and K.L. Bost
University of Alabama at Birmingham Department of Physiology and Biophysics Birmingham, Alabama Received
March
20,
1990
We have produced a monoclonal antibody that specifically recognizes the adrenocorticotropic receptor on rat adrenal cells. The immunogen was designed from an RNA sequence complementary to the mRNA coding for This complementary peptide, termed HTCA, has been shown to ACTH,-,,. specifically bind ACTH and was proposed to mimic the ACTH binding site of the hormone receptor. The monoclonal anti-HTCA antibody recognized a restricted domain of the HTCA peptide, bound to Y-l adrenal cells with a KD of 1.8 nM, and blocked the binding of 'lsI-ACTH to rat adrenal cells. These findings show that anti-HTCA competes with ACTH for binding to the ACTH receptor. 0 1990 Academic Press, Inc.
Owing to the difficult task of isolating the ACTH receptor by conventional procedures we have used a novel technique to mimic the receptor binding site. Blalock and coworkers hypothesized that a complementary copy from the mRNA coding for a peptide hormone may code for a peptide having similar binding properties as the native receptor (1). Although the physical principal for the specific binding interactions are as yet unknown, there are several examples for demonstrating this type of molecular mimicry such as insulin (2), fibronectin (3), opiates (4). luteinizing hormone releasing hormone (5,6). myelin basic protein (7), vasopressin (9), angiotensin II (lo), and ribonuclease S peptide (11). In a previous study, Bost and Blalock (12) produced a rabbit polyclonal antibody to the ACTH receptor by synthesizing the complementary peptide to the ACTH,-,, analogue. This polyclonal anti-HTCA antibody was found to specifically bind to Y-l cells and could be used to immunopurify a protein complex of 225,000 with an ACTH binding subunit of 83,000 Mr. We decided to produce a monoclonal antibody given the possibility that a even if monospecific, polyclonal antibody, may have a subpopulation of antibodies that could crossreact with several protein species. Since ACTH and its complementary peptide HTCA both consist of 24 residues, a priori there will be a heterogeneous population of epitopes recognized by the polyclonal antibody: by contrast a monoclonal antibody would recognize a single epitope. The availability of such a monoclonal antibody would provide a valuable probe for studying the biochemical and structural properties of the ACTH receptor. In this report, we describe the binding characteristics 0006-291X/90 Copyright All rights
$1.50
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
1020
Vol.
168, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
of one such monoclonal antibody that was generated against the peptide HTCA representing the complementary peptide to the peptide hormone ACTH,-,,. This monoclonal antibody was found to bind adrenal cells at an ACTH specific site. MATERIALS AND METHODS Peptide probes. The peptides PHE'-NLE'-ACTH,_,, (ACTH,-,,). HTCA1_,,. and LYS-GLU-ARG-ILE-LELJ-LEU (LERILL) were synthesized as previously described (13). Iodinated ACTH,-,, or Protein G was prepared using iodo-beads (Pierce Chemical) and Na "'1 (NEN DuPont) as previously described (13). Monoclonal antibody production. The immunogen was produced by conjugating HTCA to keyhole limpet hemocyanin (Calbiochem) using gluteraldehyde (13) with a coupling efficiency of 40% based on uncoupled peptide recovery. Several BALB/c mice were inoculated with 100 Irg of conjugated peptide prepared in an emulsification of Freund's complete adjuvant (Sigma). Mice developing antibodies capable of binding HTCA in an ELISA, rounding Y-l cells (14) and blocking "'I-ACTH binding were fused to SP2/0-Ag14 (ATCC CRL 1581) myeloma cells using polyethylene glycol (Boehringer Mannheim) as previously described (15). After 11 days, 768 wells were screened by ELISA for reactivity against HTCA, and 82 were determined to be positive as judged by at least a three-fold increase in absorbance over media controls. Cells were cloned in soft agar, and supemates from clones were retested for reactivity against HTCA and further tested for their ability to block 1"51-ACTH binding to Y-l adrenal cells. Clones most effective in blocking binding were injected into pristane primed BALB/c mice, and ascitic fluid collected. As a routine, hybridomas were periodically subcloned and selected for HTCA binding to ensure an optimal in vivo production of monoclonal antibody. One hybridoma subclone, designated 3/l-8, was chosen for further characterization of agonist activity based on optimal induction of Y-l adrenal cell rounding and is the subject of this report. Cell culture. The adrenal glands from decapitated male Sprague-Dawley rats (150-2OOg) were rapidly excised and stored in sterile RPMI-1640 plus 1% BSA on ice. The adipose tissue was removed prior to squeezing the medulla and fasciculata-reticularis layers away from the capsule. The inner adrenal tissue was suspended in 1 ml of RPMI-1640/l% BSA per two glands, then minced by opposing slices with two razor blades, and then collagenase type IV (Sigma) and DNase I (Sigma) were added to a final concentration of 1 mg/ml and 0.1 mg/ml, respectively. The digestion mix was incubated for 15 min at 37OC with mild agitation, then spun to a pellet and resuspended in fresh RPMI-1640/l% BSA, and finally poured through a sterile (50 mesh) sieve. Viable cells were isolated by centrifugation on Hypaque-Ficoll and cultured for 3-5 days in RPMI-1640/l% FCS prior to use in binding assays. Binding assays using mouse Y-l adrenal cells were performed in 24-well Cells were grown in RPMI-1640/l% fetal calf serum to plates (Costar, Mark II). a density of approximately 500,000 per well prior to measuring antibody binding. All chemicals were of reagent grade unless otherwise specified. General. Protein content was determined by the Bradford method (16). Purified immunoglobulin was quantitated by absorbance at 280 nM using 1.3 as the absorbtivity in mg/ml per absorbance units. Centrifugations were performed on either a Sorvall RT6000B refrigerated table-top unit or Sorvall RC5B refrigerated superspeed centrifuge and a Sorvall SS34 rotor. RESULTS
!Che anti-Hl'CA antibody binds specifically to the ETCA peptide. Specificity for the 3/l-8 monoclonal antibody was demonstrated using an ELISA Microtiter plates coated with BSA conjugated peptides and washed technique. Bound to remove nonbound protein were tested for binding to anti-HTCA. 1021
Vol.
168,
No.
3, 1990
2 31.0 5 . 25 L
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A
3.5
1.0
-
0.8
-
,0.5
0 20 40 BSA-HTCA
60 80 (ug/mi)
P g s
0.6~
s f z 0
0.4
-
ANTIBODY
(ug/ml)
Figure 1. Binding of anti-EICA to El!CA. Microtiter plates were coated with BSA conjugated peptides and assayed by an BLI8A procedure. Panel A shows the effects from various concentrations of soluble BSA-BTCA on the binding of anti-El'CA to solid phase BSA-BTCA. Panel B shows the bind1 3/l-8 to wells coated with BSA-BTCA (0). BSA-LBBILL (A>, BSA-A normal non-e mouse IgG was also checked for binding to BSA-ElKA Thekdndingofanti-EICA to &fferentEl!CApeptidefragmente BTCA consisting of residues l-24 (0) lo-24 (0). 15-24 6-18 a) were used in a solid p&&assay to determine us fragments of BTCA on binding to anti-ETCA.
complexes were then detected using an anti-mouse IgG antisera conjugated to alkaline phosphatase. Only the wells coated with BSA-HTCA showed significant binding to anti-HTCA with proportional binding starting at a protein concentration of 16 &ml of anti-HTCA and attaining saturation by 50 &ml (Figure 1). Soluble BSA-HTCA competed with the binding of anti-HTCA to the solid phase BSA-HTCA (Figure 1). Binding by the constant regions of anti-HTCA was deemed insignificant since a nonimmune mouse IgG showed no binding above background levels at protein concentrations of 50 &ml or less, and an additional increase to 160 &ml raised the binding to only 30% of anti-HTCA level. A binding analysis using fragments of the HTCA peptide demonstrated that the anti-HTCA monoclonal antibody recognizes a specific domain (Figure 2). Various fragments of HTCA were conjugated to BSA and used to assay for anti-HTCA binding by the ELISA method. The fragments containing only residues 1-11 and 6-18 were poor ligands for the anti-HTCA antibody, in comparison the fragments containing residues 15-24 and lo-24 bound well to anti-HTCA antibody. 1022
Vol.
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BIOCHEMICAL
AND BIOPHYSICAL
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Table 1 ANTI-HTCABINDING TO RAT ADRENALCELLS
Treatment 3/l-8
3/l-8 3/l-8 3/l-8
BSA-HTCA ACTH
NIgG
Concentration
(&ml)
Radioactivity
0.2 1.0 5.0 25.0 25.0 25.0 25.0
(CPM)
416 484
1,056
3,740 1,027
949
949
Isolated rat adrenal cells were treated with various concentrations of protein G purified antibody for 60 min at room temperature. The cells were then washed two times with RF'MI-1640 and then approximately 100.000 cpm of "'I-labeled protein G wss added and incubated for an additional 60 min at room temperature. The cells were finally washedthree times with RPMI-1640 and then assayed for cell-associated radioactivity.
The mmoclonal mtibody auti-3TCA recognizes ACTH receptors. Direct binding of anti-HTCA to the cell surface was measured using "'I-labeled protein G (Table 1). This probe forms high affinity complexes with immunoglobulins including mouse IgG, (17). In preliminary experiments, we determined that protein G binding to the cell surface of rat adrenal cells was stoichiometrically related to the binding of anti-HTCA to the cell surface, and in the absence of antibody there was virtually no cell surface bound protein G (data not shown). However, a low level of cell-associated radioactivity was detected in the presence of normal mouse immunoglobulin. Both excess free ACTH and free BSA-HTCA could compete away similar amounts of anti-HTCA binding at similar protein concentrations. Presumably under these conditions the ACTH is binding to the receptor, while free HTCA is binding to anti-HTCA thus prohibiting antibody binding to the cell. The binding of "'I-protein G was proportional to the amount of anti-HTCA. A nonimmune IgG at 25 fig/ml showed only 17% of the cell-associated radioactivity compared to an equivalent amount of anti-HTCA. The anti-HTCA titers in ascities fluid could be increased by 180-fold A further purification after eluting over a protein G sepharose column. using an HTCA sepharose column recovered 34% of the total protein with a This protein G corresponding enrichment of blocking activity by 3-fold. to isolated rat purified material competitively blocked 'a51-ACTH binding adrenal ce.lls in a dose dependent manner with a 50% blocking titer at 1.6 purified antibody, the dissociation N/ml (Figure 3). Using an HTCA-affinity constant was calculated to be 1.8 nM and the antibody recognized 64,000 dissociation binding sites per cell (Fig. 4).In comparison, the high affinity constant of la'I-ACTH was 0.69 nM and recognized 85,000 of the high affinity sites on Y-l adrenal cells (data not shown). DISCUSSION Observations made during earlier polyclonal antisera to HTCA suggested 1023
studies on the development of a that the development of an ACTH
vol. 168,
No.
3. 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
25
t m
I
0
3
I
0.16 0.016 ANTIBODY
I
&$I-;
I
16.0
10-
04 0 O
100 BOUND
I, 200
1
300
(fmol/106
*
'00 P
cells)
Fiaure 3. Anti-H!KA blocks the binding of r*'I-ACTE to the ACT8 receptor. Isolated adrenal cells were incubated with "'I-ACT8 at 800 nM. in the presence of various concentrations of anti-8TCA for 30 min at room temperature. The cells were then washed prior to assaying for cell-associated radioactivity. Each symbol represents the mean of three values plus the S.E.M. Figure 4. adrenal cells.
A Scatchard analysis
of "'I-anti-EEA
binding
to
moue.
Y-l
mimicking antibody would be better accomplished using monoclonal antibodies. the anti-receptor titer from one animal to the next varied First, considerably due in part to genetic variability since the rabbits were Second, anti-HTCA antibodies could interact with rabbit ACTH outbred. induce an autoimmune response (unpublished receptors and possibly observation). Considering the findings of ACTH receptors on lymphocytes (18) and that high concentrations of ACTH will down-regulate antibody production (19) would suggest that the anti-ACTH receptor antibodies may limit antibody the anti-HTCA antibodies that are production. In support of this notion, specific for the receptor were found to transiently appear and then disappear even though the titers for anti-HTCA remained high suggesting a selective down regulation was occurring. This last observation should be viewed in light of the HTCA used as an immunogen was a 24 residue peptide and would, therefore, generate antibodies of differing specificity and affinities. The production of monoclonal antibodies therefore provides an unlimited source with known specificity. The anti-HTCA monoclonal antibody was found to be selective towards HTCA The binding while showing no binding towards ACTH nor an Irrelevant peptide. of anti-HTCA was freely competitive since soluble BSA-HTCA blocked binding to the solid phase BSA-HTCA in a dose-dependent pattern. Also binding was through the epitope and not by some structural domain of the immunoglobulin 1024
Vol.
168, No. 3, 1990
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since a nonimmune IgG showed very little interaction with BSA-HTCA. The important property for anti-HTCA is that the antibody does recognize the ACTS receptor as demonstrated by: 1) the direct competition of anti-HTCA with '"I-ACTH binding to rat adrenal cells; 2) a concentration dependent binding of "'I-anti-HTCA to Y-l cells: and 3) indirect determination by binding "'I-protein G to anti-HTCA labeled adrenal cells. Also the binding of anti-HTCA could be disrupted by the presence of BSA-HTCA to block antibody binding or ACTH to block ACTH receptor binding, and therefore negating any arguments that surface entrapment may be occurring. In addition, this monoclonal antibody was able to recognize an anti-ACTH antibody in an fashion idiotypic/anti-idiotypic (manuscript in preparation). ThiS observation is consistent with investigations where anti-idiotypic antibodies made against a particular anti-ligand antibody have been used to recognize that ligand's receptor (20). The anti-HTCA antibody bound to Y-l cells with relatively high affinity; lasI-ACTH binding was only about a 2.6 times greater affinity than the In addition to having comparable anti-HTCA antibody for ACTH receptors. binding KD's, both ACTH and anti-HTCA recognize similar numbers of surface the competitive inhibition study receptors and therefore supports demonstrating a common binding site. Developing antibodies directed against a receptor binding site is, in general, a difficult task. Here we have used a novel technique to generate monoclonal antibodies against the adrenal ACTH receptor. Although this antibody will certainly be useful for further characterizing of ACTH receptors, a more important consideration is the application of the molecular The ability to make anti-receptor antibodies without recognition hypothesis. first purifying the receptor offers a significant advantage to conventional methods. Furthermore, this body of work may have implications about the in general, and the possible encoding nature of protein-protein interactions, of this information in the mRNA. We would like to thank Diane Weigent for help in Acknowledgments. preparation of this manuscript, Todd McBurnett for expert technical assistance, and David Pascual for insightful suggestions. This research was supported by NIDDKD grant DK 39299. REFERENCES
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