Reprod. Fertil. Dev., 1992, 4 , 223-30
Monoclonal Anti-idiotypic Antibody to Progesterone with Internal Image Properties
Vrinda Khole and Umashashi ~ e ~ d e * Institute for Research in Reproduction, J.M. Street, Parel, Bombay 400012, India. TO whom correspondence should be addressed.
A
Abstract An auto-anti-idiotypic approach was used to generate mouse monoclonal anti-idiotypic antibody E9FllF6 (Ab2) to progesterone. A conjugate of 4-pregnane 3,20 dione and bovine serum albumin was used as the immunogen. E9FllF6 bound to a rabbit anti-progesterone antibody in a linear concentration-dependent manner. It inhibited the binding of [3~]progesterone to a monoclonal anti-progesterone antibody, E9D5 (Abl); this inhibition was concentration dependent. Results from chase experiments showed that this inhibitory activity of Ab2 was not due to steric hindrance but was a result of direct binding to the ligand combining site. Indirect immunofluorescence studies also showed that Ab2 and progesterone bound to similar binding sites on Abl. Overall, the results suggest that E9F1 IF6 contains an internal image of at least part of the progesterone molecule, and that it can be classified as an Ab2P antibody. This anti-idiotypic antibody may be of value in further studies of endocrine and reproductive physiology.
Introduction An anti-idiotypic antibody (Ab2) is an antibody directed against the antigen-combining site of another antibody (Abl). These anti-idiotypic antibodies can have three different specificities depending on the idiotopes they recognize on Abl. Anti-idiotypes that mimic antigens are termed Ab2P antibodies and those that do not mimic antigens are Ab2a antibodies. Those of the third type, Ab2y, recognize idiotypic determinants located close to the binding site of Abl and interfere with the binding of antigen through steric hindrance (Bona and Kohler 1984). Of these, the Ab2P antibodies have been of considerable theoretical and practical interest because they carry the internal image of the antigen. Ab2P antiidiotypic antibodies have been extensively used in relation to diseases (Monroe and Green 1986). The most practical source of anti-idiotype would be from a hybridoma that is secreting the appropriate monoclonal antibody in large quantities. In this report we describe the production and characterization of a monoclonal anti-idiotypic antibody (Ab2P) that binds to the steroid binding region of anti-progesterone antibodies (Abl). This antibody was raised through an auto-anti-idiotypic approach (Cleveland and Erlanger 1986) using a progesterone derivative coupled to bovine serum albumin. The auto-anti-idiotypic strategy does not require prior purification of the receptor and has been successfully used in the preparation of polyclonal or monoclonal antibodies to several receptors including insulin, acetylcholine, thyrotropin and aldosterone (Sege and Peterson 1978; Wassermann et al. 1982; Hill and Erlanger 1988; Lombes et al. 1989). 1031-3613/92/O2O223$O5.00
V. Khole and U. Hegde
Materials and Methods Reagents were obtained as follows: [3~]progesterone(specific activity 96 Ci per mmol) from Amersham International, Amersham, UK; the progesterone conjugate and other steroids from either Steraloids, Wilton, USA or Sigma, St Louis, USA; RPMI 1640 and fetal calf serum (FCS) from Gibco Laboratories, New York, USA; polyethylene glycol (PEG) (mol. wt 1500) from the J. T. Baker Chemical Co., Phillipsburg, USA; and rabbit anti-mouse(1g)-HRP (horseradish peroxidase) conjugate from Dakopatts, Glostrop, Denmark.
Preparation of Monoclonal Antibodies Balb/c mice were hyperimmunized with 4-pregnane-3,20-dione (Sigma) conjugated to bovine serum albumin (Sigma). The antigen (30 pg) in 0.1 mL phosphate buffered saline (PBS 0.01 M, pH 7.4) was emulsified with 0 . 2 mL of Freund's complete adjuvant (FCA, Sigma) and injected subcutaneously into 2-3-month-old adult male mice at multiple sites. Booster injections were given subcutaneously (30 pg of progesterone conjugate emulsified in 0.2 mL Freund's incomplete adjuvant (FIA, Sigma) every 4 weeks. Mice were immunized over a period of more than 5 months. The final booster inoculation was given interaperitoneally as 30 pg of the conjugate in saline. Splenocytes of the hyperimmunized mice were fused with mouse myeloma (SP2/0) cells grown in RPMI 1640 containing 10% FCS. PEG (30%) was used as the chemical fusogen. The fusion was carried out by the method described by Kohler and Milstein (1975). The hybrid cells were plated out into 96-well plates in RPMI 1640 containing 20% FCS and hypoxanthine, aminopterin and thymidine (HAT). The supernatants from hybrids were screened for anti-progesterone activity. Only one hybrid (E9) was positive for antiprogesterone activity. It was further subcloned. All the resulting clones were screened for anti-progesterone activity and those that were negative were screened for anti-idiotypic activity. Clones that were positive for anti-idiotypic activity and anti-progesterone activity were expanded in vitro and in vivo. Detection of Anti-progesterone Antibodies by a Direct Binding Assay The radioimmunoassay (RIA) involved the method described by Fantl et al. (1981). Briefly, 100-pL aliquots of the culture supernatants were incubated with [3~]progesterone[ l o 000 disintegrations per minute (dpm)] dissolved in PBS containing 0.1% gelatin. Incubation was at 3 7 T for 20 min followed by 4OC for 20 min. An aliquot of 200 pL of dextran-coated charcoal (6.25 g activated charcoal and 0.625 g dextran per litre of steroid assay buffer) was added to each tube and the contents were mixed with a vortex mixer. This reaction mixture was incubated at 4OC for 20 min and the tubes were centrifuged at 700 g at 4OC for 10 min. The supernatant was decanted into vials containing 5 mL scintillation fluid and counted in a 6 counter (Kontron, Basel, Switzerland). Purifcation of Monoclonal Antibody to Progesterone Culture supernatants from E9D5 hybrid cells secreting anti-progesterone antibody were precipitated with a 50% saturated ammonium sulfate solution. The resultant precipitates were dissolved in PBS and dialysed overnight against PBS. Anti-progesterone antibody E9D5 has been well characterized (Khole and Hegde 1991). Detection of Anti-idiotypic Antibodies by Competitive Inhibition Assay The RIA described earlier was slightly modified. Culture supernatants (100 pL aliquots) from wells growing clones showing no anti-progesterone activity were added to [3~]progesterone(10 000 dpm) dissolved in PBS containing 0.1% gelatin and 100 pL of a suitably diluted (50-60% binding) antiprogesterone antibody E9D5. The reaction mixture was incubated at 37OC for 20 min and then at 4°C for 20 min. An aliquot of 200 pL of dextran coated charcoal was added to each tube and the contents were mixed with a vortex mixer. The reaction mixture was incubated at 4OC for 20 min; the tubes were then centrifuged at 700 g at 4'C for 10 min. The supernatant was decanted into vials containing 5 mL of scintillation fluid and counted in a 6 counter. Those clones that inhibited the binding of [3~]progesteroneto E9D5 were taken to be positive for anti-idiotypic antibody activity. Production of Ascites Fluid Balb/c mice primed with 0.5 mL FIA were injected 1-2 days later with an intraperitoneal inoculum of hybrid cells (1 x lo6). After 3 weeks 5 mL sterile 0.15 M NaCl solution was injected into the peritoneal cavity. Mice were allowed to rest for a few minutes before the fluid was withdrawn from
Anti-idiotypic MAb to Progesterone
the peritoneal cavity (Coll 1987) and assessed for antibody activity. Ascites fluids were recovered once a week and precipitated with 50% saturated ammonium sulfate, dialysed against PBS and stored until required.
Characterization of Monoclonal Antibody E9FIIF6 Effects of progesterone on the binding of E9FIIF6 to a polyclonal anti-progesterone antibody For enzyme linked immunosorbent assays (ELISA), 96-well polystyrene microtitre plates were coated with 100 pL polyclonal anti-progesterone antibody suitably diluted in 0.1 M sodium bicarbonate (pH 9.3) and were incubated at 4OC overnight. After being washed three times with PBS (0.1 M, pH 7.4) containing 0.1% Tween 20 (PBS-T2O) the plates were incubated with various concentrations of progesterone at room temperature (27-29°C) for 30 rnin and then with suitably diluted ammonium sulfate-precipitated E9FllF6 ascites fluid at 30°C for 2 h. After five washes with PBS-T20, rabbit anti-mouse immunoglobulin (Ig) coupled to HRP (1 : 1000) was added and the reaction mixture was incubated at room temperature for 90 min in the dark. The plates were then washed five times with PBS-T20, and 200 pL of substrate (8 mg 0-phenylene diamine dichloride in 10 mL of 0.1 M citrate phosphate buffer, pH 4.8, containing 50 pL of 30% HzOz) was added and the plates were incubated at 30°C for 15 min. The reaction was stopped by the addition of 100 pL 4 N H2S04 and the optical density of the reaction mixture was determined at 492 nm in a Titertek ELISA reader. The results are expressed as the means of duplicate points. The absorbance at 492 nm in the absence of any steroid is taken as 100% binding and the percentage of binding in the presence of steroid has been calculated. Inhibition of [3~]progesterone binding to murine monoclonal anti-progesterone antibody E9D5 by monoclonal anti-idiotypic antibody E9FIIF6 The inhibition of binding was assessed by RIA. Anti-progesterone monoclonal antibody E9D5 was incubated at 37'C for 30 min with [3~]progesterone in the presence of serial dilutions of anti-idiotypic monoclonal antibody E9F11F6. The reaction mixture was then incubated at 4'C for 20 min after which time 200 pL of dextran-coated charcoal was added. The reaction mixture was incubated for a further 20 min at 4°C and the tubes were then centrifuged at 700 g at 4OC for 10 min. The supernatants were decanted into vials containing 5 mL scintillation fluid and were counted in a fi counter. Dissociation kinetic studies of [3~]progesteroneantibody complexes with E9FIIF6 To explore the possibility that the inhibitory activity of the anti-idiotypic antibody was due to steric hindrance instead of a direct binding to the combining site, a chase experiment was done. Anti-progesterone antibody (Abl) was preincubated with [3~]progesteroneat 37OC for 30 min after which time Ab2 was added and the incubation continued at 37OC for 5, 10, 20, 40, 60, 80 or 100 min before binding was measured by RIA. Effect of increasing concentrations of [3~]progesteroneon the inhibition of its binding to idiotype (E9D5) by anti-idiotype (E9FIIF6) In these studies monoclonal anti-progesterone antibody E9D5 and anti-idiotypic antibody E9F11F6 were incubated together with increasing concentrations of [3~]progesterone at 37OC for 30 min followed by incubation at 4'C for 20 min. Dextran-coated charcoal (200 pL) was added and the incubation continued at 4OC for 20 min. The reaction mixture was centrifuged at 700 g at 4OC for 10 min. The supernatant was decanted into vials containing 5 mL scintillation fluid and was counted in a P counter. Indirect Zmmunofluorescence Preparation of fixed hybridoma cells Actively growing hybridomas secreting the anti-idiotypic antibody E9FllF6 were harvested, washed and resuspended in PBS (0.01 M, pH 7.4) at a cell density of 1 x lo7 cells per mL. Cells were fixed either with glutaraldehyde (0.025-0.2%) or with methanol. The reaction mixture was incubated at room temperature (26-30°C) for 30 min. Glycine was added to the glutaraldehyde-fixed cells to a final concentration of 0.5 M. After an additional incubation period a t room temperature for 10 min the cell suspension was centrifuged at 400 g for 10 min. The cell pellet was washed once with PBS, resuspended in PBS containing 0.1% sodium azide, and used for membrane labelling. In the first set of experiments the cells were incubated either with normal rabbit serum or with rabbit polyclonal anti-progesterone antibody, for 1 h at room temperature followed by three washes in PBS.
V. Khole and U. Hegde
These cells were then incubated with a 1 :50 dilution of goat anti-rabbit antibody conjugated to fluorescein isothiocyanate (GaR FITC conjugate) at room temperature for 1 h. Goat anti-rabbit-FITC conjugate was prepared in the laboratory by the method of Biggs et al. (1960). The cells were again washed three times in PBS and the slides were prepared for examination under a Zeiss fluorescent microscope. In the second set of experiments, the inhibition studies, polyclonal anti-progesterone antibody was preincubated with different concentrations of progesterone (200 pg, 400 pg and 2000 pg) at 37OC for 30 min. This preincubated mixture was then added to the fixed hybridoma cells, and the incubation continued at room temperature for 1 h. The cells were washed thrice with PBS and incubated with a 1 :50 dilution of GaR-FITC conjugate at room temperature for 1 h. The cells were washed three times with PBS and the slides were prepared for examination under a Zeiss fluorescent microscope. FITC fluorescence was assessed on a qualitative scale: -(negative); +(weakly positive); ++(positive); +++(strongly positive) and ++++(very strongly positive) by two independent observers.
Results Production and Characterization of Murine Monoclonal Antibodies Out of a total of 384 wells plated there were 30 visible hybrids of which only one (E9) was positive for anti-progesterone activity. This hybrid E9 was further subcloned and the resulting clones were screened for anti-progesterone activity. In this screening only one clone, E9D5, had a high titre of anti-progesterone antibodies. The remaining clones without any anti-progesterone activity were screened for anti-idiotypic activity. Of these one clone, E9Fl1, showed anti-idiotypic activity and was further subcloned. On subcloning, a clone E9FllF6 showed very high anti-idiotypic antibody activity and this antibody was used for these studies. Hybridoma cells secreting anti-idiotypic antibody, E9F11F6 were expanded in vivo by injecting the hybridoma cells into FIA-primed Balb/c mice. The method described by Coll (1987) for the recovery of ascites was superior, in terms of quantity of ascites fluid and frequency of recovery, to that described by Mueller et al. (1986). The monoclonal anti-idiotypic antibody E9FllF6 binds to rabbit anti-progesterone antibodies in a linear concentration-dependent manner (Fig. 1). Binding is inhibited by preincubation with progesterone (Fig. 2). These results suggest that E9FllF6 is an Ab2P anti-idiotypic antibody.
.,
Fig. 1. Binding of monoclonal anti-idiotypic antibody E9FllF6 to polyclonal antiprogesterone antibody: control ascites fluid; 0, monoclonal anti-idiotypic antibody E9Fl lF6.
Ascites fluid (reciprocal dilution) Anti-idiotypic antibody E9FllF6 did not bind to [3~]progesteronein a direct binding assay and therefore was not competing with anti-progesterone antibodies for binding to progesterone. On the contrary, E9FllF6 inhibited the binding of [3~]progesterone to antiprogesterone antibody E9D5 in a dose-dependent manner (Fig. 3). These results confirm that this antibody has the properties that one would expect in an antibody that contains an internal image of progesterone. Dissociation kinetic studies (Fig. 4), revealed that the inhibitory activity of anti-idiotypic antibody E9F11F6 was due not to steric hindrance but to its direct binding to the combining site of Abl. In the presence of anti-idiotypic antibody E9F11F6, [3~]progesterone binding
Anti-idiotypic MAb to Progesterone
decreased and reached a plateau of inhibition after 20 min. Acceleration of the dissociation of [3~]progesteronefrom Ag-Ab complexes clearly shows that the inhibition brought about by the anti-idiotypic antibody E9FllF6 is not due to steric hindrance.
Fig. 2. Effect of progesterone on the binding of anti-idiotypic antibody E9F11F6 to polyclonal antiprogesterone antibody.
Competitor concentration (logM)
80
Fig. 3. Inhibition of [3~]progesterone binding
..-
to monoclonal anti-progesterone antibody E9D5 by monoclonal anti-idiotypic antibody E9FllF6: a, control ascites fluid; 0, monoclonal anti-idiotypic antibody E9FllF6.
0
K
Ascites fluid (reciprocal dilution)
Fig. 4. Dissociation kinetic studies of [3~]progesterone antibody complexes with anti-idiotypic antibody E9FllF6: a, control ascites fluid; 0, monoclonal anti-idiotypic antibody E9F11F6.
0
20
40
60
Time (min)
80
400
V. Khole and U. Hegde
If progesterone and the anti-idiotypic antibody E9FllF6 bound to similar sites on the anti-progesterone antibody, then in accordance with the general rule of equilibrium they should compete reversibly with each other. For a fixed amount of anti-progesterone antibody in the presence of a constant concentration of anti-idiotypic antibody E9FllF6, increasing the concentration of ~ ~ ~ l ~ r o ~ e s tled e r to o na edecrease in the percentage inhibition (Fig. 5). For immunofluorescence studies the method of cell fixation is crucial. Gluteraldehyde (0-025%, 0.1% and 0.2%) did not give satisfactory results, possibly because of the destruction of antigenic sites, whereas fixation with methanol was satisfactory and hence was used throughout the study. The results of these studies are summarized in Table 1. No immunofluorescence was seen when normal rabbit serum was used. The intensity of fluorescence was at a maximum when the cells were incubated with anti-progesterone antibody alone. The intensity decreased as the concentration of progesterone was increased.
Fig. 5. Effect of increasing concentrations of [3~]progesteroneon the inhibition of its binding to idiotype (E9D5) by anti-idiotypic antibody E9Fl lF6.
- 40
-9
-8
[3H]Progesteroneconcentration log^)
Table 1. Immunofluorescence studies showing inhibition of interaction between idiotype (antiprogesterone antibody) and anti-idiotype (anti-idiotypic antibody) by progesterone GaR FITC, goat anti-rabbit antibody conjugated to fluorescein isothiocyanate; NRS, normal rabbit serum
Treatment
Intensity of fluorescence
Fixed cells + NRS Fixed cells + (anti-progesterone antibody) + GaR FITC Fixed cells + (anti-progesterone antibody + 200 pg cold progesterone) + GaR FITC Fixed cells + (anti-progesterone antibody 400 pg cold progesterone) + GaR FITC Fixed cells + (anti-progesterone antibody + 2000 pg cold progesterone) + GaR FITC
- (negative) ++++(very strongly positive)
+
+++ (strongly positive) ++ (positive) + (weakly positive)
Discussion The production of a monoclonal anti-idiotypic antibody to progesterone is described. The antibody was raised by the use of an auto-anti-idiotypic strategy that exploits the network theory of Jerne (1974). This approach used antigen exposure t o induce an Abl immune response in vivo, which in turn induces an auto-Ab2 immune response (Cleveland and Erlanger 1986). The immunogen used in the present study was a progesterone-BSA conjugate. The study showed that a single fusion can yield both anti-progesterone and antianti-progesterone monoclonal antibodies. In mice, immunization with insulin causes the spontaneous appearance of anti-idiotypic antibodies to insulin (Schechter et al. 1982). Similarly, auto-idiotypic antibody has been detected during a normal immune response to tetanus toxoid (Geha 1982). Lombes et al. (1989 have successfully used the auto-anti-idiotypic approach for raising anti-idiotypic antibodies to aldosterone. All these studies have shown that the idiotype network is functional during the normal immunization process. In fact, it has been shown that the idiotype network is functioning very actively as early as five days
Anti-idiotypic MAb to Progesterone
after the first booster injection (Binion and Rodkey 1982). The present study has shown that prolonged immunization results in the formation of anti-idiotypic antibody-forming clones and this is consistent with the cited investigations. As far as is known, this is the first report of the production of a monoclonal antiidiotypic antibody to progesterone. The production of a polyclonal anti-idiotypic antibody to progesterone has been reported earlier (Taussig et al. 1986). The polyclonal response is diverse, whereas the monoclonal antibody is homogenous, stable and available in large quantities. The monoclonal anti-idiotypic antibody (E9FllF6) bound to rabbit antiprogesterone antibody. This interaction was inhibited by progesterone, indicating that binding of the Ab2 antibody probably occurred at or close to the combining sites of the Abl antibody. The inhibition of [3~]progesteronebinding to the monoclonal anti-progesterone antibody E9D5 by the anti-idiotypic antibody E9Fl lF6 provides additional evidence for its anti-idiotypic nature. The results suggest that the monoclonal antibody E9Fl lF6 has ligandlike properties, contains the internal image of a progesterone epitope and can therefore be classified as an Ab2P anti-idiotypic antibody. In the chase experiments, dissociation of [3~]progesteronefrom the antigen-antibody complex was accelerated on addition of the anti-idiotypic antibody E9FllF6. Thus, the inhibitory activity of Ab2 was not due to steric hindrance but was a result of direct binding to the combining site. The results discussed show that both progesterone and the antiidiotypic antibody E9FllF6 bind to the same site on the Abl antibody. Additional evidence for this comes from experiments where an increase in the concentration of [3~]progesterone in a mixture containing a constant concentration of anti-progesterone antibody (E9D5) and anti-idiotypic antibody (E9Fl lF6) led to a decrease in the percentage of inhibition. Indirect immunofluorescence showed that the anti-idiotypic antibody E9FllF6 and progesterone used similar binding sites on the idiotype. In this study the anti-idiotypic antibodies were immobilized in their native form during active biosynthesis on their own native carriers. Several groups have shown the association of MAbs with their hybridoma cells (Sedgwick and Holt 1983). It is felt that such fixed cells could be used as a new solid phase for MAbs in immunoassays such as RIA and in a homogenous fluorescent immunoassay (Wang et al. 1986). The concept of antigenic mimicry was conceived as a stereo-chemical molecular mimicry but now it has been shown that anti-idiotypic antibodies can mimic any molecule in an organism and this mimicry does not necessarily require shared regions of common primary structures (Erlanger 1989). The present study shows that the immunoglobulin (anti-idiotypic antibody) E9FllF6 mimics progesterone in its reaction with the antibody. This function does not necessarily require an identical primary sequence. In our preliminary studies with the monoclonal antibody E9Fl lF6 it was encouraging to see that the anti-idiotypic antibody bound to the progesterone receptors from MCF-7, a breast cancer cell line, as well as to the chick oviduct progesterone receptors. Further studies along these lines are in progress. This antibody may, therefore, serve as an immunoreagent to purify progesterone receptors by affinity chromatography, and could be used as a probe for mapping their functional domains. It could also be used to study the localization and regulation of progesterone receptors in physiological situations and in the study of the changes in progesterone receptor that are associated with reproductive disfunctions. Another relevant application of this anti-idiotypic antibody would be to use it as a surrogate antigen to induce an anti-progesterone antibody response. Mirror-image antiidiotypic antibodies have been widely used as antigen mimics and as experimental vaccines (Zanetti et al. 1987; Dalgleisch and Kennedy 1988). However, in most of these studies, the anti-idiotypic antibodies were either of xenogeneic or allogeneic origin. Acknowledgment The technical assistance of Mrs Sudha Premchandran is gratefully acknowledged.
V. Khole and U. Hegde
References Biggs, J. L., Loh, P. C., and Eveland, W. G. (1960). A simple fraction method for preparation of fluorescein-labelled gamma globulin. Proc. Soc. Exp. Biol. (NY) 105, 655-8. Binion, S. B., and Rodkey, S. L. (1982). Naturally induced auto-anti-idiotypic antibodies. J. Exp. Med. 156, 860-72. Bona, C., and Kohler, H. (1984). Anti-idiotypic antibodies and internal images. In 'Monoclonal and Anti-idiotypic Antibodies: Probes for Receptor Structure and Function'. (Eds J . C. Venter, C. M. Eraser and J. Lindstrom.) pp. 141-50. (Alan R. Liss: New York.) Cleveland, L. W., and Erlanger, B. F. (1986). The auto-anti-idiotypic strategy for preparing monoclonal antibodies to receptor combining sites. In 'Methods in Enzymology'. (Eds J. L. Langone and H. V. Vunakis.) Vol. 121, Part I. pp 95- 106. (Academic Press: New York.) Coll, J. M. (1987). Injection of physiological saline facilitates recovery of ascitic fluids for monoclonal antibody production. J. Zmmunol. Methods 104, 219-22. Dalgleish, A. G., and Kennedy, R. C. (1988). Anti-idiotypic antibodies as immunogens: idiotypic based vaccines. Vaccine 6, 215-20. Erlanger, B. F. (1989). Some thoughts on the structural basis of internal imagery. Zmmunol. Today 10, 151-2. Fantl, V. E., Wang, D. Y., and Whitehead, A. S. (1981). Production and characterization of a monoclonal antibody to progesterone. J. Steroid Biochem. 14, 405-7. Geha, R. F. (1982). Presence of auto-anti-idiotypic antibody during the normal human immune response to tetanus toxoid antigen. J. Zmmunol. 129, 139-44. Hill, B. L., and Erlanger, B. F. (1988). Monoclonal antibodies to the thyrotropin receptor raised by an auto anti-idiotypic protocol and their relationship to monoclonal auto antibodies from Graves' patients. Endocrinology 122, 2840-50. Jerne, N. K. (1974). Toward a network theory of the immune system. Ann. Inst. Pasteur Zmmunol. (Paris) 125C, 373-89. Khole, V., and Hegde, U. C. (1991). Production and characterization of a monoclonal antibody to progesterone and its effect on fertility. Indian J. Exp. Biol. 34, 6-11. Kohler, G., and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (Lond.) 256, 495-7. Lombes, M., Edelman, I. S., and Erlanger, B. F. (1989). Internal image properties of a monoclonal auto-anti-idiotypic antibody and its binding to aldosterone receptor. J. Biol. Chem. 15, 263-86. Mueller, U. W., Haves, C. S., and Jones, W. R. (1986). Monoclonal antibody production by hybridoma growth in Freunds's adjuvant primed mice. J. Zmmunol. Methods 87, 193-6. Sedgwick, J. D., and Holt, P. G. (1983). A solid-phase immunoenzymatic technique for the enumeration of specific antibody secreting cells. J. Zmmunol. Methods 57, 301-9. Sege, K., and Peterson, P. A. (1978). Use of anti-idiotypic antibodies as cell-surface receptor probes. Proc. Natl Acad. Sci. USA 75, 2443-7. Shechter, K., Maron, R., Elias, D., and Cohen, I. R. (1982). Autoantibodies to insulin receptor spontaneously develop as anti-idiotypes in mice immunized with insulin. Science 216, 542-4. Taussig, M. J., Brown, N., Ellis, S., Holliman, A., Peat, D., Richardson, N., Heap, R. B., and Feinstein, A. (1986). Anti-idiotypic sera against monoclonal anti-progesterone antibodies: production in rabbits and rats and characterization of specificity. Immunology 58, 445-52. Wang, L., Feingers, J., Gorsky, Y., Calatano-Sherman, J., and Inbar, M. (1986). Monoclonal antibodies embedded in their hybridoma cells: an immunodiagnostic concept. Hybridoma 5, 237-42. Wassermann, N. H., Penn, A. S., Freimuth, P. I., Treplow, N., Wentzel, S., Cleveland, W. L., and Erlanger, B. F. (1982). Anti-idiotypic route to anti-acetylcholine receptor antibodies and experimental myasthenia gravis. Proc. Natl Acad. Sci. USA 79, 48 10-14. Zanetti, M., Serarz, E., and Salk, J. (1987). The immunology of new generation vaccines. Zmmunol. Today 8, 18-25.
Manuscript received 1 May 1990; revised and accepted 23 December 1991
Monoclonal Anti-idiotypic Antibody to Progesterone with Internal Image Properties
Vrinda Khole and Umashashi ~ e ~ d e * Institute for Research in Reproduction, J.M. Street, Parel, Bombay 400012, India. TO whom correspondence should be addressed.
A
Abstract An auto-anti-idiotypic approach was used to generate mouse monoclonal anti-idiotypic antibody E9FllF6 (Ab2) to progesterone. A conjugate of 4-pregnane 3,20 dione and bovine serum albumin was used as the immunogen. E9FllF6 bound to a rabbit anti-progesterone antibody in a linear concentration-dependent manner. It inhibited the binding of [3~]progesterone to a monoclonal anti-progesterone antibody, E9D5 (Abl); this inhibition was concentration dependent. Results from chase experiments showed that this inhibitory activity of Ab2 was not due to steric hindrance but was a result of direct binding to the ligand combining site. Indirect immunofluorescence studies also showed that Ab2 and progesterone bound to similar binding sites on Abl. Overall, the results suggest that E9F1 IF6 contains an internal image of at least part of the progesterone molecule, and that it can be classified as an Ab2P antibody. This anti-idiotypic antibody may be of value in further studies of endocrine and reproductive physiology.
Introduction An anti-idiotypic antibody (Ab2) is an antibody directed against the antigen-combining site of another antibody (Abl). These anti-idiotypic antibodies can have three different specificities depending on the idiotopes they recognize on Abl. Anti-idiotypes that mimic antigens are termed Ab2P antibodies and those that do not mimic antigens are Ab2a antibodies. Those of the third type, Ab2y, recognize idiotypic determinants located close to the binding site of Abl and interfere with the binding of antigen through steric hindrance (Bona and Kohler 1984). Of these, the Ab2P antibodies have been of considerable theoretical and practical interest because they carry the internal image of the antigen. Ab2P antiidiotypic antibodies have been extensively used in relation to diseases (Monroe and Green 1986). The most practical source of anti-idiotype would be from a hybridoma that is secreting the appropriate monoclonal antibody in large quantities. In this report we describe the production and characterization of a monoclonal anti-idiotypic antibody (Ab2P) that binds to the steroid binding region of anti-progesterone antibodies (Abl). This antibody was raised through an auto-anti-idiotypic approach (Cleveland and Erlanger 1986) using a progesterone derivative coupled to bovine serum albumin. The auto-anti-idiotypic strategy does not require prior purification of the receptor and has been successfully used in the preparation of polyclonal or monoclonal antibodies to several receptors including insulin, acetylcholine, thyrotropin and aldosterone (Sege and Peterson 1978; Wassermann et al. 1982; Hill and Erlanger 1988; Lombes et al. 1989). 1031-3613/92/O2O223$O5.00
V. Khole and U. Hegde
Materials and Methods Reagents were obtained as follows: [3~]progesterone(specific activity 96 Ci per mmol) from Amersham International, Amersham, UK; the progesterone conjugate and other steroids from either Steraloids, Wilton, USA or Sigma, St Louis, USA; RPMI 1640 and fetal calf serum (FCS) from Gibco Laboratories, New York, USA; polyethylene glycol (PEG) (mol. wt 1500) from the J. T. Baker Chemical Co., Phillipsburg, USA; and rabbit anti-mouse(1g)-HRP (horseradish peroxidase) conjugate from Dakopatts, Glostrop, Denmark.
Preparation of Monoclonal Antibodies Balb/c mice were hyperimmunized with 4-pregnane-3,20-dione (Sigma) conjugated to bovine serum albumin (Sigma). The antigen (30 pg) in 0.1 mL phosphate buffered saline (PBS 0.01 M, pH 7.4) was emulsified with 0 . 2 mL of Freund's complete adjuvant (FCA, Sigma) and injected subcutaneously into 2-3-month-old adult male mice at multiple sites. Booster injections were given subcutaneously (30 pg of progesterone conjugate emulsified in 0.2 mL Freund's incomplete adjuvant (FIA, Sigma) every 4 weeks. Mice were immunized over a period of more than 5 months. The final booster inoculation was given interaperitoneally as 30 pg of the conjugate in saline. Splenocytes of the hyperimmunized mice were fused with mouse myeloma (SP2/0) cells grown in RPMI 1640 containing 10% FCS. PEG (30%) was used as the chemical fusogen. The fusion was carried out by the method described by Kohler and Milstein (1975). The hybrid cells were plated out into 96-well plates in RPMI 1640 containing 20% FCS and hypoxanthine, aminopterin and thymidine (HAT). The supernatants from hybrids were screened for anti-progesterone activity. Only one hybrid (E9) was positive for antiprogesterone activity. It was further subcloned. All the resulting clones were screened for anti-progesterone activity and those that were negative were screened for anti-idiotypic activity. Clones that were positive for anti-idiotypic activity and anti-progesterone activity were expanded in vitro and in vivo. Detection of Anti-progesterone Antibodies by a Direct Binding Assay The radioimmunoassay (RIA) involved the method described by Fantl et al. (1981). Briefly, 100-pL aliquots of the culture supernatants were incubated with [3~]progesterone[ l o 000 disintegrations per minute (dpm)] dissolved in PBS containing 0.1% gelatin. Incubation was at 3 7 T for 20 min followed by 4OC for 20 min. An aliquot of 200 pL of dextran-coated charcoal (6.25 g activated charcoal and 0.625 g dextran per litre of steroid assay buffer) was added to each tube and the contents were mixed with a vortex mixer. This reaction mixture was incubated at 4OC for 20 min and the tubes were centrifuged at 700 g at 4OC for 10 min. The supernatant was decanted into vials containing 5 mL scintillation fluid and counted in a 6 counter (Kontron, Basel, Switzerland). Purifcation of Monoclonal Antibody to Progesterone Culture supernatants from E9D5 hybrid cells secreting anti-progesterone antibody were precipitated with a 50% saturated ammonium sulfate solution. The resultant precipitates were dissolved in PBS and dialysed overnight against PBS. Anti-progesterone antibody E9D5 has been well characterized (Khole and Hegde 1991). Detection of Anti-idiotypic Antibodies by Competitive Inhibition Assay The RIA described earlier was slightly modified. Culture supernatants (100 pL aliquots) from wells growing clones showing no anti-progesterone activity were added to [3~]progesterone(10 000 dpm) dissolved in PBS containing 0.1% gelatin and 100 pL of a suitably diluted (50-60% binding) antiprogesterone antibody E9D5. The reaction mixture was incubated at 37OC for 20 min and then at 4°C for 20 min. An aliquot of 200 pL of dextran coated charcoal was added to each tube and the contents were mixed with a vortex mixer. The reaction mixture was incubated at 4OC for 20 min; the tubes were then centrifuged at 700 g at 4'C for 10 min. The supernatant was decanted into vials containing 5 mL of scintillation fluid and counted in a 6 counter. Those clones that inhibited the binding of [3~]progesteroneto E9D5 were taken to be positive for anti-idiotypic antibody activity. Production of Ascites Fluid Balb/c mice primed with 0.5 mL FIA were injected 1-2 days later with an intraperitoneal inoculum of hybrid cells (1 x lo6). After 3 weeks 5 mL sterile 0.15 M NaCl solution was injected into the peritoneal cavity. Mice were allowed to rest for a few minutes before the fluid was withdrawn from
Anti-idiotypic MAb to Progesterone
the peritoneal cavity (Coll 1987) and assessed for antibody activity. Ascites fluids were recovered once a week and precipitated with 50% saturated ammonium sulfate, dialysed against PBS and stored until required.
Characterization of Monoclonal Antibody E9FIIF6 Effects of progesterone on the binding of E9FIIF6 to a polyclonal anti-progesterone antibody For enzyme linked immunosorbent assays (ELISA), 96-well polystyrene microtitre plates were coated with 100 pL polyclonal anti-progesterone antibody suitably diluted in 0.1 M sodium bicarbonate (pH 9.3) and were incubated at 4OC overnight. After being washed three times with PBS (0.1 M, pH 7.4) containing 0.1% Tween 20 (PBS-T2O) the plates were incubated with various concentrations of progesterone at room temperature (27-29°C) for 30 rnin and then with suitably diluted ammonium sulfate-precipitated E9FllF6 ascites fluid at 30°C for 2 h. After five washes with PBS-T20, rabbit anti-mouse immunoglobulin (Ig) coupled to HRP (1 : 1000) was added and the reaction mixture was incubated at room temperature for 90 min in the dark. The plates were then washed five times with PBS-T20, and 200 pL of substrate (8 mg 0-phenylene diamine dichloride in 10 mL of 0.1 M citrate phosphate buffer, pH 4.8, containing 50 pL of 30% HzOz) was added and the plates were incubated at 30°C for 15 min. The reaction was stopped by the addition of 100 pL 4 N H2S04 and the optical density of the reaction mixture was determined at 492 nm in a Titertek ELISA reader. The results are expressed as the means of duplicate points. The absorbance at 492 nm in the absence of any steroid is taken as 100% binding and the percentage of binding in the presence of steroid has been calculated. Inhibition of [3~]progesterone binding to murine monoclonal anti-progesterone antibody E9D5 by monoclonal anti-idiotypic antibody E9FIIF6 The inhibition of binding was assessed by RIA. Anti-progesterone monoclonal antibody E9D5 was incubated at 37'C for 30 min with [3~]progesterone in the presence of serial dilutions of anti-idiotypic monoclonal antibody E9F11F6. The reaction mixture was then incubated at 4'C for 20 min after which time 200 pL of dextran-coated charcoal was added. The reaction mixture was incubated for a further 20 min at 4°C and the tubes were then centrifuged at 700 g at 4OC for 10 min. The supernatants were decanted into vials containing 5 mL scintillation fluid and were counted in a fi counter. Dissociation kinetic studies of [3~]progesteroneantibody complexes with E9FIIF6 To explore the possibility that the inhibitory activity of the anti-idiotypic antibody was due to steric hindrance instead of a direct binding to the combining site, a chase experiment was done. Anti-progesterone antibody (Abl) was preincubated with [3~]progesteroneat 37OC for 30 min after which time Ab2 was added and the incubation continued at 37OC for 5, 10, 20, 40, 60, 80 or 100 min before binding was measured by RIA. Effect of increasing concentrations of [3~]progesteroneon the inhibition of its binding to idiotype (E9D5) by anti-idiotype (E9FIIF6) In these studies monoclonal anti-progesterone antibody E9D5 and anti-idiotypic antibody E9F11F6 were incubated together with increasing concentrations of [3~]progesterone at 37OC for 30 min followed by incubation at 4'C for 20 min. Dextran-coated charcoal (200 pL) was added and the incubation continued at 4OC for 20 min. The reaction mixture was centrifuged at 700 g at 4OC for 10 min. The supernatant was decanted into vials containing 5 mL scintillation fluid and was counted in a P counter. Indirect Zmmunofluorescence Preparation of fixed hybridoma cells Actively growing hybridomas secreting the anti-idiotypic antibody E9FllF6 were harvested, washed and resuspended in PBS (0.01 M, pH 7.4) at a cell density of 1 x lo7 cells per mL. Cells were fixed either with glutaraldehyde (0.025-0.2%) or with methanol. The reaction mixture was incubated at room temperature (26-30°C) for 30 min. Glycine was added to the glutaraldehyde-fixed cells to a final concentration of 0.5 M. After an additional incubation period a t room temperature for 10 min the cell suspension was centrifuged at 400 g for 10 min. The cell pellet was washed once with PBS, resuspended in PBS containing 0.1% sodium azide, and used for membrane labelling. In the first set of experiments the cells were incubated either with normal rabbit serum or with rabbit polyclonal anti-progesterone antibody, for 1 h at room temperature followed by three washes in PBS.
V. Khole and U. Hegde
These cells were then incubated with a 1 :50 dilution of goat anti-rabbit antibody conjugated to fluorescein isothiocyanate (GaR FITC conjugate) at room temperature for 1 h. Goat anti-rabbit-FITC conjugate was prepared in the laboratory by the method of Biggs et al. (1960). The cells were again washed three times in PBS and the slides were prepared for examination under a Zeiss fluorescent microscope. In the second set of experiments, the inhibition studies, polyclonal anti-progesterone antibody was preincubated with different concentrations of progesterone (200 pg, 400 pg and 2000 pg) at 37OC for 30 min. This preincubated mixture was then added to the fixed hybridoma cells, and the incubation continued at room temperature for 1 h. The cells were washed thrice with PBS and incubated with a 1 :50 dilution of GaR-FITC conjugate at room temperature for 1 h. The cells were washed three times with PBS and the slides were prepared for examination under a Zeiss fluorescent microscope. FITC fluorescence was assessed on a qualitative scale: -(negative); +(weakly positive); ++(positive); +++(strongly positive) and ++++(very strongly positive) by two independent observers.
Results Production and Characterization of Murine Monoclonal Antibodies Out of a total of 384 wells plated there were 30 visible hybrids of which only one (E9) was positive for anti-progesterone activity. This hybrid E9 was further subcloned and the resulting clones were screened for anti-progesterone activity. In this screening only one clone, E9D5, had a high titre of anti-progesterone antibodies. The remaining clones without any anti-progesterone activity were screened for anti-idiotypic activity. Of these one clone, E9Fl1, showed anti-idiotypic activity and was further subcloned. On subcloning, a clone E9FllF6 showed very high anti-idiotypic antibody activity and this antibody was used for these studies. Hybridoma cells secreting anti-idiotypic antibody, E9F11F6 were expanded in vivo by injecting the hybridoma cells into FIA-primed Balb/c mice. The method described by Coll (1987) for the recovery of ascites was superior, in terms of quantity of ascites fluid and frequency of recovery, to that described by Mueller et al. (1986). The monoclonal anti-idiotypic antibody E9FllF6 binds to rabbit anti-progesterone antibodies in a linear concentration-dependent manner (Fig. 1). Binding is inhibited by preincubation with progesterone (Fig. 2). These results suggest that E9FllF6 is an Ab2P anti-idiotypic antibody.
.,
Fig. 1. Binding of monoclonal anti-idiotypic antibody E9FllF6 to polyclonal antiprogesterone antibody: control ascites fluid; 0, monoclonal anti-idiotypic antibody E9Fl lF6.
Ascites fluid (reciprocal dilution) Anti-idiotypic antibody E9FllF6 did not bind to [3~]progesteronein a direct binding assay and therefore was not competing with anti-progesterone antibodies for binding to progesterone. On the contrary, E9FllF6 inhibited the binding of [3~]progesterone to antiprogesterone antibody E9D5 in a dose-dependent manner (Fig. 3). These results confirm that this antibody has the properties that one would expect in an antibody that contains an internal image of progesterone. Dissociation kinetic studies (Fig. 4), revealed that the inhibitory activity of anti-idiotypic antibody E9F11F6 was due not to steric hindrance but to its direct binding to the combining site of Abl. In the presence of anti-idiotypic antibody E9F11F6, [3~]progesterone binding
Anti-idiotypic MAb to Progesterone
decreased and reached a plateau of inhibition after 20 min. Acceleration of the dissociation of [3~]progesteronefrom Ag-Ab complexes clearly shows that the inhibition brought about by the anti-idiotypic antibody E9FllF6 is not due to steric hindrance.
Fig. 2. Effect of progesterone on the binding of anti-idiotypic antibody E9F11F6 to polyclonal antiprogesterone antibody.
Competitor concentration (logM)
80
Fig. 3. Inhibition of [3~]progesterone binding
..-
to monoclonal anti-progesterone antibody E9D5 by monoclonal anti-idiotypic antibody E9FllF6: a, control ascites fluid; 0, monoclonal anti-idiotypic antibody E9FllF6.
0
K
Ascites fluid (reciprocal dilution)
Fig. 4. Dissociation kinetic studies of [3~]progesterone antibody complexes with anti-idiotypic antibody E9FllF6: a, control ascites fluid; 0, monoclonal anti-idiotypic antibody E9F11F6.
0
20
40
60
Time (min)
80
400
V. Khole and U. Hegde
If progesterone and the anti-idiotypic antibody E9FllF6 bound to similar sites on the anti-progesterone antibody, then in accordance with the general rule of equilibrium they should compete reversibly with each other. For a fixed amount of anti-progesterone antibody in the presence of a constant concentration of anti-idiotypic antibody E9FllF6, increasing the concentration of ~ ~ ~ l ~ r o ~ e s tled e r to o na edecrease in the percentage inhibition (Fig. 5). For immunofluorescence studies the method of cell fixation is crucial. Gluteraldehyde (0-025%, 0.1% and 0.2%) did not give satisfactory results, possibly because of the destruction of antigenic sites, whereas fixation with methanol was satisfactory and hence was used throughout the study. The results of these studies are summarized in Table 1. No immunofluorescence was seen when normal rabbit serum was used. The intensity of fluorescence was at a maximum when the cells were incubated with anti-progesterone antibody alone. The intensity decreased as the concentration of progesterone was increased.
Fig. 5. Effect of increasing concentrations of [3~]progesteroneon the inhibition of its binding to idiotype (E9D5) by anti-idiotypic antibody E9Fl lF6.
- 40
-9
-8
[3H]Progesteroneconcentration log^)
Table 1. Immunofluorescence studies showing inhibition of interaction between idiotype (antiprogesterone antibody) and anti-idiotype (anti-idiotypic antibody) by progesterone GaR FITC, goat anti-rabbit antibody conjugated to fluorescein isothiocyanate; NRS, normal rabbit serum
Treatment
Intensity of fluorescence
Fixed cells + NRS Fixed cells + (anti-progesterone antibody) + GaR FITC Fixed cells + (anti-progesterone antibody + 200 pg cold progesterone) + GaR FITC Fixed cells + (anti-progesterone antibody 400 pg cold progesterone) + GaR FITC Fixed cells + (anti-progesterone antibody + 2000 pg cold progesterone) + GaR FITC
- (negative) ++++(very strongly positive)
+
+++ (strongly positive) ++ (positive) + (weakly positive)
Discussion The production of a monoclonal anti-idiotypic antibody to progesterone is described. The antibody was raised by the use of an auto-anti-idiotypic strategy that exploits the network theory of Jerne (1974). This approach used antigen exposure t o induce an Abl immune response in vivo, which in turn induces an auto-Ab2 immune response (Cleveland and Erlanger 1986). The immunogen used in the present study was a progesterone-BSA conjugate. The study showed that a single fusion can yield both anti-progesterone and antianti-progesterone monoclonal antibodies. In mice, immunization with insulin causes the spontaneous appearance of anti-idiotypic antibodies to insulin (Schechter et al. 1982). Similarly, auto-idiotypic antibody has been detected during a normal immune response to tetanus toxoid (Geha 1982). Lombes et al. (1989 have successfully used the auto-anti-idiotypic approach for raising anti-idiotypic antibodies to aldosterone. All these studies have shown that the idiotype network is functional during the normal immunization process. In fact, it has been shown that the idiotype network is functioning very actively as early as five days
Anti-idiotypic MAb to Progesterone
after the first booster injection (Binion and Rodkey 1982). The present study has shown that prolonged immunization results in the formation of anti-idiotypic antibody-forming clones and this is consistent with the cited investigations. As far as is known, this is the first report of the production of a monoclonal antiidiotypic antibody to progesterone. The production of a polyclonal anti-idiotypic antibody to progesterone has been reported earlier (Taussig et al. 1986). The polyclonal response is diverse, whereas the monoclonal antibody is homogenous, stable and available in large quantities. The monoclonal anti-idiotypic antibody (E9FllF6) bound to rabbit antiprogesterone antibody. This interaction was inhibited by progesterone, indicating that binding of the Ab2 antibody probably occurred at or close to the combining sites of the Abl antibody. The inhibition of [3~]progesteronebinding to the monoclonal anti-progesterone antibody E9D5 by the anti-idiotypic antibody E9Fl lF6 provides additional evidence for its anti-idiotypic nature. The results suggest that the monoclonal antibody E9Fl lF6 has ligandlike properties, contains the internal image of a progesterone epitope and can therefore be classified as an Ab2P anti-idiotypic antibody. In the chase experiments, dissociation of [3~]progesteronefrom the antigen-antibody complex was accelerated on addition of the anti-idiotypic antibody E9FllF6. Thus, the inhibitory activity of Ab2 was not due to steric hindrance but was a result of direct binding to the combining site. The results discussed show that both progesterone and the antiidiotypic antibody E9FllF6 bind to the same site on the Abl antibody. Additional evidence for this comes from experiments where an increase in the concentration of [3~]progesterone in a mixture containing a constant concentration of anti-progesterone antibody (E9D5) and anti-idiotypic antibody (E9Fl lF6) led to a decrease in the percentage of inhibition. Indirect immunofluorescence showed that the anti-idiotypic antibody E9FllF6 and progesterone used similar binding sites on the idiotype. In this study the anti-idiotypic antibodies were immobilized in their native form during active biosynthesis on their own native carriers. Several groups have shown the association of MAbs with their hybridoma cells (Sedgwick and Holt 1983). It is felt that such fixed cells could be used as a new solid phase for MAbs in immunoassays such as RIA and in a homogenous fluorescent immunoassay (Wang et al. 1986). The concept of antigenic mimicry was conceived as a stereo-chemical molecular mimicry but now it has been shown that anti-idiotypic antibodies can mimic any molecule in an organism and this mimicry does not necessarily require shared regions of common primary structures (Erlanger 1989). The present study shows that the immunoglobulin (anti-idiotypic antibody) E9FllF6 mimics progesterone in its reaction with the antibody. This function does not necessarily require an identical primary sequence. In our preliminary studies with the monoclonal antibody E9Fl lF6 it was encouraging to see that the anti-idiotypic antibody bound to the progesterone receptors from MCF-7, a breast cancer cell line, as well as to the chick oviduct progesterone receptors. Further studies along these lines are in progress. This antibody may, therefore, serve as an immunoreagent to purify progesterone receptors by affinity chromatography, and could be used as a probe for mapping their functional domains. It could also be used to study the localization and regulation of progesterone receptors in physiological situations and in the study of the changes in progesterone receptor that are associated with reproductive disfunctions. Another relevant application of this anti-idiotypic antibody would be to use it as a surrogate antigen to induce an anti-progesterone antibody response. Mirror-image antiidiotypic antibodies have been widely used as antigen mimics and as experimental vaccines (Zanetti et al. 1987; Dalgleisch and Kennedy 1988). However, in most of these studies, the anti-idiotypic antibodies were either of xenogeneic or allogeneic origin. Acknowledgment The technical assistance of Mrs Sudha Premchandran is gratefully acknowledged.
V. Khole and U. Hegde
References Biggs, J. L., Loh, P. C., and Eveland, W. G. (1960). A simple fraction method for preparation of fluorescein-labelled gamma globulin. Proc. Soc. Exp. Biol. (NY) 105, 655-8. Binion, S. B., and Rodkey, S. L. (1982). Naturally induced auto-anti-idiotypic antibodies. J. Exp. Med. 156, 860-72. Bona, C., and Kohler, H. (1984). Anti-idiotypic antibodies and internal images. In 'Monoclonal and Anti-idiotypic Antibodies: Probes for Receptor Structure and Function'. (Eds J . C. Venter, C. M. Eraser and J. Lindstrom.) pp. 141-50. (Alan R. Liss: New York.) Cleveland, L. W., and Erlanger, B. F. (1986). The auto-anti-idiotypic strategy for preparing monoclonal antibodies to receptor combining sites. In 'Methods in Enzymology'. (Eds J. L. Langone and H. V. Vunakis.) Vol. 121, Part I. pp 95- 106. (Academic Press: New York.) Coll, J. M. (1987). Injection of physiological saline facilitates recovery of ascitic fluids for monoclonal antibody production. J. Zmmunol. Methods 104, 219-22. Dalgleish, A. G., and Kennedy, R. C. (1988). Anti-idiotypic antibodies as immunogens: idiotypic based vaccines. Vaccine 6, 215-20. Erlanger, B. F. (1989). Some thoughts on the structural basis of internal imagery. Zmmunol. Today 10, 151-2. Fantl, V. E., Wang, D. Y., and Whitehead, A. S. (1981). Production and characterization of a monoclonal antibody to progesterone. J. Steroid Biochem. 14, 405-7. Geha, R. F. (1982). Presence of auto-anti-idiotypic antibody during the normal human immune response to tetanus toxoid antigen. J. Zmmunol. 129, 139-44. Hill, B. L., and Erlanger, B. F. (1988). Monoclonal antibodies to the thyrotropin receptor raised by an auto anti-idiotypic protocol and their relationship to monoclonal auto antibodies from Graves' patients. Endocrinology 122, 2840-50. Jerne, N. K. (1974). Toward a network theory of the immune system. Ann. Inst. Pasteur Zmmunol. (Paris) 125C, 373-89. Khole, V., and Hegde, U. C. (1991). Production and characterization of a monoclonal antibody to progesterone and its effect on fertility. Indian J. Exp. Biol. 34, 6-11. Kohler, G., and Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (Lond.) 256, 495-7. Lombes, M., Edelman, I. S., and Erlanger, B. F. (1989). Internal image properties of a monoclonal auto-anti-idiotypic antibody and its binding to aldosterone receptor. J. Biol. Chem. 15, 263-86. Mueller, U. W., Haves, C. S., and Jones, W. R. (1986). Monoclonal antibody production by hybridoma growth in Freunds's adjuvant primed mice. J. Zmmunol. Methods 87, 193-6. Sedgwick, J. D., and Holt, P. G. (1983). A solid-phase immunoenzymatic technique for the enumeration of specific antibody secreting cells. J. Zmmunol. Methods 57, 301-9. Sege, K., and Peterson, P. A. (1978). Use of anti-idiotypic antibodies as cell-surface receptor probes. Proc. Natl Acad. Sci. USA 75, 2443-7. Shechter, K., Maron, R., Elias, D., and Cohen, I. R. (1982). Autoantibodies to insulin receptor spontaneously develop as anti-idiotypes in mice immunized with insulin. Science 216, 542-4. Taussig, M. J., Brown, N., Ellis, S., Holliman, A., Peat, D., Richardson, N., Heap, R. B., and Feinstein, A. (1986). Anti-idiotypic sera against monoclonal anti-progesterone antibodies: production in rabbits and rats and characterization of specificity. Immunology 58, 445-52. Wang, L., Feingers, J., Gorsky, Y., Calatano-Sherman, J., and Inbar, M. (1986). Monoclonal antibodies embedded in their hybridoma cells: an immunodiagnostic concept. Hybridoma 5, 237-42. Wassermann, N. H., Penn, A. S., Freimuth, P. I., Treplow, N., Wentzel, S., Cleveland, W. L., and Erlanger, B. F. (1982). Anti-idiotypic route to anti-acetylcholine receptor antibodies and experimental myasthenia gravis. Proc. Natl Acad. Sci. USA 79, 48 10-14. Zanetti, M., Serarz, E., and Salk, J. (1987). The immunology of new generation vaccines. Zmmunol. Today 8, 18-25.
Manuscript received 1 May 1990; revised and accepted 23 December 1991
