Immunochemical studies of guinea pig progesterone-binding plasma protein.

0013-7227/78/1035-1678$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society

Vol. 103, No. f>

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Immunochemical Studies of Guinea Pig ProgesteroneBinding Plasma Protein* MARTINE PERROT AND EDWIN MILGROM Groupe de Recherches sur la Biochimie Endocrinienne et la Reproduction (INSERM U-135), Faculte de Medecine Paris Sud, 94270 Bicetre, France ABSTRACT. An antiserum specific for guinea pig progesterone-binding plasma protein (PBP) has been prepared. Using a very sensitive immunoenzymatic assay, PBP could be detected not only in pregnant guinea pig plasma (970 jug/ml of plasma at day 40-60 of pregnancy), but also in the plasma of fetuses (2.77 jug/ml), umbilical arteries (1.79 ^g/ml), umbilical vein (2.90 jug/ml), and in amniotic fluid (0.47 jug/ml). The protein was also found in low concentration in the plasma of nonpregnant females (2.10 jug/ml) and males (1.56 jug/ml).

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SPECIFIC progesterone-binding protein (PBP) has been described in the plasma of the pregnant guinea pig (1, 2). This protein has been purified (3-6), and its physiochemical (3-5) and steroid binding (2, 7-9) properties have been studied. However, very little is known concerning the biological aspects of its function. It has been previously thought that PBP is present only in the maternal circulation during pregnancy and absent in fetal plasma and in males or nonpregnant females (3). However, all of these studies were based on assays using the steroid binding characteristics of PBP. Thus, it remained possible that the protein had escaped detection because of low concentration or the presence of a "nonactive" (nonsteroid-binding) state. The species distribution of PBP has also been studied through its progesterone-binding properties: specific proteins binding progesterone have been described during pregnancy in various hystricomorphs (10), whereas they were not found in pregnant women or rat plasma (3). Structural and immunological similarities between plasma proteins and intracellular steReceived January 4, 1978. Address reprint requests to: Dr. Edwin Milgrom, Hopital de Bicetre, 78 rue du General Leclerc, 94270 KremlinBecetre, France. * This work was supported by INSERM and CNRS.

The antiserum was used to search for immunological similarities between various steroid-binding proteins. No cross reaction was found with cavian or human corticosteroid-binding globulin and human sex steroidbinding plasma protein. There was no cross reaction between guinea pig PBP and PBP of other pregnant hystricomorphs (viscacha, degu, and coypu). Moreover, no evidence was found of an interaction between guinea pig uterine progesterone receptor and the anti-PBP antiserum. (Endocrinology 103:1678, 1978)

roid-binding receptors have also been a subject of controversy (11-13). To answer these various questions it was necessary to prepare a specific antiserum to PBP in order to detect and quantitate the protein through properties different from those of binding steroids. Such an antibody could also be useful in studying the important and unresolved problems of the origin of PBP and of its distribution in different tissues and eventually within cells. Materials and Methods Materials Steroids. [l,2-3H]progesterone (New England Nuclear Corp., SA 47.9 Ci/mmol) was purified by alumina column chromatography with a benzene ethanol gradient (14). Its purity was periodically checked by silica gel thin layer chromatography (benzene 3, ethylacetate 2, vol/vol). Chromatographically pure unlabeled progesterone and cortisol were obtained from Roussel Uclaf. Buffers. Tris 0.01 M, KC10.11 M, HC1, pH 7.4 buffer (Tris-KCl buffer) was used in PBP purification from pregnant guinea pig plasma. Tris 0.01 M, NaCl 0.15 M, HC1, pH 7.4 buffer (Tris-NaCl buffer) was used in sucrose gradient experiments. Sodium phosphate 0.05 M, NaCl 0.2 M, pH 7.4 buffer (sodium phosphate buffer) was used in equilibrium dialysis studies. Sodium phosphate 0.01 M, NaCl 0.15 M, pH

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PROGESTERONE-BINDING PLASMA PROTEIN 7.4 buffer (phosphate-buffered saline or PBS) was used in the immunoenzymatic assay of PBP. Plasma and serum. Forty- to 60-day pregnant Hartley guinea pigs were anesthetized with ether, and blood was collected into heparinized vessels from the blood vessels of the neck. After centrifugation at 1,200 X g for 15 min, the plasma was separated and kept frozen at —20 C. Blood was collected from cord vessels by puncturing the umbilical vein and arteries. Blood was collected from fetuses by decapitation. Purification of PBP and corticosteroid-binding globulins (CBG). PBP was purified to homogeneity as previously described (3). Guinea pig and human CBG were purified to homogeneity by affinity chromatography by the method described by Le Gaillard et al. (15) for human CBG. Purified sex steroidbinding plasma protein was a gift from C. MercierBodaird and M. Renoir (16). Preparation of antisera Antisera to purified PBP. Male New Zealand rabbits (3 months old) were immunized according to Vaitukaitis (17). Pure PBP (300 jag/rabbit dissolved in 1 ml saline) was emulsified with 1 ml Freund's complete adjuvant (Calbiochem, La Jolla, CA) and dried tubercle bacilli (75 mg/rabbit) (Institut Pasteur, Paris). The solution was injected intradermally into 15-20 sites on the back of the rabbits. At successive 14 and 9-week intervals, booster injections (150 ng PBP in 1 ml Freund's Incomplete adjuvant) (Difco laboratories, MI) were given. Ten days later, blood was taken by puncture of the marginal ear vein. Most of the antisera showed two bands on immunoelectrophoresis when reacted against pregnant guinea pig serum: a main band corresponding to PBP and a slight contaminating band, which was also present with nonpregnant guinea pig plasma. To remove these contaminating antibodies, adsorption was performed with nonpregnant guinea pig plasma proteins previously fixed on polyacrylamide beads by the method of Temynck and Avrameas (18). The adsorbent contained 2.5 mg protein/ml, and adsorption was allowed to occur for 60 min at room temperature. Antisera to pregnant guinea pig plasma. These were prepared with a similar technique using 100 jug protein. However, no adsorption was performed. Goat antisera to nonpregnant guinea pig plasma proteins. These were obtained from Microbiological Associates (Bethesda, MD) Sheep immunoglobulin G fraction. IgG tagged with

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glucose oxidase of antisera prepared against rabbit y-globulins was obtained from Pasteur Production (Paris). Double immunodiffusion and immunoelectrophoresis Double immunodiffusion was performed as described by Ouchterlony (19) and immunoelectrophoresis according to Grabar (20) as modified by Scheidegger (21) using agarose 1.2% in 0.025 M barbital buffer, pH 8.6. The electrophoresis was run at 4 C for 120 min with 6 V/cm. Staining was performed with Coomassie Brillant Blue (22). Immunoenzymatic assay of PBP PBP was assayed by the immunoenzymatic technique of Guesdon and Avrameas (23). Plates were prepared with 10 ml agarose, 1.2% in phosphatebuffered saline, containing 1% of the anti-PBP antiserum. Radial diffusion of various concentrations of antigen was then performed for 48 h. After washing, the plates were incubated for 2 h with a sheep y-globulin fraction of antisera to rabbit y-globulin (5 ju,g/ml). These antibodies were tagged with glucose oxidase. After washing, the enzymatic activity was detected as previously described (24). Background staining was reduced by adding cobalt chloride (0.2 mg/ml) to the tetrazolium salt. The squared diameters of the stained precipitin ring were linearly proportional to the concentration of PBP. The standard concentrations of PBP were run on every immunoplate. Equilibrium dialysis and sucrose gradient experiments, protein assay, and radioactivity measurements were performed as previously described (3, 25).

Results Preparation of a specific antiserum against PBP A preparation of PBP which appeared to be homogenous by various biochemical criteria (3) was used to immunize the rabbits. This preparation was also pure by immunological criteria. In immunodiffusion and immunoelectrophoresis (Fig. 1) it gave a single precipitin line with an antiserum elicited against pregnant guinea pig plasma. However, the antiserum obtained with this preparation of PBP, when reacted against pregnant guinea pig plasma, displayed two precipitin lines: one due to PBP and a supplementary one which was

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FIG. 1. Immunoelectrophoretic control of the purity of PBP. Electrophoresis was performed on 2 jul PBP (5 mg/ml) (central well), 2 /xl pregnant guinea pig plasma (upper well) and 2 /nl nonpregnant guinea pig plasma (lower well). Rabbit antiserum elicited against pregnant guinea pig plasma (100 jul) was then added to the troughs and diffusion was performed for 12 h at 25 C and 24 h at 4C.

also present in nonpregnant guinea pig plasma. These contaminating antibodies were then removed by adsorption with nonpregnant guinea pig plasma (see Materials and Methods). The adsorbed antiserum was studied by double immunodifrusion: it yielded a single precipitin line of complete identity with pure PBP and pregnant guinea pig plasma (Fig. 2, top). There was no reaction with nonpregnant guinea pig plasma (up to a dilution of 1:2048). Immunoelectrophoresis of this antiserum with PBP or pregnant guinea pig plasma showed a single precipitin line in the a-globulin region (Fig. 2, bottom). No line was formed when nonpregnant guinea pig plasma was tested. Within the limits of sensitivity of these two methods, therefore, the adsorbed antiserum was specific for PBP. Study of serial dilutions of pregnant guinea pig plasma by the Ouchterlony technique with this antiserum indicated that PBP could be detected at a plasma dilution of 1:32 (not shown).

FIG. 2. Specificity of the anti-PBP antiserum. Top: Ouchterlony immunodiffusion. Twenty microliters of the following reagents were introduced into the wells: antiPBP antiserum (central well), pure PBP (100 Mg/ml) (wells 1 and 4), pregnant guinea pig plasma (wells 2 and 6), nonpregnant guinea pig plasma (well 3), and rabbit serum (well 5). Bottom: immunoelectrophoresis. AntiPBP antiserum (100 jul) was present in both troughs and reacted with pure PBP (1.5 mg/ml) (central well), nonpregnant guinea pig plasma (upper well), and pregnant guinea pig plasma (lower well).

Concentrations of PBP in pregnant females, fetuses, amniotic fluid, nonpregnant females, and males

FIG. 3. Immunoenzymatic assay of PBP. PBP, at various concentrations, was placed in the wells (8 /AI) of a plate containing anti-PBP antiserum. After diffusion, washing and staining (as described in Materials and Methods), the squared diameters (d2 X mm2) of immunoprecipitin rings were plotted against PBP concentration. Regression line: y = 3.57X + 21.09 (r = 0.97).

As shown in Fig. 3, the immunoenzymatic assay (23) was linear for PBP concentrations ranging from 0.8-16 jtig/ml. Parallel lines were obtained with pure PBP and pregnant guinea pig plasma which contains PBP. When the same pool of plasma was assayed in six different experiments a coefficient of interassay variation (26) of 4.9% was obtained. This assay was used to measure PBP in

various physiological situations. In the plasma of 40- to 60-day pregnant guinea pigs the concentration of PBP was 970 ± 4 jiig/ml (five determinations ± SEM). In fetal plasma the concentration of PBP was 2.77 ± 0.32 /xg/ml (n = 5), whereas it was 1.79 ± 0.40 jug/ml (n = 5) in the plasma of umbilical arteries, 2.90 ± 0.88 jug/ml (n = 5) in the plasma of umbilical


PROGESTERONE-BINDING PLASMA PROTEIN vein, and 0.47 ± 0.03 /xg/ml (n = 3) in amniotic fluid (1.2 mg protein/ml, concentrated to 24 mg/ml before the assay). In previous experiments (3), we have not been able to detect PBP in fetal plasma. However, dilutions comparable to those used for maternal plasma were then employed to study [ :t H]progesterone binding to fetal plasma. Because the immunological assay showed a 350fold difference in P B P concentration, equilibrium dialysis experiments were repeated using fetal plasma at markedly higher concentration (only 10-fold dilution). Under these conditions, binding of [ 'H]progesterone to a protein having a similar affinity to that of maternal P B P was observed (Fig. 4). Binding site concentrations were 65 nM in fetal plasma and 10,500 nM in maternal plasma. Thus PBP is present in fetal plasma but at a concentration 160- to 350-fold lower than in maternal plasma. When plasma from nonpregnant females or males was analyzed by the immunoenzymatic method, a positive reaction was observed but

FIG. 4. Binding of [:tH]progesterone to pregnant guinea pig plasma and to fetal plasma. Plasma was obtained from three pregnant guinea pigs and their fetuses. Pools were prepared and diluted 300-fold and 10-fold, respectively. Dialysis was performed on l-ml samples against various concentrations of [lH]progesterone in sodium phosphate buffer for 64 h at 4 C. B: bound hormone; U: unbound hormone; Scatchard plot (33); , Scatchard plot corrected for nonspecific binding according to Rosenthal (34). a, pregnant guinea pig plasma; b, fetal plasma. The equilibrium association constants (KA) were 1.1 X 10H M ' and 1.0 X IOH M ' for maternal and fetal plasma, respectively.

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at the very limit of sensitivity of the method. Pooled plasmas were therefore enriched about 2-fold in PBP by ammonium sulfate precipitation (50-80% saturation, the recovery was 67.4% ± 2.9, n = 5, see Ref. 3). PBP could then be precisely quantitated and values of 2.10 ± 0.13 jiig/ml (means of four samples ± SEM) and 1.56 ± 0.24 /xg/ml were obtained (after correction for recovery) for nonpregnant females and males, respectively. Attempts were made to confirm the presence of PBP in the plasma of males and nonpregnant females by showing high affinity binding of [3H]progesterone. Unfortunately, high concentrations of albumin did not allow the detection of such low concentrations of PBP in nondiluted or slightly diluted plasma. Comparison of the antigenic properties of steroid binding plasma proteins in various species CBG and sex steroid-binding plasma protein. CBG were purified to homogeneity from human and cavian plasma (15). They were tested by immunodiffusion and immunoelectrophoresis (Fig. 5, top) against anti-PBP antiserum. No precipitation was observed. The same negative result was observed with purified human sex steroid-binding plasma protein (Fig. 5, bottom). PBP from pregnant hystricomorphs. It has been shown by Illingworth and Heap (10) that

FIG. 5. Cross reactivity of various steroid-binding plasma proteins wth anti-PBP antiserum. Immunoelectrophoresis was performed with the following proteins: pure cavian CBG (2 mg/ml) (well 1), pure PBP (2 mg/ml) (wells 2 and 5), pure human CBG (2 mg/ml) (well 3), and pure SBP (0.5 mg/ml) (well 4). Anti-PBP antiserum was introduced into the troughs.


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during pregnancy various hystricomorphs exhibit plasma proteins that bind progesterone with high affinity and are similar to cavian PBP. We have studied the binding of [3H]progesterone to plasma proteins from viscacha, coypu, and degu using sucrose density gradient ultracentrifugation. The experiments were designed to detect proteins similar to PBP (i.e. present in pregnant animal plasma and having more affinity towards progesterone than towards cortisol). Plasmas from the pregnant guinea pig, viscacha, degu, and coypu were diluted 20-fold, incubated with either 10 nM [3H]progesterone or 10 nM [3H]progesterone plus 2 /XM unlabeled cortisol or progesterone, and submitted to ultracentrifugation through a sucrose gradient. In all cases A

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Rndo « 1378 Vol 103 • No 5

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FIG. 6. Binding of [^HJprogesterone to plasma proteins of pregnant hystricomorph rodents. Plasmas of pregnant guinea pig (A), viscacha (Lagostomus maximus) (B), coypu (Myocastor coypus) (C), and degu (Octodon degus) (D) were analyzed. Plasma was diluted 20-fold with Tris NaCl buffer, pH = 7.4, and incubated 3 h at 4 C with 10 nM [3H]progesterone (a) or with 10 nM [3H]progesterone plus a 200-fold excess of unlabeled progesterone (b) or cortisol (c). A 0.2-ml portion of each incubate was layered onto a 5-20% sucrose gradient and centrifuged 18 h at 45,000 rpm in a SW 50.1 rotor; 0.2 ml bovine serum albumin solution (10 mg/ml) was run as reference.

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FIG. 7. Cross reaction of hystricomorph PBP proteins with anti-PBP antiserum. Ouchterlony immunodiffusion was performed with anti-PBP antiserum (20 /xl) in the central wells and plasma (20 fil) obtained from the following animals: pregnant viscacha (well 1), pregnant guinea pig (wells 2 and 4), pregnant coypu (well 3), pregnant degu (well 5), nonpregnant viscacha (well 6), nonpregnant guinea pig (wells 7 and 9), nonpregnant coypu (well 8), and nonpregnant degu (well 10).


PROGESTERONE-BINDING PLASMA PROTEIN in the uteri of estrogen-primed ovariectomized guinea pigs), immunodiffusion, immunoelectrophoresis or even immunoenzymatic techniques could not be used. It was possible to study the receptor only by utilizing its steroidbinding properties. In a control experiment (Fig. 8A), diluted pregnant guinea pig plasma was incubated with [ 3 H]progesterone and then ultracentrifuged on a sucrose gradient. The radioactivity was associated with PBP in the 4.5S region. If the incubation mixture was treated with antiPBP antiserum, most of the bound radioactivity was either displaced to a higher S value or precipitated at the bottom of the tube. No

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displacement of the [3H]progesterone bound to PBP was observed when anti-PBP antiserum was replaced with preimmune serum. A parallel experiment was carried out with uterine cytosol containing progesterone receptor instead of plasma (Fig. 8B). [3H]Progesterone receptor complexes were observed in the 6-7S region (28). When anti-PBP antiserum was added, no displacement towards a higher S value and no precipitation of radioactivity was observed. The slight broadening of the peak of bound radioactivity towards the 4.5S region was due to some binding of [3H]progesterone to the albumin present in the anti-PBP antiserum. Addition of preimmune serum, of course, gave the same negative result as that of anti-PBP antiserum (not shown). Thus, under strictly parallel conditions, when care has been taken to have similar concentrations of PBP and receptor (receptor concentration was measured as previously described (29)), [3H]progesterone-PBP complexes interacted with anti-PBP antiserum, whereas [3H]progesterone-receptor complexes did not. Discussion

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FIG. 8. Comparison of the antigenic properties of PBP and of the uterine progesterone receptor. A, Pregnant guinea pig plasma was diluted 110-fold with Tris (0.01 M) EDTA (1.5 mM), HC1 (pH 7.4) buffer and incubated 2 h at 0 C with 2 nM ["^progesterone. One-tenth volume of anti-PBP anti-serum (a), preimmune serum (b), or buffer (c) was then added, and the incubation was resumed for 3 h at 0 C. Aliquots (200 jul containing 18 pmol PBP) were analyzed by a 5-20% sucrose gradient ultracentrifugation (16 h at 45,000 rpm in a SW 50.1 rotor). In exp d, incubation conditions were identical to exp a except that a 1,000-fold excess of unlabeled progesterone was added. B, Uterine cytosol was prepared (29) from guinea pigs ovariectomized 1-3 weeks before the experiment and treated with 5 fig estradiol/day for 3 days. It was incubated first with 2 nM [3H]progesterone then with anti-PBP antiserum (a) or buffer (c). Incubations d and e were identical to incubations a and c, except that 2 JUM unlabeled progesterone was also present. Incubation conditions were as in A. Aliquots (200 /txl containing 13.2 pmol of receptor and 4.4 mg protein) were analyzed by sucrose gradient ultracentrifugation as described in A. In all experiments (A and B) 1 jtM unlabeled cortisol was present in order to prevent [3H]progesterone binding to CBG. Thus saturable binding (compare a and d or c and e) in plasma could be ascribed to PBP and in uterine cytosol to receptor.

The preparation of PBP which was used for immunization of rabbits appeared to be homogenous by various biochemical and immunological criteria. However, antibodies were obtained not only against PBP but also against another plasma protein. The latter was probably present as a trace impurity in the PBP preparation but was strongly immunogenic. Since this contaminating protein was probably present in high concentration in nonpregnant guinea pig plasma where PBP was found in extremely low concentration, the antiserum could be made specific by adsorption to nonpregnant guinea pig serum. An immunoenzymatic assay for PBP was developed according to the method of Guesdon and Avrameas (23) in which 0.8 /xg/ml PBP (about 1/1000 of the concentration of PBP in pregnant guinea pig plasma) could be detected. With this sensitive assay it was possible to show, contrary to previous reports (3), that PBP was actually present in fetal plasma, but at a concentraction 160- to 350-fold lower than in maternal plasma. Using relatively con-


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centrated fetal plasma it was also shown that this PBP was able to bind progesterone with a nonmodified equilibrium dissociation constant. PBP was also found to be present in nonpregnant females and in males but at very low levels (about 1/1000 of that of pregnant guinea pig). Measurements of PBP by immunological techniques were in all cases parallel to measurments using its progesterone binding properties. Thus no evidence was obtained of the existence of an inactive (nonhormone binding) form of the protein. Comparative studies were performed to investigate the degree of immunological specificity of PBP with regard to some steroid binding proteins. Pure guinea pig CBG did not react immunochemically with the antiserum prepared against PBP. Thus, there was no evidence for any common structural properties in these two proteins of different hormonal specificity from the same species. Furthermore, neither human sex steroid-binding protein nor CBG reacted immunochemically with anti-PBP antiserum. Inasmuch as human CBG binds progesterone with high affinity (30), there was no evidence for any common antigenic determinants related to a similarity of function (i.e. binding of the same steroid). More surprisingly, plasma from pregnant hystricomorph rodents which contain progesterone binding proteins did not show immunoreactivity against the anti-PBP antiserum. Thus proteins similar to PBP by biochemical criteria are immunologically different from PBP. Similar absence of cross reaction was observed for CBG by Muldoon and Westphal (31) when plasma from the rabbit, rat, and guinea pig were tested with anti-human CBG antiserum. In contrast, antisera against human 25 - hydroxycholecalciferol - binding protein did cross react with dog, cat, horse, goat, sheep, cow, and monkey plasma (32). In our study, anti-PBP antiserum did not react with guinea pig uterine progesterone receptor. This result does not preclude the possibility of structural similarities between the two proteins. It is possible that differences in carbohydrates on the surface of these molecules are responsible for immunological differences.

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The specific antiserum which has been obtained against PBP may now be used to study the distribution of this protein in various tissues and eventually within cells. It will also become possible to try to determine the site of synthesis of PBP.

Acknowledgments We wish to thank Dr. J. Uriel (CNRS, Villejuif) for helpful discussions, Drs. R. B. Heap and N. Awkland (Agricultural Research Council, Cambridge) for the gift of plasma from viscacha, coypu, and degu. D. Saltarelli has helped in some immunological studies.

References 1. Milgrom, E., M. Atger, and E. E. Baulieu. Progesterone binding plasma protein (PBP). Nature 228: 1205, 1970. 2. Burton, R. M., G. B. Harding, N. Rust, and U. Westphal, Steroid protein interaction. XXIII Non identity of cortisol-binding globulin and progesterone-binding globulin in guinea pig serum. Steroids 17: 1, 1971. 3. Milgrom, E., P. Allouch, M. Atger, and E. E. Baulieu, Progesterone-binding plasma protein of pregnant guinea pig. Purification and characterization, J Biol Chem 248: 1106, 1973. 4. O. A. Lea, Isolation and characterization of a progesterone- and testosterone-binding globulin from pregnant guinea pig serum, Biochim Biophys Acta 317: 351, 1973. 5. Burton, R. M., G. B. Harding, W. R. Aboul-Hosn, D. T. Mac Laughlin, and U. Westphal, Progesterone binding globulin from the serum of pregnant guinea pigs, a polydisperse glycoprotein, Biochemistry 13: 3554, 1974. 6. Stroupe, S. D., and U. Westphal, Conformational changes in the progesterone binding globulin-progesterone complex, Biochemistry 14: 3296, 1975. 7. Lea, O. A., On the steroid specificity of the pregnant guinea pig progesterone-binding globulin, Biochim Biophys Acta 322: 68, 1973. 8. Kontula, K., O. Janne, E. Rajakoski, E. Tanhuanpaa, and R. Vihko, Ligand specificity of progesterone-binding proteins in guinea pig and sheep, J Steroid Biochem 5: 39, 1974. 9. Tan, S. Y., and B. E. P. Murphy, Specificity of the progesterone-binding globulin of the guinea pig. Endocrinology 94: 122, 1974. 10. Heap, R. B., and D. V. Illingworth, The maintenance of gestation in the guinea pig and hystricomorph rodents: changes in the dynamics of progesterone metabolism and the occurrence of progesterone binding globulin (PBG), Symp Zool Soc London 34: 385, 1974. 11. Mac Laughlin, D. T., and U. Westphal, Steroid-protein interaction. XXX. A progesterone-binding protein in the uterine cytosol of the pregnant guinea pig. Biochim Biophys Acta 365: 372, 1974.


PROGESTERONE-BINDING PLASMA PROTEIN 12. Uriel, J., D. Bouillon, C. Aussel, and M. Dupiers, Alpha-fetoprotein: the major high-affinity estrogen binder in rat uterine cytosols, Proc Natl Acad Sci USA 73: 1452, 1976. 13. Radanyi, C, C. Mercier-Bodard, C. Secco-Millet, E. E. Baulieu, and H. Richard-Foy, a-Fetoprotein is not a component of the estradiol receptor of the rat uterus, Proc Natl Acad Sci USA 74: 2269, 1977. 14. Lakshmanan, T. K., and S. Lieberman, Improved method of gradient elution chromatography and its application to separation of urinary ketosteroids, Arch Biochem 53: 258, 1954. 15. Le Gaillard, F., A. Racadot, N. Racadot-Leroy, and M. Dautrevaux, Isolement de la transcortine humaine par chromatographie d'affinite, Biochimie 56: 99, 1974. 16. Mercier-Bodard, C, J. M. Renoir, L. Fox, and E. E. Baulieu, Physicochemical properties of sex steroid binding plasma protein (SBP), Fifth International Congress on Endocrinology Hambourg July 18-25, 1976, Excerpta Medica, Amsterdam. 17. Vaitukaitis, J., J. B. Robbins, E. Nieschlag, and G. T. Ross, A method for producing specific antisera with small doses of immunogen, J Clin Endocrinol 33: 988, 1971. 18. Avrameas, S., and T. Ternynck, Biologically active water-insoluble protein polymers: their use for isolation of antigens and antibodies, J Biol Chem 242: 1651, 1967. 19. Ouchterlony, O., Antigen-antibody reactions in gels, Ark Kemi Mineral Geol 26B: no. 14, 1949. 20. Grabar, P., and C. A. William, Methode permettant l'etude conjuguee des proprietes electrophoretiques et immunochimiques d'un melange de proteines: application au serum sanguin, Biochim Biophys Ada 10: 193, 1953. 21. Scheidegger, J. J., Une micromethode de l'im- muno electrophorese, Int Arch Allergy Appl Immunol 7: 103, 1955. 22. Uriel, J., Analyse immunoelectrophoretique, Masson, Paris, 1960, p. 33. 23. Guesdon, J. L., and S. Avrameas, An immunoenzymatic method for measuring low concentrations of

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"Human guinea pig" asks for animal studies.

Immunochemical localization of aminopeptidase M in the alimentary tract of the guinea pig and rat.

Purification of plasma membrane of guinea pig peritoneal macrophages.

Further studies of immunoglobulin synthesis by guinea pig leukaemic lymphocytes.

Differential centrifugation studies of guinea pig lung proteases.

Pharmacological studies on CCKB receptors in guinea pig synaptoneurosomes.

Protein profiles of perilymph and endolymph of the guinea pig.

Immunochemical detection of the Thomsen-Friedenreich antigen (T-antigen) on the pig lymphocyte plasma membrane.

Nicotinamide mononucleotide adenylyltransferase, a nonhistone chromatin protein. Immunochemical comparative studies.

Constitutive protein secretion by guinea-pig seminal vesicle epithelial cells.

Immunochemical and immunohistochemical evidence of estrogen-mediated collagenolysis as a mechanism of cervical dilatation in the guinea pig at parturition.

Immunochemical studies on lens protein-protein complexes I. The heterogeneity and structure of complexed alpha-crystallin.

Characteristics of guinea pig IgE.

Guinea pig models of asthma.

Immunochemical studies of the interaction between myelin basic protein and S-100 protein.

Expression of heat shock protein, HSP72, in the guinea pig and rat cochlea after hyperthermia: immunochemical and in situ hybridization analysis.

Immunochemical studies on factor V.

Immunochemical heterogeneity of calcitonin in plasma.

Comparison of guinea pig cytomegalovirus and guinea pig herpes-like virus: growth characteristics and antigentic relationship.

Suppression of guinea pig ileum induced contractility by plasma albumin of hibernators.

Verapamil stimulates Ca(++)-uptake and Ca(++)-ATPase in plasma membrane vesicles of guinea pig spermatozoa.

Comparison of plasma, erythrocyte and brain lithium concentrations in the guinea-pig and rat [proceedings].

Guinea pig ciliary muscle development.

[Guinea-pig asthma (author's transl)].