[51] Assay of antibodies directed against cell surface receptors

656

ANTIBODIES IN HORMONE ACTION

[51]

[51] Assay o f Antibodies Directed against Cell

Surface Receptors

By

SIMEON I. TAYLOR, LISA H . U N D E R H I L L , and BERNICE MARCUS-SAMUELS

Antibodies directed against cell surface receptors for hormones and neurotransmitters play an important role in the pathogenesis of human disease (see the table)) -16 In some disease states, the interaction between the antibody and receptor may lead to biological effects identical to those of the natural ligand. In other conditions, anti-receptor antibodies may antagonize the bioactivity of the natural ligand. In addition to their role in pathogenesis of human disease, anti-receptor antibodies play a central role as reagents in studies of receptor structure and function. 4'13'i4'14a'17-19 1 M. Zakarjia and J. M. McKenzie, J. Clin. Endocrinol. Metab. 47, 249 (1978). 2 B. R. Smith, Recept. and Recognition, Ser. B 215 (1981). 3 M. Kishihara, Y. Nakao, Y. Baba, N. Kobayashi, S. Matsukura, K. Kuma, and T. Fujita, J. Clin. Endocrinol. Metab. 52, 665 (1981). 4 C. R. Kahn, K. L. Baird, J. S. Flier, and D. B. Jarrett, J. Clin. Invest. 60, 1094 (1977). 5 j. S. Flier, R. S. Bar, M. Muggeo, C. R. Kahn, J. Roth, and P. Gorden, J. Clin. Endocrinol. Metab. 47, 985 0978). 6 S. I. Taylor, G. Grunberger, B. Marcus-Samuels, L. H, Underhill, R. F. Dons, J. Ryan, R. F. Roddam, C. E. Rupe, and P. Gorden, N. Engl. J. Med. 307, 1422 (1982). 7 L. Tardella, L. Rossetti, R. DePirro, A. Camagna, S. Leonetti, G. Tamburrano, S. RossiFanelli, M. Merli, and R. Lauro, J. Clin. Lab. Immunol. 12, 159 (1983). 8 D. B. Drachman, C. W. Angus, R. N. Adams et al., N. Engl. J. Med. 298, l l l 6 (1978). 9 D. B. Drachman, R. N. Adams, L. F. Josifek, and S. G. Self, N. Engl. J. Med. 307, 769 (1982). l0 j. Lindstrom, Ciba Found. Syrup. 90, 178 (1982). N j. S. Flier, C. R. Kahn, D. B. Jarrett, and J. Roth, J. Clin. Invest. 58, 1442 (1976). 12 C. R. Kahn, J. S. Flier, R. S. Bar, J. A. Archer, P. Gorden, M. M. Martin, and J. Roth, N. Engl. J. Med. 294, 739 (1976). 13 F. A. Karlsson, E. Van Obberghen, C. Grunfeld, and C. R. Kahn, Proc. Natl. Acad. Sci. U.S.A. 76, 809 (1979). J4 C. Grunfeld, E. Van Obberghen, F. A. Karlsson, and C. R. Kahn, J. Clin. Invest. 66, 1124 (1980). J4a S. I. Taylor and B. Marcus-Samuels, J. Clin. Endocrinol. Metab. 58, 182 (1984). 15 j. C. Venter, C. M. Fraser, and L. C. Harrison, Science 207, 1361 (1980). ~6 V. Chiauzzi, S. Cigorraga, M. E. Escobar, M. A. Rivarola, and E. H. Charreau, J. Clin. Endocrinol. Metab. 54, 1221 (1982). 17 M. Kasuga, E. Van Obberghen, K. M. Yamada, and L. C. Harrison, Diabetes 30, 354 (1981). 18 E. Van Obberghen, M. Kasuga, A. Le Cam, J. A. Hedo, A. Itin, and L. C. Harrison, Proc. Natl. Acad. Sci. U.S.A. 78, 1052 (1981). 19 j. A. Hedo, M. Kasuga, E. Van Obberghen, J. Roth, and C. R. Kahn, Proc. Natl. Acad. Sci. U.S.A. 78, 4791 (1981).

METHODS IN ENZYMOLOGY,V()L. 109

Copyright (,3 1985by AcademicPress, Inc. All rightsof reproductionin any form reserved. ISBN 0-12-182009-2

[51]

657

ANTIBODIES AGAINST CELL SURFACE RECEPTORS ANTI-RECEPTOR ANTIBODIES IN HUMAN DISEASE

Disease Graves' disease Hypoglycemia Myasthenia gravis Type B extreme insulin resistance Asthma Hypergonadotropic amenorrhea

Bioeffect of anti-receptor antibody Mimics thyrotropin Mimics insulin Antagonizes acetylcholine Antagonizes insulin Antagonizes effects of epinephrine Antagonizes effects of folliclestimulating hormone

References 1-3 4-7 8-10 11-14a 15 16

Identification of anti-receptor antibody requires demonstration (1) that the antibody binds to a receptor and (2) that the same receptor binds to the ligand. These two properties can be demonstrated using several different assay techniques. No single assay method is perfect. Rather the different approaches complement one another in definitive identification of antireceptor antibodies. In this chapter, we will describe the three principal assay techniques which are employed to detect antibodies to the insulin receptor. Similar approaches may be applied to the detection of antibodies directed against other cell surface receptors.

Binding-Inhibition Assay Antibodies directed against the insulin receptor were first demonstrated using an assay based on the ability of the antibodies to inhibit insulin from binding to the receptor.ll Cells are incubated with antibody and then washed; subsequently, 125I-labeled insulin binding to the cells is assayed. This method continues to be the most useful technique for analyzing clinical samples because of its simplicity, specificity, and resistance to interference from other substances which may be present in the sera of insulin-treated patients (e.g., anti-insulin antibodies, insulin, etc.). All of the reported examples of human autoantibodies to the insulin receptor inhibit insulin binding although some anti-receptor antibodies raised by immunization of laboratory animals are not detectable using this technique.20, 21 20 S. Jacobs, K. Chang, and P. Cuatrecasas, Science 200, 1283 (1978). 21 F. C. Kull, Jr., S. Jacobs, Y. F. Su, and P. Cuatrecasas, Biochem. Biophys. Res. Commun. 106, 1019 (1982).

658

ANTIBODIES IN HORMONE ACTION

[5 1]

Reagents IM-9 lymphoblastoid cells: Supplied by American Type Tissue Collection (Rockville, Maryland). Cultivated in RPMI-1640 medium supplemented with 10% (v : v) fetal bovine serum Phosphate-buffered saline, pH 7.4 Binding assay buffer: NaCI, 120 mM; MgSO4, 1.2 mM; KCI, 2.5 mM; Na acetate, 15 mM; glucose, 10 mM; EDTA, 1 mM; HEPES, 50 mM; bovine serum albumin, 10 mg/ml; pH 7.8 lzSI-labeled insulin: Porcine insulin labeled with 1251to a specific activity of 100-150 Ci/g. 22'23 [125I]Monoiodoinsulin (receptor grade) supplied by New England Nuclear (Boston, MA) is also satisfactory Porcine insulin. Supplied by Elanco, Inc. (Indianapolis, IN). Stock solution (1 mg/ml) in 0.01 N HCI.

Procedure 1. Sediment cultured lymphocytes (IM-9 lymphoblastoid cells) by centrifugation (200 g, 10 min, room temperature). After washing the cells once with phosphate-buffered saline, resuspend them at a density of 10 7 cells/ml in binding assay buffer. 2. Place aliquots (0-0.05 ml) of the serum to be tested in plastic culture tubes (12 x 75 mm), set up in duplicate. Adjust the total volume in the tube to 0.05 ml using binding assay buffer. A range of dilutions of serum may be employed if it is desired to titer the antiserum. A parallel set of tubes with normal serum instead of test serum should be employed as a control. 3. Add aliquots (0.45 ml) of cells suspended in binding assay buffer to the tubes containing serum samples. Incubate the cells for 2 hr at room temperature with intermittent shaking to keep the cells suspended. After addition of 2 ml of binding assay buffer, sediment the cells by centrifugation (200 g, 10 min, room temperature). Aspirate and discard the supernatant fluid, taking care not to aspirate cells. 4. Add aliquots (0.5 ml) of assay buffer containing nSI-labeled insulin (-0. I ng/ml). To estimate nonspecific binding of 125I-labeled insulin, add unlabeled insulin (I0/xg/ml) to one of each pair of duplicate tubes. Incubate cells for 3 hr at 15° with intermittent shaking to keep the cells suspended. 5. At the end of the incubation, layer aliquots (0.2 ml) of cells over ice cold binding assay buffer (0.15 ml) contained in microcentrifugation tubes 22 p. Freychet, J. Roth, and D. M. Neville, Jr., Biochem. Biophys. Res. Commun. 43, 400 (1971). 23 j. Roth, this series, Vol. 37, p. 223.

[51]

659

A N T I B O D I E S AGAINST CELL SURFACE RECEPTORS

A REMISSION ,~ 1 0 0 X ~s

8o

z

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-- 1000

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FiG. 1. Identification of anti-receptor antibody in patient's serum. Serum samples were obtained from a patient with fasting hypoglycemia resulting from autoantibodies to the insulin receptor. 6 One sample was obtained at the time she manifested hypoglycemia; the other sample was obtained at the time when the antibody titer had fallen and the clinical symptoms had remitted. The serum samples were assayed using two techniques: binding inhibition (A) and immunoprecipitation (B).

(0.4 ml capacity). After sedimenting the cells by centrifugation (-10,000 g, 30 sec), aspirate, and discard the supernatant fluid. Excise the tip of the tube and measure cell-associated radioactivity in a gamma counter. 6. Calculate specific lZSI-labeled insulin binding by subtracting nonspecific binding from total binding. Plot specific ~25I-labeled insulin binding as a function of the dilution of antiserum (Fig. 1A). Comments

1. Ordinarily, we do not incubate cells in concentrations of serum exceeding 10%. So long as the concentration of serum is kept below 10%, preincubation in normal serum has little if any effect upon insulin binding. Plasma derived from heparinized blood or purified immunoglobulin fractions may be substituted for serum in these studies. However, it has been reported that heparin in sufficiently high concentrations may inhibit t25Ilabeled insulin binding to insulin receptors from some (but not all) tissues. TM 2. In the original description of this method, H the cells were washed three times with phosphate-buffered saline after preincubation with serum. This wash was intended to eliminate anti-insulin antibodies as well 23a K. Kriauciunas and C. R. K a h n , Clin. Res. 32, 401A ( a b s t r a c t ) (1984).

660

ANTIBODIES IN HORMONE ACTION

[51]

as insulin which might have been present in the serum sample. In most cases, our simplified washing procedure is sufficient. However, in an occasional sample, the titer of anti-insulin antibody is so high (e.g., detectable at a dilution of greater than 1 : 1000) that it is necessary to employ the original procedure for washing the cells. In addition, if the concentration of insulin in the incubation medium were to exceed 10-8 M, a more extensive washing procedure would be necessary. 24 3. We have never observed a major effect of anti-receptor antiserum upon nonspecific binding of insulin to IM-9 cells. Thus, nonspecific binding need not be determined at each concentration of serum. 4. This assay method will detect only those anti-receptor antibodies which inhibit 125I-labeled insulin binding to the receptor. If anti-receptor antibodies bound to the receptor without inhibiting 125I-labeled insulin binding, they would not be detected in this assay. While the latter class of anti-receptor antibodies has not been definitively demonstrated in clinical samples, this type of antibody has been raised by immunizing animals with solubilized insulin receptors. 2°,21 Immunoprecipitation Assay The major application of the immunoprecipitation technique is to detect anti-receptor antibodies which do not inhibit insulin binding. The assay as originally described 25 was based upon the ability of anti-receptor antibodies to immunoprecipitate ~25I-labeled insulin bound to receptor. Unfortunately, anti-insulin antibodies can interfere in this assay because of their ability to immunoprecipitate ~25I-labeled insulin directly. Thus, the presence of anti-insulin antibodies in the majority of insulin-treated patients greatly restricts the utility of this assay in analyzing clinical samples. In recent years, other methods have been devised for labeling the receptor either biosynthetically or by covalent modification. ~7-19,26,27 These labeling methods combined with the immunoprecipitation technique have greatly expanded our understanding of receptor structure. However, because the methods are relatively cumbersome and insensitive, they have not been utilized as a routine method for the assay of antireceptor antibodies. 24 F. C. Kosmakos and J. Roth, J. Biol. Chem. 255, 9860 (1980). 25 L. C. Harrison, J. S. Flier, J. Roth, F. A. Karlsson, and C. R. Kahn, J. Clin. Endocrinol. Metab. 48, 59 (1979). 26 p. F. Pilch and M. Czech, J. Biol. Chem. 254, 3375 (1979). 27 M. Kasuga, C. R. Kahn, J. A. Hedo, E. Van Obberghen, and K. M. Yamada, Proc. Natl. Acad. Sci. U.S.A. 78, 6917 (1981).

[51]

ANTIBODIES AGAINST CELL SURFACE RECEPTORS

661

Reagents Triton X-100, 20% (v : v) Buffer A: NaCI, 150 mM; HEPES, 50 mM; pH 7.8 Buffer B: Sucrose, 250 mM; HEPES, 50 raM; pH 7.6 Buffer C: HEPES, 50 mM, pH 7.6; bacitracin, 100 U/ml; aprotinin, 1 TIU/ml; phenylmethylsulfonyl fluoride, 1 mM ~25I-labeled insulin: Porcine insulin labeled with ~zsIto a specific activity of 100-150 Ci/g. 22'23 [125I]Monoiodoinsulin (receptor grade) supplied by New England Nuclear (Boston, MA) is also satisfactory. Porcine insulin: Supplied by Elanco, Inc. (Indianapolis, IN) Sheep anti-human y-globulin antiserum: Supplied by Miles Laboratories, (Elkhart, Indiana) Triton extract of human placental membranes: Prepared as described below.

Preparation of Solubilized Insulin Receptors Preparation of Placental Membranes (A ) I. A fresh human placenta (within 4 hr postpartum) is rinsed in ice cold buffer B. Trim away and discard large blood vessels as well as amniotic and chorionic membranes. Rinse until most of blood is washed away. 2. Homogenize the tissue at 4° (3 × 30 sec in a Waring blender or similar homogenizer) in 1.5 volumes of ice cold buffer B supplemented with bacitracin (100 U/ml), aprotinin (I TIU/ml), and phenylmethylsulfonyl fluoride (1 mM). 3. Centrifuge the homogenate (600 g for 10 rain at 4°). Save the supernatant fluid and resuspend the pellet in one-half the original volume of buffer. Repeat centrifugation step. 4. Discard the pellets and centrifuge (12,000 g, 30 rain, 4°) the combined supernatants from step 3. 5. Discard pellet and add NaC1 (5.8 mg/ml) and MgSO4 (0.05 mg/ml) to the supernatant medium (final concentrations, 100 mM NaCl and 0.2 mM MgSO4). 6. Sediment placental membranes by centrifugation (40,000 g, 40 min, 4°). Discard the supernatant and wash the pellet two times with buffer C until the supernatant fluid is colorless.

Solubilization of Placental Insulin Receptors (B) 1. Suspend placental membranes at a protein concentration of 3-10 mg/ml in ice cold buffer C.

662

A N T I B O D I E S IN H O R M O N E A C T I O N

[51]

2. Add 0.05 volumes of ice cold Triton X-100 (20%, v : v) to give a final concentration of I% (v : v) detergent. Incubate for 2 hr at 4 ° with stirring. 3. Clarify the extract by centrifugation (200,000 g, 90 min, 4°). 4. Discard the pellet and the lipid layer floating on top. Save the clear red-brown infranatant which contains the solubilized insulin receptors. 5. The solubilized insulin receptors may be stored in aliquots at - 7 0 ° for a period of 6 months. Procedure

1. Distribute aliquots (0.16 ml) of buffer A containing 125I-labeled insulin (0.1 ng/ml) among microcentrifugation tubes (1.5 ml capacity). Add the Triton-solubilized extract (0.0 ml) of human placental membranes containing solubilized insulin receptors to one set of tubes (series A). In a paired set of tubes (series B), substitute a solution of Triton X-100 (1%, v : v in buffer C) for the placental extract. Incubate the tubes overnight at 4 °. 2. Add serum samples (0.02 ml) to paired tubes (one from series A and one from series B). To titer the antiserum, employ varying dilutions of serum diluted in normal human serum. Use normal human serum as a control to assay for "nonimmune" precipitation. Extend the incubation for an additional 6-8 hr at 4°. 3. Add an appropriate volume (vide infra) of sheep anti-human y-globulin containing Triton X-100 (0.1%, v" v) to each tube and continue the incubation for an additional 16-18 hr at 4 °. 4. Sediment immune complexes by centrifugation (I0,000 g for 5 min at 4°). Aspirate and discard the supernatant fluid. Excise the tips of the tubes and measure the radioactivity in the immunoprecipitate using a gamma counter. 5. Plot specifically immunoprecipitated 125I-labeled insulin as a function of the dilution of antiserum (Fig. 1B). Comments

1. This assay will detect anti-receptor antibodies which bind to the receptor in such a way that the antibodies do not inhibit binding of 1251labeled insulin to the receptor. The assay can also detect anti-receptor antibodies which do inhibit 125I-labeled insulin binding. However, the property of the latter class of anti-receptor antibodies to inhibit 125I-labeled insulin binding does interfere with this assay. To the extent that an anti-receptor antibody inhibits 125I-labeled insulin binding to the receptor, the antibody may preferentially immunoprecipitate unoccupied receptors.

[51]

ANTIBODIES AGAINST CELL SURFACE RECEPTORS

663

2. 125I-labeled insulin which is immunoprecipitated in the absence of insulin receptors (series B) represents 125I-insulin which has been immunoprecipitated by anti-insulin antibodies. In the presence of insulin receptors (series A), 125I-labeled insulin may be immunoprecipitated either directly by anti-insulin antibodies or indirectly by anti-receptor antibodies. 3. It is possible to modify this technique to minimize the interference by anti-insulin antibodies. This is accomplished by a two step assay. In the first step, insulin receptors are immunoprecipitated with anti-receptor antiserum plus protein A (Pansorbin; Calbiochem). In the second step, the insulin receptors remaining in the supernatant may be measured by a technique utilizing precipitation with polyethylene glycol to separate bound from free ~25I-labeled insulin. 28,29 4. The concentration of sheep anti-human y-globulin must be chosen so as to give complete immunoprecipitation of the immunoglobulin contained in 0.02 ml of human serum.

Insulin-Like Bioactivity of Anti-Receptor Antibodies Autoantibodies to the TSH receptor were the first recognized examples of an anti-receptor antibody. 1-3 These autoantibodies were identified by virtue of their bioactivity as "long-acting thyroid stimulators." In much the same fashion, autoantibodies to the insulin receptor mimic the bioactivity of insulin both in vitro and in v i v o . 4-7 Despite the biological importance of the insulin-like bioactivity of anti-receptor antibodies, the technical complexity of this bioassay has interfered with the routine use of this method to assay for anti-receptor antibodies. Two other considerations also limit the utility of this assay method. First, hormonal effects may be mimicked by antibodies directed against components other than the receptor. For example, antibodies directed against intrinsic proteins of adipocyte plasma membranes may have insulinomimetic effects upon isolated fat cells. 3° Second, some anti-receptor antibodies may fail to mimic the bioactivity of the natural ligand. For example, Roth et al. 31 have obtained a monoclonal antibody which binds to the insulin receptor but fails to mimic insulin action. 28 S. I. Taylor, in "Regulation of Target Cell Responsiveness" (K. W. McKerns, ed.), p. 133. Plenum, New York, 1984. 29 p. Cuatrecasas, Proc. Natl. Acad. Sci. U.S.A. 69, 318 (1982). 3o D. J. Pillion and M. P. Czech, J. Biol. Chem. 253, 3761 (1978). 3~ R. A. Roth, D. J. Cassell, K. Y. Wong, B. A. Maddux, and I. D. Goldfine, Proc. Natl. Acad. Sci. U.S.A. 79, 7312 (1982).

664

ANTIBODIES IN HORMONE ACTION

[51]

Reagents Bovine serum albumin: Supplied by Reheis Biochemical Department (Kankakee, IL). Must be tested and shown to be free of insulin-like bioactivity Collagenase (type I). Supplied by Worthington Biochemical Corporation (Freehold, N J). Must be tested and shown to succeed in producing dispersed adipocytes which retain insulin responsiveness Stock solution I: In 300 ml of water, dissolve NaCI (35.04 g), KH2PO4 (2.73 g), and M g S O 4 ( | . 2 3 g). Separately dissolve CaC12 (0.55 g) in 100 ml of water. Combine the two solutions and adjust the total volume to 500 ml Stock solution II: Dissolve NaHCO3 (4.2 g) in 500 ml of water Stock solution III: Dissolve HEPES (37.75 g) in 500 ml of water. Adjust to pH 7.4 with NaOH Glucose (10 mg/ml): Make up fresh daily Krebs-Ringer bicarbonate buffer (KRBHA): Combine 100 ml of water, 20 ml each of stock solutions I-III, and 1 ml of glucose solution. Dissolve bovine serum albumin (2 g). Adjust to pH 7.4 and adjust total volume to 200 ml. Make up fresh daily [3-3H]Glucose: Specific activity, 10-20 Ci/mmol. Supplied by New England Nuclear (Boston, MA) Econofluor. Supplied by New England Nuclear (Boston, MA)

Procedure 1. Prepare isolated adipocytes by collagenase digestion of epididymal fat pads from 3 male rats (-80-120 g) according to the method of Rodbell. 32 Resuspend cells in 50 ml of KRBHA at 37°. 2. Place aliquots (0.1 ml) of either the serum to be tested or normal human serum into 12 x 75-mm plastic tubes. To titer the serum, employ varying dilutions of the serum to be tested (diluted in normal human serum). Aliquots (0.9 ml) of suspended adipocytes are added to each tube. (Take great care to stir the cells so as to maintain a uniform cell suspension.) Incubate the cells at 37° for 15 min. 3. Separate the cells from the medium by centrifugation. Aspirate and discard the infranatant medium. Wash the cells three times with KRBHA (1 ml) at 37 °. Take great care not to aspirate the cells. In practice, it may be necessary to leave a small volume of medium in order to avoid aspirating cells. 4. Resuspend the cells in K R B H A (1 ml) at 37 °. Transfer the resus32 M. Rodbell, J. Biol. Chem. 239, 375 (1964).

[51]

ANTIBODIES AGAINST CELL SURFACE RECEPTORS

100

665

IN SULIN -..-~,~. ~

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ANTI-RECEPTOR ANTIBODY (H) (C.-..-O)

(dilution)

FIG. 2. Bioassay of insulin-like activity of anti-receptor antibody, lmmunoglobulin G was purified from the same samples of serum that were utilized in Fig. 1. Immunoglobulin G obtained from the patient during the time she was hypoglycemic stimulated lipogenesis by isolated adipocytes. In contrast, this insulin-like bioactivity was markedly decreased at the time her clinical symptoms had remitted. For comparison, a bioassay curve for porcine insulin is also included.

pended cells to 20 ml plastic scintillation vials containing [3-3H]glucose (0. ! /zCi). Incubate at 37 ° for 2 hr with shaking. 5. Terminate the incubation by addition of scintillation fluid (Econofluor). Cap vials tightly and shake vigorously. Allow vials to incubate 4-16 hr at room temperature to complete the extraction of 3H-labeled lipid into the organic phase. The "blank" is determined by addition of scintillation fluid to two vials at zero time. Quantitate the 3H in the lipid using a scintillation counter. The results of a representative bioassay are shown in Fig. 2. Comments

1. Ordinarily, we carry out a second bioassay in parallel in which adipocytes, preincubated with normal human serum, are subsequently incubated with varying concentrations of porcine insulin (0.03-10.0 ng/ml).

666

ANTIBODIES IN HORMONE ACTION

[51]

This control serves two functions: first, to verify that the adipocytes respond to insulin; second, to allow for quantitation of the bioactivity of anti-receptor antibody relative to the bioactivity of insulin. 2. It is important to use a scintillation fluid which is immiscible with water so that the labeled lipids will be extracted into the scintillation fluid which serves as an organic phase; the [3-3H]glucose remains in the aqueous phase. 33 Scintillation fluids which accept aqueous solutions will not be satisfactory in this method. 3. We have employed the ability of insulin to stimulate lipogenesis in rat adipocytes as a bioassay for insulin-like activity. This particular bioassay has many advantages including simplicity and sensitivity. Other bioassays using either adipocytes 4 or other systems 13,14,34 have been employed successfully. 4. Some samples of serum or plasma may agglutinate adipocytes. This agglutination may be minimized by dilution of the sample. Of course, the need to dilute the sample decreases the sensitivity of the assay to detect anti-receptor antibodies. 5. A "nonspecific" effect of human immunoglobulin to stimulate lipogenesis in rat adipocytes has been described. 35-37However, this "nonspecific" effect of immunoglobulin may be differentiated from the "specific" effect of antibodies tO the insulin receptor: a. Difference in assay methodology. In the original report by Kahn et al., 4 a two-step assay technique was employed (similar to the one described above). Cells were first exposed to antisera. Then after the cells were washed, the in vitro bioassay was carried out. The wash procedure appeared to remove the majority of insulin-like factors in serum with the exception of those antibody molecules which were tightly bound to the cell surface. Using this method, Kahn et al. 4 did not detect insulin-like bioactivity in normal human serum or in serum from patients with a variety of diseases. The studies which have reported an insulin-like bioactivity of normal human immunoglobulin G have employed a one-step assay in which the cells were not washed prior to bioassay. b. Specificity ofbioactivity. In the case of the "nonspecific" bioactivity of human immunoglobulin G, the insulin-like activity is proportional to the concentration of immunoglobulin G. 35-37In contrast, with anti-receptor antibodies, the bioactivity is not related simply to the immunoglobulin 33 A. J. Moody, M. A. Stan, M. Stan, and J. Gliernann, Horm. Metab. Res. 6, 12 (1974). 34 y . Le Marchand-Brustel, P. Gorden, J. Flier, C. R. Kahn, and P. Freychet, Diabetologia 14, 311 (1978). 35 M. A. Khokher, L. G. Coulston, and P. Dandona, Diabetologia 21, 290 (1981). 36 M. A. Khokher, P. Dandona, L. G. Coulston, and S. Janah, Diabetes 12, 1068 (1981). 37 M. A. Khokher and P. Dandona, J. Clin. Endocrinol. Metab. 56, 393 (1983).

[52]

PROLACTIN RECEPTOR ANTIBODIES

667

G concentration. Rather, the bioactivity is related (albeit in a complex way) to the concentration of anti-receptor antibody. 14 Thus, when patients with anti-receptor antibodies enter clinical remission, this is associated with a marked fall in the antireceptor antibody titer (e.g., see Fig. 1) as well as marked fall in the insulin-like activity of the immunoglobulin G fraction (e.g., see Fig. 2). c. Effect of enzymatic digestion of antibody molecules. When antireceptor antibodies are digested with pepsin, the insulin-like bioactivity is associated with the bivalent F(ab)2 moiety; the Fc moiety is devoid of bioactivity. 4,38 In contrast, Khoker and Dandona 37 found the "nonspecific" bioactivity of human immunoglobulin G to be associated with the Fc rather than the F(ab)2 fragment. 38 C. R. Kahn, K. L. Baird, D. B. Jarrett, and J. S. Flier, Proc. Natl. Acad. Sci. U.S.A. 75, 4209 (1978).

[52] C h a r a c t e r i z a t i o n o f A n t i s e r a to P r o l a c t i n R e c e p t o r s

By PAUL A. KELLY, MASAO KATOH, JEAN DJIANE, LOUIS-MARIE HOUDEBINE, and ISABELLE DUSANTER-FOURT The action of prolactin, as is true for other peptide hormones, is mediated by a specific receptor located in membrane components of the cell. The PRL receptor has been well characterized and has been shown to respond to both a down- and an up-regulation by the hormone. 1-4 This receptor was partially purified 5 and an antibody against the partially purified receptor was obtained several years ago. 6 Antibodies to the PRL receptor have been shown to inhibit the capacity of prolactin to support the synthesis of casein and the uptake of a-isoaminobutyric acid by rabbit mammary explants in culture 7 and to attenuate the action of prolactin on 1 j. Djiane and P. Durand, Nature (London) 266, 641 (1977). 2 p. A. Kelly, L. Ferland, and F. Labrie, in "Progress on Prolactin Physiology and Pathology" (C. Robyn and M. Harter, eds.), p. 59. Elsevier/North-Holland Biomedical Press, Amsterdam, 1978. 3 j. Djiane, H. Clauser, and P. A. Kelly, Biochem. Biophys. Res. Commun. 90, 1371 (1979). 4 j. Djiane, P. Durand, and P. A. Kelly, Endocrinology 100, 1348 11980). 5 R. P. C. Shiu and H. G. Friesen, J. Biol. Chem. 249, 7902 (1974). 6 R. P. C. Shiu and H. G. Friesen, Biochem. J. 157, 619 (1976). 7 R. P. C. Shiu and H. G. Friesen, Science 192, 259 (1976).

METHODS IN ENZYMOLOGY, VOL. 109

Copyright © 1985 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-182009-2